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Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Cholera: Symptoms, Diagnosis and Treatment : Symptoms, Diagnosis and Treatment, edited by Evelyn L. Melbourne, Nova Science Publishers,

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Cholera: Symptoms, Diagnosis and Treatment : Symptoms, Diagnosis and Treatment, edited by Evelyn L. Melbourne, Nova Science Publishers,

TROPICAL DISEASES - ETIOLOGY, PATHOGENESIS AND TREATMENTS

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CHOLERA: SYMPTOMS, DIAGNOSIS AND TREATMENT

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Cholera: Symptoms, Diagnosis and Treatment : Symptoms, Diagnosis and Treatment, edited by Evelyn L. Melbourne, Nova Science Publishers,

TROPICAL DISEASES - ETIOLOGY, PATHOGENESIS AND TREATMENTS

CHOLERA: SYMPTOMS, DIAGNOSIS AND TREATMENT

EVELYN L. MELBOURNE

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York

Cholera: Symptoms, Diagnosis and Treatment : Symptoms, Diagnosis and Treatment, edited by Evelyn L. Melbourne, Nova Science Publishers,

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. Library of Congress Cataloging-in-Publication Data

Cholera : symptoms, diagnosis, and treatment / editor, Evelyn L. Melbourne. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61122-151-0 (e-book) 1. Cholera. I. Melbourne, Evelyn L. [DNLM: 1. Cholera. 2. Disease Vectors. 3. Vibrio cholerae--pathogenicity. 4. Virulence Factors. WC 262] RC126.C557 2011 614.5'14--dc22 2010046998

Published by Nova Science Publishers, Inc. † New York

Cholera: Symptoms, Diagnosis and Treatment : Symptoms, Diagnosis and Treatment, edited by Evelyn L. Melbourne, Nova Science Publishers,

Contents

Preface Chapter 1

Current Perspectives on the Pathogenesis of Vibrio cholerae Non-O1, Non-O139 Prasanta K. Bag

Chapter 2

Cholera: Current African Perspectives E. Madoroba

Chapter 3

Emergence of New Virulence Factors of Vibrio Cholera as Potential Vaccine Candidates Sujata Ghosh and Shalmoli Bhattacharyya

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vii

Methods Based on Binding Affinity of the B-Subunit of Cholera Toxin to Gangliosides in Biochemistry and Histology František Šmíd, Jana Ledvinová, Tomáš Petr, Jaroslava Šmídová and Libor Vítek

Chapter 5

Quorum Quenching: Imminent Targets in Vibrio cholerae S. Princy Adline, R.Vinothkannan, Rekha Arya and Sabu Thomas

Chapter 6

Vibrio Cholerae Biofilm and Virulence Gene Expression: A Perception Towards Quorum Sensing S. Princy Adline, N.Srinath, Shrivastava Sajal, and Sabu Thomas

Chapter 7

Chapter 8

Ability to Neutralize Cholera Toxin of Mammalian Milk through Expression of Its Receptor, Ganglioside GM1 Masao Iwamori The Environmental Reservoirs and Vector of Vibrio Cholerae Malka Halpern and Ido Izhaki

Cholera: Symptoms, Diagnosis and Treatment : Symptoms, Diagnosis and Treatment, edited by Evelyn L. Melbourne, Nova Science Publishers,

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63

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vi Chapter 9

Contents Impact of Climate and Environmental Factors on the Epidemiology of Vibrio cholerae in Aquatic Ecosystems Violeta Trinidad Pardío Sedas

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Index

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159 193

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Preface Cholera is a life-threatening diarrheal disease that can spread rapidly and in explosive epidemics from one region to another, affecting large numbers of people. V. cholerae, a gramnegative motile bacterium, is the causative agent of this intestinal disease. This book presents topical research from across the globe in the study of cholera; its symptoms, diagnosis and treatment. Some topics discussed, herein, include emergence of new virulence factors of vibrio cholera as potential vaccine candidates; possible targets for quorum quenching of vibrio cholerae; the environmental reservoirs and vector of vibrio cholerae; and current African perspectives of cholera. Chapter 1- Vibrio cholerae, a Gram-negative bacterium is known as the causative agent of cholera, acute watery diarrhoea. Out of approximately 206 serogroups of V. cholerae, only O1 and O139 are thought to be capable of causing epidemic cholera. These serogroups contain genes for cholera toxin (CT) and toxin-coregulated pilus (TCP). Strains belonging to about 204 other than O1 and O139 serogroups are collectively referred to as non-O1, nonO139 strains. The majority of non-O1, non-O139 strains do not contain genes for CT and/or TCP, although the gene for toxin regulatory protein (ToxR) is genaerally present in these strains. These strains are ubiquitous in aquatic environments and have been recognized as the causative agents of sporadic cholera-like disease and outbreaks. Over the passed two decades, a sharp increase in the number of non-O1, non-O139 infections worldwide has been observed. Furthermore, non-O1, non-O139 V. cholerae is also associated with extra-intestinal infections such as inguinal skin, soft tissue, ear, wound, and primary pulmonary infections, bacteraemia causing hypotensive shock, leukopenia, decreased platelet counts, severe hypoalbuminemia, peritonitis and septicaemia. Although V. cholerae O10, O12, O31, O37, O53, and O141 serogroups are reported to be associated with cholera-like epidemics, it is yet to be established whether a definite sets of serotypes of non-O1, non-O139 are intrinsically more pathogenic than others. Furthermore, the pathogenic mechanisms by which these enteropathogens cause diarrhoea are not yet well established and the pathogenesis of invasive non-O1, non-O139 V. cholerae infections is not yet well understood. Recently, several evidences of occurrences of the toxin-coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, TCP island alone making the non-O1 strains susceptible to transduction with CTX phage, Vibrio seventh pandemic islands (VSP-1 and VSP-2), type III secretion system (TTSS) genes, and putative accessory virulence genes (mshA, hlyA, and rtx) among the non-O1, non-O139 isolates have been documented. Since some of the pathogenic strains of V. cholerae O1 and O-139 retain these genes, they might

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Evelyn L. Melbourne

play an important role in the emergence of newer V. choleare pathogens most likely depending on assembling a grouping of genes for both ecological suitability and virulence to attain epidemiological prevalence. In addition, a high proportion of reported V. cholerae nonO1, non-O139 isolates from clinical and/environmental sources are multiple antibiotic resistant. Thus it has become increasingly clear that the non-O1, non-O139 serogroups are involved in the emergence of newer variant of V. cholerae. The present review will focus on the current perspectives on the disease spectrum, ecology and environmental studies, virulence factors and pathogenicity, biochemical characteristics, diagnosis and treatment of non-O1, non-O139, to clarify the public health significance and epidemiological potential of V. cholerae non-1, non-O139 infections. Chapter 2- Cholera, which is caused by pathogenic strains of Vibrio cholerae, continues to threaten public health and socio-economic development in many developing countries, particularly those that lack access to potable water and adequate sanitation. The cumulative total number of cholera cases reported to the World Health Organization (WHO) during 2004 to 2008 (838 315) showed an increase of 24% when compared to the period 2000 to 2004. Of the 5 143 cholera fatalities that occurred worldwide in 2008, 98% were from Africa. The emergence of new V. cholerae strains and the severity of recent cholera outbreaks that began in August 2008 in Zimbabwe, coupled with climate change and elevated antimicrobial resistance, highlighted the dynamics of cholera. For these reasons, cholera was placed at the forefront of world public health agenda. Transmission of V. cholerae is through the faecaloral route after ingestion of contaminated water and food. Previously, it was assumed that cholera was only spread by infected people to susceptible hosts through faecal contamination of water and food. However, it is now known that V. cholerae are normal inhabitants of surface water and can survive and multiply independent of the human host. These strains may then cause cholera depending on environmental factors, hence a thorough understanding of the ecology of V. cholerae is inevitable to minimise contact with this pathogen. Laboratory diagnosis of cholera involves classical microbiological methods, serotyping and molecular based techniques that target specific virulence genes of V. cholerae. However, these methods alone may not reflect the true burden of cholera; hence, the WHO standard case definition must be used alongside laboratory diagnosis of this disease. The primary treatment of cholera involves electrolyte replacement through oral rehydration. Whilst antibiotics are often indicated for treating severe cholera cases, the standard medical approach for prophylaxis against cholera entails vaccination against pathogenic V. cholerae strains. Nevertheless, the protection of cholera vaccines against HIV-positive individuals is yet to be determined. Despite the advantages of treatment and prophylaxis, cholera transmission can be contained effectively by moving from a reactive to a prevention approach. For this purpose, holistic principles that encompass a coordinated multidisciplinary approach, which ensures potable water supply, protection of water distribution systems, improved sanitation and health education must be used. In this review, I discuss the recent cholera outbreaks in Africa, with particular emphasis on the causes, epidemiology, virulence factors, diagnosis, treatment and possible methods of curbing V. cholerae-induced epidemics in future. Chapter 3- Microbes were the first organisms on earth and preceded animals and plants by more than 3 billion years. They are the foundation of the biosphere from both environmental and evolutionary perspective (Staley et al., 1997). It has been estimated that microbial species comprise about 60% of the earth‘s biomass. They show far more genetic, metabolic and physiological diversity as compared to plants and animals. So far only 1% of

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the estimated 2-3 billion microbial species have been identified. The biological diversity of those species is extraordinary, having adapted to grow under extremes of temperature, pH, salt concentration and oxygen levels (Fraser et al., 2000). It is important to remember that the vast majority of microbial species cannot be cultivated at all and these organisms, which live in microbial communities, are essential to the overall ecology of the planet. Some microbes are beneficial for the human beings while there are many which are harmful and cause infectious diseases and were the leading cause of death worldwide at the beginning of the twentieth century. In US, three diseases, tuberculosis, pneumonia and diarrheal diseases – caused 30% of deaths (CDC, Atlanta, 1994). Surprisingly, by 1900, morbidity and mortality from infectious diseases had already considerably improved in much of the developed world. The improvement in life-span and decrease in mortality from infectious disease were attributed to a series of factors such as better nutrition and housing, safer food and water and improved hygiene & sanitation combined with the use of antibiotics. Between 1900 and 1980, mortality from infectious disease fell from 797 to 36 per 100,000 (Armstrong et al., 1999). It seemed that the long struggle for control over infectious diseases was almost over. However, the situation has changed drastically in the last two decades. The dawn of the new millennium has also heralded an infectious disease crisis of global proportions threatening hard-won gains in health and life expectancy. Rapid urbanization across the globe without safe water and sanitation and general deprivation has increased the incidence of infectious diseases. The end of twentieth century saw the emergence of new infectious diseases e.g. Legionnaire‘s disease, Lyme disease, toxic shock syndrome, AIDS and many others. Diseases such as cholera, tuberculosis, dengue fever, yellow fever and malaria, which had once been controlled in many parts of the world, started re-emerging. In 1998, the World Health Organization estimated that infectious diseases caused over 13 million deaths, almost a quarter of the 54 million deaths worldwide (WHO, 1999). Resistance to antimicrobial agents became a serious global problem (Cohen, 2000). Chapter 4- Cholera toxin is a heat labile enterotoxin produced by Vibrio cholerae. The toxin consists of an A-subunit (with enzymatic activity) and B-subunit (CTB) which have very high binding capacities to ganglioside GM1(Gal1-3GalNAc 1-4(NeuAc2-3)Gal14Glc1-1Cer); the main receptor molecule on the surface of the target cells in a cholera infection (e.g. epithelial cells of the small intestine). Several other cross-reactive gangliosides have been described as CTB receptors (e.g. GD1b ganglioside); however, their affinity is low, and therefore high concentration of receptor ganglioside is necessary. Additionally, other CTB-reactive gangliosides, e.g. Fucosyl GM1 ganglioside (Fuc-GM1), are usually present in negligible amounts in the tissues; however, producing very little contribution towards the reaction. The binding affinity of CTB to GM1 is used for the detection of GM1 or the other gangliotetraose-type gangliosides, produced by Clostridium perfingens neuraminidase treatment via the following methods: (a) the ELISA assay, with gangliosides adsorbed by the ceramide portion of the polystyrene microwells, and (b) TLC overlay methods, with or without pre-treatment with neuraminidase. Fluorochrome labeled CTB targeting of GM1 is widely used in the analyses of raft lipids using ELISA, TLC overlay, and flow cytometry. The subcellular distribution of GM1, as a representative of the ganglioside family, can be done similarly; however, its binding affinity in histochemical or cytological applications is not strictly specific for GM1 and thus some cross-reactive gangliosides (e.g. GD1b) can cause

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additional staining. Therefore, for critical analysis, TLC is recommended as a complementary step for in situ detection of cells or tissue sections. In our laboratory, CTB detection was applied, in order to study the changes of the content and localization of GM1 ganglioside in both normal and cholestatic rat liver. In normal rat liver, GM1 was localized in the canalicular and sinusoidal hepatocyte membranes, in both the peripheral and intermediate zones of the hepatic lobules; it was nearly absent in the central zone. In rats with cholestasis, induced by 17-ethinylestradiol, GM1 was also expressed in the central lobular zone, and a shift of GM1 staining from intracellular to sinusoidal localization was observed. These changes were correlated with the concentration of serum bile acid. The GM1 content together with the GM1-synthase mRNA expression remained unchanged. The redistribution of GM1 into the sinusoidal membrane in a situation of limited biosynthesis could be responsible for the protection of hepatocyte membranes against the strong detergent effects of bile acids, accumulated during cholestasis. Chapter 5- The rapid emergence of microbes that resist most commonly used and even newly developed antibiotics has emphasized the need for the development of new strategy against infectious diseases. As similar to many opportunistic pathogens Vibrio cholerae also rely on Quorum sensing (QS), a bacterial cell-to-cell communication system for biofilm formation and virulence character expressions. Since the QS is crucial for execution of pathogenesis called Quorum quenching or Anti-pathogenic approach which will be the promising alternative strategy to contain infectious diseases that abolishes the communication networks. As QS does not unswervingly involved in the elementary processes of bacterial growth, inhibition of QS does not enforce harsh selective pressure which brings forth resistance as like as with antibiotics. This article discussed about diverse possible targets of Quorum quenching of Vibrio cholerae for the attenuation of Quorum sensing. Chapter 6- Vibrio cholerae has a very peculiar quorum sensing mechanism working at a low cell density observed with respect to biofilm formation and virulence factors production in contrast to other Vibrio species where quorum sensing works at higher cell density. There are two basic mechanism along with an unknown mechanism involved in this quorum sensing, that is, production of Cholera Autoinducer-1 (CAI-1) whose synthesis depends on CqsA sensed by CqsS and Autoinducer-2 (AI-2) synthesized by luxS interacting with luxP/Q sensors, responsible for intraspecies and interspecies communication respectively. These interaction results in exopolysaccharide production [EPS] leading to biofilm formation triggering virulence gene expression. Thus, the appropriate environment adapted by these aquatic pathogenic organisms causes a differential shift in the cell-to-cell communication making it as an essential aspect for understanding. Chapter 7- Ganglioside GM1 is a well characterized receptor for cholera toxin, and participates in the ganglio-series ganglioside a-pathway, which is the major route for ganglioside synthesis in neural tissues, but is only present at a trace level in non-neural tissues of man. For example, in human milk, two major gangliosides, GM3 and GD3, comprise 7090% of the total gangliosides in the milk fat globule membrane, which arises from the apical surface of epithelial cells in mammary glands, and GM1 is present in a trace amount, which is not detectable with convenient chemical procedures. The major ganglioside, comprising 70% of the total lipid-bound sialic acid in the colostrums, is GD3, which gradually decreases during the course of lactation, being only present in a trace amount in later milk. Alternatively, GM3, as a precursor glycolipid for the synthesis of GD3, is the major one in the later milk, indicating that GD3 synthase belonging to the b-pathway is suppressed during

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Preface

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the process of lactation. Also, changes in the amounts of IV6NeuAcα-nLc4Cer and GT3, belonging to the neolacto-series and ganglio-series c-pathways, respectively, occur in the similar manner as for that of GD3. In contrast to the metabolic changes of the major gangliosides in human milk during the lactation period, the concentration of GM1 is maintained at a constant level amounting to 0.01-0.08% of the total lipid-bound sialic acid throughout the lactation periods examined. The binding of 30 ng of cholera toxin B-subunit to GM1 on a TLC plate is inhibited by 93% with the total gangliosides from 1 ml of human milk containing 4 pmol of GM1, indicating that GM1 in human milk is strongly reactive with cholera toxin. GM1 is also present in the milk of cows (Holstein) and goats (Japanese Saanen) in the amounts of 0.72 and 8.12 nmol per 100 ml of milk, respectively, indicating that the synthesis of GM1 is expressed in the mammary glands of several mammalian species for the protection of neonates from cholera toxin-induced diarrhea. Chapter 8- Cholera is a life-threatening diarrheal disease that can spread rapidly and in explosive epidemics from one region to another, affecting large numbers of people. V. cholerae, a gram-negative motile bacterium, is the causative agent of this intestinal disease. To date, more than 200 serogroups of V. cholerae have been recorded, of which only two (O1 and O139) have been associated with major epidemics. Cholera spreads in pandemics; however, the mechanism that enables V. cholerae to cross water bodies, including oceans, is still puzzling. V. cholerae proliferates while attached to or associated with eukaryotic organisms in the aquatic environment, particularly copepods (Crustacea). Chironomids (Diptera) were also found to serve as intermediate 'host' reservoirs for V. cholerae. Recently, it was found that both copepods and chironomids are dispersed by migratory waterbirds, which either consume them (endozoochory) or carry them externally (epizoochory). Evidence on epidemic V. cholerae strains that were isolated from waterbirds was published about twenty years ago but failed to attract the attention of the scientific community. Hence we conclude that waterfowl might be responsible for the dissemination of V. cholerae between continents, and thus for the pandemicity of cholera. Better understanding of the species of waterfowl that carry V. cholerae and their migration patterns might therefore be useful in predicting future outbreaks of cholera. Chapter 9- The global and anthropogenic climate change and variability, mainly global warming, is having measurable effects on ecosystems, communities, and populations. The combination of climate change and environmental degradation has created ideal conditions for the emergence, resurgence and spread of infectious diseases, and has led to growing concerns due to the effects of climate on health. Diverse environmental factors affect the distribution, diversity, incidence, severity, or persistence of diseases and other health effects something that has been recognized for millennia. An important risk of climate change is its potential impact on the evolution and emergence of infectious disease agents. Evidence is indicating that the atmospheric and oceanic processes that occur in response to increased greenhouse gases in the broad-scale climate system may already be changing the ecology of infectious diseases. Ecosystem instabilities brought about by climate change and concurrent stresses such as land use changes, species dislocation, and increasing global travel could potentially influence the genetics of pathogenic microbes through mutation and horizontal gene transfer, giving rise to new interactions among hosts and disease agents. Recent studies have shown that climate also influences the abundance and ecology of pathogens, and the links between pathogens and changing ocean conditions, including human diseases such as cholera. Vibrio cholerae is well recognized as being responsible for significant mortality and

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economic loss in underdeveloped countries, most often centered in tropical areas of the world. Generally, V. cholerae is transmitted through contaminated food and water in communities that do not have access to proper sewage and water treatment systems, and is thus called, ― the disease of poverty‖. During the last three decades, extensive research has been carried out to elucidate the virulence properties and the epidemiology of this pathogen. Within the marine environment, V. cholerae is found attached to surfaces provided by plants, filamentous green algae, copepods, crustaceans, and insects. The specific environmental changes that amplified plankton and associated bacterial proliferation and govern the location and timing of plankton blooms have been elucidated. Several studies have demonstrated that environmental non-O1 and non-O139 V. cholerae strains and V. cholerae O1 El Tor and O139 are able to form a three-dimensional biofilm on surfaces which provides a microenvironment, facilitating environmental persistence within natural aquatic habitats during interepidemic periods. Revealing the influence of climatic/environmental factors in seasonal patterns is critical to understanding temporal variability of cholera at longer time scales to improve disease forecasting. Recently, researchers have also been elucidating the environmental lifestyle of V. cholerae, and ecologically based models have been developed to define the role of environment, weather, and climate-related variables in outbreaks of this disease. This chapter provides current evidence for the influence of environmental factors on Vibrio cholerae dynamics and virulence traits of this organism, and the urgent need for action to prevent the consequences of climate change contributing to cholera.

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Chapter 1

Current Perspectives on the Pathogenesis of Vibrio cholerae Non-O1, Non-O139 Prasanta K. Bag*

Department of Biochemistry, University of Calcutta, Kolkata, India

Abstract Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

Vibrio cholerae, a Gram-negative bacterium is known as the causative agent of cholera, acute watery diarrhoea. Out of approximately 206 serogroups of V. cholerae, only O1 and O139 are thought to be capable of causing epidemic cholera. These serogroups contain genes for cholera toxin (CT) and toxin-coregulated pilus (TCP). Strains belonging to about 204 other than O1 and O139 serogroups are collectively referred to as non-O1, non-O139 strains. The majority of non-O1, non-O139 strains do not contain genes for CT and/or TCP, although the gene for toxin regulatory protein (ToxR) is genaerally present in these strains. These strains are ubiquitous in aquatic environments and have been recognized as the causative agents of sporadic cholera-like disease and outbreaks. Over the passed two decades, a sharp increase in the number of non-O1, non-O139 infections worldwide has been observed. Furthermore, non-O1, nonO139 V. cholerae is also associated with extra-intestinal infections such as inguinal skin, soft tissue, ear, wound, and primary pulmonary infections, bacteraemia causing hypotensive shock, leukopenia, decreased platelet counts, severe hypoalbuminemia, peritonitis and septicaemia. Although V. cholerae O10, O12, O31, O37, O53, and O141 serogroups are reported to be associated with cholera-like epidemics, it is yet to be established whether a definite sets of serotypes of non-O1, non-O139 are intrinsically more pathogenic than others. Furthermore, the pathogenic mechanisms by which these enteropathogens cause diarrhoea are not yet well established and the pathogenesis of *

Corresponding author: E.mail address: [email protected], Fax: 91-33-2461 4849, Telephone: 91-3324615445

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2

Prasanta K. Bag invasive non-O1, non-O139 V. cholerae infections is not yet well understood. Recently, several evidences of occurrences of the toxin-coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, TCP island alone making the non-O1 strains susceptible to transduction with CTX phage, Vibrio seventh pandemic islands (VSP-1 and VSP-2), type III secretion system (TTSS) genes, and putative accessory virulence genes (mshA, hlyA, and rtx) among the non-O1, non-O139 isolates have been documented. Since some of the pathogenic strains of V. cholerae O1 and O-139 retain these genes, they might play an important role in the emergence of newer V. choleare pathogens most likely depending on assembling a grouping of genes for both ecological suitability and virulence to attain epidemiological prevalence. In addition, a high proportion of reported V. cholerae non-O1, non-O139 isolates from clinical and/environmental sources are multiple antibiotic resistant. Thus it has become increasingly clear that the non-O1, non-O139 serogroups are involved in the emergence of newer variant of V. cholerae. The present review will focus on the current perspectives on the disease spectrum, ecology and environmental studies, virulence factors and pathogenicity, biochemical characteristics, diagnosis and treatment of non-O1, non-O139, to clarify the public health significance and epidemiological potential of V. cholerae non1, non-O139 infections.

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Introduction Cholera, acute watery diarrhoea is a serious epidemic disease caused by the toxigenic strains of a Gram-negative bacterium Vibrio cholerae. Cholera may have been derived from the Greek words; chole (bile) and rein (flow), meaning the flow of bile in that language. The nature of the diarrhoea is characterized by voluminous watery stools, often accompanied by vomiting and resulting in hypovolemic shock and acidosis. There has been a sharp increase in the number of cholera cases worldwide. Seven distinct pandemics of cholera have occurred since the onset of the first pandemic in 1817. During the fifth pandemic, Robert Koch isolated the causative organism of cholera, referred to as ― comma bacilli‖ from rice water stools of patients in Egypt in 1883 and in India in 1884. The pandemics arose in the Indian subcontinent, usually the Ganges delta, and spread to other continents except for the seventh pandemic, which originated on the island of Sulawesi in Indonesia (Faruque et al., 1998). V. cholerae serogroup O1 are classified into two biotypes, El Tor and classical, on the basis of several phenotypic characteristics. In addition V. cholerae O1 is classified into two serotypes, Inaba and Ogawa, based on agglutination in antiserum. A possible third serotype, Hikojima, has been described, but it is very rare. Six pandemics were due to classical biotype and seventh pandemic had been caused by El Tor biotype. The seventh pandemic began in 1961 and is caused by a specific clone of El Tor O1 V. cholerae (Faruque et al., 1998). In 1991, the El Tor O1 seventh pandemic strain was introduced from Asia into S. America and caused a major cholera epidemic (Wachsmuth et al., 1993). Currently, the El Tor biotype is responsible for virtually all of the cholera cases throughout the world, and classical isolates are not encountered outside of Bangladesh. Out of approximately 206 serogroups of V. cholerae, only O1 and O139 are thought to be capable of causing epidemic cholera. These serogroups contain genes for cholera toxin (CT) and toxin-coregulated pilus (TCP). Within the O1 and O139 serogroups, the ability to produce CT is a major determinant of virulence. Strains belonging to about 204 other than O1 and O139 serogroups are collectively referred to as non-O1, non-O139 strains. The majority of non-O1, non-O139 strains do not contain genes

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Current Perspectives on the Pathogenesis of Vibrio Cholerae Non-O1, Non-O139

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for CT and/or TCP, although the gene for toxin regulatory protein (ToxR) is ubiquitously present in these strains (Faruque et al., 1998). These strains are ubiquitous in aquatic environments and have been recognized as the causative agents of sporadic cholera-like disease and outbreaks. However, non-O1, non-O139 V. cholerae is also associated with extraintestinal infections such as inguinal skin, soft tissue, ear, wound, and primary pulmonary infection, bacteraemia, septicaemia etc. Cholera surveillance is in progress in many countries, based primarily on detection of V. cholerae O1 and O139 and determining the presence of cholera toxin using biological and molecular methods (Rivera et al., 2001). However, virulence-associated factors in V. cholerae isolates including non-O1, non-O139 from environmental sources are of concern (Rivera et al., 2001). The emergence of serogroup O139 as a second etiologic agent of cholera epidemics, along with the possible conversion of non-O1 to O1 serotype (Colwell et al., 1995), and emergence of V. cholerae O10, O12, O31, O37, and O53 as bacterial serogroups associated with cholera-like epidemics (Bagchi et al., 1993; Dakin et al., 1994; Dalsgaard et al., 1995; Morris et al., 1990; Mukhopadhyay et al., 1995; Rudra et al., 1996; Sharma et al., 1998) has provided further attention on the non-O1, non-O139 V. cholerae strains. However, the pathogenic mechanisms by which these enteropathogens cause diarrhoea are not yet well established. It has become increasingly clear that the non-O1, non-O139 serogroups are involved in the emergence of newer variant of V. cholerae, such as the genesis of V. cholerae O139 which is believed to have evolved as a result of horizontal gene transfer between the O1 and the non-O1 serogroups. Bik et al. (1996) reported that the O139 strain is closely related to the epidemic O1 El Tor biotype and the O37 Sudan strain was genetically closely related to classical O1 strains. In addition, similar to O139 Bengal, O37 Sudan lacked most of the O1 antigen cluster but did contain flanking genes. The O139 Bengal emerged in 1992 replacing the existing O1 El Tor biotype, rapidly spread through all areas in India and its neighbouring countries with unprecedented event occurred in the history of the disease cholera and were suspected as the new pandemic of cholera. The O37 strain was responsible for a large cholera outbreak in Sudan in 1968 and was classified as a noncholera vibrio (Bik et al., 1996). Thus, O37 Sudan represents a second example of an epidemic V. cholerae strain carrying non-O1 antigens. The purpose of the present review is to present a current overview of V. cholerae non-O1, non-O139 and to focus on current perspectives on the disease spectrum, ecology and environmental studies, virulence factors and pathogenicity, biochemical characteristics, diagnosis, and treatment of these serogroups, to elucidate the public health significance and epidemiological potential of V. cholerae non-1, non-O139 infections.

Non-O1, Non-O139 Disease Spectrum Although only a minority of the strains of V. cholerae non-O1, non-O139 seems to be enteropathogenic, these strains have been recognized as the causative agents of sporadic cholera-like disease and outbreaks. The relative number of non-O1, non-O139 infections is on the increase worldwide. In 1993, an epidemic of a cholera-like disease occurred in Thailand that was associated with non-O1, non-O139 (Bagchi et al., 1993). V. cholerae non-O1, nonO139 strains were isolated as the causative agent of acute diarrhoea from the hospitalized patients in Kolkata, India (Ramamurthy et al., 1993). In 1994, an outbreak of diarrhoea

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Prasanta K. Bag

caused by non-O1, non-O139 occurred among volunteers in a vaccine trial study area in Lima, Peru (Dalsgaard et al., 1995). Clinically, 95% of the patients presented with watery diarrhoea with either no or mild dehydration and isolates recovered from diarrhoeal patients belonged to seven different serogroups, with serogroups O10 (21%) and O12 (65%) being predominant. Most of these isolates were susceptible to a variety of antimicrobial agents. The distribution and virulence of V. cholerae non-O1, non-O139 in India before, during and after the advent of O139 serogroup was investigated (Mukhopadhyay et al., 1995). A total of 68 strains belonging to 31 different 'O' serogroups were identified during this study period. With the exception of O53, there was no spatial or temporal clustering of any particular non-O1, non-O139 serogroup at any given place. A surveillance study on cholera had been conducted with hospitalized patients admitted to the Infectious Diseases Hospital, Kolkata, India, from 1993 to 1995 (Mukhopadhyay et al., 1996). Although the O139 and O1 serogroups dominated in 1993 and 1994-1995, respectively, the isolation rate of non-O1 non-O139 was around 4.9% throughout this study period. In 1996, an inexplicable rise in the incidence of non-O1 non-O139 V. cholerae occurred in Kolkata, India (Sharma et al., 1998) and the rate of isolation of non-O1, non-O139 strains of V. cholerae exceeded that of O1 and O139 serogroups during this period (between February and March, 1996). These strains were found to be devoid of the ctx filamentous phage (CTXф) (Waldor and Mekalanos, 1996) and some other virulence genes such as zot [encodes zonula occludens toxin (Zot)], ace [encodes accessory cholera enterotoxin (Ace)], and tcpA [encodes toxin-coregulated pilus subunit A (TcpA)] genes etc. (Sharma et al., 1998). Some strains of non-O1, non-O139 can cause clinically cholera-like diarrhoea by a mechanism that could be distinct from that employed by the toxigenic V. cholerae O1 and O139 strains. The nomenclature ‗Enteropathogenic V. cholerae’ (EPVC) was proposed to include these serotypes (Sharma et al., 1998). The incidence of EPVC had shown an upward trend from 1997 that continued into 1998 and in 1998, the EPVC strains continued one-third of the V. cholerae strains isolated from hospitalized patients in Kolkata (Garg et al., 1998). Sporadic cases of a severe gastroenteritis associated with V. cholerae serogroup O141 has been reported and these O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes (Dalsgaard et al., 2001). In another study, the occurrence of V. cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhoea constituted 27.4% (n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India (Chatterjee et al., 2009). In a previous study, between 1995 and 2000, microbiological records of children aged 0-15 years with cholera, who were treated at a hospital, Bangkok, Thailand, were retrospectively reviewed with respect to the serogroups, and antimicrobial susceptibility of the V. cholerae isolates (Pancharoen et al., 2004). Of the 95 stool specimens that were positive for V. cholerae, O1, and non-O1, non-O139 were found in 52.6% and 47.4%, respectively. The prevalence and antimicrobial susceptibilities of V.cholerae O1, O139, and non-O1, nonO139, isolated from cholera patients admitted to Infectious Disease Hospital, Delhi, India for nine years (1992-2000), were analyzed to determine the changing trends in their isolation and drug-resistance patterns (Das and Gupta, 2005). Of the 29, 196 stool samples, 13, 730 (47%) were positive for V. cholerae: 11, 091 for V. cholerae O1 (80.7%), 1, 943 (14%) for V. cholerae O139 and 696 (5%) were non-O1 and non-O139. In recent studies, non-O1, nonO139 V. cholerae has been recovered from food borne disease outbreaks (Wei et al., 2008) and from children with acute diarrhoea (González et al., 2009).

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Current Perspectives on the Pathogenesis of Vibrio Cholerae Non-O1, Non-O139

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V. cholerae non-O1, non-O139 infection caused mostly acute diarrhoeal disease. Recently, several reports indicated that non-O1, non-O139 V. cholerae is also associated with invasive extra-intestinal infections such as inguinal skin and soft tissue infection, bacteraemia causing hypotensive shock, leukopenia, decreased platelet counts, severe hypoalbuminemia, peritonitis and septicaemia. Furthermore, non-O1, non-O139 strains may cause ear, wound, and primary pulmonary infection. On the other hand, the pathogenesis of invasive non-O1, non-O139 V. cholerae infections remains to be determined. In recent study, the clinical manifestations and predisposing factors between bacteraemic and non-bacteraemic non-O1, non-O139 V. cholerae infections have been compared (Lee et al., 2007). Compared to patients with non-bacteraemic infections, patients with non-O1, non-O139 bacteraemia were significantly more likely to have cirrhosis and thrombocytopenia (0.0% vs 77.8% and 5.9% vs 72.2%, respectively; p