Biofilms Control: Biomedical and Industrial Environments 1783323930, 9781783323937

BIOFILM CONTROL gives a concise picture of advancements in the field and about newer approaches. Biofilms are ubiquitous

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Table of contents :
Cover
Title page
Full title page
Copyright
Preface
Contents
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Chapter 6
Chapter 7
Chapter 8
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BIOFILM CONTROL in

Biomedical

and

Industrial Environments

BIOFILM CONTROL Biomedical

in

and

Industrial Environments

Editors P. Sriyutha Murthy Raju Sekar Vengatesan Thiyagarajan

α Alpha Science International Ltd. Oxford, U.K.

Biofilm Control in Biomedical and Industrial Environments 258 pgs.

Editors P. Sriyutha Murthy Biofouling and Thermal Ecology Section Water and Steam Chemistry Division Indira Gandhi Centre for Atomic Research Kalpakkam Raju Sekar Department of Biological Sciences Xi’an Jiaotong-Liverpool University Suzhou Vengatesan Thiyagarajan School of Biological Sciences The University of Hong Kong Hong Kong SAR Copyright © 2019 ALPHA SCIENCE INTERNATIONAL LTD. 7200 The Quorum, Oxford Business Park North Garsington Road, Oxford OX4 2JZ, U.K. www.alphasci.com ISBN 978-1-78332-393-7

Printed from the camera-ready copy provided by the Authors.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the publisher.

Preface Microorganisms originally were thought to exist as planktonic forms; investigations over the years have shown bacteria to predominately form biofilms in virtually all aquatic ecosystems. In nature biofilms offer a protective niche that allows bacteria to survive in hostile environments. Even though the basic factors for biofilm formation like temperature, surface properties, nutrient availability and hydrodynamic conditions, are common, each aquatic body is unique with respect to its physiochemical properties and constituent microorganism’s. Hence biofilms growing in drinking water distribution systems, wastewater and sewer systems, oral cavity, on dairy equipment, cooling water systems, and desalination plants, medical and prosthetic structures, shipping industry and petroleum wells are all unique with respect to their community structure and composition thus differing in their physiological conditions and interaction with surfaces. It is appropriate to understand their community structure and physiological condition for developing a suitable control measure in the case of detrimental biofilms. In most cases control measures needs to be tailor made for a specific condition and process depending on the severity of the problem and environmental variables. Further application of biocides for biofilm control and environmental issues form two faces of the same coin. Designers and operators will have to choose on the best available technology taking production and environment into consideration. Conventionally biofilm control measures have been by treating the bulk water by biocides which is an ongoing economically unviable practice in several industrial systems. Technological advancements in polymer sciences have now enabled intelligent biocide delivery system at the surface, using porous polymeric materials. In principle biofilms are a surface associated phenomenon and such control measures would ensure a cleaner surface, reduced biocidal requirements and minimize the impact on the environment. It is now evident that there is no such thing as a universal solution to the problem of biofilms as well as complete control of biofilms in industrial system is practically impossible. Operators are now tuning to the concept of maintaining biofilms within threshold limits such that it does not interfere with normal operation of plants. Again the cost cleanliness factor seems to prevail on the choice of biocide for industrial applications. In contrast, the beneficial use of biofilms is gaining importance in the field of bioremediation of waste, right from treating wastewater to hazardous nuclear wastes. In the last two decades numerous studies have been carried out on biofilm properties, its development, interactions and species characterization with respect to each of the industrial systems described above. It is important to bring out these latest developments in this volume. Further methods involved in characterization of biofilms have significantly developed over the years with the advent of confocal, Raman and atomic force microscopy coupled with fluorescent dyes. Each of the techniques has been shown to have limitations in characterizing environmental biofilms which have been discussed at length in this volume. It is becoming increasingly evident that biofilms are indeed ubiquitous. Field level studies have thrown much light into the nature of biofilms growing in specialized environments and the levels of disinfection achieved using conventional approaches. Studies on alternate approaches for biofilm control have also shown promising results. This book gives a concise picture of advancements in the field and about the newer approaches. P. Sriyutha Murthy Raju Sekar Vengetesan Thiyagarajan

Contents Preface v 1. Attacking Multispecies Biofilms n Industrial Environment P. Sriyutha Murthy

1.1

2. Microbial Biofilms and Human Health associated with Indwelling Devices Fabian Davamani, Wong Woan Jiun and Ebenezer Chitra

2.1

3. Gut Biofilms: Tug of War between Good and Bad Bacteria in Human Health Asit Ranjan Ghosh

3.1

4. Candida Biofilms: Characteristics in Environment Settings and Novel Therapeutic Options in Clinics Chaminda Jayamphath Seneviratne, Preethi Balan and Nhat Quynh Thuyen Truong

4.1

5. Current Strategies to Reduction of Marine Biofilm Formation

5.1

T. G. Vladkova and D. T. Akuzov 6. Biofouling and Biofilm Prevention on Aquatic Sensors T. Sullivan, A. Barrett, A. C. Power and F. Regan

6.1

7. Environmentally Benign Marine Antifouling Coatings Sitaraman Krishnan

7.1

8. Nanoparticulates and Nanocomposites as Antibiofilm Agents: Evolving Perspectives Prasana K. Sahoo, Richard Janissen, Arindam Das, D. Inbakandan and P. Sriyutha Murthy

8.1

    

  

ATTACKING MULTISPECIES BIOFILMS IN INDUSTRIAL ENVIRONMENT 

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1.2 

Biofilm Control

  Biofilms have been found to occur 3.5 million years ago [1], and about 95-99% of microbial populations exists in the form of biofilms [2]. However, biofilm formation was only reported as early as 1936 by Zobell and Anderson [3] who observed bacteria on sand particles in seawater. In nature, biofilms constitute the mode of life for microorganisms with a planktonic phase aiding in dispersion and recolonization. Some features or characteristics of biofilms which we have come to understand is that biofilms are a community of microbes enclosed in an extracellular matrix either attached to a surface or co-aggregate with each other forming substratum less biofilms (granules / flocs / mats). In this article, we will deal mainly with detrimental biofilms occurring on abiotic or manmade surfaces in industrial systems. The purview of this article is limited to biocidal control of bacterial and algal multispecies natural biofilms in industrial environments. Four typical industrial environments have been taken up for discussion namely 1) industrial cooling water systems (CWS), 2) drinking water distribution systems (DWDS), 3) diary & food processing industries, 4) specialized environments (dental unit water system; hospital and biomedical equipment). The purpose of this article is to bring out the unique nature of each of these industrial environments in which these biofilm forms, review the progress made in the application of biocides and recommend a fair control practice for different industrial systems based on know-how. Further literature on biocidal concentrations on mono and dual species cultures and mechanistic aspects of biocides is also reviewed for better understanding of disinfection efficacy.

        

     It is of interest to know why bacteria form biofilms and what advantages biofilms offer to its residents. However, a direct answer to the first question is still elusive due to the multifaceted nature of biofilm formation and adaptive plasticity shown by bacteria to environmental variables. On the other hand, we have been able to understand the advantages to microbes in a biofilm mode. One of the simplest answers to the first question attributes the presence of a suitable substratum to trigger biofilm formation. Bacteria are known to sense surfaces and this sensing triggers a signalling cascade which initiates gene regulation necessary for biofilm formation [4, 5]. Once again the sensing mechanisms utilized by bacteria are not clearly understood / investigated. Once the bacteria are near the surface various structures like flagella, fimbriae, outer membrane proteins (OMP), curli and EPS aid in adhesion and biofilm formation [6]. In comparison, a volume of literature exists on involvement of expression of certain genes in the biofilm mode which has been observed in the phenotypic switching from a planktonic to a biofilm mode [7, 8]. Analysis of such gene expressions has revealed several reasons for bacteria to adopt the biofilm mode. Biofilm formation may be a cause of 1) protection against harsh environmental conditions (viz: a stress response or defence against a host); 2) nutrient immobilization from bulk phase; 3) division of labour (metabolic burden) [8]. Others investigators have attributed biofilms as a survival strategy for bacteria [6]. Alternately it has been proposed that biofilm mode of growth may be the default mode of growth and instead studies onto what triggers planktonic mode needs to be investigated [8]. To support this concept a natural example of bacterial existence in the biofilm mode can be observed in oral Streptococci which are specific to the oral cavity. Planktonic mode of existence of these microbes would have resulted in washing by saliva, swallowed and destroyed by gastric juices [9]. Studies like these reiterate that bacteria in nature generally exist in biofilms.

Attacking Multispecies Biofilms in Industrial Environment

1.3 

  Multi species or polymicrobial biofilms are the predominant form of lifestyle of microorganisms in nature occurring both on biotic and abiotic surfaces. Biofilms occur on the river / stream bed, oil rigs, ship hulls, heat exchanger surfaces, sea or fresh water pipelines, manmade submerged structures in fresh and seawater, sub tidal and intertidal rock surfaces, DWDS, medical equipments, and tooth surfaces. Biofilms formation on these surfaces is all unique and influenced by nutrient levels, hydrodynamics, species availability and composition and physiochemical properties [10]. The microbial consortia of natural multi species biofilms vary from archae, bacteria (aerobic and anaerobic), protists and higher (Eukaryotic) organisms in mature biofilms. Understanding the mechanism of multi-species biofilm formation and their microbial composition will facilitate the development of methods for their control. The phototrophic components of natural biofilms are comprised of cyanobacteria, green algae and diatoms [11] which produce organic substrates and oxygen in the matrix which in turn supports the growth of heterotrophs. The phototrophic components also play an important role in cycling of carbon and nutrient and the mixed consortia are also involved in biogeochemical recycling in aquatic benthic environments. Similar to bacterial species, phototrophic organisms adhere by producing extracellular polymeric substances (EPS) comprised of polysaccharides, proteins and humic substances [12]. Their diversity varies depending on the natural aquatic body viz: fresh water streams, source water reservoirs, marine and brackish water environments. In natural multispecies biofilms diatoms are found to co-exist with bacteria [13]. These bacteria in natural biofilms may have stimulatory or inhibitory effect on the algal community [14]. Such interactions were demonstrated for two diatom species Amphora coffeaeformis and Cylindrotheca closterium with the bacterium Pseudoalteromonas sp. [15]. Pseudoalteromonas sp. used in the study was found to adhere strongly with C. closterium rather than A. coffeaeformis. In general, multi-cellularity in natural biofilms, offer several advantages to the members which will be unfolded with respect to disinfection strategies in the following paragraphs.

         Natural multispecies biofilms is a wide ranging area which involves a multitude of prokaryotic and eukaryotic diversity. Each industrial environment is unique with an extremely diverse native microbiota. Biofilms developing on heat transfer surfaces viz: condenser tubes (cupro-nickel, aluminium brass) are rather unique as they have to face adverse environmental conditions like biocides dosed for biofouling control, an underlying toxic metal surface, elevated surface temperatures of the equipment as well as bulk fluid. Analysis of microbial community in biofilms developed on CuNi 70:30 and aluminium brass (Al brass) using denaturing gradient gel electrophoresis (DGGE) revealed bacterial taxa belonging to Marinobacter, Alteromonas and Pseudomonas were dominant [16] in biofilms on these surfaces. Bacterial analysis on condenser surfaces (Titanium) from a nuclear power plant revealed novel taxa (Pseudoalteromonas, Pseudoalteromonas carrageenovora, Hyphomonas, Planctomycetes), resistant to chlorination [17, 18]. Rhodobiaceae and Rhizobiales were the two rare OTU’s detected in the study. Hyphomonas sp. is a common colonizer in marine habitats and has been reported to persist in chlorinated environments [19]. Pseudoalteromonas ruthenica has been isolated from chlorinated cooling water systems of a coastal nuclear power station [20]. Legionella pneumophila along with other heterotrophs is a common bacterium occurring in biofilms in cooling towers using recirculating fresh water [21]. This bacterium was found to occur in equal densities with other heterotrophs in biofilms in cooling tower fill material. Screening of different materials revealed accumulation of L. pneumophila was the lowest on polyvinyl chloride (PVC), polypropylene, and polyethylene surfaces compared to stainless steel in this study. Maukonen and Saerela (2009) [22] have catalogued beneficial and detrimental microbes present in food processing industrial environment. Listeria monocytogenes, Bacillus cereus, Clostridium

1.4 

Biofilm Control

perfringens are the major species present [23]. For microbial species in marine sediments, chronic wounds, urinary catheter, dental plague, sludge see review by Yang et al., (2011) [24]. In contrast, biofilms in paper board industry constitute Bacilli, Enterobacteria, Pseudomonads, Clostridia, moulds, yeast and sulphate reducing bacteria [25]. In seafood processing industries, the major bacterial genera involved in contamination are Salmonella sp., Bacillus sp., Aeromonas sp., Pseudomonas sp., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus are the chief contaminants [26, 27, 28]. P. fluorescens and P. putida are the main cause of spoilage of fresh fish [29]. In dairy environments, the chief bacterial genus involved is Enterobacter, Lactobacillus, Listeria, Micrococcus, Streptococcus, Bacillus, and Pseudomonas [30]. Thermoduric Streptococci and Bacillus sp. in pasteurized milk are indicators of contamination caused by dispersion of biofilms [31]. For a detailed list of species colonizing dental unit water systems (DUWS) sampled from 107 dental reservoirs refer Szymanska and Sitkowska (2013) [32]. Major groups belong to the family Burkholderiaceae, Pseudomonadaceae, Ralstoniaceae and Sphingomonadaceae recorded in this study. In addition, studies by Singh et al., (2003) [33], revealed members of bacterial genera such as Leptospira, Sphingomonas, Bacillus, Escherichia, Geobacter and Pseudomonas occurred in DUWS. In DWDS pipelines, major bacterial groups isolated include Acinetobacter, Aeromonas, Alcaligens, Arthrobacter, Corynebacterium, Bacillus, Burkholderia, Citrobacter, Enterobacter, Flavobacterium, Klebsiella, Methylobacterium, Sphingomonas and Xanthomonas [30]. Molecular analysis of biofilms in DWDS revealed -, -, and gamma-Proteobacteria and Gram positive bacteria [34]. In DWDS, the most commonly occurring pathogens include Burkholderia pseudomallei, Campylobacter spp., Escherichia coli, Helicobacter pylori, Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio Cholerae, Klebsiella pneumonia [35,30]. In addition, DWDS also have a large proportion of enteric viruses (adenovirus, rotavirus, norovirus) and parasitic protozoans (Cryptosporidum parvum) [36]. Long term application of biocides (free chlorine, chlorine dioxide, hydrogen peroxide, silver and silver with peracetic acid) in DWDS caused a population shift in biofilms which were treated and regrown indicating a selection pressure by biocides [37]. 

   

 Biofilms cause significant damage to equipment, contamination of products, energy loses, infection to humans in engineering and biomedical systems. In engineering systems, biofilms cause physical damage e.g. blockage of condenser tubes, cooling tower fill material [38], reduction in heat transfer [39] increase pressure drop across pipelines affecting flow and heat transfer [40], biocorrosion and pitting [41], turbine power loses in hydroelectric pipelines [42] reservoir of potential pathogens in drinking water pipelines and distribution systems [36], increase frictional drag on ship hulls [43]. Thermodynamic analysis and energy balance studies for power stations revealed that an increase in condenser fouling factor from 0.00015-0.00035 m2 K/W resulted in a decrease in plant output power and thermal efficiency of 1.36% and 0.4448% respectively [44]. For a detailed literature on cooling water system failure analysis refer to the Nalco guide [45]. In cooling water systems, reduction in heat transfer by biofilm formation in heat transfer equipment and microbiologically influenced corrosion (MIC) are major operational problems to be overcome. Each type of bacteria causes corrosion by creating new reactions at the anode / cathode due to their metabolites. Some of these are sulphate reducers like Desulfovibrio, Desulfomonas and Desulfotomaculum, the three major anaerobic forms influencing corrosion in CWS. Another type of bacteria is acid producers belonging to the genera Thiobacillus and Clostridium sp., which produces metabolites in addition to sulphuric and organic acids. Other iron oxidizers include Sphaerotilus, Crenothrix, and Leptothrix species. Other groups of acid producers include the nitrifying bacteria which oxidize ammonia to nitrate. Nitrobacter species reduces the pH by oxidizing the nitrite to nitrate producing nitric acid in the process which leads to

Attacking Multispecies Biofilms in Industrial Environment

1.5 

corrosion. The third variety is metal depositors viz: a bacterium (Gallionella sp.) which oxidizes ferrous iron (Fe++) to ferric iron (Fe+++) leading to formation of ferric hydroxide at surfaces. Bacterial genera belonging to Pseudomonas, Rhodotorula, Flavobacterium, Acidovorax delafieldii, Cytophaga johnsonae, Micrococcus kristinae, Acidovorax sp. and Sphingomonas sp., have been reported from copper surfaces and are known to be involved in microbial influenced corrosion of copper pipes [46]. In food processing industry bacteria form spores that contaminate process equipment and products [47]. In addition, biofilms cause problems in granular activated carbon columns, degasifiers, reverse osmosis membranes, storage tanks and micro porous membrane filters [48]. In the paper board industry, boards which are used for food packaging are contaminated by aerobic and anaerobic bacteria such as Bacilli sp. and Clostridia sp. which is not killed during drying process, poses a health risk [49]. Biofilms pose a problem in cleaning and hygiene in food industrial environments e.g. biofilms components have been demonstrated to protect Bacillus sp. by improving their heat resistance and extending the autoclaving time required for sterilization upto several hours [50]. In the manufacture of paper, operation of paper machine, life and quality of final product [51] are some of the problems caused by deposit + slime formation [52]. Chemical dosing to control non-biological deposits serve as nutrients for microbial growth.

      

It is now understood that multispecies natural biofilms are different and complex and extrapolation of laboratory results from monospecies biofilms are imprecise [53]. Interactions in multispecies biofilms are vital as they influence the volume and function of biofilms both quantitatively and qualitatively [53]. Roder et al., (2016) [54] have stressed “the need for getting closer to the natural scenario” in conducting laboratory experiments, by using strains isolated from natural environment for studying interactions between bacterial species. Basic investigations on complex bacterial communities point to either cooperation or competition to play a vital role in shaping multispecies biofilms. Indirect interactions involve the presence of some species facilitates biofilm formation by others. In addition, direct cell-cell interactions (co-aggregation) of genetically distinct strains have been reported to occur in multispecies biofilms in DWDS, the human oral cavity, urinogenital tract [55]. Alternatively, certain species of bacteria have been shown form auto and co-aggregates [56]. Detailing into the social nature of biofilms is not within the scope of this article which is dealt in individual articles in this book. However, we would like to bring out certain interactive features in multispecies biofilms which respond to environmental stress (biocides). Interspecies interactions in multispecies biofilms offer several advantages for the residing species as increased tolerance to biocides, protection against grazing [57, 58], increased virulence [59], cometabolism and syntrophy where one species feed on the by-product of the other [60]. A typical example of one such interaction (co-operation) in multispecies biofilms is with respect to the stress produced by the surrounding environment may highly affect the fitness and losses in a community [53]. A species even though faces a reduction in biomass may benefit by increased protection from various stresses thereby establishing itself in a wider niche resulting in overall fitness gain. This scenario may be one of the best plausible reasons for the observed increased antimicrobial resistance of biofilms to biocides. In contrast to cooperation, competition also exists in multispecies biofilms. An example of this is the production of biosurfactants by bacteria altering the surface properties such as wettability and charge [61]. These weaken bacteria-surface; bacteria-bacteria interactions therefore reducing the ability of certain strains to form biofilms [62]. Apart from inhibiting certain bacteria to colonize and form biofilms other interactions like inhibition of biofilm maturation; jamming of communication to newcomers; degradation of protein & polysaccharide components of matrix are other non-biocidal activities performed by species in multispecies biofilms [62]. A major lacunae for

1.6 

Biofilm Control

carrying out studies related to multispecies biofilms is lack of experimental systems mimicking natural conditions which is one of the reason for limited number of studies in this field. Currently studies at pilot scale levels are needed for a more realistic characterization of biofilms occurring in the environment.

     

Fresh / seawater are an essential component in industrial processes. For a detailed literature on different aspects of CWS such as engineering design, structures, biofouling surveillance, operational problems and environmental issues refer to reviews / reports by [63, 64, 65, and 66]. The primary use of water in industries is that of a heat sink. Requirement of water for cooling purposes in a typical thermal power plant is around 65 m3 s-1 [67]; a nuclear power plant uses about 30 to 45 m3 s-1 for 1000 MWe generation [68] and in an average about 27 billion m3 is used annually by power plants in Germany [69]. Other uses of water are for washing or conditioning of equipment and products in the paper board & food / dairy industry which are very miniscule compared to the usage for cooling purposes. The nature of fouling differs according to the design of the CWS and process parameters as well as it is governed by the presence of candidate organisms in the incoming water. Industries are located besides large water bodies either in, inland / coastal regions based on the water availability. Most of industrial cooling water systems invariably fall under two major categories: a) Open / closed recirculating systems with cooling towers and b) once through seawater cooling systems [70]. The open / closed recirculating systems are predominantly fresh water with rare instances of seawater being used in a recirculating mode. In these recirculating systems around 70% of the heat is removed by evaporation through cooling towers. In recirculating freshwater systems, biofilm formation, bacterial growth in recirculating water, scaling, microbial corrosion, fouling of cooling tower fills, aqueous corrosion due to increase in cycles of concentration (COC) are all problems to be tackled. Evaporative losses and blow down are compensated by periodic replenishments of fresh water which brings along with it a biological load which needs to be treated. In addition to oxidizing biocides, several QAC (e.g. isothiazalones) have also been used for improved efficiency and cost effectiveness compared to oxidizing biocides. Dispersants, surfactants have also been used in these systems for cleaning in place purposes which again pose a problem by increasing the nutrient levels in the residing water. Scale / corrosion inhibitors such as phosphate, phosphonates, nitrites, silicate and molybdates are dosed which form a passive layer on the surfaces. Residues accumulate and active ingredients favour microbial growth as they increase the nutrient load in recirculating waters.

     

Choice of biocide for a cooling water system depends on 1) type of organism whose growth needs to be controlled, 2) regime (concentration and contact time), 3) interaction with fresh / seawater, 4) disinfection efficacy, 5) by-product generation, 6) cost, 7) availability and ease of handling and 8) environmental issues. In turn, the efficacy of a biocide depends on 1) diffusion, 2) penetration and 3) interaction at target site [71]. Every cooling water system is unique and each industry draws water from a local source which may have unique water quality as well as ecology. Hence, a biocidal regime has to be tailor made for a cooling water system [72]. Dosing insufficient levels of biocide will lead to increase in biofilm accumulation [73]. Hence it is important to optimize the biocidal regime to prevent biofilm growth such that it does not hinder with operation of the equipment (keeping biofilms within threshold limits).

1.7 

Attacking Multispecies Biofilms in Industrial Environment

Biocidal regime has to be optimized on trial basis at a given industrial site and system using either a pilot scale or side stream monitoring device. Different biocidal dosing strategies have been recommended [73] based on extent of biofouling problem. 1) Continuous dosing (where copious biofilm development occurs in large systems due to continuous supply of nutrients and organisms), 2) shock dosing (short burst of high concentration of biocides), in this case the duration of each burst depends on the extent of fouling and disinfection achieved. This method is adopted to meet the biocide discharge criteria as well as achieve reasonable control on biofilms. 3) Pulse dosing (similar to shock dosing but on more frequent application). Continuous monitoring using side stream device and frequent adjustments /switching between modes, synergistic use of biocides need to be followed for effective biofilm control. Compared to other oxidizing biocides like chlorine dioxide and ozone a large number of laboratory studies have investigated the effects of chlorine / sodium hypochlorite (NaOCl) / monochloramine as antimicrobial agent on planktonic cells and biofilms. Few studies are discussed here to understand the concentrations of the biocide required to achieve disinfection with various bacterial genera. [72, 74, 75].

  

Oxidizing biocides like chlorine, chlorine dioxide, bromine chloride, ozone, hydrogen peroxide and peracetic acid have all been used in industrial systems. A general feature observed with oxidizing biocides is recovery and regrowth of bacteria upon cessation of dosing in industrial systems. Chlorine has been used from the 1900’s and is the main workhorse biocide for drinking water treatment and also in industrial CWS for biofouling control [76]. For detailed literature on chlorine chemistry, production engineering, handling, toxicity, by-product formation, storage, safety and applications refer “Handbook of Chlorination” by White (1985) [77]. Advantages of chlorine are its industrial reliability, large scale applicability, broad spectrum of antimicrobial activity and rapid decay in environment. A major disadvantage of chlorine is that it reacts with nitrogenous compounds in water to form substitution products such as chloramines. 0.2 mg/l

0.5 mg/l

1.0 mg/l

E. coli

S. aureus

Control

Figure 1: Scanning electron microscopic (SEM) images of 12 hour old biofilms of S. aureus and E. coli developed on silicon wafers subjected to sodium hypochlorite after 60 minute exposure to the biocide.

1.8 

Biofilm Control



Figure 2: Fluorescent micrographs of 12 hour old biofilms of S. aureus and E. coli developed on silicon wafers subjected to hypochlorite after 60 minute of exposure to the biocide. Cells in biofilms have been stained by using BacLight® Live/Dead staining kit. Cells whose membranes compromised are stained red.



Most of the oxidizing biocides disinfect by 1) oxidizing the chemical moieties of the cell membrane and 2) gain access to the interior of the cell by passage through the membrane. In general, neutral molecules can pass through the cell wall than charged ions. Hypochlorous is an effective disinfectant than hypochlorite ions. Figure 1 shows the susceptibility of biofilms of Gram-positive Staphylococcus aureus as well as Gram-negative Escherichia coli to different concentrations of sodium hypochlorite. Damage to cell wall is more pronounced in E. coli compared to S. aureus which have been shown not to lose their morphology. Figure 2 shows staining by BacLight(R) Live/Dead stain which shows both green and red fluorescence indicating the levels of live and dead bacteria in biofilms, respectively [78] of both species after exposure to hypochlorite for 60 minutes. In S. aureus the tested concentrations of hypochlorite did not cause damage to the cell wall components but were able to pass through the membranes whereas with E. coli cell wall damage was observed with leaking of intracellular material. Studies on antibacterial activity of chlorine on planktonic as well as monospecies are discussed herewith. Hypochlorite has been reported to be an effective antimicrobial agent against S. aureus, Prevotella intermedia, Peptostreptococcus miros, Streptococcus intermedius, Fusobacterium nucleatum and Enterococcus faecalis [79, 80]. 6% NaOCl significantly killed E. faecalis biofilms. Concentrations of 100 mg/l free chlorine for 30s was required to eliminate planktonic cells (L. monocytogenes, S. xylosus, P. fragi) but only 2 log reductions were achieved for L. monocytogenes biofilms at concentration of 1000 mg/l and exposure times of 20 minutes [81]. Studies by Lomander et al., (2004) [82] revealed concentration of 50 mg/l NaOCl was required to eliminate biofilm cells. Chlorine residuals at 1.0 mg/l were effective in killing protozoa isolated from cooling tower biofilms within 4 hours of exposure [83]. Comparison of killing using isothiazolinones (blend of 2-methyl-4isothiazolin-3-one and 5-chloro-2- methyl-4-isothiazolin-3-one) required concentrations of 150 mg/l for inactivating trophozoites of the species Acanthamoeba. Comparison of different biocides (hypochlorite, monochloramine - NH2Cl, bromo-chlorodimethylhydantoin, STABREX®- stabilized hypobromite biocide, mixed solution of NH4Br and HOCl) on P. fluorescens, P. aeruginosa and K. pneuomoniae were examined [84]. Results showed biofilms were more resistant to all the oxidizing biocides tested compared to their planktonic counterparts. Hypochlorite at concentration of 1.5 mg/l caused injury in P. fluorescens whereas lower concentrations (0.8 mg/l) inflicted injury in P. aeruginosa cells. NH2Cl was less effective on suspended cells than HOCl but was effective as HOCl on biofilms. Biocidal efficacy was again reported to be influenced by biofilm type. NH2Cl, mixed solution of NH4Br and HOCl were effective on P. fluorescens biofilms whereas STARBEX® showed weak disinfection on biofilms of P. fluorescens and K. pneumonia.

1.9 

Attacking Multispecies Biofilms in Industrial Environment

Again HOCl and 1-Bromo-3-chloro-5, 5-dimethylhydantoin (BCDMH) showed maximum biocidal activity in initial exposure. But both of these biocides were found to be effective on biofilms of K. pneuomoniae in this study. Helicobacter pylori was observed to be more resistant to chlorine than E. coli with 0.3 and 0.9 log reductions observed at 0.1 mg/l of chlorine [85]. Similar results were observed with the biocide ozone in this study. Table 1: Types of biocides and their dosing in industrial systems [86, 87, and 88]. Oxidizing biocides Chlorine (gaseous, hypochlorite)

Concentrations (residuals) / regime 0.1 – 0.2 ppm continuous

Chlorine dioxide Bromine chloride Ozone Hydrogen peroxide 1-Bromo-3-chloro-5,5- dimethylhydantoin Peracetic acid (Peraclean®) Electrochemically activated water Non oxidizing biocides Isothiazolinones 2,2-Dibromo-3-nitrilopropionamide (DBNPA) 2-Bromo-2-nitro-propan-1,3-diol (Bronopol) Glutraldehyde Quaternary ammonium compounds Alkyl dimethyl benzylammonium chloride Methylenebisthiocyanate S-Triazine Dodecylguanidine hydrochloride Polyhexamethylene biguanide (PHMB) Benzalkonium chloride Conditioners & auxiliary additives Phosphonates Phosphate Polycarboxylates Zinc Molybdate Triazole derivatives Fatty amines (Mexel® 432)

0.05 – 0.1 ppm continuous 0.1 - 0.2 ppm continuous 0.01 – 0.2 ppm continuous 1-5 ppm intermittent 2 - 7 ppm intermittent Product recommendation Nascent trials Concentrations 100 µl slug 10 ppm slug 35 ppm slug 30 ppm slug 50µl slug 10 ppm slug 2-6 ppm slug Product recommendation Product recommendation Product recommendation Product recommendation Concentrations 3 – 8 ppm cont / intermittent 12 – 15 ppm cont / intermittent 6 – 12 ppm cont / intermittent 1 – 3 ppm cont / intermittent 5 – 7 ppm cont / intermittent 1 – 2 ppm cont / intermittent Product recommendation

In comparison to chlorine; concentration of one order of less in magnitude is required by chlorine dioxide for reacting with cell components of microbes. Chlorine dioxide, is a powerful oxidizing agent which is 2.5 fold effective than chlorine, non-corrosive, broad working range of pH (3.0 - 8.0) [89, 90]. Chlorine dioxide was effective against two enteric bacteria such as Yersinia enterocolitica and Klebsiella pneuomoniae at concentrations of 0.25 mg/l [91]. Similarly chlorine dioxide was active against Vibrio parahaemolyticus associated with oyster tissues and concentrations of 20 mg/l with exposure times of six hours were required for complete disinfection [92]. Choi and Kim (2010) [18] compared efficacy of different biocides like free chlorine, ozone, chlorine dioxide and UV irradiation

1.10 

Biofilm Control

on E. coli. All the biocides inactivated cells at varying degrees. Cell surface damage was more pronounced with ozone compared to chlorine and chlorine dioxide shown inactivation between these two disinfectants for these bacteria.

   

QAC constitute a major class of non- oxidizing biocides which are classified as anionic, cationic or zwitterionic [93]. A general feature of these biocides is development of resistance by microbes [71] in a CWS. They are effective in preventing algal growth and control of algal biofilms. Copper sulphate, QAC and photosynthesis inhibitors like Triazine derivatives are generally used in recirculating CWS. Aldehydes, like glutraldehyde is effective against algae and used in recirculating CWS [94]. Their only disadvantage is they are only active at alkaline pH range of 7.5 - 8.5 [95]. Comparison of different biocides using a continuous flow heat exchanger loop revealed similar levels of disinfection efficacy [71]. The biocides tested were alkyl dimethyl benzyl ammonium chloride (ADBAC 30 ppm; intermittent regime), 5-chloro-2-methyl-3-isothizolone (CMI) / 2 methyl-3-isothiazolone (MI) mixtures at 3:1 ratio (4 ppm; intermittent regime), glutraldehyde (100 ppm; intermittent regime), halohydantoin (10 ppm; intermittent regime), isothiazalones (4 ppm + DIDMAC 12 ppm; intermittent), disinfectant cleaner 500 ppm and 2000 ppm slug treatment. Isothiazolinones are effective against a wide variety of bacteria. Bronopol (2-bromo-2-nitropropane-1, 3-diol) an isothiazolone acts on thiol groups of amino acids (cysteine and glutathione) and on enzymes. Other isothiazalones which have demonstrated antibacterial activity are 5-chloro-2-methyl-3-(2H)-isothiazolinone (CMIT), 2-methyl-3(2H)-isothiazolinone (MIT) which are used in a ratio of 3:1 in industrial CWS are known to be broad spectrum in their efficacy against bacteria, algae and fungi [96]. Another member of this group benzisothiazolone (BIT) which is commonly used in industrial systems act on bacterial cell membranes and cause leakage of K+ ions [97]. These biocides act as electrophilic agents, reacting with critical enzymes to inhibit growth and metabolism of cells. These have been reported to inhibit ATP production in bacterial cells [95] and hence are considered very effective. Bisphenols like hexachlorophene and 2, 2’-methylenebis (4-chlorophenol) which are membrane active agents have also been used for biofilm control in CWS [98]. In comparison to phenols, methylene bis thiocyanate methylenebisthiocyanate were found to ineffective against biofilm formation [88]. Generally nonoxidizing biocides are administered in small quantities in fresh water open or closed recirculating systems and are very effective at low concentrations. Porins present on the outer membrane of Mycobacteria have been attributed to their increased resistance to isothiazolinone biocides as well as lipophilic biocides [99]. Benzalkonium chloride (BAC) adapted P. aeruginosa exhibited a higher ability to adhere to surfaces and develop biofilms [100] on plain as well as the biocide conditioned polystyrene surface. Campanac et al., (2002) [101] have demonstrated that increase in C-chain length of the biocide benzalkonium chloride (BAC) increased the level of resistance in P. aeruginosa CIP A22 strain.

      ­€€‚€

A hallmark of biofilms is the formation of EPS matrix comprised of proteins, DNA and cells attached to the surface. Dispersants and surfactants are agents primarily used for breaking the biofilm matrix and removal of biofilms. Chemical Dispersants / surfactants widely used are based either on anionic (pH plastic > glass > stainless steel [279]. Chlorine exposures at 200 µg/ml for 15 minutes were effective on stainless steel and glass compared to plastic and wood in this study. In comparison, lesser concentration of chlorine dioxide 50 µg/ml was required for control on same surfaces. Biofilm control strategies for the dairy and food processing industry pose a challenge and methods are being researched and being improved.

                

   ­€‚ƒ€­„…„­„  † Dental unit water lines are used for washing and cleaning of oral cavity during treatment. Source of contamination are biofilms colonized on the inner surfaces of polyurethane / plastic tubing as well as rubber joints. In addition, the flow rates in these tubing’s is generally around 60 – 100 ml/minute and also infrequent which keep these tubing’s in a moist wet condition. European Union (EU) guidelines prescribe tap water with less than 100 cfu/ml at 22oC and 20 cfu/ml at 37oC for DUWS [280]. The American Dental Association (ADA) have prescribed a threshold value of 95% removal of biofilms. A major study was carried out across European countries by Schel et al. 2006 [287] to assess which of the biocides comply with ADA standards using different DUWS. Biocides tested were Alpron BRS solution (sodium hypochlorite & citric acid; 1-2% Once); Alpron Mint (Sodium-p-toluolsulfonechloramide, EDTA 30,000,000

Rate of infection (%)

Risk of mortality

10-30

Low

600,000 15,000 5,000,000

1-3 1-3 3-8

Low Low Moderate

85,000

1-3

High

Among all hospital acquired infections in humans, 65% are estimated to be biofilm origin [6]. Biofilms are a significant threat that enormously affects human health [4]. The extensive effects on human health are often related to prolonged hospital stay, economic burden, and increases patient morbidity and mortality rate [4,6]. Microbial biofilms often associated with indwelling medical devices such as endotracheal tube, heart valves, urinary catheters, contact lenses, central nervous system shunts,

2.2 

Biofilm Control

orthopedic prostheses, peritoneal dialysis catheters, central venous catheters, artificial voice prostheses, and penile prostheses and lead to infections (Table 1) [7].

   Biofilms are communities of multi-species bacteria (Figure 1) [6, 8]. The first step of biofilm formation (Figure 2) is the irreversible attachment of cell to a surface which greatly depends on cell properties (Table II) [8]. In comparison with planktonic cells, biofilm show different phenotype especially in the sense of gene transcription and interaction [9]. Biofilm Matrix (water + EPS + exudates)

   Act as an outer layer where planktonic organisms colonize and are released. Acts as a medium where nutrients are trapped from bulk water. Coci shaped bacteria 



Rod shape bacteria

Cocci shape bacteria

   1. Composed of multi-species microorganisms 2. Cocci shape bacteria

 Composed of organics primarily colonizing the surface immediately upon contact with bulk medium.

Figure 1: General composition of biofilm formation on surfaces. Table II: Adherence properties of different microorganisms: [7, 10]. Microorganisms Staphylococcus epidermidis

1.

2.

Staphylococcus aureus

1. 2.

Klebsiella pneumonia

1.

Adherence properties Adherence of S. epidermidis is an evolving process. Firstly, bacteria rapidly attach to surface of device that is mediated by nonspecific factors or specific adhesins. Nonspecific factors include surface tension, hydrophobicity, electrostatic forces while specific adhesins include proteinaceous autolysin encoded by atlE gene and capsular polysaccharide intercellular adhesin (PSA) possibly encoded by ica operon. Secondly, S. epidermidis adherence is continued by accumulative phase where bacteria adhere to each other and form a biofilm. The process is mediated by polysaccharide intercellular adhesin (PIA) encoded by ica operon. Adherence of S. aureus depends on presence of host-tissue ligands such as fibronectin, fibrinogen, and collagen. S. aureus adheres to host-tissue ligands through microbial surface components recognizing adhesive matrix molecules (MSCRAMM). Important MSCRAMMs are FnbpA and FnbpB (bind to fibronectin), clumping factor (binds to fibrinogen), and collagen adhesin (bind to collagen). Intercellular adhesion and growth through excretion of specific polymers Express type 3 fimbriae that act as appendages for cellular attachment to a surface.

Microbial Biofilms and Human Health associated with Indwelling Devices

2.3 

             

   Rate of cell attachment depends on physicochemical characteristics of the exposed surface, number and types of cells exposed to the surface, electrostatic forces, shear stress and the flow rate of liquid through the device [11]. The rate of growth will be affected by nutrient availability of medium, flow rate, antimicrobial agent concentration, and temperature [11]. The source of device material, types, surface and shape of device (Table III) may also affect the adherence of bacteria to the devices [7].

Figure 2: Formation of biofilm on surfaces. Table III: Table showing the different materials of device that affects the adherence of bacteria to the device. Indwelling device Source of device material Type

Surface of device

Shape of device

Material properties Synthetic materials promote bacterial adherence more in comparison with biomaterial. Polyvinyl chloride favors bacterial adherence more in comparison with teflon. Latex favors bacterial adherence more than silicone. Polyethylene favors bacterial adherence more than polyurethane. Stainless steel favors bacterial adherence more than titanium. Hydrophobic surface favors bacterial adherence more than hydrophilic surfaces. Smooth surface favor less bacterial adherence. Polymeric tubing favors more bacterial adherence.

   Bacteria in biofilms possess ability to communicate among each other through biochemical signaling known as quorum sensing [9]. In signaling, bacteria in biofilm are able to sense the density and numbers of microorganisms in a biofilm [9]. The quorum sensing system in gram positive bacteria are based on peptide molecules while gram negative bacteria are based on acyl-homoserine lactones [8]. Quorum sensing is an essential element in biofilm development [9]. For example, the quorum sensing

2.4 

Biofilm Control

signal 30 C12-HSL produced by P. aeruginosa cells are necessary for cell to cell communication and for a stable biofilm development [9].

 Biofilms act as source of infection as it provide a reservoir for microorganisms in which after detachment phase, the microorganisms can infect the host [12]. A protective cell layer acts as a first line of defense in preventing microorganisms and other foreign materials from entering the host system [12]. Nevertheless, microorganisms may release toxins, using virulence factors or mechanically damage the cell layer [12]. As the microorganisms penetrate the protective cell layer, they gain entry to the tissue underneath and spread to lymphatic organs [12]. The human immune system recognizes and initiates immune response [12].

       Urinary catheters are widely used to facilitate urethra repair after surgery, manage urinary retention and incontinence in patients [6]. Biofilms development on indwelling urinary catheters made of silicone or latex differs from non-urinary devices due to the presence of urine components [13]. The first step is the attachment of urinary components on the surface of catheter and lead to the development of a protein film which enhances the adhesion of microorganisms and form biofilm [14]. Microorganisms may gain contact with the catheter through extra luminal route (outer surface of catheter) or through intraluminal route (inner surface) (Figure 3) [12]. Escherichia coli often form biofilm on urinary catheter. Adherence of E. coli depends on the device location and also the bacterial strains [7]. E. coli strains that express type I fimbriae is more common in bladder while E. coli strains that express P fimbriae usually infect kidneys [7]. Proteus mirabilis express urease which hydrolyses urea presence in urine and produces ammonia. Ammonia results in elevated pH of urine which allows mineral precipitation and leads to catheter blockage and infection [6]. Other microorganisms regularly associated with biofilms formation are Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter spp. [15]. The risk of catheter associated infection elevates by approximately 10% every day the catheter is placed [12].

Figure 3: Pathways of microbial colonization of urinary catheters



2.5 

Microbial Biofilms and Human Health associated with Indwelling Devices

 Heart valves (Figure 4) are reconstituted when the functions are impaired due to congenital or acquired defects [16]. Implantation of mechanical heart valves causes tissue damage and lead to accumulation of circulating platelets and fibrin to the valves and hence microorganism tends to adhere and develop biofilms and lead to prosthetic valve endocarditis [11]. Microorganisms might originate from skin, other indwelling devices for example central venous catheters or from invasive procedures such as dental work [11]. Primary organisms responsible for prosthetic valve endocarditis include S. epidermidis, Staphylococcus aureus, Streptococcus spp., gram negative bacilli, diphtheroids, and enterococci [11]. Biofilms are commonly formed on the tissue surrounding the device or the sewing cuff fabric which is for the attachment of device to tissue [11].







Figure 4: Mechanical heart valve May be composed of pyrolytic carbon-coated discs, polymers, tantalum, and chromium-cobalt alloy housing which can likely last for up to 25 years [16].

   

Endotracheal tube is an essential component in mechanical ventilation which is a life-saving medical approach for medically debilitated patients [17]. Ventilator-associated pneumonia (VAP) is a frequently acquired infection in intensive care units [18]. Endotracheal tubes are installed through highly colonized oropharynx and larynx into sterile tracheobronchial tree. Microorganisms gain access to the habitually sterile lower tract where they adhere to the mucosa and cause infection [19]. The origin of biofilm is also hypothesized to be from colonization of aspirated secretions from upper respiratory tract [20]. Biofilms formed as early as 12 hours after intubation may be found at the inner or outer surfaces of endotracheal tube [21]. Commonly identified microorganisms from endotracheal tubes of short intubation period are Streptococcus, Prevotella, and Neisseria while Pseudomonas aerugonisa is identified from endotracheal tubes of longer intubation period [17]. In comparison with other gram positive bacteria, Pseudomonas aeruginosa, a gram negative bacterium which possess several virulence factors (Table IV) tends to cause more severe infection [22].

2.6 

Biofilm Control

Table IV: virulence factors identified from Pseudomonas aeruginosa: [2, 23]. Virulence factor Flagella

Pili

Lipopolysaccharides

Alginate

Description Flagella adheres the bacterium to epithelial cells. Flagella also facilitate the early stages of attachment of pathogens to abiotic surfaces. Pili enable gliding motility of Pseudomonas aerugonisa across surfaces. Gliding motility plays important role in the dissemination of biofilm bacteria to new surface for colonization. Lipopolysaccharides on surface of Pseudomonas aerugonisa cells stimulate antigenic response within human body. Presence of lipopolysaccharides is also known to necessary for adhesion to silica and other surfaces. Up regulation of alginate promoter (Alg C) is essential for biofilm development of Pseudomonas aerugonisa cells on surfaces.

   Silicone voice prostheses are inserted in non-sterile habitat in throat cancer patients after laryngectomy for the rehabilitation of speech [24]. Due to the non-sterile habitat, voice prostheses are prone to microbial colonization [25]. Biofilm often forms on the valve of the voice prostheses as microorganisms from the upper airway tract adheres to the surface of the valve and multiplies [26]. As microbial biofilm develops on the esophageal side of the voice prostheses, salivary leakage through the prosthesis valve or around the prosthesis may occurs, difficulty in speaking due to intraluminal obstruction of the prosthesis may occurs, lifespan of the prostheses is reduced, and often require frequent replacement [25, 26, 27]. Anaerobic and microaerophilic pathogens such as Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, and Eikenella corrodens are identified as part of biofilms on voice prostheses [26]. Staphylococcus aureus is another common microorganism associated and its presence often causes malfunction of silicone voice prostheses rapidly [25].

       Subarachnoid screws, cup catheters, and intraventricular catheters are monitoring devices used to monitor and control elevated cerebrospinal fluid pressure or for administration of therapeutic substances [28]. Ventriculoperitoneal or ventriculoatrial shunts are often applied in the treatment of hydrocephalus [28, 29]. Shunts comprised of a catheter inserted into the cerebral ventricles, aims to divert cerebrospinal fluid (CSF) from cerebral ventricles to either heart or abdomen where it is reabsorbed [29]. It is widely used but the risk of infections elevates if implant frequency increases or duration of implantation is prolonged [29, 30, 31]. Staphylococcus epidermidis, Bacillus cereus and S. aureus adhere to the surface of implanted devices and lead to shunt infections [32, 33, 34]. In response to the biofilms, microglia plays an important role in producing pro-inflammatory chemokines and cytokines and display bactericidal activity [31]. Astrocytes potentially able to recognize and respond to PIA from S. epidermidis and hence may function in recognizing biofilm components in the central nervous system [31].

Microbial Biofilms and Human Health associated with Indwelling Devices

2.7 

 Peritoneal dialysis is an alternative way of hemodialysis for patients suffering from end stage renal disease but formation of microbial biofilms on the peritoneal catheters often lead to peritonitis [35, 36]. The quick exchange of dialysate fluid causes intraperitoneal medium to lack of complement and immunoglobulin’s [28]. Infections may also occur if contaminated dialysate fluid is used or the external tubing and bags are contaminated [28].

   Penile prostheses are effective for erectile dysfunction treatment [37]. The most dreadful complication of implanted penile prostheses is infections associated with the device [38]. S. epidermidis is the commonest organism associated with penile prosthesis infection followed by Staphylococcus aureus and Pseudomonas aeruginosa. [38] Staphylococcus species often produce a protective mucin coat after adhering to the device and survive at lowered metabolic rate [37].

    Central venous catheters (CVCs) include various vascular access devices used for pain management, chemotherapy, antibiotics delivery, and monitoring central venous pressure [39]. Common routes of CVCs contamination are contamination of catheter hub by contact with hands, contaminated fluid or devices, and migration of skin microorganisms to catheter insertion site and subsequently colonize the catheter tip [39]. CVCs are exposed to blood stream and hence, the surface of the catheters become coated with platelets, plasma, and tissue proteins which act as conditioning films for adherence of microorganisms [1]. S. aureus adheres to proteins while S. epidermidis adheres only to fibronectin [1]. Catheters placed less than 10 days show more extensive biofilm formation on the external surface while long term catheters show more extensive biofilm formation on internal lumen [1]. As biofilm is formed on the catheters, bacteria may flow into systemic circulation and lead to bloodstream infections [39].

   Orthopedic devices include hip, knee prostheses and screws, pins, plates, and rods for stabilization [28]. Prosthetic joint infections (PJI) are classified according to the occurrence of infections after implantation [40]. Early (within 3 months of implantation) and delayed (3-12 months after implantation) are probably due to invasion of microorganisms during surgery whereas late (after 12 months of implantation) infection is commonly hematogenously acquired [40]. The main microorganisms that cause orthopedic prosthetic device associated infection are S. aureus, coagulase negative staphylococci and other microorganisms (Table V) [28, 41].

2.8 

Biofilm Control

Table V: Table showing the examples of common microorganisms involved in PJI. [28, 41, 42, 43, 44] Microorganism Staphylococci Staphylococci Streptococcus species Enterococcus species & Streptococcus agalactiae Enterococcus species Pseudomonas aeruginosa Enterobacter sp. Enterobacteriaceae Nonfermenters Anaerobes

Remarks Oxacillin susceptible Oxacillin resistant Except Streptococcus agalactiae Penicillin-susceptible Penicillin- resistant Quinolone-susceptible Example such as Pseudomonas aeruginosa Propionibacterium acnes

   Traditionally, the viable microbial load in a biofilm was quantified by counting the colony forming units (CFU) by serial dilution [9, 45, 46]. Although this method gives an estimation of the number of bacteria in a biofilm, it is not an estimate of the actual biofilm, which also includes the extracellular matrix materials as well as dead bacteria. Direct visualization of biofilms by microscopy provides information about the biofilms per se. Biofilm can be visualized by light microscopy, fluorescent microscopy as well as electron microscopy. High resolution electron microscopy provides information about the ultrastructure of bacteria as well as the biofilm matrix [46]. Molecular methods of bacterial identification provide information about the specific bacteria present in the biofilms [47]. Different methods can be used to visualize biofilms formed. Scanning electron microscopy allows visualization with a three-dimensional appearance. However during the process of the sample preparation like fixation, dehydration and coating with a conductive material might destroy some finer details and cause artifacts, however the environmental scanning electron microscope (ESEM) allows investigation of bacteria without any dehydration, fixation or coating of bacteria in the natural state [48]. This is a clear advantage. However, some species such as Fusobacterium nucleatum cannot be optimally visualized with this technique, still a lacunae persists in real time visualization of biofilms in vivo [48]. Table VI: Table showing different methods available for identifying biofilms Methods Conventional plate counting

Confocal scanning laser microscopy

Description Microbiological method Quantify swabs or scrapings of a surface on agar and express as colony-forming units (CFU). The advantage of this method is it is cost effective. However, conventional plate counting is unable to reveal whether microorganism originate from a biofilm or show the biofilm’s current developmental stage Microscopy A three-dimensional method utilizing fluorescent molecular probes and laser beams to study the interaction between bacteria and the surfaces. Computer reconstructions of the chemical and physical conditions within the micro-environments of bacterial communities

Microbial Biofilms and Human Health associated with Indwelling Devices

Scanning electron microscopy (SEM) Atomic force microscopy (AFM)

Fluorescence insitu hybridisation Others Matrix-assisted laser desorption ionization coupled with time of flight analysis mass spectrometry (MALDITOF/MS)

can be created using digital imaging methods. Observe morphology of bacterial biofilms on surfaces. Applied in visualizing biofilm formation of S. epidermidis on contact lenses, biofilms on catheters, and ventilation tubes. Non-invasive microscopic technique observing biofilms at nanometer resolutions. Biofilms are observed in situ as no stains or coatings are required. Proteins and other cell surface properties possibly involved in biofilm development can be studied. Molecular methods Fluorescent probes specific to a nucleotide sequence with bacterial RNA or DNA give identity to live and dead bacteria in complex samples. Utilize laser ionization of bacteria to detect peptides and peptide ions on cell surface. The laser ionization can be compared to a database and hence enable identification of bacteria.

Table VII: Methods utilized to detect biofilm formation on different indwelling devices Indwelling devices Urinary Catheters Heart Valves Endotracheal tube

Artificial voice prostheses

Peritoneal dialysis catheters

Penile prostheses Central venous catheters

Orthopedic prostheses

Methods to visualize biofilms Tube adherence method, Congo Red agar method [15] Blood cultures Microscopy [49] Quantitative PCR (qPCR) and gene surveys targeting 16S rRNA genes are utilized to identify and quantify bacterial community present from endotracheal tube samples [17]. Polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) Fluorescence in situ hybridization (FISH) [27, 50] The 16S rRNA gene sequencing technique is used to identify cultured bacteria Confocal laser scanning microscopy [35, 51, 52]. Confocal scanning laser microscopy [37]. Roll-plate technique (a semi- quantitative procedure where the distal tip of the catheter is removed aseptically and rolled over the surface of a medium) [1]. As roll-plate technique has low sensitivity, biofilm quantification is done using sonication plus vortexing of catheter tips [1]. Scanning and transmission electron microscopy [1, 39, 53]. Microbiological examination of synovial fluid Isolation from blood cultures Sonication of removed implants. Molecular methods [42, 54].

2.9 

2.10 

Biofilm Control

Ϯ͘ϴ,KtdKWZsEdΘKEdZK>/K&/>D&KZDd/KE/E/Et>>/E's/͍ Formation of biofilms begins with the initial attachment of bacteria to the device. Prevention of biofilm formation therefore can be achieved by inhibiting bacterial attachment to the indwelling device by coating the device with either an inert polymer or with antimicrobial agents [55]. Polymer coating prevents physical interaction between bacteria and the surface of the device whereas antimicrobial agents offer chemical resistance and kill the incoming bacteria. Majority of efforts attempted to prevent and eradicate the biofilm have to face some difficulties since biofilms are well protected by their architecture effectively by producing extra cellular polysaccharide armoring the bacterial colonies, the mere presence of extracellular layer renders the biofilm insensitive to antimicrobial agents. Microbial cells within biofilm colonies are also much less susceptible to host immune mechanisms. Key antigens are either repressed or concealed from effector immune cells , and bacteria in colonies are highly resistant to phagocytosis by immune system phagocytes [56]. Deposition of complement C3b and IgG on bacterial surfaces has also been shown to be prevented as demonstrated for Staphylococcus epidermidis [57], contributing to protection of bacteria from killing by polymorphonuclear leukocytes. Furthermore, in airways of cystic fibrosis patients the presence of polymorphonuclear leukocytes has even been found to enhance Pseudomonas auruginosa biofilm formation due to bacterial binding to F-actin and DNA polymers [58]. Thus, the various arms of antimicrobial immunity are neutralized by the biofilm exopolysaccharide protective matrix, leaving affected patients fully vulnerable to the problem. At present, there is no ‘gold standard’ available to reveal the presence of device-related biofilm infections. However, adequate sample collection and logistics, standardized diagnostic methods, and interpretation of results by experienced personnel are important steps in efficient diagnosis and treatment of these infections [59]. The knowledge and prophylactic strategies will enable to prevent and control biofilms, this will undoubtedly assist in understanding new targets in device-related infections. Table VIII: Strategies to prevent and control biofilm formation on indwelling devices. Indwelling devices Urinary Catheters

Heart Valves

Prevention and control strategies Effective strategies to prevent UTI are using closed drainage system, antimicrobial agents in collection bags, impregnation of catheters with antimicrobial agents, avoid unnecessary catheterization, prevent prolonged catheterization if possible, maintain unobstructed urine flow, and insert catheters using aseptic technique [1,12, 60]. Antimicrobial agents are administered during valve replacement to prevent initial attachment of microorganisms while silver-coated sewing cuff is also known to produce less inflammation than uncoated ones [1]. In many cases, the device is removed and replaced to cure the infection [1].

Ϯ͘ϵ&KZDh>d/KE^'/E^d/K&/>D^͗ In terms of drug delivery strategies, it is the EPS that presents the greatest barrier to diffusion for drug delivery systems and free antimicrobial agents alike. In addition to EPS synthesis, biofilm-based micro-organisms can also produce small, diffusible signaling molecules involved in cell densitydependent intercellular communication, or quorum sensing. Not only does quorum sensing allow

Microbial Biofilms and Human Health associated with Indwelling Devices

2.11 

microbes to detect critical cell density numbers, but it also permits co-ordinated behavior within the biofilm, such as iron chelation and defensive antibiotic activities. Against this backdrop of microbial defense and cell density-specific communication, a variety of drug delivery systems have been developed to deliver antimicrobial agents and antibiotics to extracellular and/or intracellular targets , or more recently, to interfere with the specific mechanisms of quorum sensing [61][Martin et al., 2015]. The results of chitosan propolis nanoformulation study revealed that the natural nano particle can be used as a potential anti-biofilm agent in resisting infections involving biofilm formation, the formulation inhibited E. faecalis biofilm formation and reduced the number of bacteria in the biofilm by ~90% at 200 g/ml concentration [62]. . Table IX: Strategies to control biofilms by using different formulation. Antimicrobials Fluconazole

Ketoconazole

Formulations

Antimicrobial Activity Antifungal formulations • Lauryl alcohol An optimized formulation of 2 % fluconazole, 10 •Labrasol % lauryl alcohol (oil phase),20 % Labrasol / (caprylocaproyl ethanol (1:1, surfactant / cosurfactant) and 68 % macrogol-8water (w/w) was developed to give the greatest glyceride) permeation rate. This microemulsions formulation • Ethanol gave the highest percutaneous absorption of • Fluconazole fluconazole due to the increased lauryl alcohol and • Water water contents, and decreased surfactant / cosurfactant component.

• Lauryl alcohol • Labrasol (caprylocaproyl macrogol-8glyceride) • Ethanol • Ketoconazole • Water

Antifungal activity was assessed against C. albicans using a disc diffusion assay and confirmed that the microemulsion zone of inhibition was almost twice that of unencapsulated fluconazole. Empty microemulsions (no fluconazole) showed no antifungal activity. An optimised formulation of 2 % ketoconazole, 10 % lauryl alcohol (oil phase),10 % Labrasol/ethanol (1:1, surfactant/cosurfactant) and 68 % water (w/w) was developed to give the greatest permeation rate. This microemulsions formulation gave the highest percutaneous absorption of ketoconazole due to the increased lauryl alcohol and water contents, and decreased surfactant/ cosurfactant component. Antifungal activity was assessed against C. albicans using a disc diffusion assay and confirmed that the microemulsion zone of inhibition was almost twice that of unencapsulated ketoconazole. Empty microemulsions (no ketoconazole) showed no antifungal activity.

Ref [63]

[64]

2.12  Antibiotics Nadifloxacin

Biofilm Control

• Oleic acid • Tween 80 • Ethanol • Nadifloxacin • Water

Rifampicin

Rifampicin, isoniazid And / or pyrazinamide

Tween 80 Ethanol Oleic acid Phosphate buffer Rifampicin, or Isoniazid, or Pyrazinamide, or Combination of rifampicin, isoniazid and pyrazinamide

Antimicrobial activity was assessed against the obligate-anaerobic bacterium Propionibacterium acnes using the disc diffusion method. Varying concentrations of nadifloxacin microemulsions (5, 7, 10 and 14 g/ml) were compared against clindamycin as a positive control. Whilst clindamycin showed greater therapeutic efficacy against P. acnes, the antimicrobial activity of nadifloxacin microemulsions was shown to be highly concentration dependent, with higher drug concentrations resulting in larger zones of inhibition. A stable microemulsion could be formed upon incorporation of rifampicin(there were no changes in optical texture or phase separation) Controlled release of rifampicin could be achieved from the o/w emulsion droplets Individual microemulsion formulations of the three anti-tuberculosis drugs were successfully prepared despite their widely differing solubilities Drug release from the microemulsions followed the order: isoniazid > pyrazinamide> rifampicin. Analysis of release kinetics (using KorsmeyerPeppasequation) revealed that isoniazid and pyrazinamide release is non-Fickian,whilst rifampicin release follows Fickian principles. A formulation was developed which enabled different anti-tuberculosis drugs to be encapsulated in the different compartments of the microemulsion, using their varying solubilities as the driving force. This allows not only a multidrug treatment approach for tuberculosis treatment, but could also reduce the issues associated with drug deactivation and degradation (rifampicin is degraded in the presence of isoniazid). In common with release behavior from single drug microemulsion formulations analysis of release kinetics indicated that isoniazid and pyrazinamide release is non-Fickian, whilst rifampicin release follows Fickian principles.

[65

[66]

[67

[68]

Antimicrobials

Cetylpyridinium chloride (CPC)

Soybean oil CPC

Nanoemulsion improved antimicrobial efficacy after 72 h exposure against dental unit waterline microorganisms including Staphylococcus spp. Nanoemulsion improved antimicrobial efficacy (compared to nanoemulsion components alone) against biofilm-forming microorganisms. Nanoemulsion were antimicrobials active against

[69]

[70]

Microbial Biofilms and Human Health associated with Indwelling Devices

Triton X-100 Water

Soybean oil Triton X-100 Tributyl phosphate Water

Tributyl phosphate (BCTP) ± CPC

n-pentanol (TEOP)

Soybean oil Triton X-100 Tributyl phosphate Water CPC

Ethyl oleate Tween 80 n-pentanol Water

planktonic Streptococcus mutans, Lactobacillus casei, Actinomyces viscosus and C. albicans both individually and in mixed culture Bacterial counts in mature, mixed culture biofilms of the four microorganisms could also be reduced by the nanoemulsion Nanoemulsion was antimicrobials active against planktonic Streptococcus mutans Bacterial counts in mature, Streptococcus mutans biofilms could be reduced by the nanoemulsion 1 log reduction (log CFU ml-1) against S. aureus for tributyl phosphate component after 30 mins when used alone; no discernible antimicrobial activity associated with other emulsion ingredients when used alone S. aureus > P. aeruginosa 2 log reduction (log CFU ml-1) against S. aureus for BCTP-CPC emulsion P. aeruginosa > S. aureus. More effective at P. aeruginosa biofilm eradiation versus BCTP

2.13 

[71]

[72, 43]

 Studies had been done to perceive the knowledge of biofilm formation, microorganisms associated with different indwelling devices, identification of potential mechanisms of antimicrobial resistance, and others [73]. The future directions of research in biofilms may likely to focus on imaging of biofilms in situ, in vitro and in vivo models of biofilms, antimicrobial resistance in multispecies biofilms, define methods for diagnosing and quantifying biofilm infection and possibly faster, advanced, convenient, and affordable testing kits or methods for biofilms detection [3, 73].

2.14 

Biofilm Control

 In short, indwelling devices plays an important role in the modern medical care which aims to treat diseases. Sadly, the formation of biofilm on indwelling devices is an important issue where continuous studies of biofilms on indwelling devices are necessary and hopefully reliable and effective prevention and control strategies for all different indwelling devices will be available in order to bring the medical care of mankind to a better future. The genetic and molecular mechanisms underlying biofilm formation are being elucidated in the recent literature; these inevitably lead to the identification of new targets for drug delivery systems. Ultimately, it is these targets, combined with developments in novel antimicrobials, which will lead to more efficacious pharmaceutical strategies for biofilm infection treatment in the future. Moreover, many bacteria are not culturable, it was estimated that about 50 % of micro-organisms are not culturable, even though they are vital or viable. The challenge of eradicating biofilms in indwelling devices should be approached in multiple aspects like, treatment measures, the design, use characteristics, materials used, and duration of use device material construction and more effective strategies and reliable measuring of biofilms, it plays an important role in public health should also result as the role of biofilms in antimicrobial-drug resistance is being investigated by many researchers across the globe and there is a cross talk established between biofilm contamination and patient infection which needs to be explored.

  1.

Donlan R, Costerton J (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical microbiology reviews, 15, 2, 167-193.

2.

Stoodley P, Hall-Stoodley L, Costerton B, DeMeo P, Shirtliff M, Gawalt E et al. (2012). Biofilms, Biomaterials, and Device-Related Infections. Biomaterials Science an Introduction to Materials in Medicine. 3rd Edition. Elsevier, 565-583.

3.

Tenke P, Kovacs B, Jackel M, Nagy E (2006). The role of biofilm infection in urology. World Journal of Urology, 24, 1, 13-20.

4.

Kart D, Tavernier S, Van Acker H, Nelis H, Coenye T (2014). Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa. Biofouling, 30, 3, 377-383.

5.

Alhede M, Alhede M (2014). The biofilm challenge. EWMA journal, 14, 1.

6.

Percival S, Malic S, Cruz H, Williams D (2011). Introduction to biofilms. Springer, 41-68.

7.

Weinstein R, Darouiche R (2001). Device-associated infections: a macroproblem that starts with microadherence. Clinical Infectious Diseases, 33, 9, 1567-1572.



 



8.

Suleman L, Archer D, Cochrane C, Percival S (2014). Healthcare-Associated Infections and Biofilms. Biofilms in Infection Prevention and Control: A Healthcare Handbook, 165.

9.

Lindsay D, Von Holy A (2006). Bacterial biofilms within the clinical setting: what healthcare professionals should know? Journal of Hospital Infection, 64, 4, 313-325.

Microbial Biofilms and Human Health associated with Indwelling Devices

2.15 

10. Privett B, Youn J, Hong S, Lee J, Han J, Shin J et al. (2011). Antibacterial fluorinated silica colloid superhydrophobic surfaces. Langmuir. 27, 15, 9597-9601. 11. Donlan R (2001). Biofilms and device-associated infections. Emerging infectious diseases, 7, 2, 277. 12. Talsma S (2007). Biofilms on medical devices. Home healthcare nurse, 25, 9, 589-594. 13. Jordan R, Nicolle L (2014). Preventing Infection Associated with Urethral Catheter Biofilms. Biofilms in Infection Prevention and Control. A Healthcare Handbook, 287. 14. Djeribi R, Bouchloukh W, Jouenne T, Menaa B (2012). Characterization of bacterial biofilms formed on urinary catheters. American journal of infection control, 40, 9, 854-859. 15. Niveditha S, Pramodhini S, Umadevi S, Kumar S, Stephen S (2012). The isolation and the biofilm formation of uropathogens in the patients with catheter associated urinary tract infections (UTIs). Journal of clinical and diagnostic research: JCDR, 6, 9, 1478. 16. Goad MEP, Goad DL (2013). Biomedical Materials and Devices. Haschek and Rousseaux's Handbook of Toxicologic Pathology (Third Edition) Academic press Ch 26, 783-806. 17. Perkins S, Woeltje K, Angenent L. (2010). Endotracheal tube biofilm inoculation of oral flora and subsequent colonization of opportunistic pathogens. International Journal of Medical Microbiology, 300, 7, 503-511. 18. Agbaht K, Diaz E, Mu~noz E, Lisboa T, Gomez F, Depuydt P et al. (2007). Bacteremia in patients with ventilator-associated pneumoniaA1:A17 is associated with increased mortality: A study comparing bacteremic vs. nonbacteremic ventilator-associated pneumonia. Critical care medicine, 35, 9, 2064--2070. 19. Safdar N, Crnich C, Maki D (2005). The pathogenesis of ventilator-associated pneumonia: its relevance to developing effective strategies for prevention. Respiratory care, 50, 6, 725-741. 20. Bahrani-Mougeot F, Paster B, Coleman S, Barbuto S, Brennan M, Noll J et al. (2007). Molecular analysis of oral and respiratory bacterial species associated with ventilatorassociated pneumonia. Journal of clinical microbiology, 45, 5, 1588--1593. 21. Augustyn B (2007).Ventilator-associated pneumonia risk factors and prevention. Critical care nurse, 27, 4, 32-39. 22. Alhede M, Bjarnsholt T, Givskov M, Alhede M (2014). Pseudomonas aeruginosa Biofilms: Mechanisms of Immune Evasion. Advances in applied microbiology, 86, 1-40. 23. Kipnis E, Sawa T, Wiener-Kronish J (2006). Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Medecine et maladies infectieuses, 36, 2, 78-91.

2.16 

Biofilm Control

24. Neu T, Van der Mei H, Busscher H, Dijk F, Verkerke G (1993). Biodeterioration of medicalgrade silicone rubb.er used for voice prostheses: a SEM study. Biomaterials, 14, 6. 25. Rodrigues L, Banat I, Teixeira J, Oliveira R (2007). Strategies for the prevention of microbial biofilm formation on silicone rubber voice prostheses. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 81, 2, 358--370. 26. Bertl K, Zatorska B, Leonhard M, Matejka M, Schneider-Stickler B (2012). Anaerobic and microaerophilic pathogens in the biofilm formation on voice prostheses: a pilot study. The Laryngoscope, 122, 5, 1035--1039. 27. Buijssen K, Harmsen H, van der Mei H, Busscher H, van der Laan B (2007). Lactobacilli: important in biofilm formation on voice prostheses. Otolaryngology-Head and Neck Surgery, 137, 3, 505-507. 28. Dickinson G, Bisno A (1989). Infections associated with indwelling devices: infections related to extravascular devices. Antimicrobial agents and chemotherapy, 33, 5, 602. 29. Bayston R (2011). Cerebrospinal Fluid Shunts. Comprehensive Biomaterials, Edition: 1, Elsevier, Eds: P Ducheyne, KE Healy, DW Hutmacher, DW Grainger, CJ Kirkpatric k, Chapter: 6.629, 469-48. 30. Chi H, Chang K, Chang H, Chiu N, Huang F. (2010). Infections associated with indwelling ventriculostomy catheters in a teaching hospital. International Journal of Infectious Diseases, 14, 3, 216--219. 31. Gutierrez-Murgas Y, Snowden J (2014). Ventricular shunt infections: Immunopathogenesis and clinical management. J Neuroimmunology, 276, 0, 1–8. 32. Piette A, Verschraegen G. (2009). Role of coagulase-negative staphylococci in human disease. Veterinary microbiology, 134, 1, 45-54. 33. Bruinsma N, Stobberingh E, Herpers M, Vles J, Weber B, Gavilanes D (2000). Subcutaneous ventricular catheter reservoir and ventriculoperitoneal drain-related infections in preterm infants and young children. Clinical microbiology and infection, 6, 4, 202-206. 34. McGirt M, Zaas A, Fuchs H, George T, Kaye K, Sexton D. (2003). Risk factors for pediatric ventriculoperitoneal shunt infection and predictors of infectious pathogens. Clinical infectious diseases, 36, 7, 858--862. 35. Pihl M, Davies J, Johansson A, Svensater G (2013). Bacteria on catheters in patients undergoing peritoneal dialysis. Peritoneal Dialysis International, 33, 1, 51-59. 36. Dasgupta M, Anwar H, Costerton J (1992). Bacterial Biofilms and Peritonitis in Continuous Ambulatory Peritoneal Dialysis. International Biodeterioration & Biodegradation, 30, 167176.

Microbial Biofilms and Human Health associated with Indwelling Devices

2.17 

37. Silverstein A, Henry G, Evans B, Pasmore M, Simmons C, Donatucci C (2006). Biofilm formation on clinically noninfected penile prostheses. The Journal of Urology, 176, 3, 10081011. 38. Carson C (2004). Efficacy of antibiotic impregnation of inflatable penile prostheses in decreasing infection in original implants. The Journal of Urology, 171, 4, 1611-1614. 39. Al Akhrass F, Hachem R, Mohamed J, Tarrand J, Kontoyiannis D, Chandra J et al. (2011). Central Venous Catheter--associated Nocardia Bacteremia in Cancer Patients. Emerging infectious diseases, 17, 9, 1651. 40. Esposito S, Purrello S, Bonnet E, Novelli A, Tripodi F, Pascale R et al. (2013). Central venous catheter-related biofilm infections: An up-to-date focus on methicillin-resistant Staphylococcus aureus. Journal of Global Antimicrobial Resistance, 1, 2, 71--78. 41. Molina-Manso D, del Prado G, Ortiz-P'erez A, Manrubia-Cobo M, G'omez-Barrena E, Cordero-Ampuero J et al. (2013). In vitro susceptibility to antibiotics of staphylococci in biofilms isolated from orthopedic infections. International journal of antimicrobial agents, 41, 6, 521-523. 42. Osmon D, Berbari E, Berendt A, Lew D, Zimmerli W, Steckelberg J et al. (2012). Executive Summary: Diagnosis and Management of Prosthetic Joint Infection: Clinical Practice Guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases, 56, 1, 1-10. 43. Zimmerli W, Trampuz A, Ochsner P (2004). Prosthetic-joint infections. New England Journal of Medicine, 351, 16, 1645-1654. 44. Ramage G, Tunney M, Patrick S, Gorman S, Nixon J. (2003). Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials. Biomaterials, 24, 19, 3221-3227. 45. Hogdall D, Hvolris J, Christensen L (2010). Improved detection methods for infected hip joint prostheses. Apmis, 118, 11, 815--823. 46. Hannig C, Follo M, Hellwig E, Al-Ahmad A (2010). Visualization of adherent microorganisms using different techniques. Journal of Med. Microbiology, 59, 1–7. 47. Pantanella F, P. Valenti, T. Natalizi, D. Passeri, F. Berlutti (2013). Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques. Ann Ig.; 25: 31-42. 48. Bergmans L, Moisiadis P, Van Meerbeek B, Quirynen, M. & Lambrechts P (2005). Microscopic observation of bacteria: review highlighting the use of environmental SEM. Int Endod Journal, 38, 775–788.

2.18 

Biofilm Control

49. Bosio S, Leekha S, Gamb S, Wright A, Terrell C, Miller D (2012). Mycobacterium fortuitum prosthetic valve endocarditis: a case for the pathogenetic role of biofilms. Cardiovascular Pathology, 21, 4, 361-364. 50. Buijssen K, van der Laan B, van der Mei H, Atema-Smit J, van den Huijssen P, Busscher H et al. (2012). Composition and architecture of biofilms on used voice prostheses. Head & neck, 34, 6, 863-871. 51. Kim D, Yoo T, Ryu D, Xu Z, Kim H, Choi K et al. (2004). Changes in causative organisms and their antimicrobial susceptibilities in CAPD peritonitis: a single center's experience over one decade. Peritoneal dialysis international, 24, 5,424-432. 52. Fontan M, Rodr'iguez-Carmona A, Garc'ia-Naveiro R, Rosales M, Villaverde P, Vald'es F (2005). Peritonitis-related mortality in patients undergoing chronic peritoneal dialysis. Peritoneal Dialysis International, 25, 3, 274-284. 53. Arciola C, Montanaro L, Costerton J (2011). New trends in diagnosis and control strategies for implant infections. Int J Artif. Organs, 34, 9, 727--736. 54. Cazanave C, Greenwood-Quaintance K, Hanssen A, Karau M, Schmidt S, Urena E et al. (2013). Rapid molecular microbiologic diagnosis of prosthetic joint infection. Journal of clinical microbiology, 51, 7, 2280--2287. 55. Francolini I, Donelli G (2010). Prevention and control of bio¢lm-based medical-devicerelated Infections. FEMS Immunol Med Microbiology, 59, 227–238. 56. Mahenthiralingam E, Campbell ME, Speert DP (1994). Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis. Infect. Immunology, 62, 596–605. 57. Kristian SA, Birkenstock TA, Sauder U, Mack D, Götz F, Landmann R (2008). Biofilm formation induces C3a release and protects Staphylococcus epidermidis from IgG and complement deposition and from neutrophil-dependent killing. J. Infect. Disease, 197, 10281035. 58. Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, Saavedra MT, Fessler MB, Malcolm KC, Vasil ML, Nick JA (2005). Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils. Infect. Immunology, 73, 3693–3701. 59. Xu Y, Larsen LH, Lorenzen J, HallStoodley L, Kikhney J, Moter A, Thomsen TR (2017). Microbiological diagnosis of devicerelated biofilm infections. APMIS, 125, 4, 289-303. 60. Trautner B, Hull R, Darouiche R (2005). Prevention of catheter-associated urinary tract infection. Current opinion in infectious diseases, 18, 1, 37. 61. Martin C, LiLow W, Gupta A, Cairul Iqbal Mohd Amin, M., Radecka I, T Britland S, & Raj P (2015). Strategies for antimicrobial drug delivery to biofilm. Current pharmaceutical design, 21, 1, 43-66.

Microbial Biofilms and Human Health associated with Indwelling Devices

2.19 

62. Ong TH, Chitra E, Ramamurthy S, Siddalingam RP, Yuen KH, Ambu SP, Davamani F (2017). Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms. PloS one, 12(3), e0174888. 63. Patel MR, Patel RB, Parikh JR, et al. (2009). Effect of formulation components on the in vitro permeability of microemulsion drug delivery system of fluconazole. AAPS Pharm Sci Technology, 10, 917-23. 64. Patel MR, Patel RB, Parikh JR, et al. (2011). Investigating effect of microemulsion components: In vitro permeation of ketoconazole. Pharm Dev Technology, 16, 250-8. 65. Kumar A, Agarwal SP, Ahuja A, et al. (2011). Preparation, characterization and in vitro antimicrobial assessment of nanocarriers based formulation of nadifloxacin for acne treatment. Pharmazie, 66, 111-4. 66. Mehta SK, Kaur G, Bhasin KK (2007). Analysis of tween based microemulsions in the presence of TB drug rifampicin. Colloids Surf B, 60, 95-104. 67. Mehta SK, Kaur G, Bhasin KK (2010). Tween-embedded microemulsions – Physicochemical and spectroscopic analysis for antitubercular drugs. AAPS Pharm Sci Tech, 11, 143-53. 68. Mehta SK, Kaur G, Bhasin KK (2010). Entrapment of multiple anti-Tb drugs in microemulsion systems: Quantitative analysis, stability and in vitro release studies. J Pharma Sci b, 99, 1896-911. 69. Ramalingam K, Frohlich NC, Lee VA (2013). Effect of nanoemulsion of dental unit waterline biofilm. J Dent Science, 8, 333-336. 70. Ramalingam K, Amaechi BT, Rawls RH, et al. (2012). Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms. Arch Oral Biology, 57, 15-22. 71. Ramalingam K, Amaechi BT, Rawls RH, et al. (2011). Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans. Arch Oral Biology, 56, 437-45. 72. Wilson S, Zumbe J, Henry G, Salem E, Delk J, Cleves M (2007). Infection reduction using antibiotic-coated inflatable penile prosthesis. Urology, 70, 2, 337--340. 73. Rao V, Ghei R, Chambers Y (2005), Biofilms research-implications to biosafety and public health. Applied Biosafety, 10, 2, 83.

    

  

GUT BIOFILMS: TUG OF WAR BETWEEN GOOD AND BAD BACTERIA IN HUMAN HEALTH 

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…† ‡‡  †   ­ ­     ­†   †  ­„   ­† ‡‡  ˆ  †ˆ   ‡  ­­     „ ­­­   ‡   ‰   †  ­  „­­ † ‰ ‡ †  „

   ‡†  †­ ­„ Š    ‡ ‰   ­†  † ­­†  ‹ ‰  ˆ     †         ‰  ­†  †„…‚   ­   ­   ­†  ‰  †‹­ ­   ­ŒŽ‘’“    ­†„”†      ­„ 

  ­  ­­ ‡   † ­  Ž‘’

    How much do we know about the microbial flora associated with human system? This is not a philosophical but a microbiological question which needs food for thought. Increased scientific intervention in this direction in recent decade has resulted in studies which have investigated this aspect resulting in detail insights about the invisible members surviving with us. Studies have improved our understanding on microbial flora associated with us in unique environmental conditions, at different age, at different physiological niches, at different nutritional conditions, in infection, in other disease conditions including cancers, autoimmune disorders and in many more presentations. The answer is sought by a large number of researchers from United States to Asia, Europe and elsewhere. This investigation led to the discovery of the existence of unique relationship between the host and the microbe and status of wellbeing. We are marching ahead and we are transforming to the “Microbiome era” from the present “Genome era” [1] . Understanding of the microbiota we harbour has an important bearing on the health status at the societal level. Microbes live with us and make an association by forming biofilms on almost all parts in the human body, leaving blood, cerebrospinal fluid, brain and muscle [2]. An adult human body is estimated to be

3.2 

Biofilm Control

composed of approximately 1013 eukaryotic cells and serves as a natural habitat for 10 times this number of microbial cells (bacteria, fungi, viruses and phages), whose collective genome, microbiome, is at least 100 times more than human genome, and are often termed normal flora or commensals [1,3.4,5]. Microbes like bacteria have been on the earth for at least 2.5 billion years while modern humans have been evolved about 50,000 -100,000 years before [6]. However, the association of commensal bacteria in human host is explained by the hypothesis of co-evolution [5]. The commensal bacteria may be either residential (present for long time) or/ and transient (present for short period of time) while pathogens are mostly transient [4]. Microbes live with us in a symbiotic relation where we provide them constant source of nutrition while in return, they help us in our physiological development and subsequent functioning [7]. Microbial colonization by biofilm formation was demonstrated first by Anton Von Leeuwenhoek in his dental plaque as early as in the seventeenth century. However, the general theory of biofilm came into prominence in 1978 [8]. Biofilm formation is an essential step towards better survival in bacterial species. Microbial biofilm is a collection of microbes with community livelihood remain enclosed by extracellular polymeric substance (EPS), separated by a network of open water channels, adhere to natural (teeth, gut) or/ and manmade (metal, plastic) surfaces typically at a liquid-solid interface [9]. The embedded microbes in EPS matrices are protected from exposure to UV [10], acids [11], antibiotics [12], metal toxicity [13], dehydration, salinity [14], phagocytosis [2]. Thus the unique architecture of biofilm participates in cellcell interactions with harvesting nutrition for energy [15], exchange of genetic materials [16], communication signalling [17], elaboration and diffusion of metabolites [18]. The microbial biofilm is therefore so versatile and dynamic in maintaining phenotypic plasticity as it survive and sustain in both inhospitable and hostile environment. Majority of bacteria grow onto matrices forming biofilms in nutrient-sufficient moist ecosystem and are thus sessile in nature. Planktonic (floating) bacteria exhibit different mode of living compared to sessile species [19]. Since the declaration of human genome project (HGP, 2003), Microbiome project (2008-2012) took shape with the assistance of molecular tools to reveal near-to-real-time microbial members by metagenomic approaches and reported the highest complexity of microbiota exist at the human gut [1, 16]. Recent scientific reports unveil many facts about the role of human gut microbiota in health and diseases. In general, the human microbiota, a constituent of gastrointestinal ecosystem, maintains the homeostasis, saves and supports host from being diseased. In a healthy state, gut microflora functions in metabolism of hormones, carcinogens, xenobiotics; synthesizes vitamins B5, B6, K and biotin [20]; short chain fatty acids (SCFAs) like butyric acid, produces antimicrobial peptides, antagonizes and outnumbers pathogenic flora, stimulates secretory immunoglobulin A (sIgA) and IgG production [21, 22, 23]. In a diseased state, changed composition of microflora cause to dwindle the normal environment [24] and the host may experience (a) indigestion and mal-absorption, (b) production of vitamin B12 analogue [25] and certain amino acids due to microbial overgrowth [25], (c) saturation of essential omega 3 and 6 fatty acids [21], (d) disruption of the intestinal lining causing the leaky gut syndrome [15], (e) sensitization against translocated bacteria and their fragments to autoimmune diseases [26, 27], (f) development of microbial overgrowth syndromes [28], (g) development of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) [29, 30, 31, 32] (h) development of Clostridium difficile –associated enterocolitis [33] (i) and the deconjugation of bile acids and estrogens that might potentially induce bowel or breast cancer [21, 24, 27]. Microbial diversity has been shown to be affected between healthy and diseased condition [34]. A proteomic study using cerebrospinal fluid revealed alterations in the diversity of microbial communities in patients with chronic fatigue syndrome, wherein 738 out of 2,783 microbial proteins expressed which were found to be unique to such patients conferring microbial interaction (Interactome) to host. Likely, altered gut microbial diversity may influence our behaviour with both depression and anxiety [7]. Dietary composition has shown a strong relation with the composition of

3.3 

Gut Biofilms: Tug of War Between Good and Bad Bacteria...

gut microbiota and disease association [35]. Intake of drugs also affects this composition and results in persistence of several diseases [36].

  The microbiologically apparent sterile human fetus subsequently is colonized at birth from mother and the surrounding environment [37]. The microbiota at the beginning of life is relatively unsteady and experiences periodic changes before getting stabilized at the period of weaning [35, 38, 39] . GIT (gastrointestinal tract) is that anatomical part of our body which is approximately 25 feet long (small intestine ~ 20 feet and large intestine ~ 5 feet) and connects between oral cavity through anus and the gut starts from pylorus and ends at anus. GIT also offers a wide surface area. It is equivalent to 32 square meters, which is half of the surface area of a badminton court [39]. The GIT has three primary physiological functions: digestion of food, absorption of nutrients and keeping toxins and toxic elements out of the body [40, 41]. It also serves immunological function through gut associated lymphoid tissue (GALT) [42]. Failure in any of these functions results in defective energy production, faulty energy need, and waning of body’s reserve and culminates with disease. The gut in particular has a unique microbial ecosystem covering the mucosal layer of the intestine and thus offers a protective gut barrier. In general, the human gut microbiota is preferably infested by dominant bacteria of four major groups /phyla: Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, comprising >95% of the total microbiota. Over 200 bacterial species are found common with another 700-800 lesscommon bacterial species in the human gut [43] (Table 1). Table1: Different classes of Gut Microbiota (%) at different stages of human life Class

Fermicutes Bacteroidetes Actinobacteria Proteobacteria Others

Unb orn

-

Baby

Toddler

Adult

Elderly

Breast -fed

Formul a- fed

Soli d food

Antibioti c treatmen t

Health y

Malnutritio n

Health y

Obes e

25 25 25 25 -

25 25 25 25 -

20 70 3 7 -

20 80 -

20 70 3 7 -

30 15 45 10

70 25 3 2 -

70 15 5 10 -

65 80 yr s 80 15 4 1 -

>10 0 yrs

75 10 10 -

The bacterial composition is highly unpredictable in human gut. It remarkably varies within and between individuals [16]. Culture dependent study suggests the common occurrence of Escherichia coli among all the healthy adults. However culture independent high throughput sequencing studies reveal a huge variation in composition. In general, predominant phyla are Firmicutes, Bacteroidetes in adults usually, but Actinobacteria, Proteobacteria and Verrucomicrobia share also minor constituents. Methanogenic archae, yeasts and viruses (mainly phages) are also present in adult human gut [44, 45]. Such huge convoys of bacteria with other hitherto unknown microbial members are the part and parcel of our gut and hence it is like an organ (Figure 1).

3.4 

Biofilm Control

Figure 1. Distribution of gut microbiota along the length of human gut- the distribution of bacterial population varies with the aerobic to anaerobic part of the gut as it proceeds from stomach to anus. The “gut microbiota” constitutes this forgotten organ. The relationship between gut microbiota and human host is not however, truly commensalism (non-harmful co-existence) rather it is mutualism [46]. Its dynamic, plasticity is unique in response to several variable factors [20]. Research outcomes reveal several indications of host–microbe interactions including, spatial (e.g., skin, mouth, and gut), temporal (e.g., birth and senescence) distribution and several other determinants such as diet, genetic background, and immune status which affect the composition of the microbiota of the host [35, 47] (Table 1). Our understanding was limited and was fed with the information of cultivable bacteria till the advent of 16S rRNA sequencing. It has brought evolution in the identification and classification of new species. In recent times, National Institute of Health (NIH) supported Human Microbiome Project (2008-2012) and MetaHIT (2008-2012) has brought large and very new information about the hostmicrobe relation [1]. Besides, Genome Online Database (GOLD) (https://gold.jgi-psf.org) [48] also tenders huge reports on diversity of sequenced microbes of various sources including humans [26].

 The gut of human system is colonized by both luminal and mucosal microbiota. Luminal flora are mostly planktonic, transient or/ and pathogenic while commensal flora are sessile and mucosal in nature. The distribution of gut microbial community varies with the length and site of GIT. So far the research data mounted on the microbial composition of the lumen mainly because of sampling restrictions [49]. Majority of such data were retrieved from samples like faeces, or upper gut aspirates. However, mucosal adherent microbial population have also been studied in recent times, showing differences in microbial population at different loci. Large number of bacteria grow in the gut either as a part of pathogen or/ and commensal community. Streptococcus mutans is involved in plaque formation and initiation of dental

Gut Biofilms: Tug of War Between Good and Bad Bacteria...

3.5 

caries. Lactobacilli in the oral cavity probably contribute to acid formation that leads to tooth decay. Enterococcus faecalis, a regular member of the intestinal flora as with E. coli, Klebsiella, and Citrobacter (member of coliforms) is common in luminal content of human gut. Some strains of E. coli are pathogenic that cause multiple diseases like diarrhoea, urinary tract infections and neonatal meningitis [50]. Large numbers of bacteria are found in the lower intestinal tract, specifically in the colon where the most prevalent bacteria are the Bacteroides, a group of Gram-negative, anaerobic, non-spore forming bacteria cause colitis and colon cancer [51, 52]. Some species of Clostridium colonize the bowel. C. perfringens is commonly isolated from faeces. C. difficile may colonize the bowel and cause ‘antibiotic-associated diarrhoea’ or pseudomembranous colitis. Bifidobacteria are Gram-positive, non-spore forming, lactic acid bacteria (LAB) [34]. They have been described as ‘friendly’ bacteria (probiotics) in the human intestine. B. bifidum is the predominant bacterial species in the intestine of breast-fed infants, where it presumably prevents colonization by potential pathogens [53]. The mucosal bacteria growing in biofilms on surfaces lining the gut epithelial surface, these organisms may be important in modulating the host's immune system and contributing to some chronic inflammatory diseases [30]. Sessile microbial community form biofilm that are irreversibly attached to a substratum (mucosal layer in gut) or interface or to each other and remain embedded in microbial extracellular polymeric substances, display altered phenotypes with growth rate and gene transcription. The surface selection and exhibition of adherence onto substratum may be important and intrinsic properties of a bacterium. Enteric pathogens adhere to the mucosal layer by different ways. Several pathotypes of diarrhoeagenic E. coli (like enteropathogenic E. coli –EPEC, entero adherent E. coli -EAEC, enterotoxigenic E. coli – ETEC, entero aggregative E. coli –EaggEC) do adhere by different mechanisms with expressed colonization factors [54, 55] or by proteins (adhesions) by possessing plasmids ( bundle forming pilusbfp) [50]. In our observation, EAEC can adhere differently as EPEC, localised and diffused adherence (Figure 2) [56]. Once these microbes adhere to substratum, they colonize and multiply resulting in the formation of an established mature biofilm.

Figure 2. Different mode of adherence exhibited by Enteroadherent E. coli in cultured Hela cell linesunlike diffusely adherent (DA), localised adherence (LA) shown where bundle forming pilus (bfpA) enable to aggregate together and confers a mode of pathogenicity. Such biological events of preceding biofilm formation and formed biofilms govern their existence in harsh conditions. Gram negative bacteria like E. coli and several others possess rpoS regulon gene, sigma factor that enable bacteria to survive in nutrient limitation and in other environmental stress conditions [19]. There are many genes and their products which participate in biofilm formation.

3.6 

Biofilm Control

Members of Enterobacteriales, like E.coli, Salmonella produce a protein, bacterial amayloids, collectively called curli protein and possess the gene, called csg. These are part of PAMPs (pathogen associated molecular patterns) and are recognized by TLRs (toll-like receptors) in the host system. Besides, its role to play in pathogenicity, it helps in biofilm formation in the GI tract. The recent studies showed that these fibrilar curli proteins function like amyloids and maintain the homeostasis of gut lining by saving it from cellular damage. TLR2 activation by curli proteins (PAMP) promotes intestinal epithelial integrity and overall gut health [57]. Besides, majority of biofilm forming bacteria are resistant to several antibiotics and obviously are not eradicated by short period of antibiotic use [12]. The antibiotic resistance of bacterial biofilm can be explained by several mechanisms like, late penetration, altered growth rate, quorum sensing and other physiological changes [12]. However, long term usage of antibiotics damage the biofilm and influence opportunistic commensals to overgrow to cause a disease like antibiotic associated diarrhoea [16]. There is increased interest in the use of alternative therapeutic strategies to control potential pathogens on the mucosal surface, with reference to the applications involving probiotics (good gut bacteria) to reinstate gut microbiota [40, 58].

      Hundreds of scientific reports have compiled the existence of a diverse community in the gut [49]. It is a reservoir of ready and incessant source of nutrition to microbes. It forms a complex ecology which influences the health condition of the host. Residential indigenous bacteria survive onto the undigested food in the host gut and thereby participate in extraction of energy and thus regulate host’s metabolic functions [16]. Extracted energies are stored in host adipose tissue for future use. Following these, several mechanisms have been postulated to demonstrate the necessity of gut microbial population in energy homeostasis [15, 59, 60] . 1. 2.

3. 4.

Gut microbiota promote intestinal monosaccharide absorption by enhancing the density of capillaries in the small intestinal villus epithelium. Gut microbiota participate in energy extraction from undigested food components into short chain fatty acids (SCFAs) and ultimately contribute in hepatic de novo lipogenesis through the expression of several key enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Both ACC and FAS are controlled by carbohydrate responsive element binding protein (ChREBP) and sterol responsive element binding protein (SREBP-1) [61]. Gut microbiota were found to suppress AMP-activated protein kinase (AMPK) -driven fatty acid oxidation in the liver and in skeletal muscle [59]. Fourth pathway demonstrates involvement of SCFAs [62] which act as natural signaling molecules for two G protein-coupled receptors (GPRs), GPR41 and GPR43. It was shown that GPR43 knockout mice were resistant to diet-induced obesity [63]. Therefore, it supports the idea that specific metabolites coming from the gut (i.e., SCFAs) act in a variety of ways (e.g., as energy substrates and as metabolic regulators).

The non-digestible carbohydrates embody the prebiotic concept. SCFA production causes ‘The selective stimulation of growth and/or activity (ies) of one or a limited number of microbial genus (era)/species in the gut microbiota that confer(s) health benefits to the host’ [64]. SCFA production has been associated with changes in ingestive behavior by mechanisms linked to the modulation of gut peptide production and secretion (i.e., glucagon-like peptide-1 [GLP-1], peptide-YY and ghrelin) (Figure 3). It is imperative to say that SCFAs are multifunctional and they play important role in healthy and diseased conditions (obesity, cancer, diabetes etc.) of host [60, 65]. The other justification has been evolved also to explain why gut would have large number of bacterial species (x1012 cfu/ ml). It may be an apparent function of appendix. The role of vermiform

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3.7 

appendix in the gut of a healthy human is controversial. However, it is a known fact that inflammation in appendix may be lethal and is often subjected to surgery (appendectomy) for elimination of this socalled vestigial organ. Nevertheless, a renewed concept has been originated and correlation between the appendix and gut bacteria has been evolved recently. It is conceived that the vermiform appendix is a “ready source” and a “safe shelter” for commensal bacterial species which remains under protected biofilm and supplies the necessary bacterial population on demand to restore the gut health [66].

 The surface area of GIT is covered with mucus lining which is known to be a highly dynamic matrix, largely composed of mucin glycoproteins, secreted by intestinal goblet cells. The mucus is discontinuous in the small intestine, while there are two layers in stomach and in large intestine. Tight stacking of polymeric glycoproteins adjacent to the epithelium forms compact inner layer that is mostly sterile while outer layer is largely looser where several hundreds of bacterial species survive with amity forming biofilm. As the microbial diversity increases, so mucin thickens and restores the gut barrier [67]. The thickness and relative distribution of mucus varies with locations. The association with the mucus thickness has been correlated with local bacterial load; 103-104 organisms per gram of luminal contents in the duodenum and jejunum, about 108 organisms per gram in the ileum while 1010 1012 organisms per gram in the colon [68] (Figure 1). Besides, the gut epithelia are guarded by several secretions, antimicrobials along with mucus making a shield to bacteria and thus immune response is abated. In addition, intestinal macrophages produce much lower amounts of inflammatory cytokines than other macrophages hence reducing tissue inflammation. Instead, commensal bacteria may be tuned to be opportunistic pathogenic with the change in microenvironment of the gut and in the loss of immunity of the host (immunocompromised host) [69]. The gut epithelia are central to the orchestration of intestinal defences with multitasking abilities. These are composed of five different types of epithelium- absorptive enterocytes, goblet cells, Paneth cells, M cells and entero-endocrine cells and are developed from a common stem cells located near the base of the intestinal crypts. Maturation of the intestinal mucosa and GALT is initiated by microbial colonization. Central to sensing the colonizers of the GIT is the expression of wide range of germlineencoded pattern recognition receptors (PRRs) by intestinal epithelial cells (IECs) and residential immune cells in the gut. PRRs like, Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) are found on the cell surface or in endosome, and cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) of the intestinal mucosa can recognise the microbe associated molecular patterns (MAMPs) which are expressed by the constituents of resident microbiota and pathogens. However, the mechanisms behind the differential recognition between resident microbiota and pathogens via PRRs-MAMPs interaction are not well understood [67, 68, 69]. In general, new born adopts microbiota from mother and surrounding environment primarily. The lactate metabolizing flora, Bifidobacterium and Lactobacillus, derived mainly from vagina and breast milk lay the foundation at the gut for the development of complex microbial community in the later phase of life [68, 70] (Table 1). Adaptation of microbes which are 10 times higher compared to its cellular population is explained with the thought of co-evolution [4, 68]. Co-evolution of microbiota and emergence of adaptive immune system in humans set the advanced symbiotic relationship in evolutionary history [68, 71]. The mechanism(s) by which residential/ commensal bacteria induce immune tolerance have not yet been fully perceived. However, the occurrence of segmented filamentous bacteria (SFB) has been found to be important in inducing protective CD4+ T helper cells (Th 17) in the gut [69, 72]. Th-17 derived cytokines; IL-17, IL-21, and IL-22 induce other epithelial cellular functions of the gut and offer protection from pathogenic infection [73]. Besides SFB, Bacteroidetes can induce Treg cells which enable to maintain tolerance to commensals, suppression of immune responses against antigens, and activation of NF-kB (Nuclear factor- kappa B) dependent

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signalling pathways for epithelial wound healing. Residential microbiota thus, behaves like a mutual partner of the host for maintaining immunological homeostasis [67, 69].

   Healthy gut is highly variable and dynamic. The microbiota is widely influenced by several factors including age, diet, infection, medication (antibiotic use), lifestyle, environments and many more. The variations are unique and may be the signature in healthy and diseased conditions. A) Age: The composition of microbiota is found to be markedly changed with age. Significant changes in the gut microbiota occur within the first three years of life [74]. The microbiota in early age is relatively unstable. However, variation in microbiota and functional gene repertoires between individuals is greater in infants than in adults [16]. Microbial consortia in infants is affected by mode of feeding (breast fed or bottle fed), method of delivery (natural or Caesarian), and antibiotic use. Fint et al (2007) showed that the abundance of Anaerostipes caccae is very high in infants while Eubacterium hallii and Roseburia intestinalis are abundant in adults [75]. However, research evidences are not sufficient so far to explain the resilience of gut microbiota at different ages and relative influences in adult life. B) Diet: Diet plays a major role for determining the composition and establishing the homeostasis of gut microbiota. Ley (2010) reported that there remain distinct differences in gut microbiota between a lean and an obese [15]. The microbiota in obese is turned to the size and composition as that of lean on weight loss referring to role of microbiota in obesity. Dietary shift has direct and indirect influence on the physiology of the host as it influences both cellular composition and transcription network [68]. Diet rich in fat fed to mice showed shift in gut microbiota within 24h duration; increased level of Fermicutes, with special reference to bacteria of class Erysiipelotrichi [30, 76]. Differences in abundance of intestinal Faecalibacterium prausnitzii have been observed among obese Indian children [77]. Besides composition of gut microbiota, transcriptomes were also altered. Diet devoid of polysaccharides fed to gnotobiotic mice colonized with Bacteroides thetaiotaomicron showed degradation of host mucus polysaccharides. Besides, diet rich in animal protein, several amino acids with saturated fat which were consumed for long term showed association with abundance of Bacteroides while Prevotella was associated with carbohydrates and simple sugars. C) Infection: Infection is an invasion to the human system which disturbs the microbial entity and its host. Infections are common episode in humans where gut in particular experiences the maximum invasive challenges posed by infections [69]. Infection at the gut causes several diseases including diarrhoea. A large number of diarrhoeagenic pathogens (bacteria, viruses, fungi, protozoa) may cause diarrhoea and gastroenteritis [78]. Major bacterial pathogens are Vibrio cholera O1, O139; Shigella dysenteriae; pathogenic E. coli (EAEC, EPEC, ETEC, EAEC, EHEC, EAggEC), Campylobacter spp., Salmonella typhimurium [50, 78, 79]. All the pathogens are unique in pathogenesis with the possession of pathogenicity island (PAI) genes and virulence factors. Due to infection, gut physiology is highly disturbed which in turn perturbs the environment of indigenous biofilm. The attaching effacing character of certain E.coli dislodges the existing biofilm and promotes pathogenesis. However, allochthonous flora subsides with time and in many events they leave the host with the antibiotic interventions. Infections are also cleared by the existing microflora by antagonism by the production of antimicrobial peptides, lactic acid, hydrogen peroxide, ethanol, or by immunomodulating IgA, IgG production at the gut [2, 80].

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D) Antibiotics: Use of antibiotics influences largely the microbial community at the gut. Antibiotics pose a stern disturbance to the existing biofilm. However, gut microbiota attains an alternative stable state on antibiotic administration for short term and long term basis. Experimental evidences show the stability reaches in fecal microflora after considerable time with the use of two courses of ciprofloxacin in a 10 month period [36]. However, post-antibiotic equilibrium is resilient by nature. It is observed that clindamycin treatment for 2 years affected Bacteroides in the gut [81]. E) Lifestyle: Lifestyle brings considerable change in the composition of gut microbiota. In a recently published study, Gomez et al. (2016) showed difference in microflora between two African traditional populations: BaAka pigmey, having hunter-gatherer lifestyle and Bantu with western lifestyle [82]. BaAka live on meat, fish, vegetables and fruits representing ancient humans while Bantu rely on market economy based on growing tubers, flour, vegetables, raised goat meat, antibiotics and other therapeutic drugs with a trend towards western livelihood. The gut microbiota in BaAka community showed abundance of Prevotellaceae, Treponema, and Clostridiaceae, while Firmicutes in the Bantu community. The Bacteroidetes phylum was more dominant in the BaAka than the Bantu. On analysis, it was revealed that the Bantu microbiome composition fell on a spectrum between the BaAka and western populations. It was thus hypothesized that the Bantu might have lost some traditional microbiome features as they have adopted more westernized subsistence patterns [82].

     Gut microbiota is very important to restore good health. Though we have seen how it shows variation in its composition at different conditions. However, the core constituent microbiota participates in monitoring host health by mutualism. So, we observe that gut microbiota participate in host metabolism [83], in bile acid signalling [84], in regulation of gut hormones [18, 85], and in quorum sensing [2, 17, 86].

 ­  (S)-equol produced from soya isoflavone daidzein (diet rich in soya) has therapeutic application for the improvement of vasomotor symptoms, osteoporosis, prostate cancer and cardiovascular diseases, is digested by gut bacteria rather than human enzymes [87]. The fermentative gut microbiota participates in energy harvest by degrading complex polysaccharides to increased levels of SCFAs. Due to different microbiota composition in obese, SCFA is produced in large quantity which in turn become source of energy for several organs like, colon mucosa, the liver, some muscles and adipose tissue. Elevated SCFAs are bound to enterocyte receptor, GPR 41 result in secretion of a gut hormone, PYY which cause reduced intestinal transit, increased energy harvest and influence lipogenesis in liver (Figure 3). This has been experimentally demonstrated using mice model (Figure 3, 4) [83]. Alteration in phosphatidylcholine metabolism has also been observed by metabolomic analysis in mice with microbial conversion. Phosphatidylcholines are prerequisite for VLDL secretion. Impaired secretion results triglyceride accumulation in liver and is converted to methylamines which are found in plasma and urine. This condition possibly leads to steatosis and thus microbial composition and their metabolites may be in correlation with steatosis [88].

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Biofilm Control

Figure 3. Fate of gut microbiota in regulation of energy harvest-Gut microbiota utilize SCFA to harvest energy with the support of regulators





    

  



 

   



Figure 4. The gut microbiota are involved in the onset of metabolic disorders associated with obesitydysbiosis causes change in energy harvest mechanisms with development of metabolic endotoxemia which in turn cause develop T2 diabetes.

 ­€‚ƒ „…… †‡‚­­ ‡† The gut microbiota is an important regulator of bile acid metabolism. Bile acids are produced in the liver, stored in the gall bladder, and secreted into the duodenum upon ingestion of a meal. These are detergents required for lipid absorption potent signalling molecules, regulating several metabolic pathways. The gut microbiota regulates both the synthesis of bile acids and the production of

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3.11 

secondary bile acids and showed lower bile acids in germ free mice than colonized counterparts. As a signalling molecule, it activates several receptors including GPRs and regulates bile acid synthesis, cholesterol production, and glucose metabolism [65, 84].

  The gut is called the “second brain”. It establishes communication with the hypothalamus of the brain by neural and endocrine pathways for energy homeostasis [18]. The enteroendocrine cells are distributed along the length of the gut and account for 1% of the cells in the intestinal mucosa, thus constituting the largest population of hormone- producing cells in our body. Gut microbiota play an important role in regulation of gut hormones. As described earlier, these hormone producing cells have GPR41 or GPR43 and are activated by the ligand SCFA and produce increased amount of PYY causing decreased intestinal transit and increased hepatic lipogenesis (Figure 3). The gut hormoneIncretin (GLP-1), released by L-cells and K-cells respectively of the gut has shown a positive correlation with the increased levels of Bifidobacterium [85].

  

     Allochthonous microbial members of biofilms may involve in several infections in humans though it goes contrary to Koch’s postulates. Because, biofilm samples many a time do not prove to be a cause of visible etiology. However, an association between the presence of organisms and disease has been observed in diseases like periodontitis, and cystic fibrosis. Less association has been claimed in otitis media. All these events are happened due to cross talk of microbes among themselves in a social environment like biofilm. This communication is auto inducive or rather autocrine in nature. This is happened because of the liberation of a class of chemical family, called acyl homoserine lactones (AHLs) or autoinducers (AIs: AI-1, AI-2) or quorum sensing molecules and the event is Quorum sensing (QS). These molecules are released as a part of their defensive mechanism and thus depend on their numbers and sizes [17, 86].

    It has sufficiently been approved that the gut flora play a significant role in maintaining good health by restoring a condition called Eubiosis [2, 63, 68]. Eubiosis reinstates the homeostasis. Perturbations in its constituency due to known or unknown reasons result imbalance in the bacterial population, called Dysbiosis or dysbacteriosis. Dysbiosis is an etiology of bad health of an individual [68]. The fact is that most individual remains healthy with the big convoy of microbes since birth which is an obvious evidence of sustaining homeostasis. Numerous experimental data elucidate that this balance is unique and disaster comes on its damage. Injury in residential microbiota which causes loss or reduced microbial diversity results several common inflammatory diseases [68]. The dysbiosis is like a hole in a pot with water. Residential, opportunistic or pathogenic microbe gets entry into the system, cause infection and take the host to a disease state. Restoration of microbial diversity has thus been experimentally found effective in health establishment. Probiotics are those safe microbes which repair dysbiosis and proffer health beneficial effect to the host when it is taken in good quantity [40, 89]. We isolated a probiotic strain GS4, Pediococcus pentosaceus from fermented Indian cuisine [90] which showed multiple health promoting components like production of conjugated linoleic acid (CLA) [91], being antioxidant [92] and showed experimentally being anti-cancerous (colon cancer) [40], and can restabilise the normal gut microbiota in mice model [93]. Likely, Lactobacillus casei strain Shirota (LcS) showed multiple features in the improvement of intestinal health in human [94].

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Biofilm Control

Some of the most well-known gut problems associated with dysbiosis are IBD (inflammatory bowel diseases) [51], IBS (irritable bowel syndrome) [95], GERD (gastroesophageal reflux disease) [96], Crohn’s disease, ulcerative colitis [30], celiac disease [97] and SIBO (small intestinal bacterial overgrowth), Clostridium difficile- associated disease [16] respectively (Table 2). Gut flora also extends its influence over atopy and asthma [98, 99, 100], autism, schizophrenia [18], autoimmune disorders [28, 101] and colon cancer [52, 102]. The imbalance in the floral constitutions leads to leaky gut formation because of disruption of the gut barrier, damage in tight junctions and inflow of foreign antigens, proteins (non self) and formation of antibodies [2]. Dysbiosis developed at the early stage of HIV infection results in low abundance of Lactobacillus with Bifidobacterium at the gut [103]. Many of foreign proteins are homologous to self-proteins (molecular mimicry) of thyroid and pancreas which result in attacking its own protein due to molecular mimicry by formed antibodies and leads to autoimmune diseases like Hashimoto’s thyroiditis and type 1diabetes [104]. Some other diseases are associated with gut flora and nutritional partnership like, obesity and type 2 diabetes (T2D) [83, 105] (Table 2). Experimental evidence claims that gut microbiota mediated energy extraction from diet directs to the development of obesity and related metabolic disorders following multiple mechanisms [105]. It is postulated that dietary fatty acids (i.e., palmitic acid) may trigger an inflammatory response by acting via LPS (lipopolysaccharide) receptor (Toll-like receptor-4) (TLR-4) signaling in adipocytes and macrophages [106]. Thus, the gut microbiota-derived LPS -TLR-4 interaction initiates low-grade inflammation at the gut (Figure 4). Dietary fat shows a regulatory mode in adjusting plasma LPS level with specific changes in gut microbial community [83]. Significant shift in microbial composition has been observed in T2D condition and control (normal) group with substantial reduction in the amount of Firmicutes in T2D. The Firmicutes member, Faecalibacterium prausnitzii is reduced while inflammation is increased in T2D demonstrating its health beneficial role at the gut. It is thus an important probiotic strain for attenuation of insulin resistance [107]. It appears that diet driven change in gut microbial composition influence to cause several inflammatory diseases. Table 2. Bacterial gut flora and associated diseases with research findings Disease/ Disorders Atopy and Asthma

Celiac disease

Colon cancer

T1Diabetes

Role of microbiota Gut microbiota are developed and offer protection under the conditions of mode of delivery and nutrition uptake

Composition of Gut microbiota are different in patient and in healthy individual

Predominant Clostridial species, C. leptum and C. coccoides among patients Role in the development of

Research findings Bifidobacteria and Bacteroides spp are major bacterial members in the gut of infants born through the birth canal [53]. Delayed development of gut microbiota among newborn through Caesarian delivery with the dominance of Staphylococcus spp., Strptococcus spp., enterobacteria and Clostridium difficile [100]. Developing gut bacterial community functionally were found different between breast-fed and formula fed children [99]. Comparatively higher abundance of Bacteroides spp. and Escherichia coli in celiac disease patients than healthy individual. Ratio of Lactobacillus- Bifdobacteria spp to BacteroidesE.coli was found lower among patients [97] Higher abundance is due to the perturbation in gut microbial community among patients [52]. Bacteroides are abundant in DP while Lactobacillus with

   'ƵƚŝŽĨŝůŵƐ͗dƵŐŽĨtĂƌďĞƚǁĞĞŶ'ŽŽĚĂŶĚĂĚĂĐƚĞƌŝĂŝŶ,ƵŵĂŶ,ĞĂůƚŚ    ϯ͘ϭϯ Gut Biofilms: Tug of War Between Good and Bad Bacteria... 3.13

T2 Diabetes

insulin resistance with contrasting difference in flora composition in experimental mice model Associated with obesity

HIV

Dysbiosis in the gut

Irritabale bowel diseases [IBD]

Microbial composition contributes to inflammation; Depletion of Tregpromoting bacterial species with the overgrowth of bacteria inducing proinflammatory Th17 cells [39]

IBD-ulcerative colitis

IBS

Gastroenteritis

NEC

Obesity

Rheumatoid arthritis

Dysbiosis typically preceded by infection, change in diet or therapeutic administration (e.g., antibiotics) Pathogenic species take advantage of GI microbial community disruption to elicit infection NEC potentially due to abnormal bacterial colonization of the GI tract and lack of appropriate commensal bacteria in the gut Involved in food storage and energy harvest from food; Regulates peripheral metabolism

Treg-promoting organisms depleted; overgrowth of bacteria that induce Th17

Bifidobacterium were found higher in DR mice [101].

Direct relationship has been established between obesity, T2D and gut miroflora [83]. Contribution of gut microbiota towards insulin sensitivity [15]. Low abundance of Lactobacillus with Bifidobacterium was observed at early stage of HIV infection [103]. Dysbiosis results lower counts of Cl. leptum; Bacteroides uniformis [51], Fermicutes [95] and Bacteroides [96] while higher counts of E. coli; Proteobacteria and Bacteroides overtus [51]. Lower count of butyrate-producing Faecalibacterium prausnitzii found [31] in parallel with increased count of E. coli. Adherent-invasive E. coli found in higher abundance, diversity and in more IBDC patients than healthy individuals; higher variability of seropathotypes [31]. Higher counts of E. coli reported for IBDU patients in comparison to IBDC patients [30]. Lower levels of Bifidobacteria and Clostridium coccoides reported in comparison to healthy individuals. Absence of Lactobacillus spp. and reduced Collinsella abundance associated with IBS [29].

Suggested that Salmonella capitalizes on host disruption of GI microbiome through immune response to this infectious agent to proliferate and infect host [78] NEC patients had higher abundance Gammaproteobacteria in the GI tract [33].

of

Obese individuals exhibit lower abundance of Bacteroidetes and a higher abundance of Firmicutes compared with lean people [15] Ratio of Bacteroidetes and Firmicutes reverts back to a composition that resembles that of lean subjects following a diet and exercise regime [51] Absence or presence of specific functional groups and not bacterial species may be a more appropriate measure of the differences between obese and lean people [35]. Patients with rheumatoid arthritis had significantly less Bifidobacteria and Bacteroides–Porphyromonas– Prevotella group, Bacteroides fragilis subgroup

 3.14   

Biofilm Control  cell populations, leading to inflammation [72]. Intestinal microbes associated with etiology

and Eubacterium rectale–Clostridium coccoides group species [28]

ADP: Asymptomatic diabetes prone; CC: Colon cancer; DR: Diabetes-resistant; GI: Gastrointestinal; IBD: Inflammatory bowel disease; IBDC: Irritable bowel disease–Crohn's disease; IBDU: Irritable bowel disease–ulcerative colitis; IBS: Irritable bowel syndrome; NEC: Necrotizing enterocolitis.

  

Gut microbiota has been emerging as signature for determining the health condition of an individual [108, 109]. Many more researches are on the queue to reveal the fact that how one individual is different from other not by his genome only but by microbiome. The fact is to consider that the human genome is 1/100th part of microbial genome. The euphoria that has been evoked by the result of HGP (20,500 genes) more than a decade ago to predict, diagnose and treat the patient is still mysterious. About 10 million single nucleotide polymorphisms (SNPs) have been registered by last decade and still no significant input has been achieved for disease management. Research findings show that every human protein has interaction with bacterial motifs with wide and abundant peptide overlapping of hepatitis C virus, extensive interaction with pathogenic strains like E. coli, Salmonella typhimurium and Yersinia pestis and thus reveals the fact that proteomes of bacteria share hundreds of nonamer (nine subunit) sequences with the human proteome [26]. Genome online database (GOLD) enlisted fully completed 1,965 with partially completed 14,743 bacterial genome excluding viruses, bacteriophages and fungi which may persist not only on mucosal surfaces but also in endometrium, lung and blood respectively. These data will be of immense use in better understanding the gutmicrobe relation and health condition [48]. The microbiome of a host is found very essential and important to sustain a healthy life. Biofilms are not all bad. Microbial biofilm with variety of community members form distinctive society at different health and body conditions. The signature microbes and biofilms are the determinants of bad or good conditions of health. The good microbes (probiotics) go with the wellbeing of the host and assist in maintaining the homeostasis while bad microbes (pathogens) jeopardise the host-microbe relationship and initiate the diseases [85]. Gut, at large is a unique example of the biggest biofilm producing settlement comprising of thousands of bacterial species covering hundreds meter square area and thus build one of the largest consortia in living system, maintains good or bad health in an individual. That day is not very far when gut microbial population will be the signature of understanding the status of human’s health.



   

The author sincerely thanks the management of VIT University for supporting the infrastructural facilities for the promotion of research for society.

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33. Wang Y, Hoenig JD, Malin KJ, et al. (2009). 16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis. ISME J, 3(8), 944-954. 34. Sanders ME, Guarner F, Guerrant R, Holt PR, Quigley EM, Sartor RB, et al. (2013). An update on the use and investigation of probiotics in health and disease. Gut, 62:787–796. 35. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, et al. 2009. A core gut microbiome in obese and lean twins. Nature, 457, 480–484. 36. Dethlefsen L, and Relman DA (2011). Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci, USA, 108, 4554-4561. 37. Collado M, Buerl, Pérez-Martínez G (2012). Defining microbiota for developing new probiotics. Microb Ecol Health Dis, 23 PMCID, PMC 3747743. 38. O'Toole PW and Claesson MJ (2010). Gut microbiota: Changes throughout the lifespan from infancy to elderly. Intl Dairy J, 20(4), 281–291. 39. Helander HF and Fändriks L. (2014). Surface area of the digestive tract - revisited. Scand J Gastroenterol, 49(6), 681-689.doi:10.3109/00365521.2014.898326. 40. Dubey V, Ghosh AR, Bishayee K, Khuda-Bukhsh AR. (2015). Probiotic Pediococcus pentosaceus strain GS4 alleviates azoxymethane induced toxicity in mice. Nutr Res, 35(10), 921-929. 41. Jakoby WB, Ziegler DM. (1990). The enzymes of detoxication. J Biol Chem, 265(34), 2071520718. PMID 2249981. 42. Mowat AM and Agace WW (2014). Regional specialization within the intestinal immune system. Nature Reviews Immunology, 14(10), 667-685. Doi: 10.1038/nri3738. 43. Guarner F and Malagelada J (2003). Gut flora in health and disease. The Lancet. 361(9356), 512-519. Doi: 10.1016/S0140-6736(03)12489-0. 44. Armougom F, Henry M, Vialettes B, Raccah D, Raoult D (2009). Monitoring bacterial community of human gut microbiota reveals an increase in Lactobacillus in obese patients and Methanogens in anorexic patients. PLoS one, 4, e7125. 45. Reyes A, Haynes M, Hanson N, et al. (2010). Viruses in the fecal microbiota of monozygotic twins and their mothers. Nature, 466, 334-338. 46. Sears CL (2005). A dynamic partnership: Celebrating our gut flora. Anaerobe, 11(5):247251. 47. Benson AK, Kelly SA, Legge R, Ma F, Low SJ, Kim J, et al. (2010). Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc Natl Acad Sci USA, 107, 18933–18938. 48. Genome Online Database, (https://gold.jgi-psf.org).

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49. Probert HM and Gibson GR (2002). Bacterial biofilms in the human gastrointestinal tract. Cur Issues Intest Microbiology, 3, 23-27. 50. Vila J, Sáez-López E, Johnson JR, Römling U, Dobrindt U, Cantón R, et al. (2016). Escherichia coli: an old friend with new tidings. FEMS Microbiol Rev. 2016 Mar 8. Pii: fuw005. [Epub ahead of print]. 51. Dicksved J, Halfvarson J, Rosenquist M, et al. (2008). Molecular analysis of the gut microbiota of identical twins with Crohn's disease. ISME J, 2(7), 716-727. 52. Scanlan PD, Shanahan F, Clune Y, et al. (2008). Culture-independent analysis of the gut microbiota in colorectal cancer and polyposis. Environ Microbiology, 10(3), 789-798. 53. Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, Louis P (2009). Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii. Br J Nutr, 101, 541–550. 54. Ghosh AR, Koley H, De D, Paul M, Nair GB, Sen D (1996). Enterotoxigenic Escherichia coli associated diarrhoea among infants aged less than six months in Calcutta. India. Eur J Epid, 12, 81-84. 55. Ghosh AR, Sen D, Sack DA, Hoque ATMS. (1993). Evaluation of conventional media for detection of colonization factor antigens of enterotoxigenic Escherichia coli. J Clin Microbiol, 31(8), 2163-2166. 56. Ghosh AR, Nair GB, Naik TN, Paul M, Pal SC, Sen D (1992). Enteroadherent Escherichia coli: an important diarrhoeagenic agent in infants aged below six months in Calcutta. India. J Med Microbiol, 33 (4), 264-268. 57. Oppong GO, Rapsinski GJ, Tursi SA, et al. (2015). Biofilm-associated bacterial amyloids dampen inflammation in the gut: oral treatment with curli fibres reduces the severity of hapten-induced colitis in mice. NPJ Biofilms Microbiomes, 1, 15019. D o I : 10 .10 38 /np jbi ofilms.2015.19. 58. Trebichavsky I, Splichal I, Rada V, Splichalova A (2010). Modulation of natural immunity in the gut by Escherichia coli strain Nissle 1917. Nutr Rev, 68, 459–464. 59. Backhed F, Manchester JK, Semenkovich CF, Gordon JI (2007). Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci USA, 104, 979-984. 60. Krajmalnik-Brown R, Ilhan ZE, Kang DW, DiBaise JK (2012). Effects of gut microbes on nutrient absorption and energy regulation. Nutr Clin Pract, 27(2), 201-214. Doi: 10.1177/0884533611436116. 61. Denechaud PD, Dentin R, Girard J, Postic C. (2008). Role of ChREBP in hepatic steatosis and insulin resistance. FEBS Lett, 582, 68–73.

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62. Samuel BS, Shaito A, Motoike T, Rey FE, Backhed F, Manchester JK, et al. (2008). Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41. Proc Natl Acad Sci USA, 105:16767–16772. 63. Bjursell M, Admyre T, Goransson M, Marley AE, Smith DM, Oscarsson J, et al. (2011). Improved glucose control and reduced body fat mass in free fatty acid receptor 2 (Ffar2) deficient mice fed a high fat diet. Am J Physiol Endocrinol Metab, 300, E211–E220. 64. Roberfroid R, Gibson GR, Hoyles L, McCartney AL, Rastall R, Rowland I, et al. (2010). Prebiotic effects: Metabolic and health benefits. Br J Nutr, 104, S1–S63. 65. Mishra AK, Dubey V, Ghosh AR (2016). Obesity: An overview of possible role(s) of gut hormones, lipid sensing and gut microbiota. Metabolism, 65(1), 48-65. 66. Randal BR, Barbas AS, Bush EL, Lin SS, Parker W (2007). Biofilms in the large bowel suggest an apparent function of the human vermiform appendix. J Theor Biol, 249(4), 826831. 67. Jakobsson HE, Rodríguez-Piñeiro AM, Schütte A, et al. (2015). The composition of the gut microbiota shapes the colon mucus barrier. EMBO Rep, 16, 164-177. 68. Maynard CL, Elson CO, Haton RD, Weaver CT (2012). Reciprocal interactions of the intestinal microbiota and immune system. Nature, 489, 231-241. 69. Koboziev I, Webb CR, Furr KL, Grisham MB (2014). Role of the enteric microbiota in intestinal homeostasis and inflammation. Free Rad Biol Med, 68, 122-133. 70. Santiago GL, Cools P, Verstraelen H, Trog M, Missine G, El Aila N, et al. (2011). Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles. PloS one, 6, e28180. 71. Sachs JL, Hollowell AC (2012). The Origins of cooperative bacterial communities. mBio, 3:e00099–12. 72. Chow J, and Mazmanian SK (2009). Getting the bugs out of the immune system: does bacterial microbiota “fix” intestinal T cell responses? Cell Host Microbe, 5(1), 8-12. 73. Farkas AM Panea C, Goto Y, Nakato G, Galan-Diez M, Narushima, Honda, Ivanov II (2015). Induction of Th17 cells by segmented filamentous bacteria in the murine intestine. J Immunology Methods, 421, 104-11. Doi: 10.1016/j.jim.2015.03.020. 74. Koenig JE, Spor A, Scalfone N, et al. (2011). Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci, USA, 108, 4578-4585. 75. Fint HJ, Duncan SH, Scott KP, Louis P (2007). Interactions and competition within the community of the human colon: links between diet and health. Environ Microbiol, 9, 11011111. 76. Walker AW, Ince J, Duncan SH, Webster LM, Holtrop G, Ze X, et al. (2011). Dominant and diet-responsive groups of bacteria within the human colonic microbiota. ISME J, 5, 220–230.

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CANDIDA BIOFILMS: CHARACTERISTICS IN ENVIRONMENT SETTINGS AND NOVEL THERAPEUTIC OPTIONS IN CLINICS                  ­€‚ ƒ„…†‡ˆ‰€‡ ­Š€­‹Œ„…†‡ˆ†ŽŽ€‡ŽŠ‰ ‘’“„ ”•

   ƒ  ƒ–Œƒ  “–   ““  ƒ  —˜™˜        ƒ“ƒ    ƒ “”  –  ƒ  ƒ– “ ’ƒ“–““š ›– “œ ˜ ƒ ƒ    ƒ ƒ    – “   “  ž  ƒ –ƒ    ƒ “’ – “    “Œ’     – “  ƒƒƒ ƒ    ˜       –            “ “  –    – “    ƒ “” ƒ     – “ƒƒ ƒƒšƒƒƒ  Ÿƒ     ƒ   ƒ   ƒ  – “’  ƒ

 „– “ƒ“ƒ

   ­­€ Over the last few decades, we have witnessed the exponentially rising incidence of fungal infections due to the growing numbers of compromised populations such as HIV/AIDS patients, transplant recipients and patients on chemotherapy [1]. Candida is the major fungal pathogen in humans [2]. Candida is a dimorphic fungus that thrives in a variety of niches in the human body in harmony with the resident microbiota. Candida species inhabit approximately one-half of human oral cavities as commensals, and persist as mixed species communities intraorally. Candida is also found in other mucosal habitats such as the vagina. However, under certain circumstances, Candida cause infections

4.2 

Biofilm Control

or candidiasis, which range from superficial mucous membrane infection to life-threatening systemic disease [2]. Since the early 1960s Candida has emerged from being an infrequent pathogen to become one of the most common agents of nosocomial infection (Matthews, 1994). Hence, candidiasis is now the third or fourth leading cause of nosocomial infection in the United States, ranking even higher than some common bacterial infections [3, 4]. Moreover, mortality rates among patients with candidiasis have been increasing, and are reported to be as high as 40% to 60% [5, 6]. Major factors contributing to the virulence of Candida include its versatility in adapting to a variety of different habitats for growth, and formation of surface-attached microbial communities known as ‘biofilms’ [1]. Candida albicans, the major fungal pathogenic species is also capable of morphogenesis. Hence, C. albicans can exist as yeast, pseudohyphae or hyphal forms [2]. Yeast form of C. albicans aids adhesion and dissemination of cells whilst hyphal form facilitates the invasion of host tissues. Biofilm forming ability is regarded as one of the major virulent attributes of Candida species. In particular, Candida species are well known for their ability to adhere to indwelling medical devices and form biofilms, often leading to blood stream infections [7]. In clinical terms, Candida biofilm communities are capable of withstanding many external challenges such as antifungal treatment. Moreover, interaction of Candida and bacterial species form mixed-species biofilms causing a dilemma for clinicians. Dental plaque found on the tooth surface is an example of mixed-species biofilm where Candida co-exist with resident bacteria [8]. Understanding Candida and bacterial interactions and their underlying biofilm-forming mechanisms are pivotal for both biological sciences and clinical settings. It is imperative that the clinicians remain abreast of this evolving field of fungal biofilms and its relevance to clinical outcome. In this chapter, we discuss the characteristics of the Candida biofilms in environmental settings, its clinical relevance and novel therapeutic options.

 Biofilms are one of the earliest consortia of life forms on Earth [9]. It is defined as microbial communities encased in a matrix of extracellular polymeric substance (EPS) and displaying phenotypic features that differ from their planktonic or free-floating counterparts [10]. The fungus most commonly associated with biofilm-related human infections is C. albicans, although other nonalbicans Candida species have also been implicated in biofilm formation [11]. Fungal biofilm formation proceeds in an organised manner and in sequential stages: First, adhesion of a microorganism to a surface; second, individual microcolony formation, and organisation of cells; third, secretion of EPS; then maturation into a three-dimensional mature biofilm structure; and last, dissemination of progeny biofilm cells [Figure 1] [1]. This staging is based on growth kinetic assays and microscopic observations of developing biofilms on a surface, over a period of time. Most of the fungal biofilm follow this general sequence, indeed with subtle variation at species and strains levels [12]. There are some similarities and differences in the architecture of the Candida biofilms compared to that of common bacterial biofilms. Thickness of the Candida biofilms can either be thicker or thinner than bacterial biofilms depending on the species and the environmental conditions [13]. Candida biofilms grown on in vitro models usually consist of several layers. Biofilm thickness may range from few micrometres to few hundred micrometres. Unique feature of C. albicans species that it forms biofilms containing different morphological forms such as yeast, pseudohyphae and hyphae cells encased in EPS [1]. A summary of the events taken place during the formation of a typical Candida biofilm is given below.

4.3 

Candida Biofilms: Characteristics in Environment Settings...





Sequence of Biofilm formation Detachment of biofilm fragments

  biofilm

 Adhesion   D etachment

 colonizers

Adhesion

EPS production Adhesion S U R FA C E

Figure 1: Sequence of biofilm formation

 The first event in building up a biofilm community is the adhesion of microorganism to a biotic or abiotic surface. One of the most promising features of Candida to be a successful biofilm former is its ability to adhere strongly to various surface [14]. Candida is known to adhere to a surface within one to two hours [15]. Non-specific interactions such as hydrophobic and electrostatic forces mediate the initial adhesion between the cells and the substratum [16, 2]. This is followed by expression of specific adhesion molecules to facilitate stronger adhesion [14]. Candida adhesions are specific cell wall proteins that help the fungus to adhere to the abiotic and biotic surfaces. In the human body these adhesions interact with host cell proteins to facilitate fungal colonization. After adhesion, cells begin to proliferate and spread both horizontally and vertically which results in microcolony formation. Cell wall of the Candida is mainly composed of carbohydrates such as glucan, chitin and mannose [17]. Adhesins are glycosylated cell-wall proteins (CWP) located at the exterior side of the cell wall of Candida species. Most of the adhesion proteins have glycosyl-phosphatidyl-inositol (GPI) anchor [18]. In C. albicans the three gene families encoding for adhesion properties are ALS, HWP, and IFF/HYR. Agglutinin-like sequence (ALS) gene family is the mostly studied adhesion in C. albicans [19, 20]. Candida glabrata has adhesins encoded by the EPA (epithelial adhesion) gene family and Epa proteins are structurally similar to Als proteins of C. albicans [21]. Hwp1 and Ecap1 are two adhesins present in C. albicans that promote colonization. to surfaces. Eap1, unique to C. albicans facilitates adhesion to abiotic surfaces and biofilm formation [14]. On the other hand, Ywp1, a yeastspecific protein of C. albicans is known to negatively regulate the adhesion [22, 21]. Hence, Ywp1 is not present in the hyphal or chlamydospores forms. It has been shown that yeast cells that lack Ywp1 are more adhesive and form thicker biofilms implying an anti-adhesive activity for Ywp1.

4.4 

Biofilm Control

    Second step of building a biofilm community is formation of microcolonies. Adherent blastospores undergo division and repeated aggregation over each other (co-adherence) to form microcolonies, which eventually lead to formation of a complex heterogeneous structure. The highest architectural stability of the C. albicans biofilm reaches a developmental plateau between 24-48 h [12]. By about 12-30 h, microcolonies in the biofilm are bridged by intertwining hyphal elements forming a bilayered structure comprising yeast, germ tubes and young hyphae.

           Secretion of EPS is a unique property of microbial biofilms. EPS displays a three-dimensional, gellike consistency and consists of polysaccharides, proteins and extracellular DNA (eDNA). EPS is known to be instrumental in maintaining the integrity of biofilm by providing a scaffold to sustain the biofilm architecture. EPS is also directly linked with the higher resistance of biofilm communities as it provides a protective barrier against antimicrobials and phaygocytic cells. There are several studies in the literature demonstrating the critical role of EPS in antifungal resistance of the Candida biofilms [23, 24]. For instance, it was shown that when EPS is removed, the survival of Candida biofilms cells against amphotericin B is also decreased. Poor penetration of antifungal drugs through the EPS is not the only mechanism that contributes to the higher resistance. EPS may also have antifungal neutralising agents. Hence, a Candida biofilm cells grown statically in the presence of minimal matrix exhibited the same level of resistance to the antifungals flucytosine, fluconazole, and amphotericin B as cells grown in a shaker with a large amount of matrix [24]. EPS is an attractive area of research in the field of Candida biofilms due to its medical importance. If one is able to devise a strategy to impede EPS formation in Candida biofilms, it will be a step closer to find a solution for biofilmassociated infections.

      C. albicans mature biofilm consists of a dense and heterogeneous network of yeast, pseudohyphae and hyphae, which can be observed by 48 h under in vitro conditions [12]. Microscopic observations of in vitro C. albicans biofilm architecture on abiotic surfaces have shown an adherent yeast cell layer at the base, and multi-layers of hyphal elements embedded in EPS on the top [25]. However, the C. albicans biofilm in vivo is arranged in an unorganised manner with yeast cells randomly interspersed with hyphae, in the matrix additionally including other cells of the host [26]. The heterogeneous buildup of mature biofilms is also accompanied with water channel architecture which is seen surrounding the microcolonies and between hyphal elements (Figure 2). These water channels facilitate influx of water and nutrients through the biomass to the bottom layers in mature biofilms and also allowing waste/metabolite disposal to the surrounding environment. [27].

      Dispersal is the least understood and perhaps the most complicated process involved in both fungal and bacterial biofilms. As a part of their life cycle, members of the Candida biofilm community singly or as a group may detach from the biofilm, and disseminate through a fluid phase to seed new sites. Genome-wide studies have shown that Set3, a NAD-dependent histone deacetylation complex, modulates the NRG1 [28], a transcriptional regulator of biofilm dispersal [29]. Nrg1 is a transcriptional repressor of filamentation [30] and Set3 complex mutants are hyperfilamentous [31]. The typical dispersal of cells of C. albicans from biofilms is found to be in the yeast form, possibly

4.5 

Candida Biofilms: Characteristics in Environment Settings...



due to the effect of Nrg1 and Set3 complex [29]. Thus, manipulations that increase filamentous cells and decrease yeast-form cells may reduce biofilm dispersal [32]. Studies by [33] revealed that dispersal stage of the fungal biofilm has an association with disease progression. Namely, in C. albicans deletion of Nrg1 genes completely attenuates virulence in a murine model of systemic candidiasis [33]. However, it is noteworthy that deletion of Nrg1 also results in morphological changes in Candida cells [34], therefore, it is not certain that the effect is solely due to biofilm dispersal. If targeted well, biofilm dispersal stage may provide an alternative strategy for developing novel therapeutic options in future for controlling Candida biofilm associated infections.

(A)

(B) Top

Bottom  Figure 2: Scanning electron microscopy (SEM) and confocal laser scanning microscopy images of 24 hour biofilm of Candida albicans SC5314. (A) SEM: scale bar 20 m and 10 m, respectively. (B) Confocal: scale bar 20 m



4.6 

Biofilm Control

      Researchers have developed some in vitro and in vivo models in order to understand the growth kinetics and properties of Candida biofilms [12]. These experimental model systems have been used to successfully study the basic properties of Candida biofilms such as growth kinetics, architecture, antifungal resistance, intra and inter-species interaction using standard laboratory assays such as XTT assay, spectrometry, colony forming unit (CFU) counting, scanning electron microscopy (SEM) and confocal laser scanning microcopy (CLSM).

  In vitro model systems such as multi-well polystyrene plates serve as a standard method for studying biofilm development under controlled conditions with the advantage of being simple. Conventionally polystyrene 96-well plates are widely used to determine the growth kinetics and antifungal susceptibility of fungal biofilms [35]. In addition, more advanced flow cell models such as the modified Robbins device, which simulate the host environment that fungal cells would normally encounter, have also been used for C. albicans biofilm studies [27]. In vitro biofilm models are very useful for antifungal susceptibility testing against Candida biofilm mode of growth [36]. Moreover, in vitro models can also be used for high throughput screening assays of new antifungal agents against Candida biofilm mode of growth [37].

 ­€­‚ƒ€­  There are various ex vivo reconstituted epithelial models for studying Candida mucosal infection [38]. Reconstituted human oral epithelium (RHOE) is a commonly used model to examine oral candidiasis and host response [39]. To develop ex vivo C. albicans biofilm, commercially available oral epithelium is reconstituted by incubation for 24 h in serum-free, MCDB 153 defined medium in tissue culture plates [40]. RHOE is then infected with Candida strains and incubated at 37°C, in 5% CO2. Candida strains would form detectable biofilm within 24-72h.

Figure 3. Comparison of biofilms formed by C. albicans haploid and diploid on ex vivo reconstitute human oral epithelial cells (Adapted from [42].

Candida Biofilms: Characteristics in Environment Settings...

4.7 



Using this model, it was found that oral epithelia possess a mechanism that sense the Candida hyphae and secrete beta-defensins against the invading fungal pathogen [41]. Moreover, recently it was found that IRA2 is a major regulator of C. albicans biofilm formation on ex vivo oral epithelial tissue models [42]. Hence, ex vivo models are very useful for understanding the host response occuring during mucosal Candida infections without involving live animals. Therefore, researchers working on this field can consider models such as RHOE to decipher molecular mechanism of Candida-host interaction before processing for animal studies.

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In vivo biofilms show true phenotypic traits and approximately similar structure to the biofilms observed in clinical practice. However intrinsic interactions are complex and require meticulous control over process parameters to generate accurate and reproducible biofilms [43, 44]. In-spite of such limitations studies by Nett and Andes (2006) have developed successful Candida biofilm models using mouse and rat. The most commonly used in vivo model is the central venous catheter biofilm model which mimics vascular catheter infection in patients [45]. Mucosal biofilm formed on tongue surface is another in vivo model that simulates oral candidiasis in the clinical settings. Immuno-supression in mice is induced by subcutaneous injections of prednisolone for a few days prior to Candida inoculation. Candida inoculum is prepared in phosphatebuffered saline and subsequently swabbed on the tongue surface. In our experience, oral candidiasis develops approximately in a week when infected with C. albicans SC5314 strain [37]. There are also some limitations associated with in vivo Candida biofilm models. Candida is a commensal organism in the human microbiota, but not in the murine microbiota, a factor that may contribute to the difference in the outcome [46]. In addition, variations between the immune systems of mice and the humans could also contribute to the different outcome observed in in vivo studies [47]. One such difference between murine and human systems can be best demonstrated by triazole, which shows faster metabolism in mice than in humans by cyto-chrome P450, thereby enhancing the in vivo efficacy of azole in mice [46]. Therefore, it is recommended that a thorough testing for the in vivo efficacy of a lead compound should be done in the early stages of antifungal drug development.

ϰ͘ϰ/EdZd/KEK&E/^W/^/EWK>zD/ZK/>/K&/>D^ Polymicrobial biofilms serve as a reservoir for multiple species to coexist in harmony, thus posing a clinically challenging health problem in today’s World [48]. Therapeutic management of infectious diseases has become a difficult proposition owing to species heterogeneity within polymicrobial biofilms. Antimicrobials directed towards one species in a mixed-species biofilm often facilitate nontargeted organisms to thrive and infect [49] . Antibiotic sore-mouth or erythematous candidiasis is an example of the oral Candida infection due to use of broad-spectrum antibiotics for a long period of time. Suppression of the commensal bacteria leads to overgrowth of the commensal Candida species in the oral mucosa in the form of oral infections. Therefore, understanding of mixed species biofilm between Candida and bacteria is essential to develop more effective treatment options. The bacteria-bacteria or bacterial-fungal interactions have been reviewed previously in opportunistic infections such as those in the oral cavity [50], intestinal tract [51], female reproductive tract [52], as well as in implant- and catheter-related infections [53]. Candida is known to readily form mixed species biofilm on indwelling medical devices and mucosal tissues [54]. Candida species such as C. albicans, C. glabrata, C. tropicalis and C. krusei have been co-isolated either in combination or with other bacterial species such as Enterobacter species, Pseudomonas aeruginosa and Klebsiella pneumonia (Table 1) [55]. This observation suggests that polymicrobial interactions play a pivotal role in influencing clinically relevant outcomes such as drug and host resistance and virulence. For

4.8 

Biofilm Control

instance, P. aeruginosa and C. albicans mixed species biofilms are common in cystis fibrosis patients [56, 57]. It has been shown that mixed species biofilms are difficult to eradicate and often results in fatal outcomes for these patients. A brief account of Candida and bacterial interaction in the biofilm community is given below.

  The interaction between bacterial and fungal organisms has received significant attention probably because of its association with infections of endotracheal tubes, biliary stents, silicone voice prostheses and acrylic dentures [58]. C. albicans polymicrobial biofilms are also implicated as the major cause for nosocomial candidemia with Staphylococcus epidermidis, Enterococcus spp., and Staphylococcus aureus being the most commonly co-isolated bacterial species. C. albicans has been known to display a synergistic interaction with commensal oral streptococci, including most species from the viridians group of streptococci [59]. C. albicans can form aggregates with microorganisms such as Streptococcus gordonii, S. oralis and S. sanguinis, involving fungal adhesins, hyphal wall proteins and polysaccharides of streptococci [60]. Interestingly, antagonistic interactions are known to exist between bacteria and fungi coexisting in a biofilm matrix (Table 1). For example, the gram-negative bacterium, Pseudomonas aeruginosa, is often co-isolated with C. albicans from patients with hospital-acquired infections, particularly those linked with colonization of medical devices such as catheters, patients with cystic fibrosis, and burn victims. P. aeruginosa can kill yeast, hyphae and biofilms of C. Albicans [61]. These two species are known to possess various signalling molecules that perform cross-talks within the biofilm community. These molecules help P. aeruginosa to attach and form biofilms on C. albicans hyphae which in turn restricts the growth of fungus [62]. Thein et al., (2006) [63] have also reported that P. aeruginosa ATCC 27853 elicits inhibition in C. albicans biofilms in a concentration dependent manner.

      Other Candida species such as C. dubliniensis, C. glabrata, C. krusei and C. tropicalis are gaining clinical interest as emerging pathogens due to their increasing association with various forms of Candida infections, resulting in morbidity and mortality especially in compromised population groups [64, 65]. The oral carriage of other Candida species in certain groups such as HIV/AIDS patients is even higher than that of C. Albicans [66]. Hence, other Candida species are currently under focus as significant pathogens of clinical importance. Pioneer studies on growth in planktonic and biofilm mode by C. albicans with other species namely C. dubliniensis was carried out by [67]. Organisms with superior growth rates were found to dominate with time in planktonic state, perhaps due to competitive pressures within suspension cultures for nutrients, dispersion of metabolic waste, temperature, and pH. Similar competitive inhibition has been observed even during the initial step of adhesion onto a substrate during dualspecies Candida biofilm development. It has therefore been postulated that the adhesion potential of individual Candida species and the competition for adhesion sites between the two Candida species regulate the existence and colony expansion in a biofilm consortium [68]. In another independent study the ability of clinical isolates to form biofilms was evaluated in four different species of Candida in both single-species and multi-species combinations on the surface of dental acrylic resin strips. The findings revealed other Candida species to have greater biofilmforming ability than C. albicans species exhibiting intra-species variation [69]. Presence of C. albicans in multi-species biofilms enhanced biofilm production of non-albicans Candida species whereas C. tropicalis retarded the biofilm production with all other non-albicans Candida

Candida Biofilms: Characteristics in Environment Settings...

4.9 



species. These results suggest that C. albicans might be able to provide a substratum to the other Candida species on acrylic prosthesis [69]. Mixed-species Candida biofilm models on denture acrylic surfaces using C. albicans and C. krusei as competing partner-organisms revealed mutual antagonistic interactions, exemplified by low abundance of C. albicans hyphae and sparse distribution of yeast cells (both C. albicans and C. krusei) than in monospecies biofilms [70]. It thus seems that at least in this combination pair, Candida species in the monospecies mode populates to form a dense biomass, but not in combination with another species. Although the exact mechanisms of such interactions in mixed-species fungal biofilms are still elusive, quantitative and qualitative features of one Candida species do modify the physiology of another Candida species during cohabitation within a biofilm [71].

       One of the major phenotypic features of Candida biofilm is their higher resistance to antifungal agents. Conventionally minimum inhibitory concentrations (MICs) of antifungal agents are based on planktonic mode antifungal susceptibility testing. These MIC values cannot be directly implicated to the biofilm mode of growth as Candida biofilms often have higher MICs than the planktonic mode [36]. This reflects the real-life situations as treatment failures are common in Candida biofilmassociated infections. Candida biofilms exhibit resistance to agents from all available, commonly used antifungal drug classes, including the azoles (fluconazole, itraconazole, voriconazole, posaconazole), the echinocandins (caspofungin, micafungin, anidulafungin), the amphotericin B formulations and flucytosine [72]. Several mechanisms have been suggested to explain the higher drug resistance in Candida biofilms. These include altered growth/metabolic rate, restricted diffusion, presence of extracellular matrix, genomics and proteomic changes, ‘persister’ cells and adaptive stress response [1]. Many recent studies have reported that the azole group of antifungals such as ketoconazole, fluconazole, including the novel variconazole are not able to eradicate Candida biofilms [73]. Polyenes class of antifungals such as amphotericin B works better for Candida biofilms than azoles; however, biofilm mode is more resistant than planktonic cells [74]. Novel echinocandin class of drugs such as caspofungin, micafungin and anidulafungin are reported to be the most effective for Candida biofilms than other classes of antifungals [73, 75, 76]. However, efficacy of caspofungin may vary depending on the targeted strains and environment [77, 78]. These findings highlight the need for the development of new antifungal agents for biofilm-associated Candida infections with minimal toxicity to the host. Therefore, researches have sought alternative antifungal approaches such as photodynamic therapy, nano-therapy, probiotics and small molecule based drug discovery [79].

  Photodynamic therapy (PDT) is a modern therapeutic strategy that applies the interactions between a light source of a particular wavelength and a photosensitizer (PS) such as methylene blue, in the presence of tissue oxygen, to generate reactive oxygen species (ROS). These ROS will cause oxidative damage to the target cells such as microbial cells including bacteria and fungi [80]. PDTinduced singlet oxygen and oxygen radicals damage the fungal cells by perforation of the cell wall and membrane, allowing the photodynamic dye to be further translocated into the cell. Subsequently, the dye in its new sites photo-damages inner organelles such as the nucleus and induces cell death [81, 82]. Candida species including C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei and C. dubliniensis are susceptible to PDT [83]. PDT, therefore, is suggested to be as potent as topical nystatin for the treatment of denture stomatitis PDT treatment of candidiasis in HIV-infected patients using combination of a low-power laser and methylene blue 450g/mL has shown to be useful in

4.10 

Biofilm Control

eliminating fungal infection and no recurrences up to 30 days [84]. PDT also shows some potentials in eliminating oral Candida species with 40% studies employing either methylene blue or toluidine blue or both as PS, nearly 33% studies using hematoporphyrin derivative and approximately 26% of them using either curcumin, erythrosine, rosebengal or malachite green [80]. However, still there is no consensus on the effective dose/concentration of the PDT to be used against C. albicans due to varied results reported in the literature [85, 87]. On the other hand, whether PDT can effectively eliminate Candida biofilms under clinical condition is yet to be confirmed. However, this is an area warranting more investigations [87].

 According to the definition from NNI (National Nanotechnology Initiative), nanoparticles are structures of sizes ranging from 1 to 100 nm in at least one dimension [88]. As nanoparticles with optimized physicochemical and biological properties are taken up by cells more easily than larger molecules, they can be successfully used as delivery tools for currently available bioactive compounds [89]. Extracellular matrix in biofilms is known to be a barrier for the penetration of drugs and hence accounts for the reduced antimicrobial activity against mature biofilms [90]. Therefore, researchers have attempted the use of nanoparticles as a strategy to generate new antifungals with better properties than the existing ones for Candida biofilms [91]. Silver nanomaterial is a good example of this technology. Silver has been described as being ‘oligo-dynamic’, meaning it has a toxic (bactericidal/ fungicidal) effect on living cells even at low concentrations [92]. Hence, silver-containing materials have been used to prevent bacterial colonisation and infections on medical devices and dressing. It inhibits fungal multiplication by interfering with DNA replication. Silver ions can also lead to protein denaturation and cell death because of their reaction with nucleophilic amino acid residues in proteins and their attachment to thiol, amino, imidazole, phosphate and carboxyl groups of membrane proteins or enzymes [93]. Silver nanoparticles have a well-tolerated tissue response with less cytotoxicity or genotoxicity and lower propensity to induce microbial resistance [94]. Recently, antifungal activity and an inhibitory effect on adhesion and biofilm formation by denture base resin containing nano-silver were demonstrated [95]. Nano-silver’s antifungal activity is higher against C. glabrata than C. albicans. Nano-silver particles are also more effective in inhibiting biofilm formation than in controlling established biofilms [91]. In vitro study has also demonstrated potent antifungal effects of nano-silver coating of denture base material, as shown by inhibition of Candida adherence to the surface and deformation of the normal morphology of Candida. Further attempts to apply silver nano-particles-coated denture base materials for clinical use are expected.

    Probiotic microbes are defined as organisms that may be ingested in various formulations to improve the health either in humans or in animals [96]. Lactic acid bacteria including bifidobacteria, lactobacilli and enterococci are the most typical probiotic bacteria. Pioneering research more than a quarter century ago has revealed the inhibitory effect of Lactobacillus acidophilus on C. albicans. [97, 98]. The strains of L. fermentum and L. acidophilus are known to interfere with Candidal colonization by producing antifungal substances, hydrogen peroxide, and antiadhesive biosurfactants [99]. These results were supported by [100], who found that 9 out of 12 strains of Lactobacilli could produce hydrogen peroxide, which inhibits C. albicans growth. Animal studies carried out to assess the biotherapeutic potential of probiotic bacteria on candidiasis, have also demonstrated growth-retardant effect of L. acidophilus on Candida [101]. Probiotic bacteria are also shown to have protective effect on animals by modulation of a variety of immunologic (thymic and extrathymic) and nonimmunologic

Candida Biofilms: Characteristics in Environment Settings...

4.11 



mechanisms. In vitro studies, especially with L. acidophilus, L. rhamnosus GR-1 and L .fermentum RC-14, have demonstrated the effectiveness of these bacteria in preventing the colonization and infection of the vagina by C. albicans [102, 103]. But results of few clinical trial conducted do not corroborate these findings [104]. Methodological problems such as small sample size, no control group (placebo) and inclusion of women without confirmed recurrent disease have been implicated as reasons for the inability to draw definitive conclusions [105]. However, more research using molecular and proteomic approaches needs to be performed to elucidate the mechanisms underlying probiotics, so that their beneficial bio therapeutic effects can be optimized.

   In antimicrobial drug discovery, small molecules are defined as non-peptide organic compounds that are synthetic or obtained from natural product extracts and are of low molecular weight (200–500 Da), thus binding to biopolymers such as proteins and nucleic acids and altering their normal functions [106, 107]. A handful of small molecule antifungals which are effective against in vitro Candida biofilms have been identified [37]. Recently, we discovered a novel antifungal agent, SM21 using high-throughput phenotype-based screening of a library of 50,240 small molecules. Subsequent in vitro assays revealed that SM21 is fungicidal against a wide range of Candida species including multidrug resistant strains. Interestingly, this molecule was effective for the in vivo murine model of systemic and oral candidiasis. Hence, small molecule based drug discovery can be an excited new avenue to explore antifungal agent against Candida biofilms.

     Biofilm formation is a major virulence attribute for mucosal and systemic Candida infections. Exceeding high level of drug resistance of Candida biofilm is directly related to the therapeutic failure. As per foregoing information, there are some exciting strategies which may be effective for Candida biofilms seen in clinical setting, although more research and evidence is needed. Future research work should focus on understanding the molecular mechanism behind higher drug resistance of Candida biofilms as well as exploring antifungals with new mechanism for this fungal pathogen, which could bring enormous benefits for the growing body of compromised host populations worldwide.

   Some of the studies mentioned in this chapter were funded by Health and Medical and Research Fund (HMRF), Hong Kong, Hong Kong and National Medical Research Council (NMRC), Singapore. Some of the text was republished with the permission of Seneviratne CJ et al., 2008, Oral Diseases and Thein et al., 2009, Mycoses.

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4.17 



73. Kuhn DM; George T; Chandra J; Mukherjee PK; Ghannoum MA; (2002); Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother; 46(6); 1773-80. 74. Tobudic S; Kratzer C; Lassnigg A; Graninger W; Presterl E; (2009); In vitro activity of antifungal combinations against Candida albicans biofilms. J Antimicrob Chemother; 65(2):271–274. 75. Bachmann SP; VandeWalle K, Ramage G; Patterson TF; Wickes BL; Graybill JR; López-Ribot JL; (2002); In vitro activity of caspofungin against Candida albicans biofilms. Antimicrobial Agents and Chemotherapy; 46(11); 3591-3596. 76. Shuford JA; Rouse MS; Piper KE; Steckelberg JM; Patel R; (2006); Evaluation of caspofungin and amphotericin B deoxycholate against Candida albicans biofilms in an experimental intravascular catheter infection model. J Infect Dis; 194(5); 710-3. 77. Jain N; Kohli R; Cook E; Gialanella P; Chang T; Fries BC; (2007); Biofilm formation by and antifungal susceptibility of Candida isolates from urine. Appl Environ Microbiol; 73(6); 1697-703. 78. Serefko A; Chudzik B; Malm A; (2006); In vitro activity of caspofungin against planktonic and sessile Candida sp. cells; Pol J Microbiol; 55(2);133-7. 79. Kasimanickam RK; Ashish Ranjan; GV Asokan; Vanmathy R Kasimanickam; John P Kastelic; (2013); Prevention and treatment of biofilms by hybrid- and nanotechnologies. International Journal of Nanomedicine; 8; 2809–2819. 80. Javed F; Samaranayake LP; RomanosGE; (2014); Treatment of oral fungal infections using antimicrobial photodynamic therapy: a systematic review of currently available evidence; Photochem. Photobiol. Sci; 13; 726. 81. Ladan H; Nitzan Y; Malik Z; (1993); The antibacterial activity of haemin compared with cobalt, zinc and magnesium protoporphyrin and its effect on potassium and ultrastructure of Staphylococcus aureus. FEMS Micrbiol Lett; 12; 173–177. 82. Stenstrom K; Moan J; Brunborg G; Eklund, T; (1980); Photodynamic inactivation of yeast cells sensitized by hematoporphyrin. Photochem Photobiol; 32; 349–352. 83.

Doria AC; Bevilacqua I; Santos T; Teodoro G; Oliveira E; Khouri S; Nicolau R; (2014); The action of photodynamic therapy on Candida species (836.10); The FASEB Journal; 28; 836.10.

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Scwingel AR; Barcessat AR; Nunez SC; Ribeiro MS; (2012); Antimicrobial Photodynamic Therapy In The Treatment of Oral Candidiasis In HIV-Infected Patients; Photomed. Laser Surg; 30; 429–432.

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Pupo YM; Gomes GM; Santos EB; Chaves L; Michel MD; Kozlowski VA Jr; Gomes OM; Gomes JC; (2011); Susceptibility Of Candida Albicans To Photodynamic Therapy Using Methylene Blue And Toluidine Blue As Photosensitizing Dyes; Acta Odontol. Latinoam; 24; 188–192.

4.18 

Biofilm Control

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Souza RC; Junqueira JC; Rossoni RD; Pereira CA; Munin E; Jorge AO; (2010); Comparison Of The Photodynamic Fungicidal Efficacy Of Methylene Blue, Toluidine Blue, Malachite Green And Low-Power Laser Irradiation Alone Against Candida Albicans; Lasers Med. Sci; 25; 385–389.

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4.20 

Biofilm Control

Table 1. Summary of reported microbial interactions in mixed-species Candida biofilms (adapted from Thein et al., 2009 with permission) NM=Not mentioned in the article Candida Bacteria or other Candida species species C. albicans Candida dubliniensis

Nature of interactions Antagonism

C. albicans Streptococcus thermophilus B, Lactobacillus and lactis, L. lactis cremoris, L. rhamnosus, L. C. tropicalis fermentum, L. casei Shirota, Enterococcus faecium, Bifidobacterium infantis C. albicans Staphylococcus epidermidis

Antagonism

C. albicans Pseudomonas aeruginosa

C. albicans Escherichia coli

Kirkpatrick et al. (2000) [46] van der Mei et al. (2000) [29]

Commensalism Adam et al. (2002) [30] Antagonism Hogan and Kolter (2002) [36] Both El Azizi et al. antagonism and (2004) [33] commensalism

C. albicans P. aeruginosa, S. epidermidis, Serratia marcescens, Klebsiella pneumoniae, Enterobacter cloacae, C. krusei, Torulopsis glabrata, C. lipolytica, C. parapsilosis, C. guillermondii C. albicans Candida kefyr, C. tropicalis, Candida famata, NM Actinomyces, Lactobacillus, Streptococcus, Veillonella C. albicans Escherichia coli, Klebsiella pneumoniae, NM Staphylococcus aureus, Bifidobacterium adolescentis, Lactobacillus, Streptococcus C. albicans S. epidermidis NM

C. albicans A. israelii, L. acidophilus, S. mutans, S. intermedius, Prevotella nigrescens, Porphyromonas gingivalis and two transient oral colonizers, P. aeruginosa, and E. coli, C. albicans Streptococci, Lactobacilli, Bifidobacter, Actinomyces, Rothia dentocariosa, Microaerophiles such as Actinobacillus actinomycetemcomitans, Aerobes such as Neisseria mucosa, Anaerobes such as Fusobacterium nucleatum, Veillonella parvula, P. nigrescens, P. intermedia, P. gingivalis C. albicans Actinomyces naeslundii, Veillonella dispar, F. nucleatum, Streptococcus sobrinus, Streptococcus oralis C. lusitaniae Enterococcus faecalis

Reference

Antagonism

Lamfon et al. (2005) [40] O'May et al. (2005) [42] Al Fattani and Douglas (2006) [38] Thein et al. (2006) [31]

NM

Filoche et al. (2007) [39]

NM

Muller et al. (2007) [41]

Commensalism van Merode et al. (2007) [34] Both ant & Thein et al. (2007) commensalism [32]

    



CURRENT STRATEGIES TO REDUCTION OF MARINE BIOFILM FORMATION                       



 

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Marine biofouling is a natural phenomenon that affects any submerged material surface, including ship hulls, under water constructions, sensors, water desalination systems, etc. It raises many problems, such as extra energy consumption, increased corrosion, negative ecological issues, and others with very high overall economic impact [1]. Biofilm formation is the initial step of the complex marine biofouling process contributing to the increased fuel consumption and having own negative impact on many submersed marine constructions and underwater devices, for example, underwater sensors and others. In the most cases it acts as a surface to which macro-foulers such as macro-algae, barnacles, mussels, tubeworms, etc. attach and develop their fouling community. Therefore, the control over the total biofouling is often attributed to the reduction of microbial biofilm formation [1, 2]. So far, the most effective control over the marine biofilms was by the application of oxidizing biocides, quaternammonium compounds and by the use of non-toxic approaches like use of low surface energy coatings. [3]. An integrated antifouling approach like the use of biocides in the bulk and surface modification is currently needed for successful control

5.2 

Biofilm Control

of biofilms. This can be visualized as the use of the toxic biocides like diuron and tolylfluanid at concentrations of 1000 µg ml-1 did not impact the growth and viability of marine micro-organisms Pseudoalteromonas and Vibrio vulnificus [4]. Since biofilms are a surface associated phenomenon there is a lot of interest in developing minimally adhesive non-sticking surfaces by deposition of low surface energy coatings, with their most promising commercial alternative, poly(siloxane) and poly(siloxane) copolymers based fouling release coatings [5, 6]. In principle, the biofilm formation could be minimized / prevented by creation of strong hydrophilic (water like) or very hydrophobic / super hydrophobic (with surface tension approaching to zero) material surfaces to which microorganisms cannot initially attach. However in practice, synthetic materials, which are capable of preventing microbial adsorption and following microbial adhesion, are still elusive, despite a large volume of research carried out up to now. Reduction as well as removal of biofilm from surfaces exposed in marine environment remains a challenge. The attachment-mediating effects of the biofilms are believed to involve a variety of substrate material attributes including surface chemistry, micro-topography, elastic modulus, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition within the biofilms and thus change the biofilm properties and dynamics [7]. Generally, the more in depth understanding of the molecular-scale and macro-scale events between the fouling species and surfaces is expected to support the combat with the marine biofilm [8]. Current strategies to the reduction of marine biofilm formation are based on the knowledge about the composition and mechanism of marine biofilm development; the microbial adhesion and protein adsorption as mediator of this process; cell-surface and cell-cell interactions; characteristics of microbes living in biofilms; extracellular polymeric substances (EPS) production and cross-linking as well as intercellular communication via quorum sensing, have all been intensively studied lately, in the context of the controlling biofilm formation [9].

    Marine biofilm is a complex, surface attached community of microorganisms, enmeshed in extracellular polymeric substances (EPSs) that create a gel matrix providing enzymatic interactions, exchange of nutrients, protection against environmental stress and increased resistance to biocides [1012]. During the last few decades, our understanding of biofilm formation was improved and a generalized concept of how biofilm is formed was created. For the most species, it happens in several stages [11-14]. Every species has a specific set of environmental signals for initiation of biofilm formation. The most antimicrobial compounds are most effective during this first stage of microbial cells attachment, because the cells are still in a vulnerable state, and have not yet formed an exopolymeric matrix which offers them protection against stress factors [15-18]. Once the micro-organisms begin to secrete EPSs, biofilm development progresses to an irreversible process due to a cross-linking and thus attaching the micro-fouling species to the surface [19, 20]. The understanding of EPSs composition is important for enzymatic antifouling strategies as well as those based on inhibiting the cross-linking, the last one providing the irreversible attachment of the microfoulers. In the mature biofilm, the cells are already engaged in an extracellular matrix composed of proteins, exo-polysaccharides and extracellular DNA (eDNA). The matrix traps nutrients and various biologically active molecules, such as cell communication signals and accumulates enzymes that are able to degrade various matrix components, any nutrients and other substrates [21]. In addition, the matrix acts as a shield against toxins, antimicrobials and predators. Gene expression, morphology, phenotype, and the stage of differentiation and development vary between the cells in a mature biofilm [22]

Current Strategies to Reduction of Marine Biofilm Formation

5.3 

Biofilm architectures, such as mushroom- or tower-like structures, often derive from a division of labour between cells in the biofilm [23]. The heterogeneous nature of the mature biofilm may be a consequence of differential gene expression by cells across different regions in the biofilm structure, as well as of gradients of nutrients, electron acceptors and waste products, but may also be caused by mutations that lead to a diversity of genotypic and phenotypic variants [24]. The complex interactions among the various species in multispecies biofilms affect the structure and function of these communities, with antagonism (competition) and synergism (metabolic cooperation) [25]. For the majority of systems the nuances of these interspecies interactions are mostly unknown. The morphology of the biofilm itself is an ideal environment for hyper mutation and increased gene transfer and higher resistance [26-28]. The bacterial metabolic activity within a biofilm is not uniform and it is highly dependent on the internal location of the bacterium, making the cell more able to withstand micro-biocides [29-31].

ϱ͘Ϯ͘ϭŝŽĨŝůŵĚŝƐƉĞƌƐĂů In recent years, much attention is directed towards understanding the biology of biofilms, searching for inhibitors of biofilm development and biofilm-related cellular processes. The activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds is discussed by Landini et al [32]. Tiller [33] discusses the general principles of antimicrobial surfaces and strategies to inhibition of marine biofilm formation by chemical, biological, and physical methods. In recent research, numerous new ways to produce so-called self-sterilizing surfaces are introduced. These technologies are discussed with respect to their mechanism, particularly focusing on the distinction between biocidereleasing and non-releasing contact-active systems. New developments in the catalytic formation of biocides and their advantages and limitations are also covered. The conclusion is that a combination of several mechanisms has considerable benefits. Recent research on different bacterial species shows that the final stage in their life cycle includes production and release of differentiated dispersal cells. The evolutionary aspect of biofilm dispersal is now explored through the integration of molecular microbiology with eukaryotic ecological and evolutionary theory, which provides a broad conceptual framework for the diversity of specific mechanisms underlying biofilm dispersal [13]. Rendueles et al [34] describe the current understanding of competitive relationships in multi-species biofilms as well as non-biocide bio-surfactants, enzymes, and metabolites produced by bacteria and other microorganisms. These molecules target all steps of biofilm formation, ranging from inhibition of initial adhesion to matrix degradation, jamming of cell– cell communications, and induction of biofilm dispersion. Available data on non-biocide molecules and a new perspective on competitive interactions within biofilms that could lead to anti-biofilm strategies are presented. Many approaches to reduction of marine biofilm formation are described in the special literature, some of them inspired by nature or mimicking natural anti fouling surfaces. Biomimetic approaches, providing new insights into the design and development of alternative, nontoxic, surface-active antifouling technologies are discussed by Salta et al [35]. Evidently, the development of antifouling strategies should take account for all possible interactions used by the microbes to adhere and develop a biofilm on an immersed surface. Any event included in the biofilm formation could be a target to its control but it seems that combating biofilms is much easier at the initial stages of its formation.

ϱ͘Ϯ͘ϮĚŚĞƐŝǀĞ^ƚƌĂƚĞŐŝĞƐŽĨ&ŽƵůŝŶŐDŝĐƌŽŽƌŐĂŶŝƐŵƐĂŶĚ^ƵƌĨĂĐĞŚĂƌĂĐƚĞƌŝƐƚŝĐƐ/ŵƉĂĐƚŝŶŐ ƚŚĞ/ŶŝƚŝĂůĚŚĞƐŝŽŶ Microbial adhesion is an important step in the biofilm formation and therefore the knowledge of microbial adhesive strategies are very important. Biological adhesion is much more complicated

5.4 

Biofilm Control

phenomenon as compared to the physical adhesion because it includes a number of biological processes, such as cell attachment, spreading, growth and differentiation. Cells do not interact directly with surfaces. This interaction is mediated by extracellular matrix (ECM) formation. At the initial step (at a first contact with a surface), the cells secret adhesive proteins and proteinaceous substances which reorganized on the surface to form ECM. The initial interaction of cells with material surfaces is largely studied not only regarding the reduction of marine biofilm formation but also at biomaterials and in biotechnologies. Currently it is well known, that it depends on substrate surface properties in addition to the properties of the adsorbed layers. Studying initial cellular interactions with different model surfaces, Altankov [36] concluded that: 1.

2. 3. 4. 5. 6.

Moderate hydrophilic surfaces (WCA ~ 500 - 600) support cell adhesion and proliferation, cell growth and organization of focal adhesion complex; correspondingly, such surfaces should be avoided in the design/selection of materials for antifouling strategies; Chemical functional groups oppress the initial cellular interaction in the following raw:- NH2 < - OH < epoxy < - SO3 < - COOH < - CF3; Relationship exists between the efficiency of the cell interaction and the total negative surface charge; Not only chemically grafted functional groups but also adsorbed ions influence this interaction; The synthesis and organization of protein matrix by cells is better on surfaces bonding weakly matrix proteins; The conformation of the adsorbed adhesive proteins plays also an important role in the cell adhesive interaction [37].

The adhesion strategies of the fouling organisms are divers and usually include two components, both reversible and permanent attachment. Despite that a general theory of bio-adhesion remains still illusive, surface properties of the substrate [38-40], such as hydrophobicity/hydrophilicity (surface free energy), water contact angle and its hysteresis, steric hindrance, surface roughness, elastic modulus and surface chemistry, are known to influence the microbial adhesion. The existence of a "conditioning layer" at the surface as well as "teta surface" as more general antifouling concept, are thought to be important in the initial cell attachment process. The effect of substratum color on the formation of micro and macro fouling communities has been emphases [41]. It is known, that substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans [42]. Accumulation of protein in the biofilm matrix of Pseudomonas putida has been observed [43]. It was found that polysaccharide portion of H. rosenbergii capsular extracellular polymeric substance was involved in the primary adhesion process [44]. Both polysaccharides and proteinaceous compounds may be involved in initial adhesion, depending on the organism [45]. Primary adhesion and gliding are based on localized secretion of EPS, and sessile adhesion can be affected by secretion of copious amounts of EPS to form a more stable matrix [46, 47]. Davies et al. [48] demonstrated that the transcription of alg C, a key gene involved in the biosynthesis of alginate, required for the synthesis of EPS, is up-regulated within minutes up to three- to five-fold in adhered cells of P. Aeruginosa, compared with their planktonic counterpart. The EPS matrix that keeps the microorganisms together in biofilms is responsible for adhesion to a given surface [49, 50]. Interactions between EPS and surfaces could be based on non-covalent bonds, such as electrostatic attraction and hydrogen bonds [51]. The binding force of these interactions is weak compared with that of the covalent C–C bond. However, these weak interactions are multiplied by the large number of binding sites available in the macromolecules, and the total binding force exceeds that of covalent C–C bonds [52, 53].

Current Strategies to Reduction of Marine Biofilm Formation

5.5 

Biofilm structure is firmly associated with production and cross-linking of EPSs, consisting mainly of poly (saccharides) and other macromolecules such as proteins, DNA, lipids and humic substances [52, 54]. Polysaccharides are often investigated with regard to bacterial adhesion. The composition of the extracellular mucilage covering the diatom Pinnularia viridis gives an impression about the complexity of diatom EPS [55]. The polysaccharide EPS is composed of pentose, hexose, 6deoxyhexose, methylated and amino hexoses. These sugars are linked in multiple ways corresponding to highly heterogeneous structure, with the most prominent linkages being 2,4-linked and terminal xylosyl, 2,3-linked rhamnosyl, terminal and 2-linked fucosyl, and a heterogeneous mixture of minor galactosyl and mannosyl residues. Various studies have investigated the roles of different biofilm components and factors influencing its development: the influence of surface energy and surface chemistry on attachment of bacterial and algal spores [56]; identification of common features during bacterial biofilm development [57]; biofilm associated proteins [58]; biological adhesives [59]; presence of amyloid adhesins in natural biofilms [60]. Although diatom EPS is a highly complex matrix, research suggests that different species share common features [61]. Jaina and Bhosle [62] presented biochemical composition of marine conditioning film: total carbohydrates (CFCHO), total proteins (CFP) and total uronic acids (CFURA) and the influence of these compounds on the adhesion of three marine bacterial cultures, (Pseudomonas sp. CE-2, Pseudomonas sp. CE-10 and Bacillus sp. SS-10). Berne, Kysela and Burn [63] showed that the proteobacterium Caulobacter crescentus extracellular DNA (eDNA) inhibits the ability of its motile cells to settle in a biofilm. Conventional physicochemical models and cell appendage-mediated cell adhesion as well as technologies for controlling microbial adhesion and biofilm formation based on the adhesion mechanisms were reviewed [64]. Conventional physicochemical approaches (DLVO theory and thermodynamic approach) based on Lifshitz-van der Waals, electrostatic and acid–base interactions provide important models of bacterial adhesion but they have limited capacity to provide a complete understanding of the complex adhesion process of real microbial cells [64]. Cell surface structures, directly affect microbial adhesion to solid surfaces, complicating in this way this phenomenon. EPS and proteinaceous cell appendages have functions for bridging between the cell body and the substrate. Many kinds of bacteria have filamentous cell appendages, flagella and pili, which are in fact nanofibers, acting as adhesion mediating species to abiotic surfaces and biofilm formation. Whereas EPS are important for development of biofilm structure, rather than for cell adhesion, proteinaceous cell appendages are often essential for the initial interaction between cells and substrates. Non-febrile autotransporter adhesins (ATADs) also attract considerable attention as a new class of virulence factors [64]. Generally, inhibition of microbial adhesion and biofilm formation is quite difficult in natural environments, even if it is possible against specific targeted species. Nevertheless, some effective methods for controlling microbial adhesion and biofilm formation have been developed recently. While much research is focused on the initial cell - surface interaction and influencing factors, all in the context of control over this phenomenon, only few studies have investigated the role of EPSs crosslinking mechanisms ensuring irreversible attachment of micro-fouler. A lot of current approaches are focused on the quorum sensing mechanisms and its restoration to the reduction of marine biofilm development.

                  The primary mechanism in the attachment of marine organisms to surfaces involves secretion of protein or glycoprotein adhesives [65-67]. Protein adsorption that happens within seconds to minutes

5.6 

Biofilm Control

following immersion, acts as a "conditioning layer", altering the physical-chemical properties of the surface and providing a nutrient source for attachment of microbes [61, 68]. While the biological cascade of "conditioning layer" and following biofilm formation begins with deposition of proteins, low protein adsorption is now accepted as the most important pre-requisite for biofouling resistance that could be used as an simple way to screen new materials for antifouling properties [38]. Identification of the type and amount of adsorbed proteins on material surfaces could provide important information for the rational development of new materials resisting micro-fouling. A number of surface characteristics, such as chemical composition, topography, hydrophilichydrophobic balance, charge of the surface, mobility of surface functional groups, thickness and density of modifying layers and their adhesion to the substrate, etc. are well known, influencing the protein adsorption [38, 39, 69-72]. Such surface characteristics could be used to control this phenomenon and irrespectively "conditioning layer" formation as an initial step in a biofilm development. Protein conformation defines functionality with respect to cell adhesion. The protein conformation is strongly influenced by both the physical and chemical properties of the surface, including hydrophilic/hydrophobic balance and electrostatic charge [37, 63]. Adsorption of adhesive proteins, secreted by different organisms and undergoing subsequent underwater curing, is a mediator of the irreversible bioadhesion and biofouling. Some investigations are focused on the study the curing mechanisms of bioadhesive proteins as well as on the mechanical properties of bioadhesives such as green alga Ulva spore adhesive glycoproteins [73, 74]. Pioneering studies of Ikada, Susuki and Tamada [75] theoretically predicted that the work of adhesion, W12 (the interfacial surface energy) in aqueous media, approaches to zero when the water contact angle, θ approaches to zero (super hydrophilic, that is water-like surface) or to 900 (surface energy, γ1w approaches to zero, that is strong hydrophobic surface). This theoretical prediction, experimentally confirmed by bovine serum albumin (BSA) adsorption on various polymer surfaces, get a start point in the development of strong hydrophilic or strong hydrophobic low adhesive protein repellent materials and non-sticking fouling release surfaces. The best protein repellent, strong hydrophilic surfaces created so far are those of PEG (electrically neutral) and zwitterionic polymers (the last one have both positive and negative domains, but remain electrically neutral overall), very intensively studied for biomedical applications [76]. Unfortunately, they are not enough stable in marine environment [76]. Lately polysaccharides are studied as a promising material for non-fouling surfaces because they are high hydrophilic and able to form waterstoring hydrogels. Bauer et al [77] investigated the non-fouling properties of hyaluronic acid (HA) and chondroitin sulfate (CS) against marine fouling organisms. Additionally, the free carboxylic groups of HA and CS have been post-modified with the hydrophobic tri-fluoro-ethylamine (TFEA) to block free carboxylic groups and render the surfaces amphiphilic. All coatings have been tested with respect to their protein resistance and against settlement and adhesion of different marine fouling species. It appears that both, the settlement and adhesion strength of the marine bacterium Cobetia marina, zoospores of the seaweed Ulva linza, and cells of the diatom Navicula incerta are reduced compared to glass control surfaces. In most cases TFEA increases or maintains the performance of the HA coatings, whereas for the very well performing CS coatings, the antifouling performance reduces after capping of TFEA. Unfortunately, all hydrophilic materials are not enough durable in marine environment. Creation of strong/super hydrophobic, low adhesive surfaces is preferable for marine bio-fouling control. Composition coatings, based on poly (siloxanes) and their fluorine containing co-polymers are the most studied in this regard [78] but only few investigations are focused on marine biofilms and possibilities for their reduction. Liu et al [79] studied the inhibition of sea water microbial adherence on a super hydrophobic (static contact angle for sea water > 150°) anti-corrosive film, fabricated on anodized aluminium foils by chemical adsorption of meristic acid [CH3 (CH2)12 COOH]. The fluoroscope and SEM have showed significantly decreased adhesion of microorganisms on the samples with the super hydrophobic film after 24h immersion into nature seawater. The study suggests that the

Current Strategies to Reduction of Marine Biofilm Formation

5.7 

super-hydrophobic film does not only decrease the corrosion currents densities (ICORR), but also inhibition of microbial accelerated corrosion (MICI) due to prevention of microbial colonization. Cordiero et al [80] demonstrated fluorinated poly (dimethylsiloxane) (PDMS) surfaces prepared by low pressure CF4 plasma to create more resistant to microbial fouling surfaces. XPS, AFM and contact angle measurements were used for their characterization. Smooth PDMS coatings with fluorine content up to 47 % were attained. The CF4 plasma treatment leaded to formation of harder, non-bridle layer at the top-most surface of the PDMS. Surprisingly, PDMS surface got more hydrophilic after the introduction of fluorine that was explained by increased exposure of oxygen containing moieties towards the surface upon re-orientation of fluorinated groups towards the bulk and/or oxidative effects associated with the plasma treatment. Experiments with strains of marine bacteria with different surface energies (Cobetia marina and Marinobacter Hydrocarbonoclasticus) showed a significant decrease of the bacterial attachment upon the fluorinated surface. Significant attention is directed currently toward development of efficient protein-resistant surfaces but prevention of the protein adsorption is very difficult because of the versatile nature of the proteins and their ability to adsorb on many mechanisms when are in front of complementary surfaces [81].

   

   While the use of toxic biocides containing antifouling paints were banned in the year 2008, a general current trend to combat marine biofilms is by use of non-toxic approaches based on engineering material surfaces by employing physical, chemical and biological methods, mostly inspired by nature or mimicking natural anti-fouling surfaces. Some combinations of them seem to be most promising and beneficial. Nature provides many examples of mechanisms for biofouling control. Variety of defense was copied (biomimetic) or tailored (bio-inspired) to solve problems of fouling on manmade structures [82]. One inspiration arises from natural surfaces resisting biofouling in marine environment. These anti-fouling surfaces appear to use a combination of chemistry and micro- and nano-topography to inhibit biofouling. Marine organisms’ anti-fouling defense and different antifouling technologies are widely discussed in the special literature [69, 83, 84]. A number of biological extracts of secreted metabolites and enzymes are anticipated to the toxic biocides to act as environmentally safe antifoulants. Physical methods such as modification of surface topography, hydrophobic properties and potential charge are also considered to prevent biofouling. The physical antifouling technologies are an ultimate antifouling solution, because of their broad-spectrum effectiveness and zero toxicity [83]. Some microorganisms have been involved in inhibition of other micro-organisms as in the case of biofilms on macro-algae surface. Inhibitory effects of bacterial biofilms and epibiotic microbial assemblages have also been investigated and a biofilms repellent effect was shown benefiting the host by preventing the fouling on its surface. [85]. Most general, the anti-biofilm approaches could be grouped according to their nature as physical, physico-chemical, chemical, biological or combined; or according to the targeted event as protein adsorption or/and cell adhesion reducing, cell-communication (QS) and EPS matrix formation restoring. Anti-biofilm agents are synthetic or natural biocides, surface active substances, enzymes and antioxidants.

   ­  Since 1914, when Harrison [86] observed the response of embryonic nervous tissue fibroblast cells to spider silk topography, surface topography modification was considered as a possible way to marine biofouling reduction. Since that time, a lot of reports on cellular responses to topographical cues on both nanometer and micrometer scales appear in the special literature. However, it is argued by a

5.8 

Biofilm Control

number of authors that these structural futures are of less significance in the initial stages of the attachment process than the intrinsic thermodynamic factors involved [5]. Scheuerman et al [87] showed that surface topography alters settlement of bacteria. The change of surface wettability that results from surface roughness, i.e. topography, is likely to be contributing factor to these responses. Prior to adhesion, the swimming spore is able to select suitable surfaces on the base of surface topography or surface physical-chemical properties, such as contact angle [88, 89]. Influence of nanoscale topography of fluoirin polymer thin films on the hydrophobicity (the contact angle and its hysteresis) was reported [90]. Surface topography for non-toxic bioadhesion control has been patented [91]. The importance of wettability models in predicting cellular contact guidance for engineered topographies has been experimentally demonstrated, but the process was not fully explained [92]. Techniques such as photo- and electron beam lithography are used currently to create moulds for producing micro- and nano-scaled topographies with various shapes and spatial arrangements [92]. Engineered micro-topographies composed of pillars or ridges with various heights (5 or 1.5 m) and spacing’s (5 or 20 m) systematically enhance settlement of the spores of Ulva when created in poly (dimethyl siloxane) elastomer. Such experiments implied that the width and spacing of topographical features necessary to deter biofouling must be tailored to the size of the organism [93]. There are few reports about studies focused on surface topography manipulation for reduction of microbial biofilms formation. Bio-inspired surface topography, Sharklet AF™, inhibiting Staphylococcus aureus biofilm formation was reported by Chung et al [94]. The application of surface roughness to alter wettability for antifouling coatings especially super hydrophobic coatings is extensively investigated. The surface wettability alteration, resulting from surface topography, i.e. roughness, was likely to be a contributing factor [63, 95]. The elastic modulus of the substratum and the hydrodynamics were other physical factors influencing the bioadhesion. For example, it was demonstrated both empirically and experimentally that E. coli was attracted to the walls of a container purely by hydrodynamic interactions. These hydrodynamic interactions may initiate the settlement process by allowing the organism to “find” the surface [96, 97]. Another approach to creation of antifouling surfaces is the utilization of the concept of fluid slip. Fluid slip is the boundary condition in which the fluid has a finite velocity at an interface. The no slip boundary condition is relevant to a fluids moving over air, which occurs in the case of super hydrophobic materials in the “non-wet” or Cassie-Baxter state. It seems possible to prevent hydrodynamic attraction of swimming organisms through the use of fluid slip [98-101]. Correlation between the attachment density of cells from two phylogenetic groups (prokaryotic Bacteria and eukaryotic Plantae) with surface roughness has been reported [5]. The results represented a shift of the paradigm in the understanding of cell attachment, which is a critical step in the biofouling process. The model predicted that the attachment densities of zoospores of the green alga, Ulva, and cells of the marine bacterium, Cobetia marina, scale inversely with surface roughness. These studies demonstrated for the first time that organisms respond in a uniform manner to a model, which incorporates surface energy and the Reynolds number of the organism. Possible control over bacterial biofilm growth by nano-structural mechanics and geometry was also presented [102]. The tunable effects of physical surface properties, including high-aspect-ratio (HAR) surface nano-structure arrays were investigated, recently reported to induce long-range spontaneous spatial patterning of bacteria on the surface. The functional parameters and length scale regimes that control such artificial patterning for the rod-shaped pathogenic species Pseudomonas aeruginosa were elucidated through a combinatorial approach. A cross-over regime of biofilm growth was reported on a HAR nano-structured surface versus the nano-structure effective stiffness. When the 'softness' of the hair-like nano-array was increased beyond a threshold value, biofilm growth was inhibited as compared to a flat control surface. This result is consistent with the mechanic-selective adhesion of bacteria to surfaces. Therefore by combining nano-array induced bacterial patterning and modulating the effective stiffness of the nano-array - thus mimicking an extremely compliant flat surface bacterial mechanic-selective adhesion can be reduced.

Current Strategies to Reduction of Marine Biofilm Formation

5.9 

Halder et al [103] employed novel approach to determine the efficacy of patterned surfaces for biofouling control in relation to its microfluidic environment. While biofouling can be controlled to various degrees with different microstructure-based patterned surfaces, understanding of the underlying mechanism is still imprecise. Researchers have long speculated that micro-topographies might influence near-surface micro-fluidic conditions, thus micro-hydro dynamically preventing the settlement of microorganisms. It was therefore very important to identify the micro-fluidic environment developed on patterned surfaces and its relation with the antifouling behaviour of those surfaces. This study considered the wall shear stress distribution pattern as a significant aspect of this micro-fluidic environment. Patterned surfaces with micro-well arrays were assessed experimentally with a real-time biofilm development monitoring system, using a novel micro-channel-based flow cell reactor. Finally, computational fluid dynamics simulations were carried out to show how the microfluidic conditions affect the initial settlement of microorganisms Chapman et al [104] investigated the role of surface topography and chemistry combined in a single material – a property that naturally exists in some common macroalgae. Saccharina latissima (sugar kelp) and Fucus guiryi (Guiry's wrack) were selected as a platform for “Bioinspiration”. The surfaces of the samples were characterised and then replicated using simple polymeric reproduction methods. Furthermore, a pre-extracted brominated furanone was doped into this matrix (0.05 g ml−1). The replicated macroalgae samples containing the brominated furanone compound were compared in a 7day marine study to investigate the effects of biofouling. The bio inspired samples directly demonstrated that combinatory approaches (using topography and chemistry) exhibits lower levels of biofouling. Here it is reported that both chemistry and topography demonstrated 40% less biofouling when compared to blanks in all of the pre-designed biochemical biofouling assays. It was found that unique surface characteristics of rice leaves and butterfly wings combine the shark skin (anisotropic flow leading to low drag) and lotus (super hydrophobic and self-cleaning) effects, producing so called rice and butterfly wing effect [105,106]. Mimicking this effect, replica rice leaf and shark skin samples received a super hydrophobic and low adhesion nano-structured coating. Antifouling properties of four micro-structured surfaces inspired by rice leaves and fabricated with photolithography and hot embossing techniques have been studied. Antifouling data were presented to understand the role of surface geometrical features resistance to fouling. Conceptual modeling provides design guidance when developing novel antifouling surfaces. Unfortunately, the conventional materials, working in a marine environment are not able to continuously keep artificial patterns that limit their practical application although the significant progress in the patterning techniques development.

   Another physical approach to the reduction of marine biofilm formation is based on employment of electrical fields which have been used for both, microbial adhesion prevention and biofilm growth inhibition [107-110]. Use of copper electrodes [111], direct electron transfer between bacteria and anode surface [110,112,113] electro- repulsive interaction between bacteria and cathode surfaces [114] have all been demonstrated to inhibit bacterial adhesion. When an anodic current or potential is applied, inactivated bacteria tend to remain on the surface. In such cases, fouling on the surface from the inactivated bacteria can provide seeds for the next bacterial adhesion [115]. Alternative methods like use of low voltage (0.5-5V) pulsed electric fields, inter digitized electrodes, variations in applied voltage, frequency and pulse duty ratio have all been demonstrated to inhibit bacterial adhesion and biofilm formation [116]. In general, the application of low-duty ratio pulses had a positive effect on preventing biofouling. Comparatively, frequency and applied voltage have less influence on biofouling. To overcome the limitations associated with the application of direct constant cathode or anodic current, the application of block current or potential, which utilizes cathode and anodic currents

5.10 

Biofilm Control

or potentials in turn, is demonstrated as an effective approach for bacterial detachment and inactivation [108]. An electrode surface that employs block current, maintaining effective antifouling ability for two years has been reported [113]. In addition, the application of block current has been suggested to be better than direct current in terms of heat dissipation. Another form of electro assisted approach is by the use of ultrasound for detachment of biofilms [117]. A threshold for detachment was defined if more than 95% of the bacteria were removed. Detachment rate was found to depend on (i) the sound intensity (threshold 2 Watt), (ii) the time of exposure (60 s) and (iii) the distance between transducer and membrane (4 cm). The shear forces in the acoustic boundary layer are considered to be responsible for the detachment of the bacteria. The first results reveal that the application of ultrasonic waves provides a tool for both: cleaning of biofouled surfaces and investigation of the adhesion forces of microorganisms to surfaces. Laser-ablation (ND:YAG lasers) of biofilms has also been demonstrated as an effective method to inhibit growth of biofilm [118]. Mechanistically, the reduction in re-colonization of surfaces is thought to be due to the lethal and sub lethal impacts of laser irradiation on bacteria. Pulsed laser irradiation seems to be an effective tool but its application is restricted to samples at the laboratory scale. Further developments in technology are necessary to effectively integrate the process for desalination membranes and cooling water systems where natural biofilms are a problem. UV radiation (UVR- 254nm) has been used as a biofouling control measure for optical sensors in marine moored instruments [119]. The efficiency of UVR in preventing biofouling increases significantly with increase in intensity and exposure time. UVR is effective even in reducing the population of micro-foulers from already developed biofilms. This technique has been used where sterilization of seawater/freshwater is required in small quantities. However this technique has limitations and cannot be applied for treating / disinfecting seawater in large industrial plants.

ϱ͘ϯ͘ϯŚĞŵŝĐĂůŶƚŝͲďŝŽĨŝůŵŐĞŶƚƐ͕^ƵƌĨĂĐĞDŽĚŝĨŝĐĂƚŝŽŶĂŶĚŽĂƚŝŶŐ Employment of antimicrobial agents, surface modification and coatings are some of the well-known approaches to combat biofilm formation. Conventionally, biocides have been used for disinfecting of a surface [120,121]. However toxic effects on non-target organisms caused by these biocides and development of resistance to them has prompted investigations on alternate antifouling methods like use of newer biocides or modification of the surfaces. Various chemical moieties have been successfully demonstrated to possess antimicrobial activity viz: N-substituted maleimides and succinimides [122], interestingly these groups of compounds have been shown to interfere with the curing of the enzyme polyphenoloxidase of the macrofoulants, Mytilus edulis; apart from those, polyphenols [123] which interfere with bacterial quorum sensing; polyethylene oxide [124] decreasing the Lifshitz-van der Waals attraction between the cells and the glass surfaces; methoxy-terminated poly(ethylene glycol) (mPEG) conjugated to the adhesive amino acid l-3,4-dihydroxyphenylalanine (DOPA) [125, 145] inhibiting diatom settlement. Incidentally this bio-inspired polymer has demonstrated to be more effective than polydimethyl siloxane (PDMS) and may be a potential candidate for future foul release coatings. Surface charge has also been implicated to inhibit biofilm formation as demonstrated by Gottenbos et al [127] wherein positively charged surfaces. Chemical moieties interfering with the bacterial cell surface hydrophobicity like 2, 4-dinitrophenol (DNP) have been identified by Jaina et al [128]. Another important aspect in disinfection of biofilms lies on the efficacy of biocides to disinfect both planktonic and sessile forms which has been elucidated by Kim et al. [129]. In the search for novel biomimetic compounds possessing antimicrobial activity, furanones, especially with a conjugated exocyclic vinyl bromide on the furanone ring, appeared to be effective against E. coli [130]. Some synthetic analogues of natural and natural-derived products were reported as anti-biofilm agents. For example, synthetic analogue of farnesol, containing 1,4-disubstituted-1,2,3-

Current Strategies to Reduction of Marine Biofilm Formation

5.11 

triazol (TFA-Z), that displays significant anti-adhesion activity without toxicity, based on specific antiadhesion mechanism [131]. Investigations on the use of herbicides triazine and isothiazoline along with and without zinc oxide nanoparticles for photocatalytic activity have revealed the later to be effective [132]. Other pesticides like Diuron and tolylfluanid, both being used as booster biocides, appeared to be more active against diatoms than against bacteria [4]. A new class of antimicrobial agents, 5-(alkylidene)-thiophen-2(5H)-ones, is also reported, one of them having greater biofilm reducing ability [133]. Inhibition and dispersion of bacterial biofilms with imidazole-triazole derivatives were patented by Melander et al [134]. In addition bioassay methods investigating antifouling activity have also been developed [135]. Subsequently a novel compound named Butenolide has been identified which inhibits marine fouling by influencing the primary metabolism of the target organisms [136]. Several substituted 2-aminopyrimidine (2-AP) derivatives from the 2aminoimidazole class, display an ability to modulate bacterial biofilm formation, exhibiting greater activity against Gram-positive than Gram-negative strains [137]. Other approach to antifouling relies on protection of the surfaces by polymeric coatings. Neoh and Kang [138] have listed the polymeric properties and the methodologies involved in immobilizing them to surfaces. Important points in the use of polymeric coatings are their antimicrobial activity, followed by high stability in aqueous medium to prevent degradation. Several studies on surface modification of polymers have been carried out. For example, ContrerasGarcia et al [139] grafted poly(2-(dimethylaminoethyl) methacrylate) (pDMAEMA) to low density polyethylene (LDPE) and silicone rubber (SR) in order to make them less susceptible to microbial biofilm formation. The direct grafting of DMAEMA using -rays appears to be an efficient and fast procedure for obtaining modified materials, which could be quaternized in a second step using methyl iodide. Raman spectroscopy show that the grafting occurs only at the surface of the LDPE, but both at the surface and in the bulk of the SR. Consequently, the grafted chains cause changes in the surfacerelated features of the LDPE (water contact angle and viscoelastic behaviour in the dry state) and in the bulk-related properties of the SR (swelling and viscoelasticity in the swollen state). The microbiological assays revealed that the grafted DMAEMA reduces Candida albicans biofilm formation (almost no biofilm on SR), while the quaternized surfaces inhibit C. albicans and Staphylococcus aureus biofilm by more than 99% compared to pristine materials. These materials are able to inhibit microbial biofilm formation and promising for anti-biofouling applications. Novel approaches [145] are based on the isolation of the amino acid 2-chloro-4,5dihydroxyphenylalanine (Cl-DOPA) from the proteinaceous glue of the sand castle worm Phragmatopoma californica which demonstrated antibacterial activity. Surface modification of stainless steel (AISI 304) by annealing and passivation [140] demonstrated inhibition of manganese oxidizing bacteria which leaded to an improvement in the corrosion resistance of the steel in aqueous environment. Surface modification by use of nano coatings like nano-TiO2 coating, prepared onto anodized aluminium surface by vacuum dip-coating of TiO2 sol–gel, inhibited marine microbial adhesion and decreased microbial influenced corrosion [141]. UV reflection spectroscopy results showed that the nano-TiO2 coatings improved the photo catalytic activity that influenced the microbial corrosion inhibition. Further, naturally occurring vanadium haloperoxidases, and vanadium pentoxide nanowires, act like them to prevent marine biofilm formation [142]. Use of metals (Ag) and metal oxide (TiO2) as nanoparticles have been investigated extensively. It was shown that Ag nanoparticles cause a shift in microbial community [143] and also when incorporated in polymeric matrices could effectively inhibit micro-fouling [78]. Gutierreza et al [146] evaluated the anti-biofilm activities of silver nanoparticles (AgNPs) against several microorganisms. The antimicrobial activity of AgNPs was tested within biofilms generated under static conditions and also under high fluid shears conditions using a bioreactor. The antibacterial activity of AgNPs on various microbial strains grown on polycarbonate membranes is reported. AgNPs effectively prevent the formation of biofilms and kill bacteria in established biofilms, which suggests that AgNPs could be used for prevention of biofilm-related problems .

5.12 

Biofilm Control

Approaches like the use of surface modification of materials with reactive organo- silanes resulted in a creation of antimicrobial surfaces [144]. Among the modifying agents examined, poly [dimethylsiloxane-co-(N,N-dimethyl-N-n-octylammonium propyl chlorid) methylsiloxane)] terminated with hydroxydimethylsilyl groups (20 %) in silicone elastomer given the most desirable results. The surface tension of the modified surface was comparable to the non-polar native surface. However, almost half of this value was due to polar forces. The antibacterial activity of the functional organosilanes was associated only with the carrier surface because no antibacterial compounds were detected in the liquid culture media that are able to inhibit cell growth.

  In a number of strategies, enzymes are proposed as a viable non-toxic alternative to biocides. The chemistry, involved in each step of the biofouling process, is a key point for determination of which event in biofouling may be affected by enzymes, and how the manipulation of this step influences the biofouling in general. Molecular and general aspects of this process, fouling biology and biological adhesives are summarized in earlier reviews [147, 148]. Generally, the chemistry of each step of the biofouling could be affected by enzymes. Enzymes have been implicated in control of micro-fouling as well as macro-fouling but the first one is within the scope of this discussion. Table 1 presents enzymes with a proved activity against biofilm formation, sorted according to their enzyme classification number (EC-number) into four classes: oxido-reductases, transferase, hydrolases and lyases [149,150]. Approaches to prevent micro-fouling (e.g. bacteria and diatoms) should interfere with the a) first contact between organisms and surfaces or b) should stop development of settled organisms to problematic levels, or c) interfere both the first contact and maturation of biofilm. Enzymes the affect microbial settlement and adhesion in four different ways determining four approaches for enzyme based combat with marine biofilms [149]: 1. 2. 3. 4.

Attack the adhesive of settling organisms; Degrade the polymers in the biofilm matrix; Catalyze the release from the surface of antifouling compounds; Obstruct the intercellular communication of microbes during colonization. Table 1. Enzymes proposed for prevention of biofilm formation EC number 1. 1.1.3 1.3 1.10.3 1.11.1 2 2.6.1 3 3.1 3.1.1 3.1.3 3.2 3.2.1 3.4.11

Enzyme Oxido-reductases Oxygen as acceptor, oxidases Acting on the CH–CH group of donors Acting on di-phenols and related substances as donors, oxygen as acceptor Peroxidases Transferase Transaminase Hydrolases Esterases Acting on carboxylic esters Phosphoric monoester hydrolases, the Phosphatases Glycosylases Hydrolysing O- or S- glycosyl compounds Aminopeptidase

References 151, 152 152 151, 153 151 151, 152 154 151 153, 154 155, 156 158 151, 158-161 156, 162-168 156

5.13 

Current Strategies to Reduction of Marine Biofilm Formation

3.4.21–25/3.4.99

Endopeptidase, protease

159, 169, 170

3.4.21 3.4.22 3.4.24

Serine-endopeptidase Cystein-endopeptidase Metalloendopeptidase Acylases. Acting on carbon–nitrogen bonds, other than peptide bonds, in linear amides Lyases Carbon–Oxygen Lyases acting on Polysaccharides

156 156 156

3.5.1 4 4.2.2

154, 171, 172 160

ϱ͘ϯ͘ϱŶnjLJŵĂƚŝĐĚĞŐƌĂĚĂƚŝŽŶŽĨĂĚŚĞƐŝǀĞƐƵƐĞĚďLJŵŝĐƌŽͲŽƌŐĂŶŝƐŵƐ The basic issue of micro-fouling is the formation of a biofilm which initial step, e.g. the primary (reversible) and secondary (irreversible) microbial adhesion could be affected by enzymes knowing the chemistry of adhesives used for settlement. Micro-foulers, such as bacteria and diatoms use proteinaceous glue for adherence to the submerged surfaces (primary, reversible adhesion) whereas their polysaccharide-based adhesives are very important for the establishment of secondary (irreversible) adhesion [149]. Hydrolases viz: proteases, glycosidases and lipase has been shown to be effective against the biofilm forming bacterium Pseudoalteromonas sp. D41 [173]. Among those Subtilisin (a broad spectrum hydrolase) has been the most effective with respect to inhibition of attachment as well as removal of adhered bacteria [156]. The action of hydrolases seems to be concentration dependent and Subtilisin has been found to act on proteins and polysaccharides of the bacteria Pseudoalteromonas sp. D41 as detected in the soluble and capsular fractions. The high antifouling potential of Subtilisin in the very first stage of fouling, the bacterial adhesion, is promising for less toxic control over biofouling than the organometallic compounds in antifouling paints. Proteolytic enzymes may also provide an effective technology for biofouling control as they are able to degrade proteinaceous nature adhesives produced by most fouling organisms. Incorporation of the protease - Subtilisin A into polymeric coatings reduced the settlement of Ulva and Navicula sp. and reduced the adhesion strength of Navicula sp [174]. The antifouling efficacy of the bioactive coatings increased with increasing the enzyme concentration on the surface, and it was superior at the equivalent amount of the enzyme in solution. The results provided a rigorous analysis of one approach to the use of immobilized proteases for reduction the marine bio-foulers adhesion. The serine protease, Esperase HPF (Subtilisin) inhibited the formation of multispecies biofilm as demonstrated by Hangler et al [175]. The effects of enzyme activity, time and application of the enzyme were tested on the density and the oxidative metabolism of biofilms. Esperase HPF did not inhibit the oxidative metabolism of the bacterial biofilm or planktonic growth, but the enzyme inhibited biofilm formation by its proteolytic activity as the inactivated enzyme has no effect. The effective concentrations of the enzyme in solution were the same regardless of time of application (i.e. before or after biofilm formation), but the immobilization of the enzyme lowers effective concentration. Esperase HPF is an attractive alternative to biocidal compounds used in antifouling paints. Some evidences that immobilized in polymeric antifouling coatings enzymes affect the biofilm formation were already reported [80].

ϱ͘ϯ͘ϲŶnjLJŵĂƚŝĐĚŝƐƌƵƉƚŝŽŶŽĨƚŚĞďŝŽĨŝůŵŵĂƚƌŝdž EPSs are the main component of the biofilm matrix and the basic reason for the resilient microbial proliferation on the submerged surfaces [176,177]. The complexity and heterogeneity of the marine biofilm EPS’s require combinations of hydrolases and lyases in order to achieve efficient degradation

5.14 

Biofilm Control

of the polymeric networks constituting the biofilm matrix [160]. The two most prominent components between the EPS, proteins and polysaccharides, are the most popular in different antifouling approaches. A lyase enzyme with potential relevance to antifouling purposes is alginase, which breaks up the polysaccharide alginate by an elimination reaction [176]. Degradation of polysaccharides is, one of the main targets of the enzymatic attack, which can be facilitated by glycosylases, and specifically hydrolyse for ester-bonds in oligo- and polysaccharides. Exoglycosylases and endoglycosylases cut off terminal saccharide units and chains at internal sites. They act on either - or -glycosidic linkages by site specific enzymes. This sub-class includes amylase, cellulase, chitinase, galactosidase, pectinase, collagenase, hyaluronidase, and others. For example, broad-spectrum glycosylase formulations have some effect on release of diatom cells release under shear [149, 150]. The polysaccharide portion of secondary adhesive is a difficult target for glycosylases, since polysaccharides have complex chemistry, as exemplified for EPS used for adhesion by diatoms [178]. Glycosylases individually attack a limited range of linkages in the polysaccharides. The variation of linkage types in a mixed biofilm is great and therefore to select the appropriate combination of glycosylases is a challenge. The same holds good for lyase enzymes, such as alginase, acting on polysaccharides. For example, a glycosylase which succeeded in inhibiting the adhesion of Pseudoalteromonas sp. bacteria, was not able to detach cells already adhered in a mature biofilm [173]. This assessment was supported by evaluation the adhesion strength of Navicula perminuta diatoms: broad-spectrum glycosylase formulations in solution have some effect in promoting diatom release from a surface under shear, but serine-proteases was superior [179]. However, the presence of amyloid protein structures may confer limitations to the comprehensiveness of proteolytic break down of biofilm matrices [180], because of the structural stability of the amyloid fibrils to most proteases [181]. The class of enzymes called alginase breaks up the polysaccharide alginate by an elimination reaction. Glycosylases individually, attack limited linkages in the polysaccharides. Proteases are efficient in both the prevention of adhesion and breaking down the biofilm matrix, because proteins are a significant part of the biofilm matrix together with polysaccharides, both being very important for the formation of its chemical architecture. The amyloid protein structures confer limitations to the comprehensiveness’ of proteolytic break down of biofilm matrices, due to the structural stability of the amyloid fibrils to most proteases [149, 150]. Another class of enzymes viz: the cellulase which are specific in degradation of  1-4 linkages of the EPS has been found very effective in removing Pseudomonas sp CE-2 biofilms from SS coupons as revealed by calcoflour and concanavalin A (Con A) assays [182]. These results indicated the ability of certain enzymes to attack specialized regions of the exopolymer of certain bacterial species which is to be considered when designing antifouling coatings. Yet another class of enzymes like the nucleases (Nuc B) which act as extracellular DNase is found to rapidly break up biofilms of both Gram positive and Gram negative strains of bacteria [183]. They also demonstrated that bacteria can use secreted nucleases as an elegant strategy to disperse established biofilms and to prevent de novo formation of biofilms of competitors. DNase therefore plays an important dynamic role as a reversible structural adhesive within the biofilm. The enzyme classes: transferases, isomerases and ligases are also proposed as antifouling properties holding enzymes [154, 184].

         Naturally produced antifouling compounds have been divided into two categories: 1) non-polar metabolites remaining on the surface of the organism and repelling invertebrate larval exploration and 2) polar metabolites liberated into overlying water detectable by larval receptors and triggering avoidance behaviour. These antifouling compounds are classified as deterrents (not toxins), since their

Current Strategies to Reduction of Marine Biofilm Formation

5.15 

modes of action may not be due to toxic effects [185]. Enzymes suggested to mimic the above by generating deterrents diffusing out of a coating include glucose oxidase [152, 186], hexose oxidase [187] and haloperoxidase [188], which all belong to the class of enzymes called oxido-reductases [150]. The oxidases are used to produce hydrogen peroxide, while haloperoxidase catalyses the formation of hypohalogenic acids. Similar to other reactive oxygen species (ROS) H2O2 may induce oxidative damage in living cells [189, 190]. Hypohalogenic acids, e.g. HOBr or HOCl, are highly reactive and are thus used as oxidants in water treatment as important disinfecting agents [191, 192]. The oxidative damage exerted by hydrogen peroxide on cells has been employed in a number of applications [193, 194]. Use of hydrogen peroxide does not pose the problem of bioaccumulation [194], which implies the high potential of this chemical as an efficient non-ecotoxic biocide, providing a considerable environmental improvement over current biocidal antifouling biocide technologies, which has been supported by a number of investigations [152, 186, 187]. The environmental fate of hypohalogenic acid is comparable to that of hydrogen peroxide [188, 192].

           Quorum sensing (QS) plays an important role in the formation of bacterial biofilms. Enzymes interfering with intercellular communication include racemases, epimerases, cis-trans-isomerases, intramolecular oxido-reductases, intramolecular transferases, and intramolecular lyases. Synthetase or synthase are synonyms for ligase. Ligase activity is defined by the joining of two molecules with concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate cofactor. An example could be a peptide-synthase [150]. N-acyl homo-serine lactones (AHL), used by Gram-negative bacteria for quorum sensing (QS), are necessary for rapid development of a biofilm [195, 196]. Acylases have been discovered degrading AHL in vitro by hydrolyses of the acyl-amide bonds between carboxylic acids and amines/amino acids. Thus the elimination of AHL prevents the development of bacterial fouling [153, 154, 171, and 172]. The most widely suggested enzyme-based approach for effective prevention of both reversible and irreversible attachment and EPM formation. This has been achieved by hydrolytic breakdown of adhesive or structural polymers based on protein and polysaccharide components. Proteases have the highest potential in this respect, although some protein structures are highly resistant to proteolysis breakdown. Degrading polysaccharide networks glycosylases targets an individual polysaccharide structure that reduces the possibility to find a single or combined glycosylases with comprehensive effect. Various peroxidases and oxidases can be applied to produce compounds at the surface deterring settling organisms, e.g. peroxide compounds. Another solution is to degrade promoting biofouling signaling compounds by AHL acylases. Several features of the enzymes limit their practical application as anti-biofilm agents: i) to carry out its function keeping their own natural activity, i.e. any enzyme needs to be structurally stable in aqueous solutions and needs to be released in water and structural mobility, both difficult to be provided when it is included in antifouling coating; ii) the enzyme activity increases whereas its stability decreases with a temperature increase and finding an optimal balance between enzymes’ activity and stability is another challenge at with enzyme-based antifouling coatings.

  It is well known, that the natural antifouling surfaces use both physical and chemical strategies in the combat with the biofouling, the last one inspiring the current chemical defense of manmade surfaces with natural anti-foulants [5, 82]. Large variety of marine organisms such as marine sponges, algae, seaweeds, bacteria, fungi, etc. are capable of bio-synthesizing a broad variety of secondary metabolites ensuring their natural chemical defense against biofouling [135, 197].

5.16 

Biofilm Control

The first formulation of antifouling paints (acrylic) that incorporate marine natural products was reported by Armstrong et al as early as in the year 2000 [198]. Large varieties of biologically active compounds have been isolated to date from various marine sources with antifouling properties. Largely unexplored sources for isolation of new microbes (bacteria, fungi, actiomycetes, micro-algaecyanobacteria and diatoms) exist that are potent producers of bioactive secondary metabolites [199]. The identified so far natural antifouling agents from marine flora and fauna involve bacteria, natural biocides, bio-surfactants/dispersals, and quorum-sensing inhibitors [200]. Problems such as accessibility of the natural sources, too low ratio bioactive substance/natural raw material, too high price, etc limit their commercialization and practical application.

     The production of antimicrobial compounds by bacteria and their living hosts work in concert to protect the hosts' surfaces. All these compounds can be potential candidates and suitable alternatives to TBT and copper compounds [201]. Production of inhibitory substances among bacterial biofilms is a common phenomenon [202]. In Table 2 are summarized some bacteria used in antifouling strategies. Bacteria, isolated from living surfaces in the marine environment, have already been considered as a promising source of natural antifouling agents. Burgess et al [203] have developed an antifouling coating based on natural marine product. A paint formulation containing extract of Pseudomonas sp. strain NUDMB50-11, has shown excellent activity. Yee et al [206] immobilize the antifouling bacterium Pseudoalteromonas tunicata in -carrageenan to demonstrate how a surface may be protected from fouling by bacteria, i.e. a ‘living paint’. Table 2 Bacteria in antifouling strategies Bacterium source Brown algae

Awaji Island, Japan

Alga Ulva australis

Species Pseudomonas NUDMB50-11

sp.

strain

Sargassum serratifolium, Sargassum fusiforme, Sargassum filicinum, Padina arborescens, Undaria pinnatifida, Petalonia fascia, Colpomenia sinuosa, Scytosiphon lomentaria and Ecklonia cava epiphytic bacteria

Active substance crude extract

Biological activity

Ref.

multispecies

203

epibiotic bacteria

antibacterial

204

inhibitory compounds

colonization inhibition

205

5.17 

Current Strategies to Reduction of Marine Biofilm Formation

Pseudo alteromonas tunicata in -carrageenan Sediments of West Pacific Ocean

Many strains from the genera of Pseudomonas, Psychrobacter and Halomonas Shewanella oneidensis SCH0402

Pseudomonas sp. Strain 3J6

“living plant”

crude ethyl acetate extracts from deepsea bacteria chloroform extract;2hydroxymyri stic acid and cis-9-oleic acid bacterium and its active exo-products

α-proteobacterium Caulobacter crescentus

selected bacterial supernatants extracellular DNA

Cyanobacterium Lyngbya majuscule

Pseudoalteromonas sp. Strain D41

Danish coastal waters

antibacterial

206

against one or more bacteria

207

against a wide range of micro-and macrofoul.

208

medical antibiofilm 209 against Vibrio spp

inhibits settling of motile progeny cells against barnacle larva settlement

210

63

inhibits strain 3J6 in mixed biofilms 211

secondary metabolites from polyketidepolypeptide structural class secrets bacteria

212

against Pseudoalteromonas sp. Strain S91 and Ulva australis zoospores independent of bactericidal activity

213

Many bacteria producing inhibitory compounds have been isolated, like: epibiotic bacteria from the surface of brown algae (Sargassum serratifolium, Sargassum fusiforme, Sargassum filicinum, Padina arborescens, Undaria pinnatifida, Petalonia fascia, Colpomenia sinuosa, Scytosiphon lomentaria and Ecklonia cava) [204]; epiphytic bacteria from the marine alga Ulva australis [205]; 28 deep-sea

5.18 

Biofilm Control

bacterial strains, belonging mainly to the genera of Pseudomonas, Psychrobacter and Halomonas, isolated from sediments of the West Pacific Ocean [207]; marine bacterium, Shewanella oneidensis SCH0402 from which have been isolated 2-hydroxymyristic acid (HMA) and cis-9-oleic acid (COA), both with antifouling activity against micro- and macro-algae, barnacles, and mussels [208]; marine bacterium Pseudoalteromonas sp. Strain 3J6 also with medical antimicrobial activity [209]; bacterial culture supernatants on biofilm formation of Vibrio spp., that demonstrated promising anti-biofilm properties and potential for application in marine aquaculture [210]; bacterial extracellular DNA (eDNA) that inhibits the ability of its motile cell type to settle in a biofilm [63]; marine cyanobacterium Lyngbya majuscule from which were isolated 12 secondary metabolites, tested against barnacle larval settlement [211]. Klein et al [212] reported that the marine bacterium Pseudoalteromonas sp. 3J6 secreted antibiofilm active substances as well as the discovery of another Pseudoalteromonas sp. strain, D41, which inhibits the development of strain 3J6 in mixed biofilms. Marine bacteria from Danish coastal waters showed antifouling activity against marine fouling bacterium Pseudoalteromonas sp. Strain S91 and zoospores of the green alga Ulva australis independent of bacteriocidal activity [213]. Carvalho and Fernandes [214] reviewed the metabolites produced by marine bacteria (biosurfactants, siderophores, fatty acids, exopolymeric substances) and illustrated how they can be used in relevant areas. Bacteria produce molecules that prevent the attachment, growth and/or survival of challenging organisms in competitive environments. Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions were investigated by Dusane et al [215]. Four marine epibiotic bacteria displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. It has important biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production. To gain a better insight into biofilm composition, Kim et al [216] studied the exopolysaccharide of the Gram-negative bacterium Vibrio vulnificus. Monosaccharide composition analysis of the wild-type and mutant V. vulnificus EPS was carried out. The influence of galactosamine on biofilm formation was studied using four bacterial species. No significant inhibition of biofilm formation was observed in bacteria that produce autoinducer type-1 signal molecules. The results of this study suggest the antifouling role of galactosamine for bacteria that produce AI-2. Papa et al [217] study the anti-biofilm activity of Antarctic marine bacterium Pseudoalteromonas haloplanktis, TAC125. The results demonstrated that supernatant of P. haloplanktis grown in static condition, inhibited biofilm of Staphylococcus epidermidis.

 Many of extracts from marine sponges [14, 212, 218-220], algae [2, 221-223], and soft corals [224], seaweeds [225], fungi [226, 227] and bacteria [205, 209] demonstrate own biocide effect. A number of compounds derived from them and their synthetic analogues display mono-specific [228, 229] or broad bactericidal activity. Between them are: 10--formamidokalihinol-A and kalihinol A [230], polymeric 3-alkylpyridinium salts [231], dihydrooroidin already tested as a component of antifouling paints [233], succinic acid [234], taurine acid substituted bromopyrrole alkaloids and di-bromophakellin derivatives [235], flustramine inspired syntheic pyrroloindoline triazole amides [236], cyclized diterpenoids [228], decadienal (polyunsaturated aldehyde) [229], or bread bactericidal activity. Between them are: 10--formamidokalihinol-A and kalihinol A 3-phenyl-2-propenoic acid, cyclophenylpropenoic and cyclovalerilpropenoic acid [237], α-proteobacterium extracellular DNA [211], 2-hydroxymyristic acid and cis-9-oleic acid [209].

Current Strategies to Reduction of Marine Biofilm Formation

5.19 

Natural compounds have lately attracted significant attention with their large variety of potential applications as antimicrobials. The chemistry and biology of various natural organic compounds (alkaloids, non-ribosomal peptides, guanidine-bearing terpenes, polyketides and shikimic acid derivatives), their isolation, structural characterization determination, synthesis, biosynthesis and biological activities have been extensively studied [238].

ϱ͘ϰ͘ϯEĂƚƵƌĂůŝŽĐŝĚĞƐĞƌŝǀĞĚĨƌŽŵDĂƌŝŶĞ^ƉŽŶŐĞƐ Generating chemical defense for their survival, marine sponges and their associated microbial consortia are a source of natural biocides, producing a diverse array of molecules with antimicrobial capacity. Many marine sponges and soft corals defend themselves against fouling directly through the production of antifouling compounds, or indirectly through regulating the epibiotic microbes that affect larval settlement. A compound -formamidokalihinol-A and kalihinol-A were insulated from the marine sponge Acanthella cavernosa (Dendy) [230]. The results indicated that both compounds inhibit the growth of bacteria isolated from the natural environment. Water-soluble polymeric 3-alkylpyridinium salts have been isolated from the Mediterranean sponge Reniera sarai and 14 related synthetic analogues against marine biofilm bacteria [231]. Pyrroleimidazole alkaloids (PIA) have been extracted from several families of sponges with focus on bromopyrrole derivatives from the Agelasidae family [239-241]. The native toxicity of the PIAs serves as a potent defense mechanism for sponges [14]. Successful anti-biofilm libraries have been created utilizing the 2-aminoimidazole moiety found in many PIA, which are studied mainly as medical antibiofilm agents [242-245]. The marine natural product bromo-ageliferin has been isolated by bioassay guided fraction and was able to inhibit the formation of Pseudomonas aeruginosa biofilms. Many terpenes and pyrrole-imidazole alkaloids have antifouling capabilities against both microfouling and macro-fouling organisms, but many of these compounds are either toxic or have limited industrial applications due to formulation difficulties. The oroidin analogue, dihydrooroidin (DHO) only from the biofilm modulators has been tested directly for marine antifouling activity. DHO is an easily synthesized oroidin variant [246] that is an active inhibitor of biofilm formation against the bacteria Halomonas pacifica. Based on its success against H. pacifica, DHO is combined with marinebased paint and subjected to mesocosm tank trials. After three weeks in the tanks, the DHO paint had 125% less biomass than the paint-only controls. These trials show that DHO is a viable antifouling agent and it is still active after three weeks in the tank. Since PIAs are notorious toxins, DHO toxicity was evaluated using the same mammalian cytotoxicity assays used to evaluate TAGE and it was found that DHO is nontoxic up to six times the dosage used in the paint trials [232, 233]. Several sponge derived polybrominated diphenyl ethers and synthetic analogues have been identified which have showed potent antifouling activity [235]: against marine bacteria, diatoms, barnacle larvae and mussel juveniles. (1) 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)-6-methylbenozoic acid methyl ester; (2) 3,5-dibromo-2-(2,4-dibromophenoxy)phenol; (3) 3,4,5-tribromo-2-(2,4-dibromophenoxy)phenol, (4) 3,4,5-tribromo-2-(2-bromophenoxy)phenol, (5) 3,5-dibromo-2(2,4-dibromophenoxy)phenol, (6) 3,4,5,6-tetrabromo-2-(2-bromophenoxy)phenol; (7) 4-phenoxyphenol, (8) 4-phenoxyaniline, (9) 1-chloro-4-phenoxybenzene, (10) 1-bromo-4-phenoxybenzene

5.20 

Biofilm Control

The naturally occurring compound 2[3,5-dibromo-2-(2,4-dibromophenoxy)phenol], showed the strongest antifouling activity combined with lack of toxicity. Overall, the naturally occurring compounds show stronger activity than the commercially available analogues and could be possible future non-toxic antifouling candidates. The marine alkaloid, oroidin has been effective in dispersion and inhibition of the common proteobacteria and has resulted in the development of library of analogues [248]. This methodology represents a significant improvement over the generality known methods to acylate substrates containing 2-aminoimidazoles and has the potential for broad application in the synthesis of more advanced oroidin family members and their corresponding analogues. Seven novel sponge-associated marine bacteria have been screened for their antibacterial and antilarval-settlement activity in order to find possible new sources of non-toxic or less toxic bioactive antifoulants [259]. New secondary metabolites (exopolysaccharides) have been investigated from two Indonesian marine sponges Agelas linnaei and A. nakamurai and afforded 24 alkaloid derivatives representing either bromopyrrole or diterpene alkaloids[219]. The new compounds include the first iodinated tyramine-unit bearing pyrrole alkaloids, agelanesins A–D. Further new compounds include taurine acid substituted bromopyrrole alkaloids and a new di-bromophakellin derivative. Bunders et al [236] develop anti-biofilm agents based upon the flustramine family of alkaloids isolated from Flustra foliacea and perform biological evaluation of pyrroloindoline triazole amides as novel inhibitors of bacterial biofilms. Novel antibacterial proteins have been identified from the microbial communities associated with the sponge Cymbastela concentrica and the Green Alga Ulva australis [250] Japanese researchers [251] found that some marine sponges (Psammaplysilla purpurea, Callyspongia) contain anti-biofilm compounds, such as benzoic acid, aeroplysinin-I, and bromoageliferin, and developed chemical substances regulating biofilm formation. The inhibitory effect has been studied of cyclic Trihydroxamate Siderophore, Desferrioxamine E on the biofilm formation of Mycobacterium Species. Antifouling activity of bioactive compounds from marine sponge Acanthella elongata (Dendy) and five species of bacterial biofilm have been studied [220]. The crude extract and partially purified fractions of A. elongata showed significant inhibition the settlement of bacterial species. Water-based coatings containing natural biocides rose as a new environmentally friendly antifouling solution. Lately, a sea anemone metabolite complex embedded into an epoxy-resin-based commercial anticorrosion coating was presented [252]. Water-based coatings were developed that use low-toxic elements and natural biocides, isolated from surfaces immersed in a marine environment. The results were new environmentally friendly antifouling coatings that are able to mitigate the problem of biofouling without affecting the surrounding medium [253].

   Marine micro- and macroalgae (seaweeds) are also a source of antifouling compounds. Some of the compounds have exhibited antimicrobial activity but have been nontoxic to larvae of oyster and sea urchins. This indicated that they are potential ingredients of antifouling coatings. Screening for antimicrobial activity of marine flora has been done by Devi et al [254]. Protocols for bioassay guided fractionation was developed by Hellio et al [255] and antipatharian colonies have been screened [223] also from species like Ceramium botryocarpum [222]. Several classes of di-terpenoids have been isolated from the Mediterranean brown alga Dictyota sp. with four new cyclized diterpenes, one xenicane (l) and three dolabellanes (2-4) along with seven previously reported metabolites: 3-hydroxydilophol, dictyols E and C, hydroxy-crenulide, 9-acetoxy-15-hydroxy-1, 6-dollabelladiene, hydroxyacetyldictyolal, and fucoxanthin which have demonstrated potent activity against Pseudoalteromonas sp. D41 [228]. Lipid extracts from the brown seaweed Sargassum muticum have shown potent anti-microfouling activity with the active molecule identified as galacto-glycero lipids

Current Strategies to Reduction of Marine Biofilm Formation

5.21 

[256]. The green alga Dicytosphaeria ocellata and its organic extracts altered natural bacterial biofilm communities. Extracts from this organism have been incorporated into a matrix PhytagelTM and screened for activity [22]. Some of the secondary metabolites released by microalgae can influence the composition of benthic communities. Leflaive and Ten-Hage [229] determined the effects of decadienal (DD), a polyunsaturated aldehyde produced by diatoms, on motility and aggregation of a benthic diatom, Fistulifera saprophila and biofilm formation. The results indicate that the presence of DD-producing diatoms in a biofilm may favour the presence of certain microalgae at the expense of others. In addition to the effects on adhesion and motility, DD induces the formation of aggregates of F. saprophila cells. Complementary experiments, performed with two other benthic diatoms, Nitzschia palea and Mayamea atomus, showed that the effects of DD on adhesion and aggregation are speciesdependent. Bioassays using algal metabolites have also been developed [257]. Well-known common problems of the natural antifoulants such as scare production, antimicrobial activity keeping when is included in coatings, etc. limit the practical application also of the antimicrobial agents derived from marine algae.

     Antimicrobial compounds have been extracted from soft corals like Sinularia sp [258]; for the first time were reported formulations of acrylic paints combined with antifouling extracts from seaweeds (Fucus serratus and Archidoris pseudoargus) bacterial strains [198]; 3-chloro-2,5-dihydroxybenzyl alcohol was obtained from a marine-derived fungus Ampelomyces species that effectively inhibits bacteria and larval settlement of the tubeworm Hydroides elegans and cyprids of the barnacle Balanus Amphitrite [259]; the potential role of a metabolite isolated from the sponge Acanthella cavernosa surface-associated fungus was investigated and succinic acid was isolate with both antibacterial and anti-larval activity [260]; antifouling and antibacterial compounds from the marine fungus Cladosporium sp. F14 were reported, among which, 3-phenyl-2-propenoic acid, cyclo-(Phe-Pro) and cyclo-(Val-Pro) showing various activities against fouling bacteria [226]; a broad spectrum antimicrobial agent was isolated from a marine fungus strain designated as Penicillium viridicatum [227]; antibacterial activity was proved also of two benthic sea anemones (Heteractis magnifica and H. aurora) collected from the southeast coast of India [252]. The cyclic trihydroxamate siderophore, Desferrioxamine E, from the culture of marine-derived Actinomycete MS67 were re-discovered as inhibitor of biofilm formation [251]. Desferrioxamine E inhibited biofilm formation of Mycobacterium smegmatis and M. bovis with minimum inhibitory concentration of 10 M, while no anti-microbial activity was observed up to 160 M. Desferrioxamine E was also able to restore the anti-microbial activity of isoniazid against M. smegmatis by inhibiting biofilm formation. Mechanistic analysis of desferrioxamine E suggested that such inhibition might be due to the depletion of iron in the medium, which is essential for biofilm formation in Mycobacterium species. Cells of the bacterium Pseudomonas aeruginosa 1242 and their metabolites were immobilized into an epoxy-resin-based commercial anticorrosion coating [261]. The experimental coatings have prevented adhesion of micro- and macro-foulers in seawaters of different climatic zones. Potent activity has been observed from extracts of the seaweeds Dictyota dichotoma and Chaetomorpha linoides from the southeast coast of India against biofilm bacteria, anti-macro-fouling activity against brown mussels, as well as feeding deterrence activity against sea angel Monodactylus kottelati. The acetone extracts showed a wide spectrum activity against biofilm bacteria. The seaweeds also were found to inhibit byssus production and attachment in brown mussels. The lower epiphytic bacterial number on the seaweed's surface compared to the surrounding seawater medium indicated selective inhibition or surface mediation [225].

5.22 

Biofilm Control

Marine organisms possess an inexhaustible source of useful chemical substances for the development of new antifouling approaches [200, 262] but the concentration of their metabolites which are potential natural biocides are produced in a scarce amount. A promising solution of this production problem is the creation of biomimetic antifouling compounds or such that are synthetic in nature but inspired by natural products, many of which could be derived from marine sponge metabolites. Anyway, their including in antifouling paints is complicated and some of them are toxic for aquatic organisms.

ϱ͘ϰ͘ϲEĂƚƵƌĂůĞƌŝǀĂƚŝǀĞƐhƐŝŶŐEŽŶͲŝŽĐŝĚĂůDĞĐŚĂŶŝƐŵƐ Some naturally produced antifouling compounds are non-polar metabolites, presenting on the surface of the organism and repealing biofoulers whereas other are polar metabolites released into the surrounding water and triggering avoidance behaviour. These antifouling compounds are classified as deterrents (not toxins), since their mode of action is not due to toxic effects [185]. Bio-surfactants or microbial surfactants are surface active amphiphilic biomolecules, produced by variety of microorganisms that play critical roles in a variety of bacterial and environmental processes due to their interfacial interactions. Natural derivatives using non-microbiocidal mechanisms such as bio-surfactants/dispersals and exopolysaccharides have lately attracted improved attention with their unique properties: high biodegradability, low toxicity and effectiveness at extreme temperatures, pH and salinity. Biosurfactants are mainly comprised of lipids and lipo-peptides. These act by reducing the surface tension, inducing swarming or coagulation of cells. Studies on identification of marine micro-organisms producing biosurfactants is on the rise. Small molecules have been isolated from sponges which have demonstrated to inhibit / disperse bacterial biofilms specifically through non-microbiocidal mechanisms [14] as well as proteobacterial biofilms [244]. Discovery of natural products and synthesis of their analogues, including marine sponge-derived compounds (ageloxime-D; manoalide; 2-aminoimidazole) as well as the initial adjuvant activity and toxicological screening of the novel anti-biofilm compounds are also discussed. Small molecules have been synthesized [232], denoted TAGE, based on the natural product bromo ageliferin and demonstrated that TAGE had anti-biofilm activity against Pseudomonas aeruginosa. TAGE did not have selective toxicity against cells within the biofilm, it inhibited biofilm development under flow conditions and dispersed preformed biofilms whereas TAGE derivatives were shown not to possess such properties. Variety bio-surfactants / dispersants were reported in the literature, such as those insulated from marine Bacillus circulans lipopeptide [265] with activity against Gram-positive and Gram-negative pathogenic and semi-pathogenic microbial strains [265,266,267]. Terpenes have a large structural diversity originating from isopren sub-units modification, and a broad range of bioactivity including anti-biofouling. Among the sponge terpenoids, only ageloxime D, manoalide and two manoalide congeners are able to infer with bacterial biofilm formation without disrupting the cellular growth [219, 268]. Two classes of marine sponge metabolites without bactericidal effects have been isolated: pyrrole-imidazols [233, 242, 244, and 245] and terpenoids [63, 219, and 268] that house non-bactericidal biofilm modulators. Dendrilla nigra sponge associated bacteria Brevibacterium casei MSA19 [269], produce glycolipid biosurfactants which disrupt biofilm formation under dynamic conditions. Another class of lipid metabolites namely galacto-glycero-lipids have been isolated from brown algae which have antibacterial activity [74]. Trehalose lipid biosurfactants have demonstrated good inhibitory activity [270]. Rhamnolipids have also demonstrated ability to inhibit adhesion and to disrupt pre-formed B. pumilus biofilms by concentrations > 1.6 mM [271]. A glycolipid surfactant produced by tropical marine bacterial strain of Serratia marcescens isolated from hard coral, Symphyllia sp. has demonstrated antimicrobial activity towards pathogens of Candida albicans and Pseudomonas aeruginosa as well as toward the marine bacterium Bacillus

Current Strategies to Reduction of Marine Biofilm Formation

5.23 

pumilus. The surfactant appeared to be a glycolipid composed of glucose and palmitic acid. The glycolipid prevents adhesion of C. albicans BH, P. aeruginosa PAO1 and B. pumilus TiO1 and also disrupts preformed biofilms [272]. eDNA from the -proteobacterium Caulobacter crescentus inhibits the motile cells ability to settle in a biofilm [63]. Different groups of biosurfactants exhibit diverse properties and display a variety of physiological functions in the microorganisms; these include enhancing the solubility of hydrophobic/waterinsoluble compounds, heavy metal binding, bacterial pathogenesis, cell adhesion and aggregation, quorum sensing and biofilm formation. Antimicrobial and anti-adhesive properties of the biosurfactant “Lunasan” produced by Candida sphaerica UCP 0995 have been evaluated with regard to its biomedical applications [273]. Anti-biofilm activity of exopolysaccharides [274, 275] had several features that provided a tool for better exploration of novel anti-biofilm compounds. Inhibiting biofilm formation of a wide range of bacteria without affecting their growth appears to represent a special feature of some polysaccharides. Further research on such surface-active compounds might help the development of new classes antibiofilm molecules with broad spectrum activity and more in general will allow exploring of new functions for bacterial polysaccharides in the environment. Secondary metabolites ranging from furanone to exopolysaccharides have been suggested to have anti-biofilm activity in various recent studies. Among these, Escherichia coli group II capsular polysaccharides inhibited biofilm formation in a wide range of organisms. Recently, marine Vibrio sp. and Kingella kingae were found to secrete complex exopolysaccharides having the potential for broad spectrum biofilm inhibition and disruption without any bactericidal effect. Anti-adhesive activity has been studied of the biosurfactant Pseudofactin II secreted by the arctic bacterium Pseudomonas fluorescens BD5 [276]. Pseudofactin II is a novel compound identified as cyclic lipopeptide with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. The C-terminal carboxylic group of the last amino acid forms a lactone with the hydroxyl of Thr3. Adsorption of biosurfactants to a surfaces, e.g. glass, polystyrene, silicone, modifies their hydrophobicity, interfering with the microbial adhesion and desorption processes. The functional roles of biosurfactants in bacterial and environmental processes [277] have been studied. Environmental isolates were identified with biosurfactant-producing capabilities and a range of functions. However, large-scale production of the above described molecules is difficult because of low yields in production processes, high recovery and purification costs, all limiting their practical application [14, 263, 264].

         Quorum sensing (QS), a form of cell to cell communication, attracts increasing interest as a new target potentially substitutive or complementary to traditional treatments for reduction of marine biofilm formation [196]. Bacterial QS is a gene regulatory mechanism in regulating expression of virulence factor that allows bacteria to coordinate swarming, biofilm formation, stress resistance as well as production of toxins and secondary metabolites in response to threshold concentrations of QS signals, accumulated within a diffusion-limited environment [278]. One route to disrupt an already established inter-relationship between micro-fouling organisms is by blocking their dual functioning signal/receptor transcriptional regulators. Different types of compounds are currently known inhibiting the QS transcriptional regulator in Gram-negative bacteria. These compounds are sub-divided into two main groups, 1) comprising structural analogs of the native signalling molecules and 2) the other comprising compounds lacking structural resemblance. Biological activity of anti-QS compounds could be rationalized on the basis of structure-activity relationship and structural insight into the targeted protein [279]. QS regulate both, microbial biofilm development and pathogenesis. QS is a

5.24 

Biofilm Control

process by which bacteria take a census of their numbers and regulate specific phenotypic expression using small signalling molecules, called auto-inducers (AIs). Interference with QS is identified as a potentially novel approach to regulate marine biofilms formation. The attenuation of the bacterial community prevents the successful biofilm establishment. Quorum sensing inhibitors have been isolated from the red alga, Ahnfeltiopsis flabelliformis [280]. Screening of many extracts of marine organisms (LuxR-derived QS screen) showed that some of them are active in the specific Pseudomonas aeruginosa QS screen. The secondary metabolites manoalide, manoalide monoacetate, and secomanoalide, isolated from the sponge Luffariella variabilis showed strong QS inhibition of a lasB:gfp (ASV) fusion, demonstrating a potential for further identification of specific QS antagonists from marine organisms [268]. Cinnam-aldehyde and substituted cinnamonaldehydes [281] reduced virulence in Vibrio spp. by decreasing the DNA-binding activity of the QS response regulator LuxR (inhibitors of V. harveyi LuxR expression) as experimentally proved. Bacterial intervention strategies [282] have been developed targeting microbial cell signalling processes. Cell-to-cell communications in bacteria mediated by small diffusible molecules termed as auto inducers (AI) are known to influence gene expression and pathogenicity. Oligopeptides and Nacylhomoserine lactones (AHL) are major AI molecules involved in intra-specific communication in Gram-positive and Gram-negative bacteria respectively, whereas boronated-diester molecules (AI-2) are involved in inter-specific communication among both Gram-positive and Gram-negative bacteria. Naturally occurring furocoumarins from grapefruit showed > 95% inhibition of AI-1 and AI-2 activities based on the Vibrio harveyi based auto-inducer bioassay. These results suggest that furocoumarins could serve as a source to develop bacterial intervention strategies targeting microbial cell signalling processes. Bacterial small molecules [283] that modulate interspecies interactions as well as complex relationships such as those between microbes and insects interactions, resulting in non-antagonistic outcomes (i.e. developmental and morphological processes) and how they were co-cultured lead to a discovery of new small molecules and the use of known compounds to evoke unexpected responses and to mediate cross-talk between microbes. Microbial QS signalling in marine biofilms could be disrupted/inhibited by different strategies, including employment of derived from marine organisms compounds [278]. eDNA from -proteobacterium Caulobacter crescentus [63] produces a motile swarmer cell and a sessile stalked cell at each cell division and showed that C. crescentus extracellular DNA (eDNA) inhibits the ability of its motile cell type to settle in a biofilm.  eDNA binds to the polar holdfast (an adhesive structure, required for permanent surface attachment and biofilm formation), thereby inhibiting cell attachment. Because stalked cells associate tightly with the biofilm through their holdfast, they hypothesize that this novel mechanism acts on swarmer cells born in a biofilm, where eDNA can be accumulated to a sufficient concentration to inhibit their ability to settle. By targeting a specific cell type in a biofilm, this mechanism modulates biofilm development and promotes dispersal without causing a potentially undesirable dissolution of the existing biofilm. Unicellular microorganisms use QS to co-ordinate their activities that allows their population in biofilms to function as multi-cellular systems. Artificial intra-species and inter-species communication were demonstrated lately through synthetic circuits incorporating components of bacterial QS systems. Engineered QS-based circuits have a wide range of applications such as production of bio-chemicals, tissue engineering, etc. as it was discussed in an overview [284] on bacterial QS system as well as on the mechanisms developed by bacteria and higher organisms to obstruct QS communications. Disruption of quorum sensing is discussed as a viable strategy for prevention the formation of harmful biofilms in membrane bioreactors and marine transportation. The chemistry and biology of formation of Gram negative bacterial biofilms (including their constituents and signaling compounds that mediate or inhibit the formation of biofilms) as well as cell–cell communication by N-acyl-l-homoserine lactones (AHLs) including their biosynthesis, identification, and role in biofilm formation was

Current Strategies to Reduction of Marine Biofilm Formation

5.25 

discussed [285]. Mechanisms of AHL antagonism and the quorum sensing cascade description in the Vibrio genus were presented. Quorum sensing signal production and inhibition by coralassociated Vibrio has also been reported [286]. Screening of a range of Vibrio, isolated from variety of healthy and diseased corals, for the production of the QS signal molecules, N-acylhomoserine lactones (AHLs) and the AI-2 (autoinducer-2), small furanone signal molecules have been studied. All examined strains activate the AI-2 biosensor, but only some of them activate an AHL biosensor. They show the effect of temperature on AHL production may vary considerably among the isolates. For the first time has been reported QS inhibition by Vibrio harveyi. This occurs only at higher temperatures but it is not due to degradation of AHLs. The large diversity of Vibrio and the different effects of temperature on signal production may partly explain the complexity of coral-associated community changes in response to environmental factors. Target-based screening for anti-biofilm agents [32], focusing on inhibitors of quorum sensing offered a novel and promising class of biofilm inhibitors. Lately, the most characteristic target for molecules with anti-biofilm activity were compounds interfering with the metabolism of the signal molecule on cyclic di-GMP metabolism as well as on inhibitors of DNA and nucleotide biosynthesis. The antibacterial activity and QSAR of chalcones have been studied against biofilm-producing bacteria, isolated from marine waters [287]. The antagonism of bacterial cell to cell signaling by metabolites of marine gram-positive bacteria has been investigated [288]. Two aryl-ethyl-amides from a Halobacillus salinus were isolated that were able to inhibit the quorum sensing systems of two other bacteria, Chromobacterium violaceum and Escherichia coli. They were competitive inhibitors of V. harveyi LuxR expression. Gram-positive bacterial isolates from a broad range of marine environments and substrata were screened for inhibition of the quorum-sensing controlled phenotype of bioluminescence in the marine bacterium V. harveyi BB120. Chemical analysis of one of the active isolates, Bacillus cereus D28, leaded to the identification of cyclic-L-proline-L-tyrosine as an antagonist of bioluminescence production by V. harveyi. Microbe-microbe interactions from marine particles, hot spots for bacterial colonization, are studied. Isolates from particles and the surrounding water column were assayed for the production of both toxic and non-toxic substances. Results from the studies demonstrated that non-toxic interactions are more prevalent than toxic interactions among all bacterial types. Recently a marine Halobacillus salinus isolate was reported [289] that secreted secondary metabolites capable of quenching quorum sensing phenotypes in several Gram-negative strains. To investigate how widespread the production of such compounds may be in the marine bacterial environment, 332 Gram-positive isolates from diverse habitants were tested for their ability to interfere with Vibrio harveyi bioluminescence, a cell signalingregulated phenotype. A total of 49 bacterial isolates interfered with bioluminescence production in the assays. Metabolite extracts were generated from cultures of the active isolates, and 28 reproduced the bioluminescence inhibition against V. harveyi. Of those 28, five extracts additionally inhibited violacein production by Chromobacterium violaceum. Chemical investigations revealed that phenethylamides and a cyclic dipeptide are two types of secondary metabolites responsible for the observed activities. The active bacterial isolates belong to either the genus Bacillus or Halobacillus. The results suggest that Gram-positive marine bacteria are worthy of further investigation for the discovery of quorum sensing antagonists. Marine biofouling inhibition was reported by bacterial quorum sensing inhibitors [290], screening for natural products from chemical libraries containing compounds from marine organisms (sponges, algae, fungi, tunicates and cyanobacteria) and terrestrial plants, using a reporter strain Chromobacterium violaceum CV017. The QS inhibitory activities of the most potent and abundant compounds were further investigated using the LuxR-based reporter E. coli pSB401 and the LasRbased reporter E. coli pSB1075. Hymenialdisin, demethoxy encecalin, microcolins A and B and kojic acid inhibited responses of the LuxR-based reporter induced by N-3-oxo-hexanoyl-L-homoserine lactone at concentrations >0.2 M, 2.2 M, 1.5 M, 15 M and 36 M, respectively. The ability to

5.26 

Biofilm Control

prevent micro fouling by one of the compounds screened in this study (kojic acid; final concentrations 330 M and 1 mM) was tested in a controlled mesocosm experiment. Kojic acid inhibited formation of microbial communities on glass slides, decreasing the densities of bacteria and diatoms in comparison with the control lacking kojic acid. The results suggest that natural products with QS inhibitory properties can be used to control of biofouling communities. For the first time, novel anti-quorum-sensing acylase activities from a B. pumilus strain of marine origin [291, 292] have been found. Several findings were reported for numerous aquatic organisms such as micro-algae, macro-algae, invertebrates, or even other bacteria that have a potential to disrupt QS. The mechanism of action varies from degradation of signals through enzymatic or chemical inactivation to antagonistic and agonistic activities. Enhancement or induction of antimicrobial biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions have been investigated [215]. Marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1) displaying antimicrobial activity against biofouling fungi (Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA, Bacillus pumilus BP) were chosen for this study. Marine epibiotic bacteria when co-cultivated with afore mentioned fungi or bacteria showed induction or enhancement of the antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in the most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production. Expression of virulence factors in many pathogens requires quorum sensing autoinducer (QSA) to reach a certain threshold concentration [293]. Quorum sensing inhibitors (QSIs) can competitively inhibit the quorum sensing signal system, which provides a novel target to combat pathogens for the development of new drugs. Bacterial extracellular polysaccharides have been shown to mediate many of the cell-to-cell and cell-to-surface interactions that are required for the formation, cohesion and stabilization of bacterial biofilms. However, recent studies identify several bacterial polysaccharides that inhibit biofilm formation by a wide spectrum of bacteria and fungi both in vitro and in vivo. Rendueles et al [294] discuss the composition, modes of action and potential biological roles of antibiofilm polysaccharides recently identified in bacteria and Eukaryote. Some of these molecules may have technological applications as anti-biofilm agents in industry and medicine.

 Marine biofilm formation is a complex dynamic process of interaction between surfaces, molecules and micro-organisms that depends on the local environmental conditions. On this reason the combating marine biofilms is a significant challenge and requires approaches adjusted to the corresponding surface in the corresponding equatorial and exploration conditions. Theoretical basis of current anti-biofilm strategies involves knowledge about the composition and mechanism of marine biofilm formation along with characteristics of the living in biofilms microbes; cell - surface interactions, microbial adhesion and protein adsorption as mediator of this process; secreted extracellular polymeric substances (EPS) and their cross-linking as well as inter- and intracellular communication via quorum sensing. Antifouling strategies should take into account all possible interactions used by the microbes to adhere and develop biofilm on an immersed surface.

Current Strategies to Reduction of Marine Biofilm Formation

5.27 

Chemical messages used for cell to cell communication, a process referred to a quorum sensing, can be effectively used to combat biofilm formation by interfering during different stages of biofilm formation viz: reversible and irreversible attachment, etc. Current search for inhibitors of biofilm and biofilm-related cellular processes are directed towards more in depth understanding the biofilm biology. The evolutionary aspect of biofilm dispersal is now explored in the combat with biofilms through the integration of molecular microbiology with eukaryotic ecological and evolutionary theory, which provides a broad conceptual framework for the diversity of specific mechanisms underlying biofilm dispersal. The activation of biofilm dispersal rises as a novel mode of action for anti-biofilm compounds. The quorum sensing mechanisms as well as cell - surface and multi-species biofilm competitive interactions understanding support the development of non-biocide approaches in combating biofilms. Most of them are based on employment of enzymes, bio-surfactants, or other microbial metabolites and target all steps of biofilm formation: from the initial adhesion inhibition to the matrix degradation, cell to cell communications disruption and biofilm dispersion induction. Replacement of the toxic biocides with naturally derived, degradable and repellent compounds or enzymes are in the focus of current anti-biofilm strategies. Some chemical inhibitors and electroassisted anti-micro fouling technologies are still under investigation. But no single technology has been demonstrated as universally effective. Many antimicrobial compounds are most effective during the first stage of microbial cells attachment, because they are still in a vulnerable state, and not themselves coated in exo-polymeric substances. Unfortunately, antimicrobial compounds that are able to stop totally the reversible adsorption of microbes are still not known. The most widely suggested enzyme-based approach for effective prevention of both, irreversible adhesion and proliferation of microorganisms in biofilms is the hydrolytic breakdown of adhesive or structural polymers based on protein and polysaccharide components. Different approach, not relying on degradation of polymers, is to produce compounds at the surface that deter settling organisms. Another possible solution is to degrade signaling compounds that promote bio-fouling. But the enzymatic alternatives suffer of some disadvantages limiting their practical application: the catalytic activity of the enzymes increases whereas their stability decreases with increasing temperature. The need in both water and structural mobility to perform their function as well as an optimal balance between activity and stability limit the practical applications of enzymes. Very active research during the last decades was focused on the study of the defense of naturally non-fouling surfaces and development of bio-mimetic or bio-inspired approaches. The natural defense includes combinations of chemical and physical strategies. A number of anti-biofilm approaches are based on the replication of specific natural surface topographies of non-fouling natural surfaces as well as on the identification of natural antifouling agents, such as natural biocides and biosurfactants/dispersants used by microbes for own defense. Surface patterning is a promising totally non-toxic approach but the conventional materials exposed in marine environment are not able to keep their surface topography for enough long period. In addition, it was shown that special surface topographies usually reduce mono-species and rarely multispecies biofilms formation. Many naturally derived, degradable and repellent compounds (natural biocides, bio-surfactants and quorum sensing inhibitors) and enzymes, also demonstrate significant activity against mono species but only few are active against multispecies biofilms. In addition, they are scarcely available and difficult for application in antifouling composition coatings. Despite the exclusively large investigations, the combat with marine biofilms remains a challenge whose surmount requires, as agreed by many researchers, a complex integrated strategy. Since the biological cascade of the biofilm formation begins with deposition of secreted by fouling microorganisms adhesive molecules, the in depth understanding of their adsorption mechanisms could be the key to identification of "universal" surface that would release all microfoulers. But the large diversity of microbes and irrespective diversity of secreted adhesive substances make difficult the total prevention of the initial reversible adhesion.

5.28 

Biofilm Control

Many surface characteristics are known to influence initial interactions of materials with microbial cells that could be used to reduce biofilm formation, such as surface roughness, topography, hydrophilic/hydrophobic balance and chemistry. The most important pre-requisite of the “clean” surface is to be low adhesive, e.g. chemically inert, non-charged, high elastic, with suitable topography and strong hydrophobic/strong hydrophilic. Its anti-biofilm performance could be improved by a presence of surfactant/bio-surfactant, inhibitor of QS or/and crosslinking of secreted EPS. Surfactant/bio-surfactant could impede the initial microbial adhesion, together with non-toxic inhibitor to disturb the crosslinking of secreted EPS and to assists the biofilm dispersal. Low adhesive antifouling coatings are only a current commercial alternative of the toxic biocide antifouling pains. Their preventive potential with respect to combating marine biofilms is still not fully explored.

 Bulgarian Scientific Fund and DAAD are gratefully acknowledged for their financial support (grant DNTS 01/6/15.11.2011.)

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Plouguerné E., Ioannou E., Georgantea P., Vagias C., Roussis V., Hellio C., Kraffe E., StigerPouvreau V., (2010). Anti-microfouling Activity of Lipidic Metabolites from the Invasive Brown Alga Sargassum muticum (Yendo) Fensholt, Marine Biotechnology, 12(1), 52-61.

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Yang LH, Lau SCK, Lee OO, Tsoi MMY, Qian PY (2007). Potential roles of succinic acid against colonization by a tubeworm, Journal Experimental Marine Biology Ecology, 349(1), 1–11.

262.

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Das P, Mukherjee S, Sen R (2008). Antimicrobial potential of a lipopeptide biosurfactant derived from marine Bacillus circulans, Journal Applied Microbiology, 104(6), 1675–1684.

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BIOFOULING AND BIOFILM PREVENTION ON AQUATIC SENSORS                                  ­    €  ‚  ƒ  „……   †     ‡ˆ …†‰Š‹Œ  Ž   ‘ 



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         … ……   ……   ‡       ‡        ……       …    ‡                 

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6.2 

Biofilm Control

 Biofouling is often defined as the unwanted attachment and growth of organisms such as bacteria, algae or barnacles on submerged artificial surfaces [1]. It is a major obstacle for successful and costeffective long-term deployment of autonomous sensing systems and data acquisition [2]. Biofouling of moored sensor systems (often those most affected by biofouling) results in (1) reduced reliability in data acquisition or (2) reduced functionality and poor data quality from affected transducers, contaminated reagents, occluded optical windows, clogged microfluidic devices and pumping components or membranes [3, 4, 5, 6, 7, 8]. The overall effect of biofouling is to reduce sensor efficiency and reliability, increasing maintenance costs, and even potentially preventing the application of certain sensor systems beyond the laboratory scale. At present, common methods used in industrial environments such as physical (e.g. back-washing, back-pulsing, air sparging) and chemical (e.g. acids, bases, oxidants, chelating agents, polymeric coagulants, surfactants) cleaning methods can often fail to adequately protect against biofouling on membranes and sensors in marine or riverine environments. There has been a concerted effort to produce novel antifouling methods and recent efforts toward environmentally benign antifouling materials have explored fluoropolymers, polysaccharides, polyethylene glycol (PEGylated) polymers, and zwitterionic polymers among others [9, 10], however the focus has generally been on antifouling coatings for structures with greater surfaces areas than those of sensors, i.e. ship hulls, ocean energy devices and aquaculture infrastructure. The resulting antifoulants can be unsuitable for application to sensors (particularly optical components and other common sensor materials), so sensor antifouling strategies may consist of sub-optimal application of generic antifouling approaches that were designed with other applications in mind. Furthermore, multiple (integrated) antifouling strategies may offer increased operational lifetime in the field for sensors, where a broad range materials for different components is often used, and where the optimum method of biofouling control may vary depending on the sensor type, application area, depth, season etc. and materials.

             Current aquatic sensors collect data on a range of physiochemical and environmental parameters such as pH, dissolved oxygen (DO), conductivity, temperature and depth (CTD), flow rates, turbidity and inorganic nutrients [11]. However, a significant need exists for more in situ sensors measuring more parameters, and improved measurement methodologies and novel sensors are continuously sought [12]. As pointed out by [12], current and future sensors need to have excellent detection limits, precision, selectivity, response time, a large dynamic concentration range, low power consumption, robustness, and less variation in instrument response with temperature and pressure, as well as be free from fouling problems (biological, physical, and chemical) [12]. Wireless data transmission and the development of a new generation of micro- and nano-mechanical sensors have made autonomous environmental monitoring an achievable goal [13]. When functioning effectively, autonomous sensor networks would be capable of communicating in real time and transmitting data directly to a remote data storage and analysis facility, thus greatly increasing the application potential of environmental sensors and revolutionising our understanding of aquatic processes [14, 15, 16]. However, in many instances biofouling remains a substantial obstacle to the realisation of this possibility at present. Modern aquatic sensors are constructed with a wide variety of material types, and all the immersed components of the sensor system viz: operational components (membranes, optical windows, and electrodes); housings (casings), and mooring components are subject to biofouling [7]. Providing adequate practical antifouling strategies for all the involved components is challenging since deployed sensors are frequently constructed of many different types of material (Table 1.), meaning that each material component might well need to be considered individually in terms of an optimal antifouling

6.3 

Biofouling and Biofilm Prevention on Aquatic Sensors

strategy. Materials are usually chosen based on their cost, mechanical and optical properties, durability and corrosion resistance rather than their antifouling ability, which is influenced by the sensor type. The challenge therefore lies in developing an antifouling system that protects all vital components of the sensor platform and system – while not affecting measurements and remaining affordable on restricted monitoring budgets. Therefore, multiple strategies can exist on any one device depending on the degree of antifouling required, local biofouling conditions (rate of growth, presence of specific fouling organisms etc.) and presence of specific factors such as increased drag resulting from high flow rates (meaning that moorings and other components should be antifouled to reduce hydrodynamic drag). Table 1: Some common materials exposed to biofouling in environmental sensor construction, and possible antifouling approaches. Material

Use

Example Sensors

Possible Antifouling Strategies and references

Metals Anodised aluminium

Sensor housings

Available in some commercial turbidity sensors.

304L steel

Stainless

Sensor housings

Widespread applications.

316L steel

Stainless

Sensor housings

Specifically marine applications and corrosive industrial applications

Stainless steel microscreens

Filtration

Titanium

Sensor housings Electrodes Antifouling

Available for particulate matter screening on some sensors e.g. conductivity and temperature. Replacement for SS housings.

Tin-free coatings or cuprous thiocyanate – caution with potential electrolysis with copper or other metal based AF [14]. Various, including novel ultrasonic methods possible [17] – not generally for marine immersed marine components. Various, surface finishing specifications may effect performance [18], caution with galvanic corrosion and in anaerobic environments. Unknown

Platinum Copper/Bronze, specific brasses Polymers

in

freshwater

Conductivity Many commercial systems

Surface finish can affect biofouling retention [19] . Unknown Often specific AF component [20].

6.4 

Biofilm Control

Polyoxymethylene (Acetal, Delrin ®)

Sensor housings

Copper(i)oxide (copper tape), Zinc pyridinethione [21, 22].

Sensor housings

Available on commercial pH, fluorimetry and ORP sensors among others. Available on some pH and ORP sensors.

Polyphenylene sulfide (PPS) (Ryton ®) FEP Teflon

Membranes

Dissolved oxygen.

Polyurethane

Cabling

Many commercial systems

Acrylonitrile butadiene styrene (ABS) Polyvinyl chloride (PVC)

Sensor housings

Used some commercially available sensor models

Various depending on materials [23] – occasionally tributyl-tin leaching components in combination with water flushing, copper surrounds. Often not protected from fouling. Non-specific commercial coatings.

Sensor housing

Some multi-parameter sondes

Various, often not protected.

Sensor Housings

Some multi-parameter sondes

Non-specific coatings.

Cabling

Many commercial systems

Largely unprotected, although patents filed regarding antifouling for geophysical cables [24]

Electronics, housing material Diaphragms

Various

Non-specific

Water level sensors.

Mechanical (wipers / brushes) Mechanical (wipers / brushes) [4, 25]. Mechanical (wipers / brushes) [9]. Mechanical and occasion biocide release, some UV irradiation [4, 26, 27], copper rings and surrounds.

Polycarbonate (and blends under various tradenames) HD polyurethane

Other Materials Epoxy resins

Silicon Sapphire PVDF membranes Borosilicate Glasses

Optical windows Filtration membranes Optical windows, conductivity cells etc.

Turbidity among others Phosphate, combined models. Turbidity, conductivity, camera and optical equipment

Commercial coatings.

Antifouling

commercial

   

   Biofilms develop on aquatic sensors in a similar manner to biofilms in other environments (e.g. clinical or industrial environment) albeit with some dependence on substrate materials [28]. The classical description of biofilm development involves initial adsorption and formation of a conditioning layer immediately following immersion of a surface, followed by attachment of microorganisms and propagules of macrofouling organisms within hours or days (depending on the season, latitude, depth,

Biofouling and Biofilm Prevention on Aquatic Sensors

6.5 

nutrient and salinity status of the surrounding medium etc.) and progresses to macrofouling by growth and further colonisation of exposed surfaces [29]. This progression is shown in the context of sensors unprotected against biofouling in Figure 1, where progression of fouling was followed in an estuarine setting (Liffey Estuary, Dublin, Ireland) during early spring (February to March 2012). The progression from a microfouling layer after 1-2 weeks to complete coverage of the unprotected sensor components after two months of exposure is shown.

Figure 1: Progression of biofouling on an environmental sensor housing from initial conditioning and colonisation of a surface (left) (including individual bacteria, lower image, arrowed) through to microfouling (middle) where initial barnacle colonisation can be seen on the sensor guard in the top of the photo and finally macrofouling (right) – where biofouling growth hides a temperature and conductivity probe.

              Many commercial sensor antifouling strategies currently rely upon a two-pronged approach consisting of mechanical methods combined with biocide release, or occasionally seawater flushing combined with biocide release. Mechanical wipers are commonly used in many commercial systems to keep optics and membrane free of biofouling, and are combined or constructed with copper alloys for additional protection in marine environments [4]. Mechanical methods have been available for decades, and the use of mechanical wipers, shutters and brushes has benefitted from improvements in software control, lower power requirements and sometimes user serviceable parts in recent years. Centrally controlled wipers appear to be increasingly common (see for example the Xylem Analytics EXO 2 sonde) in comparison to individually wiped sensors on data sondes, with the possible drawback that failure of the central wiper means complete failure of the mechanical antifouling protection on those sondes. Pairing a biocide-based antifouling with mechanical methods allows each strategy to compensate for the shortcomings of the other to some degree [4]. Mechanical cleaning removes fouling on surfaces by physically scraping biological and non-biological material off the sensor, while biocides kill or reduce the abundance of attaching organisms in the first place. The mechanical methods also remove non-biological fouling (perhaps particularly important in high turbidity environments or in anoxic zones where local chemical reactions can result in chemical precipitates accumulating on components). Biocide release, either through active release or through passive leaching from coatings, can kill

6.6 

Biofilm Control

colonising organisms, either at or in close proximity to the surface [3, 1]. The final and less commonly used strategy involves controlled biocide generation [4], sometimes generated in situ near sensors, where biocides such as chlorine or acids are injected onto or produced around the sensing area. Local chlorination is well known as a means of biofilm control in other areas [30, 31], but requires electrical power and may have specific advantages and disadvantages, discussed in detail later.

Figure 2: A commercial available sensor as part of a multiparameter environmental sensing sonde, with individual antifouling wipers for protecting single sensors – in this case the membrane of a probe for measuring dissolved oxygen. The component parts are labelled: note the use of copper alloy for construction of the wiper blade for dual mechanical/biocidal antifouling capacity. The common weakness of these systems (apart from failure of the wiper seals leading to water ingress) is the stainless steel central wiper spindle which can be a colonisation point for algae and other fouling, reducing data quality, particularly in sensors with optical windows (turbidity, algal sensors and others). An advantage of use of wipers / shutters and other mechanical methods is the absence of hindrance of the wiper or brush from the sensing area during data recording. Wipers or brushes pass over the sensing area between measurements to remove adhered material and usually have little impact on measurements taken by the sensor (a notable exception perhaps being in low-turbidity environments). A second advantage of mechanical cleaning is that the antifouling ability is nonspecific i.e. it removes both biofoulers, their products as well as non-biological fouling (silt / mud etc.) deposited on the surface [7]. Energy consumption during operation of motors, controlling devices and software is a disadvantage of the use of mechanical cleaning apparatus, and is primarily an issue for remote or inaccessible sensor installations. Despite rapid improvements, battery life is still a crucial factor in ocean sensor deployment, and careful energy budgeting is still required for remote or inaccessible sampling stations, and in many cases the deployment period is still very often limited by battery life. For example, [32] compared the performance of two dissolved-oxygen sensing technologies, the Aanderaa Instruments AS optode model 3830 and the Sea-Bird Electronics, Inc., model SBE43, both protected and unprotected from fouling [32]. The optode (or optrode, an optical sensor that uses indicator dyes mounted in a matrix or polymer layer) had an optional copper surround for antifouling around the optical window instead of the standard plastic material, while the SBE43 had tributyl-tin leaching tips on the intake and discharge of the sensor. Although both systems performed well, the flushing required for Sea-Bird SBE43 resulted in 17 times the power per sample than that of the optode.

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6.7 

Furthermore, mechanical methods can fail in cases where macrofouling is a problem, or where the forces required when removing already adhered fouling exceed those produced by the wiper/brush (particularly possible in the case of calcareous fouling e.g. barnacles). Another less obvious drawback of mechanical cleaning is that the cleaning mechanism often provides a larger surface area for biofouling (Figure 3). Without additional antifouling mechanisms, the sides and back of the wipers or sponges are very susceptible to fouling. Microfouling of these areas may not cause issues but when they support macrofoulers such as macroalgae, where algal fronds for example, while not growing directly on the sensor, may cause poor quality data and interfere with measurements. Direct failure of the wiper mechanisms during field deployment is a possibility, which may lead to complete failure of the sensor and to power supply problems for the sonde or other associated components. The presence of a wiper, and associated motor and seals (O-rings) means that there are more possible failure points in the sensor, and complete failure means that any antifouling and cleaning ability is immediately lost. This can obviously be a major and possibly expensive problem if failure occurs early in a remote deployment.

Figure 3: Biofouling associated with a commercially available multiparameter sonde recovered by the authors after several months of unattended operation in a tidal estuarine site (approximate location 53.344° N, 6.215° W), in which both mechanical wipers and a copperbased antifouling guard have been installed. While the copper guard has prevented macrofouling (left), it has not prevented the growth of macrofouling on the stainless steel (316L) mechanical wiper spindles close to the optical windows of the individual sensors (right), resulting in disruption of sensor measurements. Combining copper and zinc alloys or seawater soluble copper oxide (Cu2O) with mechanical methods in marine environments (to readily facilitate the release of Cu+ or Cu2+) is a very common antifouling strategy [33, 34, 35]. Copper occurs naturally in seawater, so copper usage as an antifoulant is seen as a practical solution for protecting sensors / equipment which have small surface areas [14]. However, elevated levels of copper in sediments in many port environments [36] has meant legislative control of release concentrations has occurred in some areas of the world (for example Canada, where AF paints containing copper have a release rate of less than 40 µg/cm2/day) and copper in antifouling

6.8 

Biofilm Control

coatings has an uncertain future [37]. What impact such regulation will have on the use of copper as antifouling in sensors remains to be seen. Several commercial formulations containing copper within epoxy resin carrier systems are available for marine leisure craft (for example Copper Coat, www.coppercoat.com) that could be applied to sensor materials, although there appear to be no published data on performance characteristics of these formulations on sensor materials. Commercially antifouling paint formations including copper (I) thiocyanate, cuprous bromide or cuprous iodine are also a possibility for sensors, however copper(II) oxide is still preferred in many applications due to solubility and toxicity [38]. Components can be machined directly from copper alloys in many cases for marine sensor deployments, provided there is little likelihood of interference with measured parameters or risk of galvanic corrosion. In marine multiparameter sondes, the individual sensors can be grouped together and protected by a machined copper guard that acts as an antifoulant and as a dead weight for orientating the sensor assembly vertically in the water column (although this may add up to 10 % to the total cost of the sonde) (personal observations of the authors). The possibility of galvanic corrosion in seawater between the copper and other less noble metal in use in other sensor components (stainless steel, aluminium) is also a concern. Therefore, sacrificial anodes (zinc or magnesium) must be installed in order to prevent corrosion on these systems, and these in turn must be replaced in a timely manner, otherwise expensive repair and perhaps catastrophic failure of the sensor/ sonde can be expected.

   While copper is very effective as an antifouling material, biofouling can still occur in the presence of copper under certain conditions [38]. For this reason commercial coatings used for protecting ships or other large structures often make use of biocides like Diuron or Irgarol 1051®, zinc, titanium or other materials in paint formulations [39, 40]. While providing excellent biofouling protection, common concerns with these compounds include their environmental impact, possible bio-accumulative potential, toxicity and the difficulties associated with providing an accurate assessment of the eventual fate of many of these materials [40]. Thus, the benefits need to be carefully considered before such materials are considered in environmental sensors. The impacts of such materials can be illustrated by Tributyltin (TBT), a well-known example of a biocide developed in the 1960s and thought to be the solution to biofouling until the 1980s, when TBT usage was connected to abnormal growth of marine life [41, 42]. These results eventually resulted in a reduction in TBT use as an antifouling additive and eventually complete prohibition of TBT in marine shipping antifouling coatings in 2008 (IMO, International Convention on the Control of Harmful Anti-fouling Systems on Ships, 2008). It must also be considered that antifouling strategies for sensors that rely only on biocides could mean that dead organisms may be retained on surfaces. As previously pointed out, “Killing, however, is not cleaning” in the context of biofilms and frequently “the presence of biomass and not its physiological activity is the problem” [43] (perhaps with the exception of biofouling on dissolved oxygen sensors where the biofilm may either consume or create oxygen, affecting measured values). Thus, inhibiting the attachment of microbes is dependent on the concentration and leaching / release of biocides at the surface, and although biocides may kill the attached micro-organisms, dead cells on the surface may not be removed in all cases, which in turn offers a source of nutrients and surfaces of increased roughness further facilitating increased attachment of micro-organisms [44]. Mechanical cleaning should also be used on these components where feasible, particularly since environmental sensors may also be subject to inorganic fouling (sediments) in turbid or anoxic environments. The advantages of mechanical cleaning in combination with carefully selected biocides can include a reduction in effective biocide concentrations, reducing impact on the environment as well as removing any dead cells. Lower mechanical cleaning frequency may also be possible, perhaps improving battery life in sensor sondes. Future autonomous battery-powered sensor systems may also

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6.9 

benefit from a feedback mechanism whereby the wiper frequency could be increased dynamically when fouling is high, and subsequently reduced in frequency with low fouling potential to save battery life, although this approach perhaps requires some type of biofouling sensor to operate effectively. In summary, combined AF approaches using both mechanical methods in combination with biocides, is a very viable strategy for minimizing and preventing, and removing fouling on both sensing components (membranes and optical windows) and on casings and other components.

      Novel biocides for antifouling applications have largely been developed and commercialised for shipping applications. This diverse group ranges from copper-containing compounds discussed previously, to natural product-based compounds [45, 46]. Application has generally consisted of coatings that actively release biocidal ingredients to kill or disable biofouling organisms (See [39] for an overview of the common approaches here). Therefore, biocides (and the coatings formulations within which they are usually incorporated) have been primarily developed with these applications in mind. This is perhaps understandable given the extensive licensing costs and toxicological and regulatory requirements surrounding the development of a biocide for widespread release under the Biocides Directive (Regulation (EU) No 528/2012) as an antifouling material, and potential access to large industrial markets are often necessary. Biocides widely used to date for antifouling have included tin-based compounds (such as tributyltin (TBT) – now prohibited by international treaty), potent synthetic herbicides (for example, diuron (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) and Irgarol 1051®, a highly specific and effective inhibitor of photosynthesis) and, recently, nanomaterial-based additives [3, 47, 48]. However, many of the biocidal additives used since the 1950s have often proven to be damaging to the health of aquatic ecosystems [49] and alternative approaches are necessary in many cases. This search has accelerated since prohibition of TBT (in 2008) as an antifouling additive (IMO, International Convention on the Control of Harmful Anti-fouling Systems on Ships, 2008), but has proven difficult and left some industries (shipping for example) without a satisfactory antifouling coating in the interim. This area therefore forms the core of antifouling materials research, and is a rapidly developing area that has attracted significant European and global research funding in the last decade (See for example European FP6 and FP7 funded projects like AMBIO, SEAFRONT and ByeFouling). It appears at present that only few commercial sensors utilise biocidal ingredients like bis (tributyltin) oxide to prevent biofouling (for example tributyl-tin leaching tips have been used in the Sea-Bird SBE43 oxygen sensor), and it would seem that there is scope for applying antifouling materials developed for the shipping industry to aquatic sensors on a trial basis (outside of specialised optical and membrane components). However, there appear to be few published reports on the effectiveness of this approach or any systematic studies on the effects on overall sensor performance. The search for novel marine natural products for antifouling purposes is an area of high research interest, but as yet appears to have resulted in few commercial successes [45]. Compounds extracted from marine algae and sponges often form the basis for the development of novel active ingredients in coatings in practise [50, 51, 52] but the outlook for these compounds as commercial possibilities is uncertain, and indeed their performance and application to sensor technology is still largely unknown. There are however other promising candidates, for example Selektope (or medetomidine) is a marine core-biocide that has recently received approval, delivering efficacy in concentrations from 0.1% w/w, and while largely marketed with shipping applications in mind, would seem to have attractive possibilities for use in sensors (see commercial company I-Tech: www.i-tech.se). Selektope has been determined to temporarily stimulate the octopamine receptor in the barnacle larvae, causing their legs to start kicking, and thus the organisms are repelled from a surface without causing death. This could have major advantages when applied and released in sensor technology, for example when

6.10 

Biofilm Control

impregnated into construction materials, but to date there have been no published performance evaluations available to the authors’ knowledge.

            Few, if any, commercially available antifouling coatings are specifically formulated for use in sensor applications, although some manufacturers supply sprays for antifouling applications (for example Cspray and C-Spray II from Xylem Analytics Inc. for use of multi-parameter sondes such as the YSI 6600 series systems). Recent growth in specific applications, such as display screens and optical sensors have prompted significant research in the development of novel protective coatings (selfcleaning/antifouling/self-healing) that also possess transparency at visible wavelengths. The development of commercial products by Detty and Bright [53] in recent years highlight the potential of silica-based coatings. In fact sol-gel technologies have been widely explored in this regard, and significant advances in self-cleaning coatings have been achieved [54, 3, 9, 48]. The employment of ORMOSIL (organically modified silica) coatings on a range of substrates has highlighted their usefulness and flexibility, particularly the application of “Aquafast” on the polycarbonate dome covers of video cameras used by Sicily’s Superintendance of the Sea to monitor the archaeological site of Cala Gadir 30 m underwater in the sea off the island of Pantelleria [55]. The authors reported a dramatic reduction in fouling following application of the coating with the camera “mildly cleaned with a towel from minor algal biofouling only once, after 3 years since application of the sol-gel paint” a dramatic effect when prior to the coatings application the camera’s “polycarbonate dome was rapidly colonized by algae and other organisms” thus requiring cleaning by divers every forty days. At present few commercial coatings are specifically targeted towards optical windows and the optics in use on aquatic sensors, their main focus being the immediate surrounds of the sensor. Consequently the focus of the literature in the recent past has shifted to the development of self-cleaning transparent materials. A myriad of techniques have been exploited in this endeavour including lithography , plasma etching [56, 57], polymer based processes and nanoparticle self-assembly [58, 59, 60] . The sol–gel fabrication approach of transparent self-cleaning materials has emerged as an attractive candidate for creating low fouling surfaces due to the unique structure and properties of silica-based coatings and of hybrid inorganic–organic silica in particular. This involves the formation of an oxide network via the hydrolysis of an appropriate precursor or colloidal dispersion and its consequent polycondensation reactions within a suitable solvent. The kinetics of which determine the pore size or roughness of the sol, and this in turn can be optimised through careful selection of precursor [61, 62, 63] and catalyst (specifically pH additives and curing temperature). Superhydrophobic (SH) surfaces have often been fabricated using silicate-based sol-gels [64, 65, 66]. Their popularity stems from their well-known chemistry, multiple precursors, the robustness of the silica network they produce and the volume of –OH sites at their surface, which allows for both further modification and adhesion (via covalent bonds) to a substrate. Advantages of sol–gel formulations include their good adhesion properties, which allow binding to all types of surfaces used in sensor platforms, such as steel, fiberglass, aluminium, and wood as well as their mechanical strength (and scratch resistance) that affords such coatings prolonged shelf lives which is very attractive for practical applications. Nakajima et al. [67] reported the fabrication of a tetraethyl orthosilicate (TEOS) based transparent SH surface. The authors describe the use of an acrylic polymer to induce phase separation of the precursor before treating with HFDS to render the material hydrophobic. The surface morphology was tuneable with the average diameter of crater-like pores on the surfaces ranging between 29 to 325 nm, depending on the relative polymer to sol wt%. The authors determined that a SH film with 90% transmittance in the visible range was produced with a 1 wt% polymer content. Further examination of the material however showed that prolonged

Biofouling and Biofilm Prevention on Aquatic Sensors

6.11 

exposure to UV light degraded the films [65]. Nakajima and colleagues also used a calcination technique to produce optically clear SH films from boehmite and/or silica containing variable amounts of TiO2. As TiO2 is a photoactive catalyst, this enabled the films to remove any organic stains from their surface in the presence of UV light. Treatment with FAS improved both the films hydrophobicity and transparency (up to 85%) which is initially dependent on the TiO2 concentration [65]. Moreover, the incorporation of organic additives into the sol gel matrix can significantly impact on the resulting materials morphology, for example, Chang et al. describe how the addition of a polyethylene glycol (PEG) to TEOS increased the materials static WCA proportionally as result of the increased concentration of C–O–Si covalent bonds within the silica network [68]. Whereas Shirtcliffe et al. describe how the use of an alternative organosilicate monomer, methyltriethoxysilane (MTEOS), produced a stable SH material that retained its inherent hydrophobicity despite mechanical damage, i.e. cutting/abrading of the film. The material was however opaque as a result of its morphology which featured facets greater than the 100 nm required to maintain optical transparency at visible wavelengths [66]. Similarly Latthe et al., fabricated a semi-transparent silica PMMA composite film that possessed a water contact angle of 159º and self-cleaning capabilities [63]. Enhanced and prolonged chemical and physical stability, ease of application, the waterborne nature of sol-gel coatings and their good performance against biofouling, all support the use of these coatings to efficiently reduce the accumulation of fouling layers on surfaces immersed in the marine environment. Furthermore, sol-gel glassy coatings are transparent and can be effectively applied to optical devices, windows, or solar panels with Hawkins et al., reporting the preparation of three modified silicone antifouling coatings via a sol-gel technique, the efficacy of the coatings in diminishing the accumulation of diatom slime was measured through a series of biofouling tests. A commercial silicone (Silastic T-2) served as a positive standard as its strong affinity to diatoms has been demonstrated including diatom biofouling formation, mixed biofouling formation and sea water microfouling. These settlement tests collectively highlighted the difference in performance of the coatings, which often became more pronounced over time. First, the percentage coverage, average thickness, and biomass of bacteria on the modified coatings were statistically lower than that on the silicone standard. Similarly following prolonged exposure of the coatings to C. closterium, a known fouler of silicone-based coatings, the modified coatings again exhibited statistically lower percentage coverage compared with silicone. Finally, as natural seawater provides simultaneous exposure to bacteria and diatoms, the coatings were also exposed to a mixture of the two where the modified coatings again exhibited reduced mixed biofilm formation against the silicone benchmark, particularly with longer exposure times (up to 23 days). This is consistent with results observed when coatings were exposed individually to bacteria and diatoms. Finally, coated panels were also immersed in the Atlantic Ocean for an extended period of 6 weeks to allow the observation of accumulated brown slime. Slime was noted on the silicone standard but not on the coatings [69]. The field of antifouling protective coatings still has considerable scope for novel expansion despite the recent advances in the fabrication (and understanding) of transparent self-cleaning/antifouling sol gel based materials. This may be attributed to the inherent challenges in maintaining transparency while obtaining the necessary topography for self -cleaning, namely that the surfaces rough features should be sub-wavelength, (preferably < 100 nm) to prevent scatter and the refractive index should be kept low to prevent reflection. Whereas lithographic techniques offer allow precision in surface design and feature size, such topdown techniques are costly and unsuitable for surfaces areas greater than a cm2. Consequently bottom up methodologies are increasing in popularity since they are applicable to multiple substrate types, including soft and non-flat, and significantly larger areas [70, 71, 72, 73]. Sol-gel coatings remain one of the more attractive methodologies of fabrication, offering versatility and simplicity of application and control of the final coatings morphology via optimisation of the systems chemistry. Such antifouling coatings have potential within an enhanced antifouling strategy for sensors via the combination of multiple antifouling approaches like doping of such coatings using biotechnological or

6.12 

Biofilm Control

nanomaterial additives or application in a dual system with alternative antifouling techniques such as UV irradiation [74, 4]. Preparation of novel and specific organic–inorganic composite materials has been attempted in recent years and biotechnology-based strategies are demonstrating increasing promise in particular applications. Among the commonly tested biological strategies for biofilm control are quorums quenching (QQ), enzymatic disruption (ED), energy uncoupling (EU) and cell wall hydrolysis. EPSdegrading enzymes include proteolytic enzymes for protein hydrolysis (e.g. proteinase K, trypsin and subtilisin), polysaccharases for the hydrolysis of polysaccharides (e.g. Dispersin B, Mutanase and dextranase) as well as DNases. Hydrolytic enzymes of cell walls such as the have been used to prevent microbial attachment and could act more specifically than traditional biocides [75]. Their application in sensor technology has been hindered due to their instability in the environment, where the enzymes efficiency would dependent on multiple parameters including pH, temperature and ionic strength of the test medium. Depletion rate and loss of effectiveness over time are also crucial considerations with these coatings (for a comprehensive overview of these technologies, see [76, 77].

ϲ͘ϯ͘ϭEŽǀĞůƐĞŶƐŽƌͲƐƉĞĐŝĨŝĐŶŽŶͲĐŽĂƚŝŶŐĂŶƚŝĨŽƵůŝŶŐĂƉƉƌŽĂĐŚĞƐ Several novel methods of preventing biofouling on sensor components have been patented in recent years. These include ultrasonic methods, methods for local injection of biocides, microbubble generation methods and electrochemical methods [78, 79, 80]. Increased power consumption on autonomous systems is often the main drawback with these approaches, although affecting measurements or simply not sufficiently effective under conditions of high fouling are also possible issues. Local injection of biocides has been attempted but has not yet seen widespread commercial adoption [4]. This appears to be a promising technique that has been utilised in seawater cooling systems (for example [31]), but there are few published studies demonstrating the advantages of this method over other more common antifouling methods, for example copper, in sensor applications. It would be of use to see a direct comparison between the operational field deployment results from field trials comparing leading methods including varying dosage patterns. The ease of operation, effects on rates of corrosion and effect on sensor measurements would all be important here. Microbubble and their use as antifouling are based upon the premise that a “curtain” of bubbles can be utilised to disrupt an attached biofilm and to reduce the likelihood of initial attachment. Commercial systems exist for use in aquaria and research includes ultrasonic generation for use on foul-release coatings [81]. However, for sensing applications, there is still a power consumption issue and bubbles are highly likely to disrupt measurements, particularly optical measurements or dissolved oxygen if air is used as gas for micro-bubble formation. The technique may also be impractical or unpredictable for application at depth or in areas of high flow. The localised injection of biocides shows promise in this area, as does ultrasonic technology in some instances, although few data directly comparing the performance of these techniques with other more conventional approaches appear to be available. With further development, these may indeed have applications in specific environments on sensors.

ϲ͘ϯ͘ϮŽŶƚƌŽůŽĨƐƵƌĨĂĐĞĐŚĞŵŝƐƚƌLJ Physicochemical methods that include alteration of surface and interfacial chemistry are attractive means of producing antifouling surfaces [82, 9, 83]. Many advances in fundamental understanding of what constitutes an antifouling surface have utilised interfacial chemistry and model surface chemistries to understand the adhesion processes occurring at the molecular level in biofilms and biofouling organisms [84, 85, 86]. Key areas of research include the effects of self-assembly and polymer brush-type coatings on biofouling and the related influence of surface energy and wettability on bioadhesion [87, 88, 89], effects of micro/nano topography on organism motility on and at the

Biofouling and Biofilm Prevention on Aquatic Sensors

6.13 

surface, surface settlement and adhesion [90, 91, 92, 93, 94] and nanomaterial-based coatings [95]. Other areas of interest include biomimetic strategies that include dual-approach systems [96, 97, 98]. Amphiphilic compounds, compounds which possess both hydrophobic and lipophilic properties are also of interest, and self-assemble into distinct phases at the nanometer-scale, preventing biomacromolecule adsorption and subsequent organism adhesion and biofilm formation [99, 100, 101]. Novel amphiphilic materials can be synthesized by copolymerising two or more monomers with different polarities and solvent affinities to produce surfaces with differing hydrophilicity, mobility, and topography [102]. Miscibility of the monomers used, and the relative amounts of each, controls the domain sizes, and when these nanometer-scale domains are on the same the length scale of biomacromolecules it is possible to disrupt adhesion mechanism [100]. At present it does not yet seem that such materials have been widely adopted as antifouling coatings for sensors. This could be due to difficulties in scalability of many of the coatings along with adhesion problems to the substrates of interest, lack of robustness, and basic difficulties in finding facile application methodologies (adhesive film-backed materials, sprays etc.). These materials are still under development in the shipping industry however, and thus should be considered as potential future candidate antifouling coatings that may have application in sensor materials and structures.

    Incorporation of nanomaterials into coatings for antifouling purposes has received widespread interest [54]. The use of nanoparticles as (replacement) biocidal agents such as copper, silver, gallium, zinc oxide and titanium dioxides have been of greatest interest [103, 104, 105]. Possible advantages of nanomaterials include reducing the amount of bulk materials required to achieve the same biocidal effect, thus reducing cost and perhaps slowing or reducing environmental release. However, issues with bioavailability and stability when incorporated into coatings and also stability at the requisite pH and salinity ranges of seawater (see for example discussions in [106, 107]) may limit widespread adoption antifouling materials based on nanomaterials for the marine environment, much less for aquatic sensors. Toxicity and release of materials into the environment, always a concern with antifouling materials and coatings, has meant that concerns have also been expressed in regard to the widespread environmental release and uncertain eventual fate of synthetic nanomaterials after use that could result from their incorporation into antifouling formulations [108, 109]. This concern is of course applicable to all antifouling coatings and materials, but there may be specific application areas in sensors where these materials pose less risk due to the small volumes used, and where leaching/release/emission is low. Specific possibilities include targeting membranes, and improved success may be possible where fouling levels are low or consist of specific target species [110]. A possible difficulty with incorporating nanomaterials into optical components is unwanted scattering or absorption at specific wavelengths or bandwidths, or changes in refractive index, effecting sensor measurements.

           Membrane-based sensors, which are numerous in monitoring technologies, are often difficult to protect from biofouling both in aquatic environments [111] and in vivo [112]. For example, pH measurements have been traditionally measured using a glass membrane proton-selective electrode, and the ultrathin glass is prone to biofilm formation which has a highly detrimental impact on such sensors operated in in situ systems. Even pH sensors based on spectroscopic measurements and other methods are difficult to protect from fouling [113]. Dissolved oxygen probes based on Clark polarographical electrodes

6.14 

Biofilm Control

[114] and nutrient sensors [115] which also exploit gas-permeable membrane exhibit similar issues. The fouling of these membranes is a major issue and significant effort has been expended in modification of membrane surfaces, Rana and Matsurra [23] provide a detailed catalogue of membrane surface modification processes currently being exploited in industry specifically in regards to the fouling of filtration membranes is also a serious issue in water treatment and methods used in pressuredriven processes like reverse osmosis (RO), nanofiltration (NF), ultrafiltration (UF), and microfiltration (MF). Hence, membrane fouling and its reduction has been a subject of research and development efforts since the early 1960s when industrial membrane separation processes initially emerged. The authors highlight how the selection of appropriate membrane materials, pre-treatment of the process fluid, adjustment of operating design, and conditions are all known to control fouling to some extent but emphasise that a keen interest from commercial industries in improving the performance of filtration and desalination membranes has led to the development, and on-going optimisation, of processes and methods for improving membrane performance against biofouling [116, 9, 112, 117]. Similarly Xiong and Liu [75] detail the effort which has been expended in the development of numerous biological fouling control methods, these include the inhibition of bacterial quorum sensing systems, nitric oxide-induced biofilm dispersal, enzymatic disruption of extracellular polysaccharides, proteins, and DNA, inhibition of microbial attachment by energy uncoupling, use of cell wall hydrolases, and disruption of biofilm by bacteriophages. Effects of temperature, ionic strength, flow rates and other factors influence the effectiveness of methods such as enzymatic and bacteriophage methods, making these methods difficult to apply in general biofouling scenarios. Consequently as yet, few commercial technologies have been successfully developed using biological methods [118]. Specific technologies in development at present include surface coatings functionalized with biocidal silver nanoparticles (AgNPs) and antifouling polymer brushes via polyelectrolyte layer-bylayer (LBL) self-assembly, which have shown potential in reverse osmosis membrane protection [119, 120]. Polymer functionalization by grafting of polymer brushes, using either hydrophilic poly(sulfobetaine) or low surface energy poly(dimethylsiloxane) (PDMS) has also been utilized and a patent coating composition comprising (i) a curable polyorganosiloxane polyoxyalkylene block copolymer having at least two reactive groups situated on the copolymer chain and (ii) an organosilicon crosslinking agent and/or a catalyst have recently been developed [121]. Blending of membrane components with uniformly dispersed of nano-sized selenium (nSe) and copper (nCu) particles; gallium etc has shown promise for protection of membranes. Akar’s study determined that in the case of nanoparticles blended PES membranes had good antifouling properties with high-rejection rate, compared to neat PES membranes for both protein separation and activated sludge filtration studies (the relative flux reduce of neat PES membrane was 93.8% while the 0.05 Se or Cu blended membranes was decreased to 52.7 and 76.2% respectively).

 Protecting optical components, or indeed producing optical components that are biofouling resistant, would significantly improve current environmental sensing with optical sensors [5, 122]. Copper has been widely used in this regard [2], while other approaches have utilised different biocide-impregnated components to leach biocides close to the surface to be protected. For example, Strahle et al. reported trials of bronzes and porous plastics impregnated with tributyltin oxide surrounding optical lenses of transmissometers and cameras [123]. Different sensor sites, seasons, depths (5 - 30 m) and porosities of impregnated materials were examined, resulting in an general increase in duration of useable data (defined as 60% light transmission) with depth, and in one case, from 33 to 61 days in the summer season and from 57 to 76 days in winter for the protected transmissometer in comparison to an

Biofouling and Biofilm Prevention on Aquatic Sensors

6.15 

unprotected system. This was mainly attributed to a reduction in macrofaunal growth, and was not as effective against algal growth [123]. As discussed previously, most commercially available sensors with optical components (e.g. turbidity, chlorophyll and other pigments, dissolved organic carbon etc.) also utilise mechanical methods (wipers, shutters, brushes) for biofouling removal or reduction at present. However, optical materials with inbuilt antifouling properties offer potential. For example, omniphobic fluorogel elastomers were recently prepared by photocuring perfluorinated acrylates and a perfluoropolyether crosslinker [10]. The antifouling performance of these elastomers was also improved by infusion with fluorinated lubricants (FC-70 or Krytox®), and their optical and mechanical properties can be tuned by controlling the crystalline state of the polymer chains. Such formulations are often targeted at medical devices, rather than for control of marine or riverine fouling. However, there is no barrier in principle to incorporation of such materials into environmental sensors, provided of course that the coatings do not adversely influence sensor measurements and or leach excessive lubricants into the environment, yet at present there does not appear to be any systematic study to demonstrate the effectiveness of these coatings in marine environments or any effect on sensor measurements. Another potential solution to biofouling on optical components of autonomous sensors involves irradiation of components with ultra-violet (UV) radiation [26]. However, UV irradiation is not used widely for autonomous sensors at present and most approaches appear to be implemented individually by sensor manufacturers, although modular universal designs (similar to some mechanical technologies) would appear feasible in some instances. This is likely due to energy consumption of these systems and, until recently, the prohibitive costs or unavailability of LEDs with the requisite wavelengths. Technical improvements in this field are expected in the near future, especially for low power requirement UV sources. Alternatively if low-powered LED technology, incorporating UV LEDs of the required wavelengths, could be utilised it would seem that there would be many advantages to this approach: the sensing components (emitters and detectors for instance) can be readily integrated with the power sources and control electronics of the sensors itself for maximum efficiency and control. A key to this is the availability of low-cost UV-LED based systems that include lower wavelengths (< 280 nm would appear to be optimal [124] for greater effect. Technologies that emit broadband UV light have long been available and utilised, such as deuterium and mercury lamps, which are impractical for usage with environmental optical sensors due to cost, size and power consumption. LEDs are a common solution to integrate light sources into sensors and although this has been relatively easy to achieve in the visible and infrared regions of the spectrum, the cost and lower power output of LEDs in the UV spectral region has until recently prevented this route being explored for antifouling purposes. However, recent advances in technology and a reduction in cost may well improve the adoption of UV LEDs of distinct wavelengths for various antifouling uses [27, 112].

           Slowly dissolving chlorine (trichlorisocyanuric acid) and bromine tablets have been utilized in closed optical systems for preventing biofouling [125]. Localized generation of biocides, and particularly local chlorination, have also been tested for antifouling effect on sensors [4] and in power plant cooling water applications [126]. This in situ generation of biocides has focused on the production of chlorine species or haloamines that effectively reduce inhibit and/or control the settlement, growth and proliferation of microorganisms that cause biofouling. For example, U.S. Pat. No. 5,976,386 and U.S. Pat. No. 6,132,628 disclose the preparation of haloamine biocides from hypochlorite and various ammonium salts for use in treating liquids to inhibit the growth of microorganisms. The active ingredients previously used for this purpose include active halogen species, such as hypohalites, which are often strong, corrosive oxidants, which makes them both difficult and dangerous to handle,

6.16 

Biofilm Control

especially in large quantities. Furthermore, these species may degrade over time, for example resulting in a continually decreasing level of active halogen concentration resulting in lowered potency of the material. Furthermore, the formation of bromamine produced by the methods requires the availability of defined concentration ratios between the two reactants, and strict pH control, with specialized monitoring equipment. These conditions make such technologies difficult to implement on sensor assemblies and little information is available at present on effectiveness in the context of aquatic sensors. Other proposed antifouling methods consist of a conductive layer of carbon fiber, graphite powder and binder as an anode, a cathode, and a power source thereby performing an electrolytic reaction to generate antifouling species (see for example US patent US6514401 B2). This design is comprised of an electrolytic cell consisting of membrane, saltwater as an electrolyte and a counter electrode that can be inserted in the saline feed solution. By periodic generation of gases (e.g., Cl2 and O2) fouling of the membrane surface can be controlled. Other proposed methods for oxygen membranes include epoxy polymers containing phosphorylcholine groups, created by copolymerization of glycidyl methacrylate and 2-methacryloyloxyethyl phosphorylcholine (MPC) [127]. The cured polymer films are reported to have excellent adhesion properties and are transparent at the wavelength of more than 300 nm, thus not interfering with sensor function. The cross-linking reaction of the polymer films increased with increasing amount of MPC units in the copolymer, and the addition of an aliphatic diamine significantly enhanced the cross-linking reaction of the copolymer films. The resulting films were tested in bioadhesion assays against E. coli JM109, where it was reported that the number of adhered E. coli adhered to the films reached a maximum at an incubation time of 6 h, and then gradually decreased, showing an almost 10-fold reduction to cells compared to glass. However, there are no data on performance against other species, and it would be beneficial to see a systematic evaluation of such coatings against marine organisms or marine performance trials on operation sensors in general. In summary, although electrochemical methods and application of films to sensors described here appear promising, yet again few published studies are available that have systematically evaluated the performance of the described materials and methods in field trials, or indeed against selected marine organisms (diatoms, barnacle cyprids for example) in laboratory assays, so few conclusions can be draw at this time regarding their feasibility in practical applications.

        Bio-inspired design is a broad research field where natural solutions evolved by organisms are studied as inspiration for novel synthetic materials or technologies. The eye, and the mechanisms involved in keeping the eyes of marine creatures clean, is an example where specialised mechanisms of antifouling have been developed by nature which can serve as inspiration for engineered synthetic materials [91]. Some organisms, in marine environments in particular, appear to have defence mechanisms against biofilm formation [128, 129]. Organisms studied for this purpose have included algal groups like kelp, crustaceans, sharks, crabs and bivalves [128]. However, the precise mechanisms by which these organisms reduce biofouling are often unclear and it is furthermore unclear how much many of the present antifouling technologies have a biological analogue, despite the speculated advantages of potentially useful and novel chemical and mechanical method devised marine species in particular. The role of surface topography (surface relief) has been widely considered in antifouling studies, since many marine organisms have specialised and intricately patterned surfaces. Surface topography and texture are now known to act as cues for biofouling on surfaces immersed in aquatic environment [130]. In recent years the ability to create artificial surfaces with precisely patterned micrometre and nanometre scale topography for testing against biofouling species in laboratory experiments has led to increased understanding of the impacts of surface topography on both isolated cells, and on biofilm formation and retention [131, 132]. This has led to the realisation of the involvement of surface

Biofouling and Biofilm Prevention on Aquatic Sensors

6.17 

properties like surface roughness and interfacial energies influence the attachment strength of marine biofouling organisms [131]. Attachment of a fouling organism to a textured surface can be related to triggering of specific settlement cues when the settling organism encounters surface features of some yet unspecified critical dimension. The effects of texture on recruitment and adhesion in this manner have been reported for barnacle cyprids of Balanus amphitrite (or Amphibalanus amphitrite) and Amphibalanus improvisus [133]. The ability of cyprids to actively choose surfaces with textures that increase the likelihood of the adult barnacle remaining attached to the chosen surface is of particular interest. Some response to microtopographic cues has now been established for a number of species from many of the major fouling groups, including algal zoospores, specific diatom species (Amphora sp, Navicula jeffreyi and Fallacia carpentariae among others) and barnacle cyprid settlement [134].

       While testing precisely engineered surface topography against the responses of key fouling organisms has led to a number of insights into settlement patterns, it is not clear if surface texture alone can be utilised in a practical manner to prevent biofouling on sensor components. Instead, it appears more likely that keeping roughness and texture within a suitable range many form a part of an overall antifouling approach. While there is strong scientific and anecdotal evidence on influence of surface topography on settlement, in marine and estuarine environments (Figure 4), there appears to be no clear consensus among researchers involved in antifouling or sensor manufacturers on the optimal surface topography configuration for best practical application. Retention of long-term antifouling performance on materials with specific nano/micro scale topographies under field conditions may also prove challenging as initial anti-settlement effects may diminish with time. Although control of surface roughness is common in the shipping industry, where specific guidelines are available hull roughness (and the requisite analysers), this appears to be more related reducing frictional resistance associated with drag reduction. Since most environmental sensor installations are moored (static) it remains unclear how much the onset of biofouling is affected by the surface finish applied or achieved on the materials of which the sensor is constructed, prior to deployment.

Figure 4: Photograph of initial barnacle fouling that began along the boundary between a manufacturer’s label and the PVC housing of a sensor sonde. It is unclear whether the initial colonising barnacle cyprids utilised the surface features as a cue for settlement or else were better protected from shear forces during initial settlement processes, nevertheless the authors have observed that initial settlement often begins along such surface features and proliferate from there.

6.18 

Biofilm Control

 Biofouling is a key barrier to long-term deployment of aquatic sensors. Tackling biofouling on these sensors is key challenge facing the environmental sensor industry and it represents a hurdle to largescale deployment and improvements in data quality. Larger markets for coatings and requirements of the shipping industry direct antifouling research at present, and antifouling methods for sensors generally still consist of established technologies like mechanical and copper-based methods. These methods often suffer from drawback in terms of power consumption, scalability, and long-term effectiveness, and novel antifouling materials are required for new sensors, for example microfluidicsbased sensor systems. Nevertheless, the sensor market offers a niche for testing several novel antifouling methods / technologies which may not meet the scales required of the shipping industry or applications not involving the protection of large surface areas. Nanomaterial-based coatings, lubricated polymer coatings or special dispensation to produce natural-product or enzyme-based coatings may also provide an opportunity. However, any approaches based on release of active ingredients from antifouling coatings will likely require specific regulatory approval (if not at present then in future). However, given the importance of aquatic sensors in understanding our environment and the relatively small surface areas involved, perhaps a debate about the cost-benefit of allowing specific antifouling technologies on sensors is required. Ultimately, effective long-term protection of complex, largely static, and unattended instruments like underwater sensors from biofouling will likely require multiple strategies integrated into one system, for example specific coatings / surface modifications of optical and membrane components, combined with broad spectrum biocides (perhaps copper if permissible) to reduce or kill fouling organisms, while mechanical methods remove inorganic / non-biological fouling and dead organisms. Without any sweeping changes in technology, all of these components still require optimisation for this task, and extensive further development in many cases, particularly with regard to specialised coatings for application to optical ports.

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Biofilm Control

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101.

Magin CM, Cooper SP, Brennan AB (2010). Non-Toxic Antifouling Strategies. Materials Today, 13(4), 36–44.

102.

Gudipati CS, Greenlief CM, Johnson JA, Prayongpan P, Wooley KL (2004). Hyperbranched Fluoropolymer and Linear Poly(ethylene Glycol) Based Amphiphilic Crosslinked Networks as Efficient Antifouling Coatings: An Insight into the Surface Compositions, Topographies, and Morphologies. Journal of Polymer Science Part A: Polymer Chemistry, 42(24), 6193–6208.

103.

Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ (2002). Metal Oxide Nanoparticles as Bactericidal Agents. Langmuir, 18(17), 6679–86.

104.

Jiang H, Manolache S, Wong AC, Denes FS (2004). Plasma-Enhanced Deposition of Silver Nanoparticles onto Polymer and Metal Surfaces for the Generation of Antimicrobial Characteristics. Journal of Applied Polymer Science, 93(3), 1411–22.

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Yong, Hong Eui, Karthikeyan Krishnamoorthy, Kim Tae Hyun, and Sang Jae Kim. (2015). Preparation of ZnO Nanopaint for Marine Antifouling Applications. Journal of Industrial and Engineering Chemistry, 29, 39–42. doi:10.1016/j.jiec.2015.04.020.

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Bour A, Mouchet F, Silvestre J, Gauthier L, Pinelli E (2015). Environmentally relevant approaches to assess nanoparticles ecotoxicity: A review. Journal of hazardous materials, 11(283), 764–77.

107.

Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao AJ, Quigg A, Santschi PH, Sigg L (2008). Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology, 17(5), 372–86.

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Lovern SB, Strickler JR, Klaper R (2007). Behavioral and Physiological Changes in Daphnia Magna When Exposed to Nanoparticle Suspensions (titanium Dioxide, Nano-C60, and C60HxC70Hx). Environmental Science & Technology, 41(12), 4465–70.

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Miao AJ, Schwehr KA, Xu C, Zhang SJ, Luo Z, Quigg A, Santschi PH (2009). The Algal Toxicity of Silver Engineered Nanoparticles and Detoxification by Exopolymeric Substances. Environmental Pollution, 157(11), 3034–41.

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Inbakandan, D, C Kumar, L Stanley Abraham, R Kirubagaran, R Venkatesan, and S Ajmal Khan. (2013). Silver Nanoparticles with Anti Microfouling Effect: A Study against Marine Biofilm Forming Bacteria. Colloids and Surfaces B: Biointerfaces, 111, 636–43.

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Chapter–7

ENVIRONMENTALLY BENIGN MARINE ANTIFOULING COATINGS SitaramanKrishnan ClarksonUniversity,DepartmentofChemicalandBiomolecularEngineering, 8ClarksonAvenue,Potsdam,NewYork,13699,USA Email:[email protected]

ABSTRACT The formation of biofilms of marine organisms on the surfaces of ship hulls, rudders, and propellers,heatexchangersincoolingwatersystems,watertreatmentmembranes,aquatic sensors, and other equipment in contact with the marine environment is a challenging problem, associated with not only increased operation and maintenance costs, but also damage and loss of functionality caused by microbially induced corrosion. Surface modification using coatings is an approach that has been widely used to control marine biofilm formation. Until recently, marine antifouling coatings incorporated biocides that killed the microorganisms, and consequently had an unfavorable effect on the marine ecosystem. Over the past two decades, however, several environmentally friendly nanostructured polymer coatings have been developed as promising alternatives to the biocidalpaints.Beginningwithabriefdiscussionoftheenvironmentalproblemsarisingfrom theuseofbiocides,thischapterexaminesthemacromolecularstructuraldesignandsurface science strategies used in the development of environmentally friendly nontoxic coatings. Fouling release coatings of block copolymers with fluorinated liquid crystalline side chains, siliconecoatingsfilledwithnanoparticles,hydrophilicantifoulingcoatings,amphiphilicblock copolymer coatings, and coatings with nanoscale surface heterogeneities are discussed, alongwithadetailedspecificationoftheirsurfaceproperties,probedusingtechniquessuch as surface wettability analysis, nearͲedge XͲray absorption spectroscopy, and atomic force microscopy.Somebiomimeticapproaches,suchastheincorporationofmicroorganismsthat producefoulingͲdeterrentsecondarymetabolites,arealsohighlighted.

7.1 INTRODUCTION Marine biofouling is the undesired attachment of microorganisms, seaweeds, and invertebrate animals on the surfaces of ship hulls and other artificial structures immersed in aquatic environments [1]. The corrosion and degradation of surfaces of ships by fouling organisms is a problem that is as old as the maritime industry. Until around mid-1800, timber planks were primarily used in ship construction. Wax, tar, and asphalt were probably the materials used by ancient mariners to protect the part of the

7.2  Biofilm Control  hull below the waterline from decay [2]. In the context of the Indian subcontinent, an intense sea-route trade between the Roman Empire and Damirica (the ancient Tamil country) is mentioned in a firstcentury text called the Periplus of the Erythraean Sea [3, 4]. Trade between mariners of the Chola kingdom, particularly during the reign of Rajendra Chola I (1012–1047 CE), and China and Southeast Asia is well-documented. Not much information is available on the materials used in the construction of these merchant vessels. However, metal-sheathed hulls are mentioned in the eleventh-century Sanskrit text, Yukti Kalpataru [5]. The timber planks used for building the ships were also smeared with a synthetic “pitch” [6], hydrophobic in nature, for preserving the wood. The preparation of one such pitch involved heating a mixture of two parts of dammar (a triterpenoid resin) with one part of oil (extracted from fish) [7]. Another coating composition prevalent in India, consisted of plant oil mixed with hemp and lime, as reported by the thirteenth century Venetian seafaring trader, Marco Polo [8]. Lime was used as protection for wood against certain sea worms that were notorious for causing damage to the ship hulls in the gulf of Khambat, and was applied at least once a year [8]. In the late 1800s, as timber supply became scarce in Europe, particularly in England, iron began to replace wood in ship construction. Anderson et al. [9] have discussed the problem of galvanic corrosion when the iron ships were cladded with copper sheathing (that was previously successful in not only protecting wooden hulls from teredo worm, but also keeping fouling at bay). The solution of the problem required the assistance of Sir Humphry Davy, and involved cladding the iron ship hull with wood and then fixing the copper sheets to the wood, to avoid contact of the two metals. Sir Humphry Davy also demonstrated that copper dissolution in sea water prevented biofouling [2].

7.1.1 TributyltinSelfͲPolishingCoatings A major milestone in the history of modern marine antifouling coatings was the introduction of the “self-polishing” copolymer paints in the early 1970s, comprising of the tributyltin (TBT) biocides [10]. These paints were acrylic or methacrylic copolymers in which 50 to 80 percent by weight of the copolymer consisted of triorganotin salt (ester) of acrylic or methacrylic acid. The self-polishing effect in these coatings was by erosion, caused by hydrolysis of the polymer in the coating by moving seawater [11]. The antifouling action is through the release of biocides into the seawater in the vicinity of the eroding polymer front. These biocidal tributyltin based coatings were highly effective in solving the marine biofouling problem. Coatings with thicknesses that were optimized in relation to the ablation rate could ensure foulant-free hulls for up to five years [9]. However, environmental problems associated with biocidal TBT coatings began to appear as early as the 1980s. By 1987, most of the European countries had restricted the use of these coatings [12]. The International Maritime Organization’s Convention on the Control of Harmful Antifouling Systems on Ships came into force in September 2008, and stated that “all ships shall not apply or re-apply organotin compounds, which act as biocides in antifouling systems”. As of 2009, the convention was ratified by 28 nations, representing about 44 % of the world fleet [13]. Other non-signatory nations have regional regulations restricting the use of TBT-based paints.

7.1.2 TinͲfreeBiocidalCoatings Tin-free self-polishing copolymers based on copper and zinc have been considered [11]. Several marine antifouling paints currently incorporate pigments such as cuprous oxide that can react with seawater to produce biocidal ions (Cu2+). Cuprous thiocyanate and zinc oxide have also been used [2]. Because certain microfoulers such as algae are tolerant (resistant) to these coatings, secondary or booster biocides are added [14]. The co-biocides include zinc and copper pyrithione (Omadine), zinc dimethyldithiocarbamate (ziram), zinc ethylene bis(dithiocarbamate) polymer (zineb), Nƍ-tert-butyl-

Environmentally Benign Marine Antifouling Coatings



7.3 

N-cyclopropyl-6-(methylthio)-1,3,5-triazine-2,4-diamine (Irgarol 1051), 3-(3,4-diclorophenyl)-1,1dimethylurea (DCMU, Diuron), 2,3,5,6-tetrachloro-4-(methylsulfonyl)pyridine (TCMS pyridine), 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil), N-dichlorofluoromethylthio-Nƍ,Nƍ-dimethyl-Nphenylsulfamide (dichlofluanid), 4,5-dichloro-2-n-octyl-4-isothiazolino-3-one (Sea-Nine 211), triphenylborane pyridine, 8-methyl-N-vanillyl-6-nonenamide (capsaicin), and (RS)-4-[1-(2,3-dimethylphenyl)ethyl]-3H-imidazole (medetomidine) [2, 14, 15]. Of these compounds, Sea-Nine 211 and Zinc Omadine are approved by the US Environmental Protection Agency (EPA) for use in marine paints [16]. Nevertheless, due to concerns regarding adverse effects of biocides on non-target organisms [15], and their toxicity and persistence in the aquatic environment [12, 17], it is clear that “greener” non-biocidal antifouling coatings will be the materials of choice in the future.

7.1.3 CuAccumulationandToxicity:NeedforNonͲBiocidalCoatings The 2016 draft criteria of the United States EPA for dissolved copper in estuarine or marine waters [18] recommends that the Cu concentrations should not exceed the acute and chronic toxicity levels of 2.0 Pg/L and 1.3 Pg/L, respectively. (These values are based on a temperature of 22 °C, a pH of 8, a dissolved organic carbon concentration of 1.0 mg/L, and a salinity of 32 parts per thousand. Concentration limits for other conditions of pH, temperature, salinity, and DOC are estimated using a “saltwater biotic ligand model”). Elevated copper concentrations, of up to 6 Pg/L of dissolved Cu and up to 10 pM of the toxic non-chelated free Cu2+, at sites with boats, in three San Diego Bay marinas (California, USA), were found to result in copper bioaccumulation in tissues of several macrobenthic species [19]. Copper accumulation in the marine environment, combined with ocean acidification due to increasing atmospheric CO2, could be an impending environmental problem, as already manifested in the form of increased copper toxicity in mussels [20]. Antifouling strategies that do not use any biocides are urgently needed. The development of such environmentally friendly alternatives is of particular relevance to India, whose large coastal population relies on fisheries production and export for food security and livelihood. Heavy metal contamination of the coastal waters of India, and in the coastal fishes, is becoming evident and is of concern [21, 22]. Numerous reviews and edited books have been published, in recent years, on the topic of environmentally friendly marine antifouling coatings [1, 2, 9, 23–41]. A goal of this chapter is to highlight some of the main classes of biocide-free coatings designed to prevent marine biofouling, with an emphasis on understanding how the surface chemistry of these coatings imparts resistance to biofouling. The focus will be on molecular properties of the surface and how these properties influence cell adhesion. A brief introduction of the mechanism of biofouling is followed by a discussion of hydrophobic fouling release coatings, hydrophilic non-biocidal antifouling coatings, amphiphilic block copolymer coatings, and coatings with nanoscale surface heterogeneities.

7.2 SETTLEMENTANDADHESIONOFMARINEFOULINGORGANISMS The first step in marine biofouling of a clean surface that is immersed in seawater is the formation of a conditioning film consisting of dissolved organic molecules such as carbohydrates and proteins. This is followed by colonization of the surface by a wide range of marine organisms. Figure 7.1 shows the diversity in type and size of marine fouling species. Unicellular organisms such as bacteria, diatoms, and protozoa form a complex biofilm consisting of organized communities of mixed microorganisms surrounded by a matrix of extracellular polymeric substances (EPS) [42]. These biofilms are referred to as “microfouling” or “slime”. Macroscopically visible algae (seaweeds) and invertebrates such as soft corals, sponges, anemones, tunicates, and hydroids are called “soft macrofouling”, and shelled

7.4 

Biofilm Control

Figure 7.1 Diversity and size scales of a representative fouling organisms. Reprinted with permission from ref. [1]; Copyright 2011 Macmillan Publishers Limited.

invertebrates such as barnacles, mussels, and tubeworms are called “hard macrofouling” [1]. Bacteria, motile spores of seaweeds, and certain larvae of invertebrates can all simultaneously colonize the surface, which results in a major challenge in creating a fouling-resistant coating that can resist fouling by organisms with a range of adhesion mechanisms (including the chemical composition of the EPS adhesive) and a range of sizes of the sensing apparatus that they use to select a surface for attachment [1]. Spores of the green alga (seaweed) Ulva use their apical papilla and barnacle cypris larva use a pair of sensory antennules to probe the surface. When a silicon surface coated with alternating stripes of hydrophobic 1H,1H,2H,2H-perfluorooctyl-trichlorosilane and hydrophilic trimethoxysilylated poly(ethylene glycol) was exposed to Ulva zoospores (spores capable of swimming using flagella), the spores settled at higher densities on the fluorinated stripes compared with the PEGylated stripes, showing a clear preference for settlement on hydrophobic surfaces (see Figure 7.2). Stripes of different widths were studied. The spores were able to differentiate between the hydrophobic and hydrophilic regions when the pattern size was larger than 20 Pm, but were unable to do so when the features were smaller than 5 Pm [43]. The marine bacterium Cobetia marina (syn. Deleya marina) shows a similar preference for settlement on hydrophobic surfaces [44–46]. After the organism has settled on a surface, whether it can remain attached to the surface and grow into a reproductive adult depends on how well the organism’s macromolecular adhesive is bonded to the surface, which is determined by the intermolecular forces of interaction between the adhesive plaque and the surface.

7.3 FOULINGRELEASE(FR)COATINGS “Fouling release” coatings are an important class of biocide-free coatings that function on the basis of weak intermolecular forces of interaction between the bioadhesive and the coating surface. These

Environmentally Benign Marine Antifouling Coatings

7.5 



Figure 7.2 Autofluorescence images of settled spores on regions of silicon wafer patterned with hydrophobic FOTS and hydrophilic PEG stripes. FOTS and PEG represent coatings of F(CF2)6CH2CH2SiCl3 and CH3O(CH2CH2O)6–9(CH2)3Si(OCH3)3, respectively. The dotted lines indicating the boundaries between PEGylated and fluorinated areas are provided to assist interpretation. Reprinted with permission from ref. [43]; Copyright 2008 American Chemical Society.

coatings do not prevent organisms from attaching, but the adhesion strength is weakened so that the attached organisms are more easily removed by the hydrodynamic shear forces generated by the relative movement of the surface in water. Using Kendall’s model, Chaudhury et al. [47] have discussed that the pull-off force, ‫ܨ‬ǡ required to remove a hard object attached to a thick elastomeric surface is proportional to ሺܹ‫ܧ‬ሻଵȀଶ , where ܹ is the work of adhesion and ‫ ܧ‬is the Young’s modulus of the surface. When the surface is nonpolar, the work of adhesion, ܹǡ between the coating and the bioadhesive is ଵȀଶ

given by ʹ൫J௦ Jௗ௟ ൯ , where J௦ is the surface energy of the coating and Jௗ௟ is the dispersive component of the surface energy of the liquid adhesive secreted by the marine organism [23]. Here, we have used the Dupré equation, ܹ ൌ J௦ ൅ J௟ െ J௦௟ ǡ for the work of adhesion, ܹ, in terms of the surface energies, ଵȀଶ

J௦ and J௟ , and the interfacial energy, J௦௟ , and the Fowkes-approximation, J௦௟ ൎ J௦ ൅ J௟ െ ʹ൫J௦ Jௗ௟ ൯ , for interfacial energy between two surfaces that adhere purely by dispersive intermolecular interactions. Thus, a 0.5 order of dependence of ‫ ܨ‬on the coating modulus and a 0.25 order of dependence on the coating surface energy is indicated by this analysis, which calls for soft (compliant) low surface-energy coatings to achieve easy fouling release. (Note that the order of dependence on the coating surface energy, based on this analysis, is lower than the commonly assumed value of 0.5 [9]). The surface energy (surface tension) values of some common liquids and polymer surfaces are compiled in Table 7.1. It is seen that, among the polymers listed, poly(tetrafluoroethylene) (PTFE) and poly(dimethylsiloxane) (PDMS) have the lowest surface energy values. PTFE is a highly crystalline polymer, with a melting temperature of about ͵ʹ͹Ԩ , and is, therefore, difficult to process by conventional techniques. Moreover, PTFE has a relatively high Young’s modulus at room temperature (؆ ͶͲͲƒሻ. These properties make the PTFE homopolymer unsuitable for practical application as marine antifouling coatings. In contrast, PDMS, a polymer with low glass transition temperature (approximately െͳʹͳԨ) is soft at room temperature, and its surface is nonpolar, with a relatively low surface energy of about 

7.6  

Biofilm Control

ʹͳ Ȁଶ . This polymer was, therefore, among the earliest successfully used nontoxic fouling release coating for ship hulls. PDMS-based coatings are crosslinked elastomers, generally obtained by the reaction of hydroxyl-terminated PDMS oligomers with tetraalkoxysilane crosslinking agents. Low-temperature vulcanization via the hydrosilylation reaction on vinyl-terminated PDMS is also possible. After 10 years in service, a crosslinked PDMS elastomer (PDMSe) applied on a Tropic Lure ship hull was found to be relatively clean, without macroalgae or barnacle adhesion, when simply maintained by high pressure washing and repair to damaged areas [9]. Some commercial silicone coatings contain small amounts of non-bonded silicone oils, such as methyl phenyl silicone, to enhance foul-release properties of the coating. It was found that these oils remained in the coating, without leaching into seawater [9]. Although PTFE is not a marine antifouling coating per se, a variety of fluoropolymer coatings making use of the low surface energy of fluoroalkyl groups have been developed. In a study comparing marine antifouling properties of two Intersleek non-biocidal silicone and fluorpolymer-modified silicone coatings (IS700 and IS900, respectively), the fluorinated coating was found to be more effective in short-term (10-day) field experiments. For a nonpolar surface interacting with water, the surface energy, J௦ , is related to the water contact angle, ߠ௪ , by ߛ௦ ൌ ͲǤʹͷߛ௪ଶ ሺͳ ൅ …‘• ߠ௪ ሻଶ ȀJௗ௪ , where J௪ and Jௗ௪ are the total surface energy of water (͹͵ Ȁଶ ) and the dispersion component of its surface energy (ʹʹ Ȁଶ ), respectively. From this equation, it is evident that surfaces with lower water contact angle values have higher surface energies. TABLE 7.1. Surface Energy of Some Common Liquids and Polymers at Room Temperature[a] Material

Chemical structure

Surface energy J (mJ/m2)

n-Perfluorohexane [49]

CF 3 (CF 2 ) 4 CF 3

12

n-Perfluorononane [49]

CF 3 (CF 2 ) 7 CF 3

15

n-Hexane

CH 3 (CH 2 ) 4 CH 3

18

Hexadecane

CH 3 (CH 2 ) 1 4 CH 3

28

Water

H2O

73

Poly(tetrafluoroethylene) (PTFE)

19

Poly(dimethylsiloxane) (PDMS)

21

C H3 Poly(n-hexyl methacrylate)

C H2 C O

[a]

C

n

O

Data compiled from references [50] and [51], and other sources.

30

7.7 

Environmentally Benign Marine Antifouling Coatings TABLE 7.1

(Continued)

Material

Chemical structure

Surface energy J (mJ/m2)

Poly(n-butyl methacrylate)

31

Polyethylene

32

Poly(n-butyl acrylate)

34

Poly(2,3,4,5,6-pentafluorostyrene)

35[b]

Poly(methyl methacrylate)

40

Polystyrene (PS)

41

Poly(ethylene oxide) (PEO)

43

Poly(4-vinyl pyridine) (P4VP)

47[c]

[b]

based on solubility parameter; a value of 33 r 3 mJ/m2 determined using contact angle measurements, is found experimentally [52] [c] based on molar parachor and solubility parameter predictions; a value of 68.2 mJ/m2 is reported in ref. [53]

The static water contact angle on IS900 is only about 76° [48]. In contrast, IS700 has a hydrophobi c surface with a water contact angle of about 99°. On the basis of this fact, IS900 would have a higher

7.8  Biofilm Control  surface energy than IS700. The superior FR performance of IS700 cannot be because of a lower J௦ , but probably due to nanoscale surface heterogeneities and/or an amphiphilic nature (which will be discussed separately in a following section). Among the early fluorinated coatings designed for marine antifouling application was the one in which reactive perfluoroalkyl polymeric surfactants were crosslinked with poly(2-isopropenyl-2oxazoline) [54]. The goal was to prepare a purely hydrophobic coating. But the advancing and receding water contact angles on these coatings were relatively low (a maximum of about 94° and 53°, respectively) due to the presence of polar functional groups at the surface. Surface reconstruction, defined as the rearrangement of chemical groups at the interface of the coating with water for the lowering of interfacial energy, is a problem when hydrophobic coatings are immersed in water. Polar groups tend to migrate to the polymer–water interface, resulting in a loss of the nonpolar character of the surface and, therefore, its “non-stick” properties. The coatings of Schmidt et al. [54], and other fluorinated polyurethane coatings discussed in ref. [23], relied on crosslinks as barriers to hinder surface reconstruction.

7.3.1 FRCoatingswithFluorinatedLiquidCrystallineSideChains Ober and coworkers [55] used the self-assembly of liquid crystalline (LC) perfluoroalkyl mesogens to design block copolymer coatings that were resistant to surface reconstruction. The block copolymers consisted of a polystyrene block and a fluorinated block, in which the perfluoroalkyl groups were attached to the polymer backbone through flexible alkyl spacers. Figure 7.3(a) shows the chemical structure of a representative block copolymer that consisted of perfluorodecyl mesogen in the side chain, connected to the polymer backbone by a nine carbon atom long alkyl spacer. A schematic of self-assembly of the mesogens is shown in Figure 7.3(b).

smectic layer ofrigid fluoroalkyl groups

flexible alkyl spacer

185

95

(a)

(b)

Figure 7.3 (a) Comblike block copolymer with hydrophobic LC semifluorinated side-chains. (b) Liquid crystalline self-assembly of the semifluorinated side-chains to form a non-reconstructing hydrophobic surface under water.

Environmentally Benign Marine Antifouling Coatings



7.9 

The synthesis and surface properties of these block copolymers (with different lengths of perfluoroalkyl mesogens and alkyl spacers) are reviewed in ref. [23]. The rigid perfluoroalkyl groups formed a smectic LC phase in the bulk, and also at the surface of the coating. The melting point (clearing temperature) of the smectic mesophase ranged from about 20 to 100 °C depending on the lengths of the fluoroalky mesogen and alkyl spacer in the side chains. Thus, when the mesogen and spacer lengths were designed to have a melting temperature of the smectic phase that was significantly higher than room temperature, underwater surface reconstruction could be prevented. Advancing and receding water contact angles as high as 123° and 112°, and a surface energy as low as 8 mJ/m2, could be achieved using this approach. Note that the surface energy of these polymers is lower than even that of perfluorohexane or PTFE (cf. Table 7.1) because of complete coverage of the surface with –CF3 groups, caused by LC self-assembly. Indeed, the contribution of different groups to the lowering of surface energy decreases in the order –CF3 > –CF2– > –CH3 > –CH2–. The –CF3 group with a hemispherical volume of 42.6 Å3, which is significantly higher than hemispherical volume of the–CH3 group (16.8 Å3) [56], shields the surface better and minimizes the interaction of water with any underlying polar groups. The fluorinated block copolymer of Figure 7.3(a) was soluble in organic solvents, and could be processed using techniques such as spin coating and spray coating. Figure 7.4(a) shows a tappingmode atomic force microscopy (AFM) phase image of a coating prepared by spraying. The AFM image is consistent with X-ray scattering results that showed the formation of cylindrical nanostructures (standing up perpendicular to the substrate) by microphase separation of the polystyrene and the fluorinated blocks [see Figure 7.4(b)]. Grazing incidence X-ray scattering studies (GISAXS) indicated self-assembly over three different length scales: microphase separation of the two blocks, smectic layer formation of the fluorinated mesogens, and hexagonal packing of the mesogens within a smectic layer [57]. The smectic layers in these coatings spontaneously self-assembled to be predominantly parallel to the coating surface, as desired. We evaluated the antifouling properties of the fluorinated block copolymer coatings for adhesion strength of Ulva sporelings [Figure 7.1(j)] and Navicula diatoms [Figure 7.1(d)], and found that these coatings resulted in only a weak adhesion of Ulva, but a strong adhesion of the diatoms [58]. Adhesion of diatoms is a long outstanding problem for most non-biocidal hydrophobic surfaces, including those of PDMSe. The green alga Ulva is resistant to copper-based antifouling paints [9] and diatom biofilms are difficult to remove from silicone coatings [31]. Therefore, new coatings that are resistant to fouling by both of these organisms are required.

Figure 7.4 (a) Tapping-mode AFM phase image of a spray-coated surface of LC fluorinated block copolymer with a chemical structure shown in Figure 7.3. (b) Model of self-assembly in block copolymer with LC semifluorinated side chains [57,58].

7.10 

Biofilm Control

Using a bilayer coating approach, the coating surface energy, ߛ௦ , and modulus, ‫ ܧ‬, could be independently varied. The modulus of the coating was controlled using a poly[styrene-block-(ethyleneran-butylene)-block-styrene] (SEBS) triblock copolymer as the base layer [58]. The Young’s modulus of the base layer depended on the weight fraction of the styrene block in SEBS. It was 18 MPa for Kraton G1652 and about an order of magnitude lower for Kraton MD6945 (1.2 MPa). A significantly improved fouling release behavior was observed when the softer (compliant) SEBS was used as the base layer [59]. Note that commercial PDMSe coatings such as RTV11 and Intersleek have a modulus in the range of 1 to 3 MPa. The use of SEBS as the base layer also solved the problem of adhesion of the “non-stick” coatings to surfaces of interest. SEBS, grafted with maleic anhydride (MA) groups, adhered strongly to glass and metals, through the reaction of MA groups with the hydroxyl groups on the substrate. In addition, the fluorinated block copolymer [Figure 7.3(a)] adhered well to the SEBS base layer, due to the presence of a common polystyrene block. In this manner, a variety of coatings [58–63] remained welladhered to the substrates, and could be tested in a turbulent flow channel at high flow rates, without the coatings peeling off from the substrates [64]. Besides modulus and surface energy, the thickness of the coating also plays an important role in fouling release performance. Theoretically, when the contact radius of an object with a coating is significantly higher than the coating thickness, the pull-off force is proportional to ሺܹ‫ܧ‬Ȁ݄ሻଵȀଶ , where ݄ is the thickness of the coating [47]. (In contrast, when the coating is significantly thicker than the size of the adhering object, there is no thickness dependence of contact force). Accordingly, Wendt et al. [65] found that the critical stress required for removing barnacles adhered to PDMSe coatings of different thicknesses (0.1 mm, 0.5 mm and 2 mm), decreased with an increase in the coating thickness.

7.3.2 DevelopmentsinSiliconeFoulingReleaseCoatings Polyureas and Polyurethanes Oil-filled PDMS elastomer coatings suffer from the problems of poor bonding to a substrate because of their low surface energy, and easy damage because of their low surface hardness. PDMS based polyurea (containing the –NHC(=O)NH– urea linkages) [66] and polyurethanes (containing the –NHC(=O)O– urethane linkages) [67], were synthesized to improve adhesion to the substrate with the help of hydrogen bonding interactions of the polar urea or urethane groups. In the case of the PDMSbased polyurea coatings, the composition containing 41 wt % of the hard polyurea segment resulted in a higher percentage removal of Navicula diatoms than the PDMS homopolymer coating, under the same water-jet pressure (؆ 20 kPa) [66]. This coating was hydrophobic, with a static water contact angle of about 100° that decreased to about 95° after 60 days of immersion in artificial seawater. The advancing and receding water contact angles were 105° and 89°, respectively. In the design of such coatings, it is important to ensure that the surfaces present a siloxane-rich surface even after immersion in water, and that the polar urea/urethane groups that migrate to the polymer–water interface do not cause strong bonding with the adhesive secreted by the marine organisms. If the surface functional groups react or interact with the organism’s EPS, the fouling release property will be lost. Consequently, in pseudobarnacle assays, the coatings containing the polyurea block showed a higher pull-off force compared with PDMS homopolymer. Carbon Nanotubes and Clay Fillers Silicone-based coatings filled with low levels (0.05 wt %) of multi-walled carbon nanotubes or sepiolite clay particles resulted in improved release of Ulva sporelings and reduction in the adhesion strength of adult barnacles [68]. No major changes in mechanical properties were observed by the low

Environmentally Benign Marine Antifouling Coatings



7.11 

amounts of filler. The observed enhancement in FR behavior was attributed to the change in the surface properties of the elastomer upon adding the filler particles. In contrast, previous studies on PDMSe coatings containing higher concentrations of fillers such as silica and calcium carbonate showed an adverse effect on FR performance, caused by the increase in the modulus of the coatings [69]. Biocidal Silicone Coatings As previously stated, the ultimate goal of the marine antifouling technology research is to develop coatings that will not kill marine organisms, but will repel or prevent them from settling on their surfaces. Such coatings would be not only self-cleaning, but also self-healing, similar to some of the inherently antifouling surfaces in nature [70]. Nevertheless, antifouling coatings based on biocidal action are also being actively researched, perhaps due to the inability of nontoxic coatings to satisfactorily solve the biofouling problem. The latter get heavily fouled within a few months of immersion in water. PDMSe [71] (and poly(methyl methacrylate) [72]) coatings, containing biocidal CuO and ZnO nanoparticles, were found to remain free of foulants even when there was no water flow past the surface. Other biocidal coatings include PDMS-based polyurethane containing the N-(2,4,6-trichlorophenyl)maleimide (TCM) biocide as pendant groups, tethered to the polymer backbone through the hydrolytically stable thioether group [73], and PMDS coatings with tethered quaternary ammonium salts [74]. The TCM-tethered PDMS-polyurethane coatings showed promising antifouling performance against barnacle cyprids, the marine bacterium Micrococcus luteus, and the diatom, Navicula. The polysiloxane coatings containing tethered quaternary ammonium salt showed antifouling behavior against the marine bacteria, Cellulophaga lytica and Halomonas pacifica, and the diatom, Navicula. In contrast, certain block copolymers with quaternary ammonium pendant groups, that we evaluated for fouling by Ulva sporelings and Navicula diatoms did not show marked algicidal activity, but they were highly effective against the airborne bacterium Staphylococcus aureus [75].

7.3.3 OtherFoulingReleaseCoatings A variety of other fouling release coatings have been reported. These include perfluoropolyether-based crosslinked elastomers [76], fluoropolymer/siloxane block copolymers and blends [77–79], fluorinated polyurethanes [80], and xerogel coatings [48, 81], including the halide permeable siloxane xerogels containing sequestered catalyst (for in situ formation of biocidal hypohalous acid through oxidation of the halide salts in seawater by the H2O2 released by organisms in the biofilm) [82]. Details on some of these coatings are available in previous reviews [23, 24].

7.4 HYDROPHILICNONͲBIOCIDALANTIFOULINGCOATINGS Hydrophilic coatings are those with surfaces that are partially or completely wetted by water and have water contact angles below 90°. The silicone and fluorinated FR coatings discussed in previous section are hydrophobic in nature. Water contact angles of up to about 126° are found for smooth compact layers of hydrophobic fluoroalkyl groups [83]. By controlling surface roughness, superhydrophobic surfaces with water contact angles as high as 175° can be obtained [84–86]. Such hydrophobic FR coatings allow easy detachment of marine organisms that settle on and adhere to the surface. However, antifouling surfaces, which can prevent biofouling right at the stage of settlement (and not after settlement and attachment, as in the case of FR coatings), are highly attractive and much required. Research in the biomedical field has shown that hydrophobic surfaces are, in fact, favorable for initial attachment of fouling species, and that hydrophilic surfaces are required for biofouling resistance [87, 88].

7.12   7.4.1 SurfaceHydrationandPolymerവWaterInterfacialEnergy

Biofilm Control

The high interfacial energy of a hydrophobic surface in contact with water is the thermodynamic driving force for adsorption of amphiphilic molecules, such as proteins, at these surfaces. Consider, for example, the fluorinated surface with a surface energy of 8 mJ/m2 and water contact angle, ߠ௪ , of 123°. The interfacial energy between the surface and water, ߛ௦௪ , is given by ߛ௦௪ ൌ ߛ௦ െ ߛ௪ …‘• ߠ௪ , which (using the surface tension of water, ߛ௪ ǡ equal to 73 mJ/m2) is calculated to be about 48 mJ/m2. Protein adsorption would be thermodynamically favored at this interface, because it would lead to a significant reduction in the interfacial energy. Consider, on the other hand, a hydrophilic surface such as a zwitterionic block copolymer coating reported in ref. [52]. For this coating, ߛ௦ was found to be about 31 mJ/m2 and the water contact angle was 66°. The interfacial energy is only about 1 mJ/m2. Hydrophilic surfaces possessing low interfacial energy with water are resistant to protein adsorption and cell attachment. They are also resistant to settlement by larger organisms. For example, hydrophilic coatings prepared using the poly(2-methyl-2-oxazoline) dimethacrylate macromonomer were found to greatly decrease the settlement of cyprids of Amphibalanus amphitrite compared with bare silicon [89]. Polymer brushes (surface-tethered coating of polymer molecules) containing the zwitterionic sulfobetaine groups are highly resistant to the settlement of Ulva zoospores [90]. Most spores remain motile (free-swimming above the surface) and do not settle on these surfaces. In the case of the few spores that do settle and release their adhesive, the adhesive is not able to bond to the surface. There are a few “rules” for a hydrophilic surface to be antifouling. For stable surface hydration, which is essential for preventing biofouling [91], at least two proton acceptor oxygen atoms must be present in the monomer structure, allowing strong hydration bonds in a double hydrogen bridge bonding configuration [92]. For more information on such rules (and exceptions), the reader is referred to ref. [23].

7.4.2 SettlementofUlvaZoosporesonOEGandPEGSAMs The preference of Ulva zoospores for settlement on hydrophobic surfaces, avoiding hydrophilic surfaces, is clearly demonstrated by the study using surfaces with micropatterned surface-wettability that was previously discussed (cf. Figure 7.2, [43]). Grunze and coworkers conducted an in-depth comparison of Ulva zoospore settlement on self-assembled monolayers of short chain oligo(ethylene glycol) (OEG) terminated alkane thiols and longer chain poly(ethylene glycol) (PEG) SAMs [92]. The SAMs were prepared using the thiols HS(CH2)11(OCH2CH2)YOX (EGYOX, where Y = 1–6 and X = H or CH3) and HS(CH2)2(OCH2CH2)YOX (PEGYOX, where Y = 44 or 112 and X = H or CH3), respectively. Glass slides coated with gold thin films were used as the substrates. Although the Ulva zoospores settled on the surfaces of EG6OH, shed their flagella, and secreted their adhesive, the adhesive was unable to irreversibly bond to the surface. The strength of adhesion of the settled spores was so low that even small shear forces (generated by small displacements of the substrates in the assay dishes, or by removal of the substrates through the water–air interface) was sufficient to remove the attached spore from the surface. The weak adhesion of the spores to the short chain length OEG SAMs was attributed to the remarkable strength of the bifurcated hydration bond of water to the OEG chain. In contrast, on the longer chain PEG surfaces, most of the spores did not settle at all. They did not secrete their adhesive and continued swimming above the surface of the SAM. The ability of PEG to inhibit spore settlement is because of steric repulsion of the loosely packed polymer layer with high water content [24]. Similarly, while the EG6OCH3 SAM, with a water contact angle of 68°, showed higher spore settlement than the EG6OH SAM, with water contact angle of 33°, there was hardly any effect of the terminal group (H or CH3) on the spore settlement density in the case of the

Environmentally Benign Marine Antifouling Coatings



7.13 

thicker PEGYOX SAMs. Both the hydroxyl- and methoxy-terminated PEG coatings prevented the spores from settling. EG 6OH SAMs also showed remarkable resistance against attachment of Cobetia marina [44].

7.4.3 AntifoulingCoatingswithPEGylatedSideChains In our initial studies on developing hydrated antifouling polymer coatings, diblock copolymers with methoxy-terminated PEG side chains, containing an average of 11 mers of ethylene glycol in each side chain, were evaluated for fouling by Ulva zoospores and Navicula diatoms [58]. Although the growth of sporelings on the PEGylated surfaces were significantly lower, and the release of Navicula diatoms was much higher, compared with the PDMSe coatings, the expected cell-repellent behavior of hydrophilic PEGylated surfaces, that is, their ability to completely prevent the settlement of Ulva zoospores, was not observed. There was no significant difference in the amount of Ulva sporelings between hydrophobic fluorinated and hydrophilic PEGylated block copolymer coatings. After a detailed investigation of the surfaces using near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, that probes surface composition in the top 3 nanometers of a polymer surface, it became clear that a significant amount of the polystyrene block was present at the surface, even after using spray-coating solvents and processing conditions that would lead to surface enrichment of the hydrophilic PEG groups. The presence of both PEG and polystyrene at the surface is due to comparable surface energies of dry PEO and polystyrene (43 and 41 mJ/m2, respectively; see Table 7.1). Because of the slightly higher surface energy of PEG, a preferential segregation of polystyrene would occur when the coating is prepared in air (or analyzed in vacuum).

7.4.4 AnalysisofSurfaceCompositionUsingNEXAFSSpectroscopy NEXAFS spectroscopy is a synchrotron-based X-ray absorption spectroscopy technique that is highly sensitive to the composition and orientation of chemical bonds at the surface. The surface is irradiated with a beam X-ray photons of different energies (e.g., 270 to 320 eV in increments of 0.1 eV, in C 1s NEXAFS spectroscopy) and the intensity of the emitted Auger electrons, called the partial electron yield (PEY), is recorded for each photon energy. Figure 7.5 shows the C 1s NEXAFS spectra of four different polymer coatings. The spectral features of each polymer in this set are remarkably distinct from those of the other ‫כ‬ resonance peak is prominently seen near 293 eV in the spectrum of the polymers. The ‫ ݏͳܥ‬՜ ߪେି୊ fluorinated block copolymer coating. The peak near 285.5 eV seen in the polystyrene spectrum is due to the ‫ ݏͳܥ‬՜ ߨ)‫ כ‬resonance. The relative intensities of the peaks in the block copolymer surfaces at this energy are proportional to the surface styrene contents. The ‫ ݏͳܥ‬՜ ߨ)‫ כ‬peak intensity, and therefore, the surface concentration of the polystyrene block is small in the case of the fluorinated block copolymer [see chemical structure in Figure 7.3(a)], whereas it is higher for the block copolymer grafted with 11-mer long methoxy-terminated PEG groups. Figure 7.5(d) is the NEXAFS spectrum of SEBS (Kraton G1652). The low intensity of the ‫ ݏͳܥ‬՜ ߨ)‫ כ‬peak, from the polystyrene block, indicates that the surface of the SEBS coating is composed mainly of the lower surface energy poly(ethyleneran-butylene) central block.

7.4.5 SurfaceDeliveryVehicles Surface delivery vehicles or surface anchor groups are used to bring functional groups with high surface energy to the surface of a coating [93]. Figure 7.6 illustrates the idea using representative cases of linear and comb-like diblock copolymers. Figure 7.6(i) shows the simplest case of a diblock

7.14  

Biofilm Control (b)

S ) 5

5

4

4

PEY(a.u.)

PEY(a.u.)

(a)

3 2

2 1

0

0

S )

Ͳ1 270

280

290 300 Energy(eV)

310

320

270

280

290 300 Energy(eV)

310

320

310

320

(d)

(c) 5

5

4

4 S ൌ

3

PEY(a.u.)

PEY(a.u.)

3

1

Ͳ1

V ԟ

S )

2

3 2

1

1

0

0

Ͳ1

V ԟ S )

Ͳ1 270

280

290 300 Energy(eV)

310

320

270

280

290 300 Energy(eV)

Figure 7.5 C 1s partial electron yield vs. X-ray photon energy NEXAFS spectra of (a) polystyrene, (b) block copolymer with LC semifluorinated side chains, (c) block copolymer with poly(ethylene glycol) side chains, and (d) polystyrene-b-poly(ethylene-r-butylene)-b-polystyrene (SEBS) coatings. All spectra were obtained using X-ray incidence angle of 55° [58].

copolymer coating, in which the surface is covered by the lower surface energy B block. However, the higher energy A block may be forced to be present at the surface by end-functionalization of the A block with a low-energy surface-anchor group C. In addition to the difference in surface energy values, the ability of the C group to drag the higher surface energy block to the surface depends on factors such as the lengths of the A and B blocks, and the polymer–polymer interaction parameter, F୅୆ . We investigated the strategy of end-functionalization, shown schematically in Figure 7.6(ii), using polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP) diblock copolymers [53]. The objective was to bring the higher surface energy P4VP block to the surface of the coating. Toward this goal, the P4VP block was end-functionalized with (heptadecafluoro-1,1,2,2-tetrahydrodecyl)-dimethylsilane groups that contained the low-energy perfluorooctyl surface anchor [94]. However, NEXAFS spectroscopy showed that the surface of the thermally annealed block copolymer film was covered mostly by the PS block. The relatively small perfluorooctyl group at the end of the polymer chain was not able to drag the P4VP block to the surface.

Environmentally Benign Marine Antifouling Coatings



7.15 

Figure 7.6 Schematic of surface-energy dependent surface composition of: (i) a diblock copolymer with surface energy of B block lower than that of the A block; (ii) a diblock copolymer with a low surface-energy surface anchor group, C, tethered to the end of the high surface energy block, A; (iii) a comb-like block copolymer in which side-chains or grafts G are present in one of the blocks; and (iv) a comb-like block copolymer in which the grafts, G, are end-capped with low surface-energy groups, F. The surface energies, J୅ , J୆ , Jେ , Jୈ , J୊ , and Jୋ are relative to the medium that the coatings of the block copolymers are in contact with. The conformation of the polymer backbone and the side chains depends on the chemical details of the constituent groups, and can be, for example, flexible random coil or rigid rod depending on how the backbone bonds can rotate under steric constraints present in the molecular unit.

Instead of end-functionalization, if the surface anchor groups were grafted to the polymer backbone—by quaternization reaction of the pyridine group with 1-bromohexane and/or 6-perfluorooctyl-1-bromohexane (see Figure 7.7)—X-ray photoelectron spectroscopy, NEXAFS spectroscopy, and water contact angle measurements showed that the high-energy pyridinium block was drawn to the surface by the low-energy hexyl and perfluorooctyl side chains. This approach is illustrated schematically in Figures 7.6(iii) and 7.6(iv), for comb-like polymers. A characteristic

7.16 

Biofilm Control

feature of such amphiphilic coatings, consisting of a combination of low and high surface energy groups, is that their surfaces exhibit a large hysteresis in water contact angles, that is, their advancing water contact angle is significantly higher than the receding water contact angle. block C H2 C H

random C H2 C H

m

0.7

CH2 CH

+

N

block C H2 C H

m

C H2 C H

0.3

+ Br

N



Br

n



n

+

F

N Br –

F F

F F

F F

F F

F

F F

F

F

(a)

F

(b)

F

F

Figure 7.7 Diblock copolymer (a) and terpolymer (b) with low-energy alkyl and fluoroalkyl surface anchor groups in the side chains [94]. Two different degrees of polymerizations were studied: (i) m = 105, n = 200 and (ii) m = 600, n = 630.

7.5 AMPHIPHILICBLOCKCOPOLYMERCOATINGS We used the approach of surface anchoring to prepared PEGylated antifouling surfaces using block copolymer coatings [60]. By attaching low-energy fluoroalkyl groups to one end of the PEG grafts, we were able to enrich the surfaces of these coatings with the higher energy PEG segments. Figure 7.8 shows the chemical structures of the block copolymers with fluoroalkyl-terminated PEGylated side chains. Two different block copolymers were initially investigated. Atomic force microscopy (AFM) of the surface was performed both in air, and under water (phosphate-buffered saline solution). The block copolymer with m = 82 and n = 23 showed lamellar morphology with a uniform surface layer of the PEGylated block (see the AFM image in Figure 7.9). The block copolymer with m = 369 and n = 43 exhibited cylindrical microdomains at the surface [95]. The water-swollen films showed a domain size of the order of 35 nm. block

n

m

O

O

F F

O x

F

F F

y

Figure 7.8 Block copolymers with fluoroalkyl-tagged PEG side groups. The number average degrees of polymerization in the block copolymers studied were: (i) m = 82, n = 23 [60] and (ii) m = 369, n = 43 [95]. The average values of x and y were about 3.5 and 3, respectively.

7.17 

Environmentally Benign Marine Antifouling Coatings



Figure 7.9 Tapping mode AFM topography (left) and phase (right) images of the block copolymer of structure shown in Figure 7.8, with m = 82 and n = 23 [60]. The dark spots are “holes” [97] formed because of the mismatch between the film thickness and the periodic lamellar domain spacing in the diblock copolymer film.

The coating with block lengths m = 82 and n = 23 showed higher removal of Ulva sporelings than PDMSe, and more remarkably, a higher removal of Navicula diatoms as well (more than 80 % removal, compared with only about 10 % removal from PDMSe) [60]. Although perfect antifouling behavior, in terms of low settlement density of the Ulva zoospores, was not demonstrated with this block polymer architecture, the block copolymer coating with m = 369 and n = 43 showed excellent resistance to protein adsorption in fluorescence microscopy assays and adhesion force measurements (using AFM) [95]. Subsequent studies, using block copolymers with different architectures, for example, an ABC triblock copolymer consisting of polystyrene, poly(ethylene-ran-butylene) and a third block grafted with the fluoroalkyl-terminated PEG side chains [59], and surface anchor groups of different compositions, e.g., alkyl groups [63], or grafts of short PDMS groups [96], demonstrated good inhibition of the settlement of Ulva zoospores as well. The settlement density of the Ulva zoospores on these surfaces was significantly lower than that on PDMSe. Other architectures, employing the low-energy fluoroalkyl groups as surface anchors, have been reported for PEGylated block copolymer coatings. The chemical structure of one such polymer is shown in Figure 7.10 [98]. This diblock terpolymer consisted of a PS block and a second PEO block in which some of the mers were derivatized with perfluorohexyl surface anchor groups. NEXAFS spectroscopy showed that there was no PS at the surface when at least 8 mol % of the mers in the PEO block were fluorinated. The settlement density of Ulva zoospores on the surfaces of the block copolymer coatings with 8 and 17 mol % of fluorinated mer, was significantly lower compared to the block copolymer without any fluoroalkyl anchor and also compared to PDMSe. Furthermore, the removal of Ulva sporelings after exposure to water flow (24-Pa wall shear stress) was high for these coatings. block m

stat x

y n

Figure 7.10 Poly(ethylene oxide) (PEO) based diblock terpolymer containing fluoroalkyl surface anchor groups: (i) m = 105, n = 1140, y = 8 mol %; (ii) m = 105, n = 840, y = 17 mol % [98].

7.18  Biofilm Control  The fluoroalkyl-terminated PEG groups were successfully used as surface delivery vehicles for high surface energy zwitterionic groups in carboxybetaine and sulfobetaine derivatized block copolymers, resulting in coatings that were resistant to both positively and negatively charged protein molecules [52].

7.5.1 SurfaceͲWettabilityofAmphiphilicCoatings Stable (non-reconstructing) hydrophobic surfaces are characterized by high advancing water contact angles and low contact angle hysteresis. In contrast, a surface of an amphiphilic polymer, such as that shown in Figure 7.7, 7.8, or 7.10 exhibits a large contact angle hysteresis, attributed to surface reconstruction when the dry coating comes in contact with water. This reconstruction is reversible and occurs over a time period that spans two different orders of magnitude [60]. Changes in the molecular composition of the surface up on immersion in water have been probed using techniques such as captive bubble contact angle measurement [60], NEXAFS spectroscopy [99, 100], and AFM [95]. The advancing and receding contact angles on the amphiphilic block copolymer surface with m = 82, n = 23 were 94q and 34q [60]. This difference in the water contact angles is caused by surface restructuring that happens over a short time scale, e.g., the flipping of the fluoroalkyl-tagged PEG side chains, such that the fluoroalkyl groups moves away from the water interface, exposing the PEG segments at the interface. The captive bubble contact angle decreased from 55q immediately after immersion in water to a value of about 31q after 2 weeks. This slower process is attributed to reconstruction of the block copolymer microstructure at the water interface. The hydrophilic block will preferentially move to the interface and the hydrophobic PS block will more away from the interface. By also measuring the equilibrium underwater octane contact angle (55q measured on the water side), it was possible to estimate the polymer௅water interfacial energy. The interfacial energy was fairly low, 4 mJ/m2, as would be theoretically necessary for the surface to be resistant to adsorption of proteins.

7.6 COATINGSWITHNANOSCALESURFACEHETEROGENEITIES 7.6.1 HyperbranchedFluoropolymerവPEGCopolymer Wooley and coworkers [101] postulated that surfaces of compositionally variant, nanoscopically resolved morphologies and topographies would be unfavorable for adsorption and unfolding of adhesive proteins if the dimensions of the topographical features are comparable to those of the proteins. The coatings, obtained by reacting a hyperbranched polyfluorinated benzyl ether polymer with bis(3-aminopropyl)-terminated PEG, were found to undergo a kinetically trapped phase segregation of the chemically incompatible PEG and hyperbranched fluoropolymer. Tapping-mode AFM showed a surface topography that was dependent on the weight percentage of PEG in the coating, and which changed upon incubation in artificial seawater due to the migration of PEG-rich domains to the water interface. Coatings with high concentrations of PEG (45 to 55 %) were effective against settlement of the Ulva zoospores.

7.6.2 MicrophaseSeparationinAmphiphilicBlockCopolymers Galli and coworkers [61] investigated the role of nanoscale surface heterogeneities on marine antifouling properties of block copolymer coatings. Block copolymers similar to that shown in Figure 7.8, but with styrenic (instead of acrylic) backbone, were synthesized with different combinations of block lengths (m and n). The surface compositions, and the microphase separation of the two blocks in the bulk of the coating, were investigated using NEXAFS spectroscopy, XPS, AFM,

Environmentally Benign Marine Antifouling Coatings

7.19 

dynamic water contact angle measurements, and GISAXS [100]. Figure 7.11 shows tapping-mode AFM images of the surfaces of representative block copolymer surfaces, wherein cylindrical and spherical nanodomains are clearly seen. In these coatings, discrete nanodomains of polystyrene were dispersed in the continuous matrix of the amphiphilic block. However, turbulent flow channel assays for adhesion of Ulva sporelings and Navicula diatoms did not show any correlation between adhesion strength and the microstructure of the dry block copolymer film, evidently because both the block copolymer chosen for the study contained the amphiphilic block at the surface. There were no significant differences in the water contact angle or surface energy values either.

Figure 7.11 Tapping mode AFM phase images of thin films of block copolymers with fluoroalkyl-tagged PEG side chains: cylindrical morphology (left) and spherical morphology (right) with nearest neighbor spacings of 20 and 22 nm, respectively [100].

In contrast, Grozea et al. [102] found that nanopatterned surfaces with hydrophobic and hydrophilic domains, prepared using polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP) and polystyreneblock-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymers, inhibited settlement of Ulva zoospores (cf. Figure 7.12). The comparison was with unpatterned surfaces of poly(styrene-ran-2-vinyl pyridine) random copolymer, and PS and P2VP homopolymers. The diblock copolymer coatings showed self-assembled cylindrical structures, which were retained after immersion in water.

Figure 7.12 (a) AFM height image of a crosslinked PS-b-P2VP film in air, after 8 days of immersion in water; 1000 u 1000 nm, z-range 40 nm. The block copolymer, with about 3:1 weight ratio of the PS and the P2VP blocks, was crosslinked using UV light and benzophenone photoinitiator. (b) The density of attached Ulva spores on

7.20  

Biofilm Control

polymers on silicon wafers on PS homopolymer. Reprinted with permission from [102]; Copyright 2009 American Chemical Society.

In addition to the studies discussed herein, using block copolymers, several different polymer architectures, incorporating hydrophobic, fluorinated or silicone polymers, and hydrophilic, PEG based polymers, have been explored as marine antifouling and FR coatings. The free-radical crosslinking of methacryloxy-functionalized hydrophobic perfluoropolyethers with methacryloxy-functionalized hydrophilic PEGs yielded elastomeric amphiphilic coatings [103]. The critical water pressure required for 50 % removal of attached Ulva sporelings was in the range of 40 to 80 kPa for these coatings. The corresponding water pressure, for a PDMSe reference, was about 25 kPa. In another study, a siliconemodified epoxy polymer, crosslinked with 2-amino propyl terminated poly(propylene glycol)-blockpoly(ethylene glycol)-block-poly(propylene glycol), was evaluated for macrofouling in a Mumbai harbor [104]. The coating was completely covered by algal slime, and other macrofouling organisms (barnacles, oysters, polychaetes, and ascidians) after 60 days of immersion. However, compared with an unmodified epoxy adipate resin that served as the reference, the silicone-modified epoxy coating exhibited good release of adhered organisms when cleaned with a water hose.

7.7 CONCLUSION In this chapter, we have discussed design principles for non-biocidal polymer coatings that can resist fouling by marine organisms. The interaction of the polymer surfaces with the adhesive pad of the marine organism is discussed in terms of molecular composition of the surface of the coating. The key conclusions can be summarized as follows: ƒ

ƒ

ƒ

ƒ ƒ

Low surface energy fluorinated or silicone elastomers are promising as fouling release coatings. The incorporation of LC perfluoroalkyl groups with a relatively high clearing temperature, as side chains in comb-like block copolymer architecture, results in the formation of a stable low-energy surface that is resistant to underwater reconstruction. The solvent-soluble and amorphous polystyrene block of these copolymers makes the coatings easier to process than fluoropolymers such as PTFE. The fluorinated and silicone FR coatings discussed in this chapter will be useful in situations where periodic cleaning, (e.g., using a water jet or scrubbing), is possible. Because of the good chemical stability of the polymers, these coatings are expected to have long service life. Current concerns with certain perfluorinated compounds as persistent drinking-water contaminants necessitate that the perfluoroalkyl groups be attached to the polymer backbone by hydrolytically stable linkages such as ether groups. The incorporation of hydrophilic groups such as PEG in fluorinated or silicone coatings results in the formation of amphiphilic surfaces that are resistant to a broader spectrum of marine organisms, including diatom slimes, compared with coatings with purely hydrophobic or hydrophilic characters. Such amphiphilic coatings are found to retain the FR properties of the fluorinated or silicone coatings. For long-term durability of the amphiphilic coating, it must not swell and soften greatly after immersion in water. The coatings must not contain surface functional groups that can irreversibly bond with functional groups such as phosphorylated serines and hydroxylated tyrosines (DOPA) present in the bioadhesives secreted by marine organisms [105]. Coatings with spontaneously formed nanoscale chemical heterogeneity at the surface have been explored as antifouling surfaces. The currently available data on the effectiveness of surface nanopatterns in imparting fouling resistance are not conclusive. No significant effect of block

Environmentally Benign Marine Antifouling Coatings



7.21 

copolymer morphology (cylinders vs. spheres) was observed in one study, whereas a decrease in the settlement density of Ulva zoospores was reported in another study.

In spite of the significant advances made over the past decade in the design of antifouling coatings, a biocide-free coating that can completely inhibit settlement of fouling organisms over long-term immersion in the marine environment is still being sought. A wide range of tin-free biocides are available, and are currently being used. Biomimetic approaches [70] such as topographically patterned surfaces mimicking the skins of sharks and whales [26, 106, 107], nontoxic self-polishing coatings which mimic the sloughing of polysaccharide mucus that some marine organisms use against fouling [108], and coatings with surface-immobilized enzymes and bioactive fouling-deterrent molecules [109–111], are some alternative strategies that are being explored. Aiming for a minimal adverse impact on the marine ecology, the secondary metabolites of certain naturally occurring marine macroand microorganisms have themselves been explored for inclusion as fouling repellents in polymer coatings. Commercial silicone based paints seeded with Pseudoalteromonas marine bacteria were found to be less fouled compared with control surfaces [112]. Such further advances in environmentally benign marine antifouling coatings are possible by fully understanding the mechanisms that nature uses in controlling adhesive interactions at interfaces.

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Martinelli E, Gunes D, Wenning BM, Ober CK, Finlay JA, Callow ME, Callow JA, Di Fino A, Clare AS, Galli G (2016). Effects of Surface-Active Block Copolymers with Oxyethylene and Fluoroalkyl Side Chains on the Antifouling Performance of Silicone-Based Films. Biofouling, 32(1), 81–93.

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Marabotti I, Morelli A, Orsini LM, Martinelli E, Galli G, Chiellini E, Lien EM, Pettitt ME, Callow ME, Callow JA, Conlan SL, Mutton RJ, Clare AS, Kocijan A, Donik C, Jenko M (2009). Fluorinated/Siloxane Copolymer Blends for Fouling Release: Chemical Characterisation and Biological Evaluation with Algae and Barnacles. Biofouling, 25(6): 481– 493.

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McMaster DM, Bennett SM, Tang Y, Finlay JA, Kowalke GL, Nedved B, Bright FV, Callow ME, Callow JA, Wendt DE, Hadfield MG, Detty MR (2009). Antifouling Character of ‘Active’ Hybrid Xerogel Coatings with Sequestered Catalysts for the Activation of Hydrogen Peroxide. Biofouling, 25(1), 21–33.

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Graupe M, Takenaga M, Koini T, Colorado R, Lee TR (1999). Oriented Surface Dipoles Strongly Influence Interfacial Wettabilities. Journal of the American Chemical Society, 121(13), 3222–3223.

7.28 

Biofilm Control

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Schilp S, Rosenhahn A, Pettitt ME, Bowen J, Callow ME, Callow JA, Grunze M (2009). Physicochemical Properties of (Ethylene Glycol)-Containing Self-Assembled Monolayers Relevant for Protein and Algal Cell Resistance. Langmuir, 25(17), 10077–10082.

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Sundaram HS, Cho Y, Dimitriou MD, Weinman CJ, Finlay JA, Cone G, Callow ME, Callow JA, Kramer EJ, Ober CK (2011). Fluorine-Free Mixed Amphiphilic Polymers Based on PDMS and PEG Side Chains for Fouling Release Applications. Biofouling, 27(6), 589–602.

Environmentally Benign Marine Antifouling Coatings

7.29 

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[100] Martinelli E, Menghetti S, Galli G, Glisenti A, Krishnan S, Paik MY, Ober CK, Smilgies D-M, Fischer DA (2009). Surface Engineering of Styrene/PEGylated-Fluoroalkyl Styrene Block Copolymer Thin Films. Journal of Polymer Science Part A: Polymer Chemistry, 47(1), 267–284. [101] Gudipati CS, Greenlief CM, Johnson JA, Prayongpan P, Wooley KL (2004). Hyperbranched Fluoropolymer and Linear Poly(ethylene glycol) Based Amphiphilic Crosslinked Networks as Efficient Antifouling Coatings: An Insight into the Surface Compositions, Topographies, and Morphologies. Journal of Polymer Science Part A: Polymer Chemistry, 42(24), 6193–6208. [102] Grozea CM, Gunari N, Finlay JA, Grozea D, Callow ME, Callow JA, Lu Z-H, Walker GC (2009). Water-Stable Diblock Polystyrene-Block-Poly(2-vinyl pyridine) and Diblock Polystyrene-Block-Poly(methyl methacrylate) Cylindrical Patterned Surfaces Inhibit Settlement of Zoospores of the Green Alga Ulva. Biomacromolecules, 10(4), 1004–1012. [103] Wang Y, Betts DE, Finlay JA, Brewer L, Callow ME, Callow JA, Wendt DE, DeSimone JM (2011). Photocurable Amphiphilic Perfluoropolyether/Poly(ethylene glycol) Networks for Fouling-Release Coatings. Macromolecules, 44(4), 878–885. [104] Rath SK, Chavan JG, Sasane S, Jagannath, Patri M, Samui AB, Chakraborty BC (2010). Two Component Silicone Modified Epoxy Foul Release Coatings: Effect of Modulus, Surface Energy and Surface Restructuring on Pseudobarnacle and Macrofouling Behavior. Applied Surface Science, 256(8), 2440–2446. [105] Stewart RJ, Ransom TC, Hlady V (2011). Natural Underwater Adhesives. Journal of Polymer Science Part B: Polymer Physics, 49(11), 757–771. [106] Cao X, Pettitt ME, Wode F, Arpa Sancet MP, Fu J, Ji J, Callow ME, Callow JA, Rosenhahn A, Grunze M (2010). Interaction of Zoospores of the Green Alga Ulva with Bioinspired MicroǦ and Nanostructured Surfaces Prepared by Polyelectrolyte LayerǦbyǦLayer SelfǦAssembly. Advanced Functional Materials, 20(12), 1984–1993. [107] Efimenko K, Finlay J, Callow ME, Callow JA, Genzer J (2009). Development and Testing of Hierarchically Wrinkled Coatings for Marine Antifouling . ACS Applied Materials & Interfaces, 1(5), 1031–1040.

7.30 

Biofilm Control

[108] Xie L, Hong F, He C, Ma C, Liu J, Zhang G, Wu C (2011). Coatings with a Self-Generating Hydrogel Surface for Antifouling. Polymer, 52(17), 3738–3744. [109] Olsen SM, Kristensen J, Laursen B, Pedersen L, Dam-Johansen K, Kiil S (2010). Antifouling Effect of Hydrogen Peroxide Release from Enzymatic Marine Coatings: Exposure Testing under Equatorial and Mediterranean Conditions. Progress in Organic Coatings, 68(3), 248–257. [110] Kristensen JB, Meyer RL, Poulsen CH, Kragh KM, Besenbacher F, Laursen BS (2010). Biomimetic Silica Encapsulation of Enzymes for Replacement of Biocides in Antifouling Coatings. Green Chemistry, 12(3), 387–394. [111] Ederth T, Nygren P, Pettitt M, Östblom M, Du C-X, Broo K, Callow M, Callow J, Liedberg B (2008). Anomalous Settlement Behavior of Ulva linza Zoospores on Cationic Oligopeptide Surfaces. Biofouling, 24(4), 303–312. [112] Bernbom N, Ng YY, Olsen SM, Gram L (2013). Pseudoalteromonas spp. Serve as Initial Bacterial Attractants in Mesocosms of Coastal Waters but Have Subsequent Antifouling Capacity in Mesocosms and When Embedded in Paint. Applied and Environmental Microbiology, 79(22), 6885–6893.

    



 



 

NANOPARTICULATES AND NANOCOMPOSITES AS ANTIBIOFILM AGENTS: EVOLVING PERSPECTIVES

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8.2 

Biofilm Control

  The rapid evolution in nanotechnology has impacted many areas of science and technology, including biomedical sciences [1,2]. The unique physicochemical properties of nanoscale materials offer remarkable advantages in applications pertaining to high throughput optoelectronics, catalysis, energy storage devices, drug delivery, biosensors, antimicrobial agents, and self-cleaning surfaces [3-9]. Apart from different possible morphologies of nanostructures, zero-dimensional nanostructures, colloidal nanoparticles, and nanocomposites are of tremendous interest in industrial applications due to their ease in fabrication and broad spectrum of commercial application [3, 5, 10]. Until recently, nanoparticles were mainly used for applications in photonics, photovoltaics and sensor applications. The biomedical industry recently started seeking out a way to effectively use nanoparticles for cancer treatment, cell tracking, marking, drug delivery, biosensing and antibacterial applications that can be applied to a wide range of cell systems. Currently, the global production of nanoparticles has substantially increased over the past decade, with ~ 1000th of tons in 2004, and it is expected to rise to over half a million tons by 2020 [10-13]. This increase in nanoparticle usage has raised several questions about the potential toxicity of such materials. In this chapter, we will focus on the potential use of nanoparticles as antibacterial agent and their recent prospective. The ample application of nanoparticles is stemmed from several unique material properties. First, nanoparticles possess a higher surface-to-volume ratio and thus promote a significant larger contact area with their environment in comparison to bulk materials at the same mass and cost. The increase in surface area effectively multiplies the number of immobilized free radicals at the nanoparticle surface, which increases the catalytic efficacy, and, in some cases, induces the formation of reactive oxygen species (ROS). This circumstance may also promote the solvation of nanoscale materials in liquids that allows the release of reactive ions to the immediate environment. The release of reactive ions often impedes the metabolic activity of certain cell types. In turn, nanoparticles that possess high surface energy, may promote interparticle agglomeration, adsorption of constituents from the environment, and alteration of organic matter and tissues at the contact interface. These characteristics at the nanoparticle surface can thus potentially change the mode of interaction with components of surrounding biological matter, such as cell membranes, extracellular or transmembrane proteins, as well as to connective tissue13-16. Second, owing to their small dimension, nanoparticles may be engulfed or retained in organs and individual cells to a larger extent, without affecting the crucial molecular mechanisms and metabolismrelated chemical reactions within a cell [4,5]. Third, the shape of nanoparticles may also play a crucial role in the uptake mechanism into a cell and interaction with surrounding biomolecules. For example, geometric effects have been shown for sharp carbon nanotubes, which perforate the bacterial cell wall, impeding subsequently the nanomaterial uptake [17]. Thus, a profound knowledge of interaction of the materials at the nano-bio interface is vital prior to the safe use of nanoparticles for health-related applications. More specifically, the use of nanoparticles for antibacterial applications is further complicated due to an ample variety in: i) morphology of nanoparticles used, ii) mode of synthesis conditions and chemical composition, iii) stability of the surface functional groups, iv) physicochemical parameters such as dimension, surface charge and geometry, v) environmental target cell conditions, vi) type of target cells, and vii) verification readout system [4-6]. This chapter is focused at providing an overview of the most recent development and advantages of nanoparticle based antibacterial agents, and evolving perspective based on recent literature. Besides the broad spectrum of possible biomedical applications, the potential of nanoparticle and nanocomposites as future generation antibacterial or anti-biofilm agents is of paramount importance due to a large number of associated diseases caused by bacterial pathogens. Over the past years, the number of infections associated with bacteria has increased, which are believed to be caused by biofilm-forming bacteria species [18, 19]. Both Gram-negative and Gram-positive bacteria can form

Nanoparticulates and Nanocomposites as Antibiofilm Agents...

8.3 

biofilms depending on the particular environmental condition. The most general biofilm-forming bacterial pathogens, which can cause severe diseases in human, are Staphylococcus aureus, Vibrio Cholerae, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus viridans, Klebsiella pneumonia, and Proteus mirabilis [20-24]. These bacteria species are the major cause of infections and disease such as tetanus, typhoid fever, diphtheria, syphilis, cholera, foodborne illness, leprosy and tuberculosis. A recently published report from National Institutes of Health enlisted that more than 80% of all microbial infections nowadays are caused by biofilm-forming bacteria [25]. In general, diseases caused by bacterial biofilms lead typically to persistent chronic infections due to the slow growth and maturing of biofilms, which become increasingly tolerant / resistant over time to host immune defense systems as well as to antimicrobial therapies. Basically, a biofilm represents a bacterial community consisting of a single or multiple bacterial species, embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that consist of peptides, proteins, DNA oligomers, and polysaccharides [18,19,26]. The EPS matrix shields the bacterial community against the host defense system and stabilizes the three-dimensional biofilm architecture, promoting bacterial adhesion and cellular communication. The biofilm formation is widely described by a five stages process (Figure 1A) that follows: 1) reversible adhesion of bacteria cells, 2) irreversible cell adhesion, 3) secretion and formation of EPS matrix, 4) biofilm maturation, and 5) dispersion of planktonic cells [18, 19, 26].

Figure 1. Life cycle of the phytopathogen Xylella fastidiosa. The life cycle (A) is described in 5 stages, starting with adhesion of planktonic cells (1), followed by irreversible adhesion (2), EPS matrix formation (3), biofilm maturation (4) and dispersion of cells (5). Polar cell (B) adhesion was observed via Spinning Disk Confocal Laser Microscopy (SDCLM). Irreversible adhesion (C) and secretion of EPS (white arrows) was observed via Scanning Electron Microscopy (SEM). SDLCM revealed agglomeration of cells (D) to small communities, which are at later stage interconnected (E) by filamentation of few cells (red arrows). Maturation of biofilms, forming large vertically growing 3D architectures (F) was monitored via SDCLM. Figure was reproduced with permission from Janissen et al., Scientific Reports, 2015. [26] Biofilms can be found on different humid surfaces such as natural aquatic systems, living tissues or organs, biomedical devices and implants as well as on industrial water pipes and sewerage systems. The process of biofilm formation, shown on the model phytopathogenic bacteria Xylella fastidiosa, is initiated by planktonic bacteria cells that attach and adhere to a foreign surface (Figure 1B). The

8.4 

Biofilm Control

transition from a planktonic to sessile lifestyle (Figure 1C) is attributed to recognition and transmission of particular signals from the environment and neighboring cells within the community (quorum sensing). At this stage in the biofilm life cycle, the cells secret EPS, covering the surrounding surface that further facilitates the successive adhesion of other planktonic cells from the environment. With increasing adhesion of cells on EPS-covered areas, the cells form eventually clusters (Figure 1D) with cell-cell junctions via the cell poles, and develop the incipient biofilm matrix. Within the EPS matrix, neighboring cell clusters are interconnected via a few elongated cells (filamentation) at the cluster boundaries (Figure 1E). The bridging of nearby cell clusters in turn provides enhanced spatial coverage of the continuously secreted EPS of the underlying surface, facilitating further adhesion of planktonic and daughter cells. Due to facilitated cell-attachment on EPS-covered areas and cell divisions of cells within the EPS matrix, the biofilm growths predominately vertically (Figure 1F), while the lateral expansion occurs at lower growth rate. This growth pattern of mature biofilms, which are anchored by only few cells, provides a floating architecture that maximizes nutrient flow. In the last stage of the biofilm life cycle, mature biofilms degrade, accompanied by dispersion of new planktonic cells to the environment, multiplying the probability of infection of other areas within a host by again forming biofilms at different new sites. Investigations on formation of biofilms allow us to identify possible target mechanisms for designing antibiofilm agents. As described previously, the first and crucial step of biofilm formation is the attachment of single cells to a surface and the transition from the motile stage to a sessile lifestyle. In host organisms, specific extracellular components often facilitate bacterial adhesion by reducing the energy barrier formed at the bacteria-surface interface. It is therefore important to investigate the specific physicochemical interactions between the bacteria and the surface upon adhesion. The ability of pathogenic bacteria to further modulate their cell adhesiveness based on specific host adaption mechanisms and the response to changing environmental conditions, the scenario of bacterial biofilm infection increases drastically in complexity. In this context, it has been shown that bacterial adhesion efficacy depends on various surface properties, such as chemical composition, wettability, compliance, and surface topography. A framework that is able to quantify cell-surface interaction forces and their dependence on the chemical surface composition, may unveil bacterial adhesiveness control mechanisms as new targets for specific antimicrobial agent design. Over the past decade, several quantitative investigations of the cell adhesion forces to surfaces have been realized using different techniques. Adhesion force measurements are mainly based on forceinduced deformation of sensing components. Among the existing techniques, 1-dimensional semiconductor nanowires pose the most accurate technique to date to measure adhesion strengths, covering a force range from pico-newtons to nano-newtons [27]. Quantitative measurements of cell adhesion forces can be obtained by observing optically the asymmetric displacement of nanowire tip positions upon attachment of bacteria. (Figure 2A, B). It has been shown for the model phytopathogen Xylella fastidiosa that adhesion forces exerting from the cell poles (~ 50 nN) are 2-fold higher than forces originating from the cell body (Figure 2C). This is in agreement with the previously described qualitative observation that the initial surface attachment of planktonic cells occurs via the cell pole (Figure 1B). Fluorescence-based in vivo force measurements in growth media have shown that adhesion forces of bacteria emanating from a biofilm (Figure 2E,F) are significantly higher (50 - 90%) than that of single isolated cells (Figure 2D,F) with similar orientation. This observation supports the assumption that biofilms are anchored by only few cells which stem the entire mass and drag forces of a semi-floating biofilm in a liquid environment. These results demonstrate the application of nanowires as force sensors for bacterial cell adhesion which can be exploited for the search and evaluation of new antimicrobial agents targeting bacterial adhesion and biofilm anchorage.

Nanoparticulates and Nanocomposites as Antibiofilm Agents...

8.5 

Figure 2. Nanowire-based bacterial adhesion force measurements. (A) FESEM image showing the deformation of nanowires in direct contact with the attached cell body (colorized in green). (B) Topview FESEM image of bacteria adhered to nanowires, showing the tip displacements; the inset exhibits a polar plot of force magnitude and orientation for each individual cell-attached nanowire. (C) Distribution of measured forces derived from the deformation of the nanowires by bacterial cells from body (blue) and polar (red) cell regions. (D) Fluorescence images show GFP-expressing X. fastidiosa cells adhered to InP nanowires invertical orientation (the images show the fluorescence of GFP and the reflected laser intensity at the nanowire tip) and corresponding polar plots of force and direction for individual nanowires (marked by dashed circles and numbered in the corresponding image). 3D fluorescence image (E) showing a small biofilm and two nanowires with vertically adhered bacteria, anchoring the biofilm to the surface. Tukey boxplot (F) shows the force values measured for the two highlighted in (E), with significant variation in force values when compared to individual single cell (D). Figure was reproduced with permissions from. Sahoo P.K, et al. Nano Letters, 2016. [27] The parameters favoring the initial surface attachment of bacteria include: i) the presence of an appropriate surface, ii) sufficient levels of extracellular iron, and iii) the presence of organic compounds, such as indole or other chemicals including polyamines, calcium, ions, which altogether enable biofilm formation and modulate growth rate and virulence. The different parameters facilitating biofilm formation and the associated societal problems were subject to many studies in the past [18,19]. Despite these studies, biofilms remain a frontier field of research in microbiology, owing to their importance associated with many medical, environmental, and industrial implications. The formation of biofilms, the regulation of inter-cellular communication, and triggers of phenotypical changes are to date not fully understood. For example, biofilms show higher resistance to toxic metals compositions than planktonic cells, which may be attributed to key mechanisms, such as quorumsensing mediated gene regulation, metal chelation by the EPS matrix, and increased persister cell formation that are inherent to the biofilm lifecycle. In addition, the presence of phenotypic and metabolic heterogeneity within a biofilm, caused by gradients of pH, oxygen, and nutrients within the environment, provides higher resistance to antibiotics than planktonic cultures. Due to the limited efficiency of antibiotics in preventing the formation or inhibition of biofilms, a number of alternative strategies have been recently developed, including the use of nanomaterials as potential antibacterial and antibiofilm agents [28]. However, the efficacy of the recently developed nanomaterials in

8.6 

Biofilm Control

combating biofilms cannot be compared as with each of the experimental conditions and concentration of nanomaterials differs as well as the type of micro-organisms. In general, antibacterial agents are designated either as bactericidal or bacteriostatic, based on their capacity to fight against infectious bacterial diseases. To date, the most used conventional antibacterial agents include: i) natural compounds such as b-lactams (like penicillins, cephalosporins or carbapenems, ii) purely natural products, such as aminoglycosides, and iii) synthetic antibiotics, such as sulfonamides [29]. In contrast, the use of organic compounds as disinfectants imply many disadvantages, including being toxic to the human body with adverse side effects. The development of alternative antibacterial agents is thus pivotal for many areas like health, surface disinfection, food packaging, and textile industry, owing to the fact that several pathogenic bacteria have developed resistance against many common antibacterial agents. As a result, infectious bacterial diseases continue to be one of the greatest health threads worldwide. In addition, high drug resistance leads to the use of high-dose administration of antibiotics, which often causes severe side effects. In the search of alternative strategies, studies on inorganic disinfectants, such as metal and metal oxide nanoparticles and nanocomposites, are increasing tremendously during the past decade [28]. For example, it has been demonstrated that several classes of nanostructures and nanoscale carriers for antibiotics delivery can be applied effectively for the rational treatment of many bacterial infections (Table 1). The efficiency of such nanoparticles and nanocomposites as potential future generation antibacterial agents is attributed to their associated quantum behavior as a result of high aspect ratio (surface-to-volume ratio) and carrier confinement. These characteristics result in appearance of unique optoelectronic, mechanical, chemical and magnetic properties of the nanostructures than that of their bulk counterpart [3,30]. Table 1. Nanoparticles and nanocomposites that have shown to be effective against different bacterial pathogens. Organism

Nanoparticles/Nanocomposites

References 31-44, 17

Escherichia coli

GaN, Ga2O3, SiO2, Ag, Cu, ZnO, TiO2, Al2O3, NiO, Fe2O3, CeO2, CuO, CdSe, Sb2O3, Carbon nanotube

Pseudomonas aeruginosa

GaN, Ga2O3, SiO2, Ag, ZnO, TiO2, CdSe/ZnS

31-35, 45

Ag, Cu, MgO, TiO2, CeO2, ZnO, CuO, NiO, Sb2O3, Carbon nanotubes, Fullerene

36-38, 41, 46, 47

Bacillus subtilis Staphylococcus aureus Vibrio fischeri Shewanella oneidensis

GaN, Ga2O3, Ag, Fe2O3 ZnO, TiO2, CuO CeO2, Cu-doped TiO2, ZnO

31, 32, 48 49 41, 50, 51

This chapter describes different modes of synthesis, characterization, and mechanism of action of inorganic nanostructure materials as potential alternative for the new generation of antibacterial agents. We focus at providing a concise summary of the most recent portfolios of inorganic nanoparticle-based antibiofilm agents and evolving prospective, rather than providing a thorough overview of all existing literature. Here, we strive to disclose the most general question, whether inorganic nanoparticles and/or nanocomposites have the capability to replace conventional organic compounds as prerogative antibacterial and antibiofilm agents based on the current pool of literature. Furthermore, we discuss several important considerations, opportunities and risks, while accessing the safe use of inorganic

Nanoparticulates and Nanocomposites as Antibiofilm Agents...

8.7 

compounds. In addition, we provide suggestions in this context on how to limit possible toxicity to biological matter for future design of inorganic antimicrobial agents.

  

The most prominent aspects of inorganic nanostructure materials exhibiting potential for antimicrobial activity can be classified in two domains: i) pristine nanostructure materials with unaltered surface chemistry, and ii) nanostructures modified with organic carriers at the surface. Proof-of-concept based applications investigate the detailed characteristics of nanoparticles, such as dimension, material quality, homogeneity, and surface chemistry, in order to understand their impact to their electronic, catalytic, optical and magnetic properties. In fact, inorganic nanoparticles provide different surface physicochemical properties attributed to their particular size and shape. For example, Au nanoparticles of nanometer dimensions show an interesting physical behavior: the smaller the diameter of the nanoparticle, the higher is the energy of its bandgap and surface energy per unit area. As a result of these phenomena, for example, the resulting size-dependent fluorescence properties of such nanoparticles are often exploited for the use as biomarkers to investigate biological processes in vitro as well as in vivo [52].

              

Inorganic nanostructure materials are mostly categorized in three different groups, namely: i) metal (Ag, Au, Pd, Cu, Ga, etc), ii) semiconductors (ZnO, Fe2O3, NiO, GaN, Ga2O3, MgO, TiO2, among others), and iii) insulators (SiO2, Ga2O3, Al2O3). Solution-based synthesis of metal nanoparticles is a very common method and relatively cost-effective, compared to many other techniques. The solutionbased synthesis relies on the reduction of metal salts in presence of chemical reducing agents or exposure to high-power lasers. The shape, size, and purity of the metal nanoparticles are usually controlled by carefully choosing the concentration of the precursors, reducing agents, and ligands [53,54]. For example, spherical metal nanoparticles are usually synthesized by the direct reduction of the metal salt in the presence of sodium borohydride as reducing agent [53] Different shapes and aspect ratios of metal nanoparticles can further be controlled via the seeded growth approach. This process exploits the assistance of initial metal seed particles, which are added to supplementary metal salt along with shape-directing ligands and weak reducing agents. However, the surface chemistry is the key parameter in the formation of particular shapes and sizes of metal nanoclusters, and can be tuned by varying the required ligand concentration during the synthesis process. In this context, it is further possible to modulate the surface physicochemistry by ligand exchange or polymer coating in a post-synthesis process [53, 55-57]. Such post-synthesis coatings can provide increased dispersion stability of nanoparticles in the liquid medium. However, the synthesis of semiconductor nanoparticles, which are often termed as quantum dots below a particular size range (below the exciton Bohr radius), have been realized by employing a variety of different synthesis approaches. Quantum dots are typically synthesized by combining different metals, reactive gases and/or chalcogenide precursors as salts, and heating to high temperatures (> 300°C) under inert atmospheric conditions [58-61]. Metal nitrides and oxides (e.g. Ga2O3, GaN) are often synthesized using via the chemical vapor deposition process under atmospheric pressure at even higher temperatures (> 700oC) in the presence of precursor gases, such as oxygen or NH3 as a source of O or N, respectively [60, 31, 32]. In contrast, some classes of metal oxides (Fe2O3, CuO, TiO2, ZnO etc) are synthesized using base hydrolysis reactions of molecular precursors (frequently acetates) in bulk scale and are more cost-effective [61]. In this process, the nanoparticle sizes and the hydrolysis rates can be controlled with the support of ligands, such as carboxylic acid or functional alcoxysilanes. Most importantly, during tuning the size and shape of semiconductor

8.8 

Biofilm Control

nanoparticles, it is necessary to retain the quality of the interactive surface, since these materials are vulnerable to native defects during the synthesis process. The presence of any lattice defects in the surface of the nanoparticles can alter the way they supposed to function, such as their fluorescence properties [62]. Finally, nanoparticles consisting of dielectric materials or insulators are typically synthesized by coprecipitation or sol-gel syntheses techniques. In these processes, the nanoparticle growth is quenched by surfactants or capping agents, such as long chain hydrocarbons [63, 64]. In this category, mesoporous silica is the most predominant nanoparticle composition. The structure is mainly achieved by changing the pH during growth around soft micellar templates that provide the typical porous structure. Such nanoparticles can be readily functionalized further using organofunctional alocoxysilanes that covalently bind in a self-assembly manner to the copious amount of surface oxide groups. Insulator-based nanoparticles, such as SiO2, do not possess any size-dependent optical or electronic property, as typically observed for metal or semiconductor-based nanoparticles [65]. However, the inert nature of these nanoparticle often prove to be more beneficial; for example, the porous silica structure can be loaded with molecular cargo as delivery vehicles for pharmaceuticals, or can be used as chemical catalyzers or biomarkers for the investigation of nano-bio interactions [65,66].

  The antimicrobial capability of inorganic nanoparticles depends strongly on the accurate assessment of their physicochemical characteristics, such as size, shape, material composition and surface chemistry. To date, a vast number of studies are corroborating the correlation of nanoparticle size and shape with their bactericidal properties [67]. It is noteworthy to mention that although the physicochemical characterization of nanoparticles are necessary for a detailed analysis of its biological uptake rate and interactions with living systems, such measurements are often not sufficiently adequate for envisaging the toxic effects [68]. In fact, this issue should be perceived in reverse by correlating the physicochemical characteristics of nanoparticles with their mode of interactions with cells [4, 31, 32, 68].

       



Transmission Electron Microscopy (TEM) is the most common technique for the direct observation of nanoparticle size, shape, and crystal structure properties. It is often necessary to use high resolution TEM (HRTEM) to predict accurately the crystal structure and material phase information. Generally, nanoparticle images are acquired in brightfield mode of TEM, which is based on the contrast generated by electron scattering of different atoms within the material. The detailed crystallographic orientation, particle dimension, and shape can be analyzed by any image analysis software, obtaining their statistical distribution by using only of few fitting parameters, such as circularity and ellipticity [69]. It is important to mention that nanoparticle properties may differ significantly, as their crystallographic orientation and structure can differ, while having similar chemical compositions. Figure 3 depicts HRTEM example images of Ag nanoparticles with clear crystal faces and distinct crystalline properties. Individual nanoparticles can thus be studied with this technique to assess their detailed crystalline properties. Typically, (HR) TEM sample preparations are readily performable for nanoparticle sizes with less than 200 nm in diameter. The limitation of the (HR) TEM-based analysis relies on three major factors: i) particle size, ii) sample preparation and iii) material structure stability upon exposition to high energy electron beams.

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Figure 3. HRTEM images of Ag nanoparticles which depict clear lattice fringes, based on their crystal structure. The figure was reproduced with permission from Sahoo PK, et al., J. Nanoparticle Research, 2013. [31] A classic sample preparation method is the casting and drying of droplets of monodisperse nanoparticles onto polymer-coated grids, e.g. amorphous carbon-coated Cu grids. This practice may return occasionally erroneous results in comparison to methods that measure particles dimensions in solution, due to particle agglomeration in the dried state. Due to this reason, size distribution analyses via (HR) TEM should always be compared with a complementary method that provides supportive information of the nanoparticle shape, size, morphology, and degree of agglomeration in their equilibrium state.

  Scanning Electron Microscopy (SEM) is typically used to produce real-space magnified images of surfaces containing information about texture, shape, and size of nanoparticles via the scattering of electron beams, similar to TEM. The main difference between the techniques TEM and SEM is that in a TEM the electrons pass through the sample, while in an SEM the electrons solely interact with the sample surface. The incident electron beam causes emission of back-scattered electrons by elastic scattering and secondary electrons by inelastic scattering. SEM images appear to be 3-dimensional because electrons near sharp corners and edges escape the sample more facilely and arrive faster the electron detector. Using SEM, it is also more facile to capture high resolution SEM images from metallic nanoparticles than from particles synthesized from insulating of semiconducting materials. However, the resolution limit of a conventional SEM is inferior to that of (HR) TEM. The addition of a Field Emission Gun (FEG) to the SEM can in turn provide high contrast images with low electrostatic distortion, reaching spatial resolutions below 2 nm. Depending on the type of information desired, one can image both bacterial cells and nanoparticles simultaneously, as shown in Figure 4.

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Figure 4. FESEM images of different planktonic bacterial cells. Wild type cells of (A) Escherichia coli, (B) Pseudomonas putida, and (C) Pseudomonas aeruginosa grown in the LB medium in absence of nanoparticles. (D-F) Changes in cell morphology of the corresponding cells shown in (A-C) grown in the presence of 1 mg/ml of nanoparticles. Scale bar depicts 2 µm. Figure was reproduced with permission from Sahoo PK, et al., J. Nanoparticle Research, 2013. [31]

 Unlike TEM and SEM, Atomic Force Microscopy (AFM) can provide complementary size and surface topology analysis of nanoparticles, individual bacterial cells, and biofilms in dried state as well as in liquid environment. AFM is a type of scanning probe microscopy with demonstrated resolution on the order of fractions of nanometers that is able to measure different forces, such as van der Waals interactions, capillary forces, chemical forces, and electrostatic forces. With this technique, different materials can be characterized without the necessity of any complex sample preparation methodology. In an AFM, a cantilever with nanometer-sized tip is typically used to scan across the sample surface. Variations in sample topography, surface viscoelasticity and chemical composition lead to changing tip-sample interaction forces, which in turn result in a detectable cantilever deflection. While AFM exhibits often lower lateral resolution than that of TEM due to limitations in the tip size and shape, it is very sensitive to changes in height, which is particularly useful for depth analysis at nanometer resolution, unlike TEM. The topological analysis via AFM is essentially based on physicochemical interactions with the sample surface and does not necessarily reflect the real dimensions of nanoscale features [70]. Depending on the application and kind of study, it is also possible to functionalize the cantilevers with organic compounds that can provide further information related to surface properties, such as mapping electrostatic and chemical interactions [71]. The main advantage in these techniques lies in the possibility to gain multiple information simultaneously.

       The Dynamic Light Scattering (DLS) method provides particle size analysis based on light scattering from the Brownian motion of particles in solution, whereas, in comparison, TEM relies on electron

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scattering process [72]. In principle, the DLS method evaluates the hydrodynamic radius of particles, which depends on translational diffusion rates of the particles in solution. Often, the real nanoparticle diameters are overestimated with this technique in comparison to TEM results. This is caused by the presence of organic surface layers around the nanoparticles, and depends further on the concentration, size distribution and degree of aggregation of the dispersed particles. However, unlike TEM, this method still provides a more accurate estimation of the effective size distribution of monodispersed nanoparticles in solution [71]. The major issues for estimating particle sizes accurately via DLS are: i) size distribution depends on particle concentration, ii) degree of particle aggregation, and iii) correct assessment of particle shape without substantial parameterization. In addition, the motional behavior of non-spherical particles needs to be interpreted carefully, as they may often lead to misinterpretation of the measured values.

 

Nanoparticles in solution attract a thin layer of ions of opposite charge and form subsequently a double layer of ions that accompanies the nanoparticle while it diffuses throughout the solution. The existing electric potential, based on the formed charged double layer, is defined as the Zeta potential, which lies typically in the range from +100 mV to -100 mV for nanoparticles. The magnitude of the zeta potential provides information about the nanoparticle dispersion stability: particles with higher zeta potential (e.g.  +25 mV or  -25 mV) exhibit increased dispersion stability, owing to the larger electrostatic repulsion among particles in proximity. Hence, the zeta potential analysis is an important parameter for understanding the composition of the nanoparticle surface as well as for the prediction of the long term dispersion stability in liquid [72]. However, the zeta potential is not the sole factor for nanoparticle dispersion stabilization in liquids. Nanoparticles can also be stabilized by coating them with repellant organic surface surfactants [73]. In general, it is important to understand the advantages and limitations of zeta potential measurements upon the interpretation of the results.

   The surface area and porosity are further important size-dependent factors in accessing nanoparticle activity, and can significantly influence the antimicrobial efficacy74. In fact, the surface area of nanoparticles is often larger compared to their bulk counterparts, which potentially causes an increase in surface reactivity, dissolution rate, bioavailability, and, most importantly, changes in their toxic properties. The latter is of strong importance for aerosolized particles. The Brunauer Emmet Teller (BET) method is the most common method to quantify the exposed specific surface area as well as to classify a material as a safe nanomaterial in respect to health and environmental safety [74]. The specific surface area is in principle determined by the amount of absorbed gas molecules to the nanostructure surface at a specific atmospheric pressure. The specific surface area is described as the ratio of the total surface area to the mass of the nanoparticle (in m2/g) [75]. Material specific surface area determinations via BET are explicitly relevant for the evaluation of the toxicological potential of nanoparticles and nanocomposites.

            

Nanomaterial-induced toxicity to bacteria and biofilms depends mostly on four major nanoparticle parameters: i) the material composition, ii) surface chemistry, iii) intrinsic electrophysical and optical, and iv) the target bacterial species [10,28]. Although there are numerous studies describing the antimicrobial effect of different nanomaterials, their mode of action / toxicity is found to differ with different bacterial species. This indicates that the mechanisms of toxicity, emanating from

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nanomaterials affecting different bacteria species, are complex and not fully understood [4]. Nevertheless, the most important antimicrobial mechanisms, based on the pool of current literature, are summarized schematically [28]. At large, most nanostructures are able to attach to the cell membranes via electrostatic interaction that can affect different types of processes and impacts the cell integrity, e.g. preventing transmembrane electron transfer processes, disrupting or penetrating the cell envelope matrix, or oxidizing cell components. Nanomaterials often form free radicals, such as reactive oxygen species or heavy metal ions, which lead to severe cell damage.

         Antibacterial activity of nanoparticles is to great extent influenced by nanoparticle properties as well as bacterial species involved. Some of the important parameters are discussed herewith.

      The bacterial cell wall is mainly divided into two main groups: Gram-positive and Gram-negative. The cell wall prevents the cell from osmotic rupture and damage, and their different architecture provides differences in mechanical strength, rigidity, and organo-chemical composition [76]. The cell wall of Gram-positive bacteria contains a lipid membrane layer with a thick outer layer of peptidoglycan (i.e. 20 – 80 nm). In contrast, cell walls of Gram-negative bacteria consist of a lipid double-layer membrane to which a thinner peptidoglycan layer is embedded in-between (i.e. 2 - 8 nm). Due to the different cell wall architectures and chemical compositions, the interaction with nanomaterials and the toxicological consequences can be substantially different [77, 78].

     The different structure and chemical composition of bacterial cell walls are not the only influencing parameters for the toxicological outcome; many other factors with respect to the nanomaterial composition and chemical surface composition can modulate the toxicological effect and severity. For example, i) Ag nanoparticles show higher antibacterial effect than Cu nanoparticles against Escherichia Coli and Staphylococcus aureus strains, ii) Staphylococcus aureus and Bacillus subtilis are less susceptible to CuO nanoparticles, whereas Escherichia coli is affected strongly, and iii) NiO and ZnO nanoparticles affect Staphylococcus aureus and Bacillus subtilis stronger than the model organism Escherichia coli [78].

  The growth rate of bacteria is yet another factor that can influence the resistance of bacteria against different nanostructure antibacterial agents. It has been shown that bacteria which exhibit faster growth rates are more susceptible than that of bacteria displaying slower growth rates to both nanoparticles and antibiotics [79]. This phenomenon occurs mostly due to the expression of stress-response genes in slowly growing bacteria cultures and indicates that the antibacterial response of bacteria to nanoparticles depends further on the bacterial target species.

   

One of the major drawbacks of many antibacterial nanoparticles and drugs is their inability to fight bacteria that have the capability to form a biofilm. Biofilms are complex communities of

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microorganisms attached to a surface, embedded within a matrix of hydrated EPS, which includes DNA oligomers, peptides, proteins, lipids and lipopolysaccharides. Once cell attachment occurs, the secreted EPS eventually accumulates, leading to changes in stiffness and elasticity of biofilms, and posing a serious challenge to their eradication. Once a biofilm is formed, it becomes highly resistant to antibiotics and nanomaterials over a period of time. Nevertheless, the electrostatic properties of both nanoparticle and biofilms can influence the degree of interaction at the nano-bio interface. Nanoparticles exposed to biofilms often prevent the further attachment of planktonic bacteria to the biofilm matrix and reduce significantly the biofilm maturation process. Moreover, studies testify that surfaces coated with ZnO nanoparticles can indeed prevent the formation of E. coli and S. aureus biofilms by generating ROS. Among various types of nanoparticles, magnetic nanoparticles with different metal surface coatings show increased antibiofilm activity due to the advantage that they can be guided towards the target via an external magnetic field, interfering more efficiently the biofilm formation process.

   

Preparation of metal / polymer nanocomposites dates back to early 18th century. A nanocomposite in general can be defined as a material made of two (biphasic) or more phases with one of the phase being in nanometer scale. Incorporation of nanoparticles into polymer matrix offers protection to the nanostructures offering ease of manipulation. In contrast nanoparticles alter the surface properties of polymers making them more robust, hydrophilic/hydrophobic, add to the mechanical strength etc. Dispersion of nanoparticles in polymer matrix is a grey area to be content with due to the aggregation of nanoparticles and currently it is a problem where more studies needs to be carried out. Nanocomposites have multiple applications for example with incorporation of gold, silver and copper nanoparticles they can be used for color filter application. Other applications include electro less plating of polymeric, ceramic and semiconductor substances. In the last decade numerous studies have investigated the antibacterial efficacy of different metal oxide nanoparticles against different bacterial species and have correlated them to their physical and chemical properties. Due to the highly toxic nature of metallic nanoparticles and as such nanomaterials cannot be applied / dispersed in any natural aquatic or terrestrial ecosystems currently restrictions on their use is being envisaged. Polymers incorporated with nanoparticles as fillers (nanocomposites) have emerged and have been applied in wide range of areas in biomedical (implants and catheters, wound dressing, protective clothing, nanomedicine, antibacterial surfaces) desalination and water treatment (PVDF and polyamide membranes impregnated with nanoparticles) antifouling coatings (PDMS foul release surfaces impregnated with nanoparticles) antimicrobial coatings (places of public hygiene). Bacterial adhesion and biofilm formation is a surface associated phenomenon wherein effective control measure would be to prevent biofilm development on the surface by immobilizing active biocides at the surface or bound in a polymeric matrix. Hence there is a profound interest in developing surfaces/coatings that a) prevent adhesion of bacteria and biofilm formation b) immobilization of biocides in coatings/surface and c) controlled release of immobilized biocide for long term antibacterial activity of these coatings. Biofilm formation is ubiquitous and constitutes an operational problem to engineered structures like ship hulls, heat exchangers, implants, biomaterials, hospital environments, medical equipment like endoscopes, power plant cooling water systems, desalination systems, and dental water units and in places of public hygiene. In general there is a worldwide increase in reports on bacterial resistance / tolerance to conventional medical antibiotics. Another issue is the high prevalence of multi-drug resistant bacteria [80]. Conventionally oxidizing biocides like chlorine, chlorine dioxide and ozone have all been used effectively to treat bulk water as well as biofilms [81]. However these biocides have limitation in that their biocidal efficiency is hindered by interaction with organics present in water and

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are neutralized by organics present in the biofilm matrix. Further formation of toxic disinfection by products [82] also places a cap on the concentrations which can be discharged into the environment. Biocides based on organic compounds like Quaternary ammonium compounds, isothiazolinones are also in use in various commercial products but microbes have been shown to develop resistance to these biocides and some of these biocides are being phased out due to environmental issues. In-lieu of these nanoparticles offers a potential solution as antibacterial agents and to overcome the problem of microbial resistance to biocides. Implants and device related infections has cost the around US$28 to US$45 billion in the United States of America as a result of prolonged hospital stays and revision surgery [83]. Interventions of device related infections often fail and carry the risk of reinfection or the development of antibiotic resistance. Hence there is a requirement for coatings/surfaces which are non-adhesive to microbes (repel or resists) or are able to control the growth of attached microbes (inactivating attached cells or cells coming into contact with surfaces). Several approaches are being tested currently with the use of low fouling polymer coatings, using antimicrobial peptides and nanoparticles into polymeric coatings [84,85]. Silver based polymers have been shown to resist bacterial colonization and have been used extensively in medical devices [86]. Liu and Kim (2012) [87] have developed nanocomposites for wound protection, inhibiting bleeding and antibacterial activity. Silver nanoparticles incorporated on titania nanotubes surfaces are being currently used as implant material instead of the conventional titanium Zhao et al., (2011) [88] which has minimized the complications due to post-operative infections. Nanocomposites of hydroxyapatite impregnated silver nanoparticles have been developed for bone implants [89] which have exhibited good antimicrobial activity. Similarly Ag-doped mesoporous hydroxyapatite which promotes formation of silver nanoparticle with high antimicrobial efficiency has also been synthesized Buckley et al., (2010) [90]. Pollini et al., (2011) [91] have developed haemodialysis catheters with silver nanoparticles by photoreduction which has shown excellent antibacterial properties. Membrane filtration is a widely used technology for water and wastewater treatment systems; however membrane fouling by biofilms in micro and ultrafiltration units poses a significant problem to be overcome [92]. Membrane modification by impregnation with nanoparticles is effective in modifying the surface characterestics, flux as well as antimicrobial properties. Polyvinyledenefluoride (PVDF) ZnO nanocomposites showed 100% water flux recovery as well as antimicrobial properties [92]. Biofilm formation on hull surfaces increases hydrodynamic drag, fuel consumption, hull corrosion [93]. Conventionally antifouling paints using organotin compounds were used for controlling slime and fouling. With the phasing out of these paints due to environmental issues foul release coatings based on polydimethylsiloxane (PDMS) came into vogue. Foul release PDMS coatings based on low surface energy, hydrophobic surfaces reduce the strength of the adhesive bond between adhesive organisms and coating. While foul release coatings prevent macro fouling they do not prevent the buildup of slime layers/biofilms comprised of diatoms and bacteria [94]. Incorporation of nanoparticles into the PDMS matrix enhances the mechanical properties of polymeric matrix. Incorporation of TiO2 and CNT into PDMS matrix inhibited larval settlement [95]. TiO2 and CNT incorporation into matrix did not inhibit bacterial density but reduced diatom density in biofilms [96]. Yet in another study incorporation of CNT in PDMS matrix did not improve the foul release properties of these coatings. Incorporation of nanoparticles as nanofillers did not affect the bulk mechanical properties of the coating with small changes in polymer surface properties [97]. Incorporation of ZnO nanoparticles onto PDMS coatings did not prevent adhesion of different diatom species (Figure 5 and 6) but were able to kill attached cells. Compared to PDMS, polymethyl methacrylate (PMMA) nanocomposites were more effective in inhibiting settlement of diatom species (Figure 7 and 8).

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Figure 5: Colonization of different diatom species on plain polydimethyl siloxane foul release coatings, PDMS-CuO and PDMS-ZnO nanocomposites. Figures represent dual fluorescence technique: Red auto fluorescence is exhibited by colonized cells which are living, whereas green / yellow fluorescence is exhibited by membrane compromised cells stained by SYTOX® green.

Figure 6: Colonization of different diatom species on plain poly methyl methacrylate coatings, PMMA-CuO and PMMA-ZnO nanocomposites. Figures represent dual fluorescence technique: Red auto fluorescence is exhibited by colonized cells which are living, whereas green / yellow fluorescence is exhibited by membrane compromised cells stained by SYTOX® green.

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Figure 7: Colonization of different marine bacterial species on poly(dimethylsiloxane) (PDMS) coatings, PDMS-CuO and PDMS-ZnO nanocomposites. Bacterial speices were stained by BacLight® live dead staining. Green stained cells represent live cells whereas red cells represent membrane compromised cells. Studies by Sankar et al., (2015) [98] have demonstrated incorporation of ZnO increased the hydrophobicity of PDMS, surface roughness as well as antimicrobial activity. The PDMS-ZnO nanocomposites were able to resist macrofouling in field exposures. Leaching of metal ions like Cu+ and Zn+ from PDMS nanocomposites was found to be comparatively low when compared with release by conventional antifouling coatings incorporating these agents. Assessment of biocompatibility of PDMS nanocomposites revealed that CuO nanoparticles capped with CTAB showed the highest antibacterial activity as well as biocompatibility [99]. Studies by Sathya et al., (2016) [100] have indicated that PMMA nanocomposites incorporating CuO and ZnO exhibited excellent antibacterial activity as well as barnacle larval settlement inhibition. Results of these studies highlight that irrespective of the polymer matrix incorporation of nanoparticles rendered the polymer as excellent antibacterial and antifouling materials.

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Figure 8: Colonization of different marine bacterial species on plain polymethyl methacrylate (PMMA) coatings, PMMA-CuO and PMMA-ZnO nanocomposites. Bacterial speices were stained by BacLight® live dead staining. Green stained cells represent live cells whereas red cells represent membrane compromised cells.

    

Currently about 1827 products comprising nanomaterials are being produced in 33 countries as detailed by the “Project on Emerging Nanotechnologies” (PEN) (2016) [101]. Evaluation of environmental risks imposed by nanomaterials used in commercial products needs to be investigated in detail with respect to their, solubility, mobility, ecotoxicity, bioavailability, bio persistence and bio magnification. Linking nanomaterial properties to nanotoxicological effects in biological organsims is a priority area which needs extensive investigations [102]. In principle many nanoparticles synthesized contain heavy metals and mitigating them in the enviornment is a challenge. Currently Ag, TiO2, ZnO, CNT, Pt, Pd, SiO2, Fe2O3, Fe3O4, Fe, Al2O3 constitute nanoparticles being produced commercially and used at an industrial scale in various processes [103]. Among these Ag and CNT have the widest range of application in products from antibacterial to disinfection and remediation of ground water. Nanoparticles are used in a variety of products such as electronics, biomedical, pharmaceutical, cosmetic, energy, environmental, coatings and catalytic applications, additive to tyres, automotive exhaust converters, ceramics, UV protection, fire-proof glass, paints, cement, sunscreen, textiles, plastics, fridges, vacuum cleaners, air conditioners, flat panel displays of televisions etc. [104,105]. In biomedical applications iron oxide, silver nanoparticles and quantum dots are the

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dominantly used nanomaterials [106]. In addition use of nanoparticles in medical procedures is also developing rapidly viz: imaging tools, phototherapeutic agents, gene drug delivery carriers [107]. Use of magnetic nanoparticles such as magnetite and ferrofluidic nanoparticles is also in the increase [108] in biomedical settings. Silver and lactose modified chitosan antibacterial nanocomposites were developed which showed excellent antimicrobial efficacy against both gram+ and gram- bacteria [109].

ϴ͘ϲ͘ϭEĂŶŽǁĂƐƚĞ Most of the currently used nanomaterial based consumer products contribute to a large source of nanoparticles into the water bodies and soil as wastes [110]. Currently nanomaterial based commercial products are marketed as i) nanomaterials fixed on substrate and ii) free nanoparticles. The antibacterial effect is achieved either by leaching of metal ions from the coatings (Ag ions from fridges, vacuum cleaners, washing machines) or controlled release of the nanoparticles themselves (Pt released from automotive exhaust converters) which are introduced into the environment. The source of nanoparticles into the environment may vary from point source (manufacturing site, landfill, wastewater plants) etc., or from non-point source like wear and tear of products made from nanomaterials, domestic wastes, accidental spillage and atmospheric emission. Investigations into the expected life time of the nanomaterial products, intensity of release of nanoparticles, life cycle assessments needs to be carried out [105]. The ultimate sink of these materials are in soil, atmosphere and water bodies. The production of nanomaterials was 2000 tons in 2004 which is expected to increase to 58,000 tons in 2020 [111]. This increase in manufacture and use of nanoparticles poses a significant threat to humans by their release into the environment. There is increasing interest in studying the behaviour of nanoparticles in the environment [112] and ecotoxicology [113]. Another aspect which needs to be investigated is with respect to lack of methods to analyze and identify nanoparticles from nanowastes as most of the wastes are disposed of in landfills. Among the characterestics of nanoparticles particle size affects the behaviour and reactivity of nanoparticles in addition to functionalization and solubility. Nanoparticles released into the environment do not remain pristine and are modified by environmental factors like organic matter, humic and fulvic acids, coatings, chemical or biological processes [104]. Further Maynard and Aitken (2007) [114], have reported that nanoparticles bind strongly to pollutants like cadmium and organics. The authors attribute this effect to surface properties of nanoparticles like large surface area, shape, size, aggregation, optical sensitivity and hydrophobicity. Another important aspect of nanoparticles released into the environment is their aggregation behaviour with natural colloids in the environment. Hence stability of and aggregation with other particles in environment is known to influence toxicity. There is a lacuna in knowledge about classification, risk assessment and quantification of nanomaterials in different environment’s which poses a problem in nanowaste management. Currently legal regulations on classifying nanowastes are the need of the hour with studies on evaluation of expected quantities and concentration of nanoparticles in the environment. The British standards institution has made the document PAS130 on products containing manufactured nanoparticles (BIS 2007) [115]. USEPA is also in the process of classifying different nanomaterials (EPA 2007) [116]. The United States and European Union have developed a framework to management of nanowastes [117]. In-lieu of the toxic nature and the large quantity of nanoproducts being manufactured, possibility of recycling of nanoproducts also needs to be thought off in the long run.

ϴ͘ϲ͘ϮdŽdžŝĐŝƚLJŽĨEĂŶŽƉĂƌƚŝĐůĞƐƚŽWůĂŶƚƐĂŶĚKƌŐĂŶŝƐŵƐ Accumulation of nanoparticles in environment poses a threat to non-target organisms/species. In general, ecotoxicty studies of nanoparticles are more on bacterial species due to their application as

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antibacterial materials. Concentrations as low as 8-80 ng/l of ZnO and TiO2 have been shown to be internalized in bacterial cells [118] resulting in toxicity. Numerous studies have been carried out on toxicity of nanomaterials on aquatic microalgae which are the primary producers in fresh and marine ecosystems. Toxicity of different nanomaterials on various marine organisms has been summarized by [102]. Metal oxide nanoparticles like CuO, ZnO, Ag and have been demonstrated to exhibit toxicity either by physical contact with the cell wall or by release of toxic metal ions like (Zn+, Ag+, Cu+). In most of the cases with these metal oxide nanoparticles it is difficult to confirm whether physical contact of nanoparticle with the cell membrane is responsible for toxicity or whether toxicity is due to dissolution, release and uptake of free ions by organisms. Studies by Singh et al., (2012) [119], demonstrate that Ag nanoparticles are internalized via scavenger receptor-mediated phagocytosis in murine macrophages which are localized in the cytoplasm, causing mitochondrial damage, induction of apoptosis and cell death. The study further reported that intracellular dissolution of Ag nanoparticles occurs about 50 times faster than in water. Concentration of nanoparticle also seems to play a role in toxicity of ZnO and TiO2 to the marine algae Thalassiosira pseudonana, Chaetoceros gracilis, Phaeodactylum tricornutum which differ in their sensitivities to these nanoparticles [120]. Comparing toxicity of different nanoparticles like ZnO, CuO and TiO2 by Aruoja et al., (2009) [121], on the microalgae Pseudokirchneriella subcapitata revealed ZnO to be more toxic followed by CuO and TiO2. The effect was attributed to the high solubility of Zn+ ions. Exposure of the green algae Chlorella vulgaris to NiO nanoparticles resulted in plasmolysis, cytomembrane breakage and thylakoids disorder [122]. Interestingly, the presence of algal cells caused aggregation of these nanoparticles and release of 0.14% ionic nickel as well as reduction of ions indicating the role of these algae in phytoremediation. Compared to nano sized Titania micro sized Titania also exhibited toxicity on the algae Scenedesmus sp. and Chlorella sp. [123]. Zooplankton like copepods constitutes the next higher level in the food chain and impacts of CuO and ZnO on toxicity to Tigriopus japonius have been investigated by Park et al., (2014) [124]. Results of this study revealed that toxicity was similar to that of metal ion toxicity. In marine and fresh water ecosystems, benthic organisms like mussels, oysters and clams have all been shown to bioaccumulate nanomaterials and have been proposed as bio indicators for nanotoxicity. TiO2, SiO2, carbon black and C60-fullerene were observed in the digestive glands of the Mediterranean mussel Mytilus galloprovincialis [125]. In addition these nanoparticles cause alterations in the haemocyte immune response, oxidative stress and lysosomal stability in digestive glands of these organsims. Similar decrease in lysosomal stability, DNA damage in coelomocytes has been reported in the burrowing polychaete worm Arenicola marina in response to TiO2 and SWCNT nanoparticles [126]. Neutrophil function and innate immune gene transcription was affected in the freshwater fish Pimephales promelas upon exposure to TiO2 and hydroxylated fullerenes [127]. Oxidative damage and GSH depletion was observed in the fish Micropterus salmonides [128] with down regulation of PMP70 inhibiting lipid repair caused by oxidative stress [129]. In another mollusc species the abalone (Haliotis diversicolor) TiO2 nanoparticles were shown to cause oxidative stress and increase in nitric oxide levels [130]. Studies on interaction of released nanoparticles in terrestrial enviornment and on plants have been discussed in detail by Jin et al., (2016) [131]. The authors have noted that laboratory and green house experiments need to be conducted with multiple species under field conditions for deriving the hazardous concentrations of nanomaterials in terrestrial ecosystems. In general plant cells carry a negative surface charge which allows the transport of negatively charged compounds into the apoplasts [104]. Aluminum oxide nanoparticles are a potential root toxicant and have been shown to effect root elongation [132]. In contrast titanium dioxide nanoparticles have shown to increase the activity of several enzymes and to promote the adsorption of nitrate and accelerate the transformation of inorganic into organic nitrogen [104]. ZnO and aluminum nanoparticles exerted toxic effects on germination and growth of roots and have been demonstrated to enter the apoplasts and stele of plants [133]. In the human body the mode of entry of nanoparticles is primarily through inhalation or dermal contact and

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secondary by ingestion through medication and foods [134]. Two independent studies carried out in 1997 by Dunford et al., (1997) [135] and McHugh and Knowland (1997) [136] revealed titanium dioxide/ZnO nanoparticles in sunscreen contributed to formation of free radicals in skin and damage DNA. Similarly these nanoparticles were shown to cause brain damage in mice [137] and were found to damage brain microglia and damage neurons in vitro. The impact of nanomaterial based products on the environment is still in its infancy. Currently we have now started to understand the mechanism of toxicity of different nanomaterials and their fate in the environment. Extensive studies / research programs / evaluations need to be carried out to generate data on the release of nanomaterials into the environments, their volumes, hazard evaluation and evaluation of potential impacts in terrestrial and aquatic ecosystems. Based on these legislative frameworks could be setup for classification as well as treatment of nanowastes from environment.

 A volume of literature exists on synthesis of various types of metal oxide nanoparticles as well as CNT and graphene nanomaterials. Most of these studies have aimed at synthesizing nanoparticles of different size, shape and charge using simple as well as complex methods. Some of these studies have correlated the antibacterial and antibiofilm activity with respect to the physico-chemical and structural properties of the synthesized nanoparticles. The antibacterial activity was found to be dependent on concentration and type of nanoparticle and type of bacterial species involved. Another paradigm is most of these studies have been conducted in laboratory using single or a couple of bacterial species. For a better understanding studies using nanomaterials / nanocomposites should be carried out carried out under in-situ conditions to assess their broad spectrum activity. Among the nanoparticles Ag and ZnO have been extensively researched and have been incorporated into commercial formulations. The release of these nanoparticles into the environment is a matter of concern. Specific studies should be carried out at the ecosystem level to assess their toxic effects. On the other hand nanocomposites have offered an effective alternative for controlled release of nanomaterials as well as prolonged antibacterial efficacy for surfaces.

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