Textbook of Applied Microbiology: Including Infection Control and Safety [Second Edition] 9348385593, 9789348385598

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Second Edition

Anju Dhir PhD (Micro), MSc (Micro), BSc (Med) Ex Lecturer (Microbiology) Shivalik Institute of Nursing Shimla, Himachal Pradesh

CBS Publishers & Distributors Pvt Ltd • New Delhi • Bengaluru • Chennai • Kochi • Kolkata • Lucknow • Mumbai • Hyderabad • Jharkhand • Nagpur • Patna • Pune • Uttarakhand

Disclaimer Science and technology are constantly changing fields. New research and experience broaden the scope of information and knowledge. The authors have tried their best in giving information available to them while preparing the material for this book. Although, all efforts have been made to ensure optimum accuracy of the material, yet it is quite possible some errors might have been left uncorrected. The publisher, the printer and the authors will not be held responsible for any inadvertent errors, omissions or inaccuracies. eISBN: 978-93-483-8559-8 Copyright © Authors and Publisher Second e Book Edition: 2023

All rights reserved. No part of this eBook may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system without permission, in writing, from the authors and the publisher. Published by Satish Kumar Jain and produced by Varun Jain for CBS Publishers & Distributors Pvt. Ltd. Corporate O ice: 204 FIE, Industrial Area, Patparganj, New Delhi-110092 Ph: +91-11-49344934; Fax: +91-11-49344935; Website: www.cbspd.com; www.eduport-global.com; E-mail: [email protected] Head O ice: CBS PLAZA, 4819/XI Prahlad Street, 24 Ansari Road, Daryaganj, New Delhi-110002, India. Ph: +91-11-23289259, 23266861, 23266867; Fax: 011-23243014; Website: www.cbspd.com; E-mail: [email protected]; [email protected].

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Representatives Hyderabad Pune Nagpur Manipal Vijayawada Patna

Dedicated to My family members for their constant support and encouragement & The Almighty God, Whose blessings enabled me to accomplish this creative work

Reviewers’ List A Maria Therese

G Dhanalakshmi

(Medical Surgical Nursing) PhD (N), MSc (N), MA (Hosp. Admin), PGDG & C, PGD in Health Counseling,

PhD, MSc (N), (Psychology)

Professor Mother Theresa Post Graduate and Research Institute of Health Sciences (Government Institute), Puducherry

Vice-Principal and HOD Department of Medical Surgical Nursing Billroth College of Nursing Chennai, Tamil Nadu

Asmat Parveen

Gagandeep Kaur

(Medical Surgical Nursing) MSc (N), Post-basic BSc, PGDHHM

MSc (N)

Associate Professor cum Principal Syed Mantaqi Memorial College of Nursing & Medical Technology (IUST) Jammu and Kashmir

Clinical Instructor Akal College of Nursing Eternal University Baru Sahib, Himachal Pradesh

Charlotte Ranadive

Ghanshyam Jangir

MSc (N), Persuing PhD

(Medical Surgical Nursing) MSc (N)

Vice-Principal Army College of Nursing Deep Nagar, Jalandhar Cantt, Punjab

Assistant Professor Government College of Nursing Bikaner, Rajasthan

D Bhuvaneshwari

Kondal Naik Megavath

(Obstetrics and Gynecology Nursing) MSc (N), (Psychology)

(Medical Surgical Nursing) MSc (N)

Principal

Nursing Officer

Rathnamma College of Nursing, Gudur, Andhra Pradesh

Niloufer Hospital for Women & Children Hyderabad, Telangana

Eenu

Leela Verma

(Community Health Nursing) MSc (N)

PhD (Education), MSc (Child Health Nursing)

Assistant Professor Maharishi Markandeshwar Deemed to be University Mullana Ambala, Haryana

Principal Gautam College of Nursing Hamirpur, Himachal Pradesh

Emin Elsa Thomas

Manju Rajput

(Community Health Nursing) MSc (N), BSc (Hon.) (Nursing)

(Obstetrics and Gynecology Nursing) MSc (OBG), PhD Scholar

Tutor (on deputation)

Vice-Principal

College of Nursing, All India Institute of Medical Science New Delhi

School of Nursing Noida International University Greater Noida, Uttar Pradesh

The name of the reviewers are arranged in alphabetical order

Monika Sisodia

Priyanka Chouhan

MSc (Microbiology)

(Medical Surgical Nursing) MSc (N)

Lecturer Dr Chaudhary College of Nursing Udaipur, Rajasthan

Assistant Professor Himalyan College of Nursing Joginder Nagar, Himachal Pradesh

Neelam Dass

Sahil George Lal

(Medical Surgical Nursing), (Critical Care Nursing) MSc (N)

MSc (N)

Associate Professor

Apex Trust Nursing College Mirzapur, Uttar Pradesh

Mata Mohan Dai Oswal College of Nursing Ludhiana, Punjab

Assistant Professor

Nidhi Rao

Shailaja M J Mathews

MSc (N)

(Obstetrics and Gynecology Nursing) MSc (OBG)

Assistant Professor St Marys College of Nursing Lucknow, Uttar Pradesh

Assistant Professor Maharshi Karve Stree Shikshan Samstha Smt Bakul Tambat Institute of Nursing Education Pune, Maharashtra

Noble Sharma

Simarjeet Kaur

(Medical Surgical Nursing) MSc (N)

(Medical Surgical Nursing) MSc (N), PhD Scholar

Faculty

Nursing Tutor

Maa Hateshwari College of Nursing Khilra, Himachal Pradesh

Akal College of Nursing, Eternal University Baru Sahib, Himachal Pradesh

Pankaj Yadav

Timsy

(Medical Surgical Nursing) (Critical Care Nursing) MSc (N), BSc (N)

(Obstetrics and Gynecology Nursing) MSc (OBG)

Lecturer

Assistant Professor

Mahatma Gandhi Nursing College Sikar, Rajasthan

Desh Bhagat University Mandi, Gobindgarh, Punjab

Praveen Suthar

Varinder Kaur

(Mental Health & Psychiatric Nursing) MSc (N)

PhD, MSc (CHN)

Assistant Professor Bhagyalaxmi Nursing College Modasa, Arvalli, Gujarat

Principal HIMCAPES’ College of Nursing Badhera, Una, Himachal Pradesh

Priyanka Chauhan

Vikas Sood

(Medical Surgical Nursing), (Cardiothoracic Nursing)

PhD, MSc (N)

MSc (N)

Lecturer

Assistant Professor

Shivalik Institute of Nursing and Shimla Nursing College Shimla, Himachal Pradesh

HIMCAPES’ College of Nursing Badhera, Haroli, Una, Himachal Pradesh

The name of the reviewers are arranged in alphabetical order

Preface Microbiology is a vast field and it is important for the students to understand the subject from nursing perspective. The direct application of microbiology in healthcare settings makes it necessary to be understood as a basic necessity. Principles of microbiology are pillars of the nursing care practice. This book is an attempt to present the vast knowledge of microbiology in a simple and easy-to-understand language for degree course and post-basic students in the field of nursing. The book has been written keeping in view the revised syllabus recommended by the Indian Nursing Council. Not even a single topic is excluded as each topic has its unique importance. I have tried to provide all the basic concepts of microbiology that are needed by a nurse, while working in a hospital. The contents are expressed in lucid language accompanied by short and long answers. Nursing interventions given in the chapters make this book more useful to nursing students. MCQs given at the end of each chapter will help students to assess their grasp on the specific topic. “Facteria” provided in each chapter, serves as a way of imparting extra knowledge related to the topic. The texts are supplemented by tables, diagrams and figures. It makes a chapter easy to be apprehended by a reader. The book contains 42 chapters divided into two sections. Section A deals with Applied Microbiology along with basic concepts and Section B contains Infection Control including Safety in a Healthcare Setting. y SECTION A: Applied Microbiology will enable students to acquire understanding of fundamentals of Microbiology, compare and contrast different microbes and comprehend the means of transmission and control of spread by various microorganisms. It will also provide opportunities for practicing infection control measures in hospital and community settings. The section comprises 4 units. ƒ ƒ ƒ ƒ

y

Unit I Unit II Unit III Unit IV

: Introduction : General Characteristics of Microbes : Pathogenic Organisms : Immunity

SECTION B: Infection Control and Safety will help students to acquire knowledge and develop competencies required for fundamental patient safety and infection control in delivering patient care. It also focuses on identifying patient safety indicators, preventing and managing hospital-acquired infections, and in the following universal precautions. The section comprises 11 units. ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ

Unit I : Hospital-Acquired Infections Unit II : Isolation Precautions and Use of Personal Protective Equipment Unit III : Hand Hygiene Unit IV : Disinfection and Sterilization Unit V : Review of Specimen Collection Unit VI : Biomedical Waste Management (BMW) Unit VII : Antibiotic Stewardship Unit VIII : Patient Safety Indicators and Incidents and Adverse Events Unit IX : International Patient Safety Goals Unit X : Safety Protocols Unit XI : Employee Safety Indicators

The applied aspects of the topics are given with the chapters to understand their clinical applications. I hope that this textbook of microbiology will serve as a standard guide for microbiology in the field of nursing education. Happy reading!

Anju Dhir

Acknowledgments



“Let difficulties know you are too difficult.” —APJ Abdul Kalam

Teachers are also learners, and in a teacher-learner relationship, knowledge is co-constructed by the two in a partnership. I have felt that my knowledge increased day by day while teaching. Creative elucidation is impossible without modest acknowledgment to one’s helping hands. It is an essential part of any innovative work to fuel passion and make people feel acknowledged and cherished at the same time. I am gratified to “The Universe” for giving me inspiration and energy to do such a big task. Writing a book requires a lot of patience and I found that “Patience is also a Form of Action”. While doing this project, I evolved within. My sincere thanks go to My Mother for her constant support, and to my son Nityam, who has always been a reason to live. I owe gratitude to my students who always liked my lectures and inspired me to write this book. Therefore, thanks are due to my lovely students for their love and encouragement. In fact, it’s not money but the acknowledgment of his/her work that motivates a writer—a craving is always there in a writer for his or her work to be appreciated. I hope that my mental creation is cherished by all the readers. I extend my special thanks to Mr Satish Kumar Jain (Chairman) and Mr Varun Jain (Managing Director), M/s CBS Publishers and Distributors Pvt Ltd for their wholehearted support in publication of this book. I have no words to describe the role, efforts, inputs and initiatives undertaken by Mr Bhupesh Aarora [Sr. Vice President – Publishing & Marketing (Health Sciences Division)] for helping and motivating me. Last but not least, I sincerely thank the entire CBS team for bringing out the book with utmost care and attractive presentation. I would like to thank Ms Nitasha Arora (Publishing Head and Content Strategist – Medical and Nursing), Ms Daljeet Kaur (Assistant Publishing Manager) for their editorial support. I would also extend my thanks to Mr Shivendu Bhushan Pandey (Sr.  Manager and Team Lead), Mr Ashutosh Pathak (Sr. Proofreader cum Team Coordinator) and all the production team members for devoting laborious hours in designing and typesetting the book.

From Publisher’s Desk Dear Reader, Nursing Education has a rich history, often characterized by traditional teaching techniques that have evolved over time. Primarily, teaching took place within classroom settings. Lectures, textbooks, and clinical rotations were the core teaching tools; and students majorly relied on textbooks by local or foreign publishers for quality education. However, today, technology has completely transformed the field of nursing education, making it an integral part of the curriculum. It has evolved to include a range of technological tools that enhance the learning experience and better prepare students for clinical practice. As publishers, we’ve been contributing to the field of Medical Science, Nursing and Allied Sciences and earned the trust of many. By supporting Indian authors, coupled with nursing webinars and conferences, we have paved an easier path for aspiring nurses, empowering them to excel in national and state level exams. With this, we’re not only enhancing the quality of patient care but also enabling future nurses to adapt to new challenges and innovations in the rapidly evolving world of healthcare. Following the ideology of Bringing learning to people instead of people going for learning, so far, we’ve been doing our part by: •  •  •  • 

Developing quality content by qualified and well-versed authors Building a strong community of faculty and students Introducing a smart approach with Digital/Hybrid Books, and Offering simulation Nursing Procedures, etc.

Innovative teaching methodologies, such as modern-age Phygital Books, have sparked the interest of the Next-Gen students in pursuing advanced education. The enhancement of educational standards through Omnipresent Knowledge Sharing Platforms has further facilitated learning, bridging the gap between doctors and nurses. At Nursing Next Live, a sister concern of CBS Publishers & Distributors, we have long recognized the immense potential within the nursing field. Our journey in innovating nursing education has allowed us to make substantial and meaningful contributions. With the vision of strengthening learning at every stage, we have introduced several plans that cater to the specific needs of the students, including but not limited to Plan UG for undergraduates, Plan MSc for postgraduate aspirants, Plan FDP for upskilling faculties, SDL for integrated learning and Plan NP for bridging the gap between theoretical & practical learning. Additionally, we have successfully completed seven series of our Target High Book in a very short period, setting a milestone in the education industry. We have been able to achieve all this just with the sole vision of laying the foundation of diversified knowledge for all. With the rise of a new generation of educated, tech-savvy individuals, we anticipate even more remarkable advancements in the coming years. We take immense pride in our achievements and eagerly look forward to the future, brimming with new opportunities for innovation, growth and collaborations with experienced minds such as yourself who can contribute to our mission as Authors, Reviewers and/or Faculties. Together, let’s foster a generation of nurses who are confident, competent, and prepared to succeed in a technology-driven healthcare system.

Mr Bhupesh Aarora

(Sr Vice President – Publishing & Marketing) [email protected]| +91 95553 53330

Special Features of the Book

Every chapter starts with a Chapter Outline that gives the glimpses of the content covered in the chapter.

A number of Images are used to simplify the concepts for the students.

Numerous Tables provide necessary data in a concise way for better understanding.

Flow Diagrams are used in-between the text to enhance your learning experience in one go.

Textbook of APPLIED MICROBIOLOGY

Extra knowledge related to the respective topic is covered under the Facteria boxes.

Applied aspects of Microbiology covered under Applied Microbiology boxes extensively.

New and latest updates in the field of microbiology are covered under Recent Updates boxes.

Clinical correlation from nursing point of view have been covered under Nursing Interventions boxes.

xvi

Special Features of the Book

Case Scenario case-based discussion of concerned topics to correlate with clinical aspect.

Assess Yourself section has been dedicated to selfassess knowledge attained by a student.

xvii

Syllabus SECTION A: APPLIED MICROBIOLOGY Theory: 20 hours Practical: 20 hours (Lab/Experiential Learning – L/E) Unit Time (Hrs) T

Learning Outcomes

Content

P

I

3

Explain concepts and principles of microbiology and its importance in nursing

Introduction • Importance and relevance to nursing • Historical perspective • Concepts and terminology • Principles of microbiology

II

10

10 • Describe structure, (L/E) classification morphology and growth of bacteria • Identify microorganisms

General Characteristics of Microbes • Structure and classification of microbes • Morphological types • Size and form of bacteria • Motility • Colonization • Growth and nutrition of microbes • Temperature • Moisture • Blood and body fluids • Laboratory methods for identification of microorganisms • Types of staining – simple, differential (Gram’s, AFB), special – capsular staining (negative), spore, LPCB, KOH mount. • Culture and media preparation – solid and liquid. Types of media – semi synthetic, synthetic, enriched, enrichment, selective and differential media. Pure culture techniques – tube dilution, pour, spread, streak plate. Anaerobic cultivation of bacteria

III

4

6 • Describe the (L/E) different disease producing organisms

Pathogenic organisms • Microorganisms: Cocci – gram positive and gram negative; Bacilli – gram positive and gram negative • Viruses • Fungi: Superficial and Deep mycoses • Parasites • Rodents & Vectors ƒ Characteristics, source, portal of entry, transmission of infection, identification of disease producing microorganisms

Teaching/Learning Activities

Assessment Methods

• Lecture cum discussion

• Short answers • Objective type

• Lecture cum discussion • Demonstration • Experiential learning through visual

• Short answers • Objective type

• Lecture cum discussion • Demonstration • Experiential learning through visual

• Short answers • Objective type

Contd…

Textbook of APPLIED MICROBIOLOGY Unit Time (Hrs) T IV

3

Learning Outcomes

Content

P 4 Explain the concepts (L/E) of immunity, hyper sensitivity and immunization

• Immunity: Types, classification • Antigen and antibody reaction • Hypersensitivity reactions • Serological tests • Immunoglobulins: Structure, types & properties • Vaccines: Types & classification, storage and handling, cold chain, immunization for various diseases • Immunization schedule

Teaching/Learning Activities • Lecture • Discussion • Demonstration • Visit to observe vaccine storage • Clinical practice

Assessment Methods • Short answers • Objective type • Visit report

SECTION B: INFECTION CONTROL & SAFETY Theory: 20 hours Practical/Lab: 20 hours (Lab/Experiential Learning – L/E) Unit Time (Hrs) T

Learning Outcomes

Content

P

Teaching/Learning Activities

I

2

2 (E) Summarize the evidence based and effective patient care practices for the prevention of common healthcare associated infections in the healthcare setting

HAI (Hospital acquired Infection) • Hospital acquired infection • Lecture & • Bundle approach Discussion ƒ Prevention of urinary tract infection (UTI) • Experiential ƒ Prevention of surgical site infection (SSI) learning ƒ Prevention of ventilator associated events (VAE) ƒ Prevention of central line associated blood stream infection (CLABSI) • Surveillance of HAI – Infection control team & Infection control committee

II

3

4 (L) Demonstrate appropriate use of different types of PPEs and the critical use of risk assessment

Isolation Precautions and use of Personal Protective Equipment (PPE) • Types of isolation system, standard precaution and transmission-based precautions (Direct Contact, Droplet, Indirect) • Epidemiology & infection prevention – CDC guidelines • Effective use of PPE

III

1

2 (L) Demonstrate the hand hygiene practice and its effectiveness on infection control

IV

1

2 (E) Illustrates disinfection and sterilization in the healthcare setting

Assessment Methods • Knowledge assessment • MCQ • Short answer

• Lecture • Demonstration & Re-demonstration

• Performance assessment • OSCE

Hand Hygiene • Types of hand hygiene. • Hand washing and use of alcohol hand rub • Moments of hand hygiene • WHO hand hygiene promotion

• Lecture • Demonstration & Re-demonstration

• Performance assessment

Disinfection and Sterilization • Definitions • Types of disinfection and sterilization • Environment cleaning • Equipment cleaning • Guides on use of disinfectants • Spaulding‘s principle

• Lecture • Discussion • Experiential learning through visit

• Short answers • Objective type

Contd…

xx

Syllabus Unit Time (Hrs) T V

1

VI

2

VII

2

VIII

3

Learning Outcomes

Content

P

2(E)

Illustrate on what, when, how, why specimens are collected to optimize the diagnosis for treatment and management.

Specimen Collection (Review) • Principle of specimen collection • Types of specimens • Collection techniques and special considerations • Appropriate containers • Transportation of the sample • Staff precautions in handling

Explain on Biomedical waste management & laundry management

Biomedical Waste Management (BMW) Laundry management process and infection control and prevention • Waste management process and infection prevention • Staff precautions • Laundry management • Country ordinance and BMW National guidelines 2017: Segregation of wastes, Color coded waste containers, waste collection & storage, packaging & labeling, transportation

• Explain in detail about Antibiotic stewardship, AMR • Describe MRSA/ MDRO and its prevention

Antibiotic Stewardship • Importance of antibiotic stewardship • Antimicrobial resistance • Prevention of MRSA, MDRO in healthcare setting

5 • Enlist the patient safety indicators (L/E) followed in a health care organization and the role of nurse in the patient safety audit process

• Captures and analyzes incidents and events for quality improvement

Teaching/Learning Activities • Discussion

• Knowledge evaluation • Quiz • Performance assessment • Checklist

• Discussion • Demonstration • Experiential learning through visit

• Knowledge assessment by short answers, objective type • Performance assessment

• Lecture • Short answer • Discussion • Objective type • Written assignment • Assessment of –Recent AMR assignment (Antimicrobial resistance) guidelines

Patient Safety Indicators • Care of vulnerable patients • Lecture • Prevention of latrogenic injury • Demonstration • Care of lines, drains and tubing’s • Experiential • Restrain policy and care – physical and learning chemical • Blood & blood transfusion policy • Prevention of IV complication • Prevention of fall • Prevention of DVT • Shifting and transporting of patients • Surgical safety • Care coordination event related to medication reconciliation and administration • Prevention of communication errors • Prevention of HAI • Documentation Incidents and Adverse Events • Capturing of incidents • RCA (Root Cause Analysis) • CAPA (Corrective and Preventive Action) • Report writing

Assessment Methods

• Lecture • Role play • Inquiry based Learning

• Knowledge assessment • Performance assessment • Checklist/ OSCE

• Knowledge assessment • Short answers • Objective type Contd…

xxi

Textbook of APPLIED MICROBIOLOGY Unit Time (Hrs) T

xxii

Learning Outcomes

Content

P

IX

1

Enumerate IPSG and application of the goals in the patient care settings

IPSG (International Patient Safety Goals) • Identify patient correctly • Improve effective communication • Improve safety of high alert medication • Ensure safe surgery • Reduce the risk of health care associated infection • Reduce the risk of patient harm resulting from falls • Reduce the harm associated with clinical alarm system

X

2

3 Enumerate the various (L/E) safety protocols and its applications

Safety Protocol • 5S (Sort, Set in order, Shine, Standardize, Sustain) • Radiation safety • Laser safety • Fire safety ƒ Types and classification of fire ƒ Fire alarms ƒ Firefighting equipment • HAZMAT (Hazardous Materials) safety ƒ Types of spill ƒ Spillage management ƒ MSDS (Material Safety Data Sheets) • Environmental safety ƒ Risk assessment ƒ Aspect impact analysis ƒ Maintenance of Temp and • Humidity (Department wise) ƒ Audits • Emergency Codes • Role of Nurse in times of disaster

XI

2

• Explain importance of employee safety indicators • Identify risk of occupational hazards, prevention and postexposure prophylaxis.

Employee Safety Indicators • Vaccination • Needle stick injuries (NSI) prevention • Fall prevention • Radiation safety • Annual health check Healthcare Worker Immunization Program and Management of Occupational Exposure • Occupational health ordinance • Vaccination program for healthcare staff • Needle stick injuries and prevention and post exposure prophylaxis

Teaching/Learning Activities

Assessment Methods

• Lecture • Role play

• Objective type

• Lecture • Demonstration/ Experiential learning

• Mock drills • Post tests • Checklist

• Lecture method • Journal review

• Knowledge assessment by short answers, objective type • Short answer

Contents Reviewers’ List ......................................................................................................................................................................... v Preface ..................................................................................................................................................................................... vii Acknowledgments ................................................................................................................................................................... ix Special Features of the Book ................................................................................................................................................... xv Syllabus .................................................................................................................................................................................... xix

Section A: Applied Microbiology Unit I

Introduction

3–20



Chapter 1 Chapter 2

Unit II

General Characteristics of Microbes



Chapter 3 Structure and Classification of Microbes............................................................................. 23–30 Chapter 4 Morphology of Bacteria....................................................................................................... 31–42 Chapter 5 Colonization......................................................................................................................... 43–46 Chapter 6 Growth and Nutrition of Microorganisms............................................................................ 47–54 Chapter 7 Blood and Body Fluids.......................................................................................................... 55–60 Chapter 8 Laboratory Methods for Identification of Microorganisms.................................................. 61–66 Chapter 9 Detection of Motility and Staining Techniques.................................................................... 67–73 Chapter 10 Culture and Media Preparation............................................................................................ 74–84

Unit III

Pathogenic Organisms



Chapter 11 Gram-Positive Cocci............................................................................................................. 87–102 Chapter 12 Gram-Negative Cocci ........................................................................................................... 103–109 Chapter 13 Gram-Positive Bacilli (Part A)............................................................................................... 110–129 Chapter 14 Gram-Positive Bacilli (Part B)................................................................................................ 130–143 Chapter 15 Gram-Negative Bacilli (Part A).............................................................................................. 144–158 Chapter 16 Gram-Negative Bacilli (Part B).............................................................................................. 159–178 Chapter 17 Spirochetes........................................................................................................................... 179–185 Chapter 18 Miscellaneous Bacteria........................................................................................................ 186–202 Chapter 19 General Virology................................................................................................................... 203–210 Chapter 20 Viral Diseases (Part A).......................................................................................................... 211–227 Chapter 21 Viral Diseases (Part B).......................................................................................................... 228–241 Chapter 22 Viral Diseases (Part C)—DNA Viruses................................................................................... 242–249 Chapter 23 COVID-19 and Omicron Viruses........................................................................................... 250–255 Chapter 24 Oncogenic Viruses................................................................................................................ 256–259

History and Principles of Microbiology................................................................................ 5–17 Importance and Relevance of Microbiology in Nursing....................................................... 18–20

21–84

85–319

Textbook of APPLIED MICROBIOLOGY

Chapter 25 Parasitology.......................................................................................................................... 260–296 Chapter 26 Rodents and Vectors............................................................................................................ 297–302 Chapter 27 Medical Mycology................................................................................................................ 303–319

Unit IV Immunity

Chapter 28 Chapter 29 Chapter 30 Chapter 31

321–373 Immunity—Types and Classification.................................................................................... 323–330 Antigen-Antibody and their Reactions................................................................................. 331–348 Hypersensitivity Reactions................................................................................................... 349–361 Immunoprophylaxis—Vaccines and Sera............................................................................. 362–373

Section B: Infection Control and Safety Unit I

Hospital-Acquired Infections



Chapter 32 Prevention of Hospital-Acquired Infections......................................................................... 379–386

Unit II

Isolation Precautions and Use of Personal Protective Equipment



Chapter 33 Role of Standard Precautions in Clinical Settings................................................................. 389–395

Unit III

Hand Hygiene



Chapter 34 Importance of Hand Hygiene............................................................................................... 399–403

Unit IV Disinfection and Sterilization

377–386 387–395 397–403 405–433



Chapter 35 Disinfection and Sterilization in Healthcare Settings............................................................ 407–433

Unit V

Review of Specimen Collection



Chapter 36 Specimen Collection and Transportation............................................................................. 437–448

Unit VI Biomedical Waste Management (BMW)

449–461

Chapter 37 Management of Biomedical Waste and Laundry................................................................. 451–461

Unit VII Antibiotic Stewardship

435–448

463–470

Chapter 38 Judicious Use of Antibiotics.................................................................................................. 465–470

Unit VIII Patient Safety Indicators and Incidents and Adverse Events

471–488



Chapter 39 Patient Safety Indicators and Incidents in Healthcare Setting.............................................. 473–488

Unit IX

International Patient Safety Goals



Chapter 40 Patient Safety....................................................................................................................... 491–496

Unit X

Safety Protocols



Chapter 41 Safety Protocols.................................................................................................................... 499–509

Unit XI

Employee Safety Indicators



Chapter 42 Employee Safety................................................................................................................... 513–518

489–496 497–509 511–518

Index .............................................................................................................................................................................. 519–529 xxiv

Section

APPLIED MICROBIOLOGY

Unit I: Introduction Unit II: General Characteristics of Microbes Unit III: Pathogenic Organisms Unit IV: Immunity

A

UNIT

I

Introduction Microbiology is the study of unseen lives present in this environment which affect every sphere of our day to day routine. Jains were the first who talked about unseen microorganisms in the 6th century BC. Lord Mahavira talked about unseen creatures that are living in water, air and on earth and it is customary for Jain saints to cover their mouth with a piece of cloth so that these microorganisms are not killed by them, unknowingly. Later on with the development in technologies, especially microscope, it was proved that there are lives in our surroundings which cannot be seen but their effects are observed in the form of disease, spoilage of food or rotting of dead bodies. In the first unit of this book, the author has tried to discuss the importance of microbiology in the field of nursing along with relevant history.

LEARNING OBJECTIVE After going through this unit, you will be able to:  Explain concepts and principles of microbiology and its importance in nursing.

UNIT OUTLINE The chapters included in this unit are as follows: Chapter 1: History and Principles of Microbiology Chapter 2: Importance and Relevance of Microbiology in Nursing

History and Principles of Microbiology

Chapter Outline

• How Microbiology Evolved? • Golden Age of Microbiology ƒ Robert Koch and Principles of Microbiology ƒ Applications of Microbiology

INTRODUCTION Most people are familiar with the concept of microbes, or “germs”, and their role in human health. Although a few hundred years ago, before the invention of the microscope, the existence of different types of microbes was impossible to prove. Historical evidence suggests that humans had some notion of microbial life since prehistoric times.

HOW MICROBIOLOGY EVOLVED? Antony van Leeuwenhoek—Father of Microbiology History of microbiology began with the invention of the microscope. Microorganisms were first seen in 1675 by a Dutchman Antony van Leeuwenhoek (Fig. 1.1). His microscope consisted of a single biconvex lens that magnified about ×200 and resolved microbodies with a diameter of about 1 micron (μm) (Fig. 1.2). He found microorganisms in materials such as water, mud, saliva and intestinal contents of healthy people and recognized them as living creatures. He called them animalcules because they moved about actively. His drawings of the observed forms

1

CHAPTER

ƒ Microbiology in Nursing ƒ Antimicrobial Therapy ƒ Virology ƒ Immunology ƒ Scope of Medical Microbiology

are now recognized as cocci (spheres), bacilli (rods) and spiral filaments (spirochetes). Leeuwenhoek observed that large number of bacteria appeared in watery infusions of animal or vegetable matter, which were left to stand for a week or two at room temperature. He believed that these huge populations were the progeny of a few particular organisms, or seeds that were originally present in the materials of the infusion or had entered it from the air. Other scientists suggested that the organisms arose by spontaneous generation, i.e., by the spontaneous conversion of dead organic matter into living microbes and with Leeuwenhoek’s suggestion a controversy began that lasted for 200 years.

Louis Joblot In 1718, an experiment by Louis Joblot, who was a French microscopist ultimately settled the matter. He boiled a flask of an infusion of hay for 15 minutes to kill any microbe originally present in it. Covered it with a parchment cap to prevent later entry of other microbes from the air. It was observed that on subsequent standing, it remained sterile, i.e., free from microbial growth.

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

Fig. 1.1: Antony van Leeuwenhoek

Fig. 1.3: Francesco Redi’s experiment 1860–1910 is considered as the period in which maximum discoveries happened and this period is called Golden Age of Microbiology.

Louis Pasteur—The Progenitor of Modern Immunology

Fig. 1.2: Leeuwenhoek’s microscope

John Needham Needham in 1749, made similar effort, though not identical experiments, in which by contrast, the heated covered infusions showed growth of organisms after a period of time and this supported spontaneous generation.

Pasteur is called the progenitor of modern immunology (Fig. 1.4). His discoveries substantiated as foundations for microbiology and immunology. He performed a series of experiments to prove that the solutions could be made free of microorganisms by boiling. If no air is left to come in contact with solutions, there is no emergence of microbial life.

Germ Theory of Disease In his swan neck experiment (Fig. 1.5), he took broth in long necked flask. He softened the neck of flask under the flame and molded it in the shape of a swan neck. Broth was boiled until steaming and then cooled. This solution of broth did not show growth of microorganisms when kept for long periods because the microbes were not able to reach the broth.

Francesco Redi It was later shown by Francesco Redi, an Italian physician in mid-17th century that theory of spontaneous generation does not exist. He performed an experiment by taking pieces of meat and placed them separately in two jars and covered one of these jars. After a few days, maggots appeared in one of the jars which was left open. Redi concluded that because flies visited freely the open jar, the maggots appeared but in other jar that was closed or sealed, flies could not visit the meat piece and no maggots appeared (Fig. 1.3). Although his findings were not accepted. The controversy about the germ theory continued.

6

Fig. 1.4: Louis Pasteur

CHAPTER 1     History and Principles of Microbiology UNIT I     Introduction

Fig. 1.5: Swan neck experiment

Fig. 1.6: Joseph Lister

This further rejected the theory of spontaneous generation. The microbes could not enter the broth due to peculiar shape of the neck of flask. This experiment supported germ theory of disease. Germ theory is also known as theory of pathogenesis. Leeuwenhoek’s findings started building a foundation for germ theory. His contemporary scientific workers had this view that disease originates due to some curse when a person displeases the God—theory of spontaneous generation. But with the invention of microscope and other laboratory techniques, many scientists discovered pathogenic organisms and it slowly disapproved theory of spontaneous generation. In the golden age of microbiology, which covered a time period of 1857 to 1914, many microorganisms were discovered by scientists from time to time. Neisser discovered gonococcus in 1879, Ogston discovered the staphylococci, Loeffler isolated the diphtheria bacillus, Frankel described the pneumococcus, Bruce identified the causative agent of Malta fever, Schaudinn and Hoffmann discovered the spirochetes of syphilis in 1905 and many more microbes were discovered.

Davaine observed that anthrax bacillus was transmitted by inoculation of infected blood. Pasteur in 1861 proved that microorganisms are present in the environment and later he associated germs with diseases. He found the cause of wine souring and related this to growth of bacteria in the wine barrels. He discovered vaccines to protect against the diseases like cholera, rabies and anthrax. In 1860, Joseph Lister (Fig. 1.6) applied a phenolic compound for dressing the surgical wounds and found that it was effective in preventing surgical wound infections. He is also known as “Father of Antiseptic Surgery”. On getting good results by the application of phenol on wounds, he also introduced spray of carbolic acid in operation theaters (OTs) so that the growth of microorganisms could be prevented. Koch contributed by giving four postulates which proved as foundation in microbiology. He discovered causative organisms for tuberculosis, anthrax and cholera. He gave many microbiological techniques like staining technique, use of solid culture media and smear fixation by heat, etc.

Contributions to Germ Theory of Disease Fracastoro in 1546 told that “the disease is caused by minute ‘seed’ and it spreads from person to person”. In 1796, Jenner used cowpox virus to immunize against smallpox. He tried to protect against smallpox by inoculating a less virulent virus and Bassi in 1835 demonstrated that silkworm disease was due to microbial infections. Oliver Wendell Holmes (1809– 1894) suggested that sepsis during child birth was due to some germs being transmitted from mother to child. In 1840, Ignaz Semmelweis observed that by hand washing, transmission of puerperal sepsis was prevented. In 1845, Barkley proved that potato blight was caused by fungus Phytophthora infestans.

Applied Microbiology Knowledge of germ theory touched the working life of nurses and other health care workers and gave the understanding as follows: • A specific disease is caused by a specific microorganism. • Transmission of a disease is due to the presence of existing microorganism in the environment. • Hygienic practices were introduced in medical technology based on the germ theory. • Sterilization and disinfection methods were introduced after understanding germ theory. • Antibiotics were used for the first time in medical treatment. • Germ theory is the foundation for diagnostic microbiology. • The epidemic spread of disease can be prevented.

7

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY Alexander Fleming discovered first antibiotic penicillin in 1928 that proved as wonder drug in Second World War and saved many precious lives.

y

Contributions of Louis Pasteur In 1856, Pasteur was called by wine makers of Lille to investigate the cause of souring of alcohol being produced. He investigated and found that there was a gray material growth in the substrate used for manufacturing alcohol. This microorganism growing in alcoholic product was the cause of souring of wine. He found that these undesirable microorganisms could be killed by heating the wine at 50°– 60°C for a short period of time. This method is now popularly known as pasteurization and involves moderate heat treatments of the product to kill microorganisms. This process is now backbone of wine industry. Nowadays, pasteurization is widely used in milk industry, food and beverage industries. We can summarize his contributions as follows: y Pasteur discovered that fermentation of a substrate is caused by microorganisms. y Pasteur performed swan neck experiment to disapprove the theory of spontaneous generation. y He was the first one to use terms like aerobics for those organisms that can live in the presence of oxygen and

y y y

y

y y y

anaerobic for those organisms that prefer to live in the absence of oxygen. He discovered the process of pasteurization that is used nowadays to keep the food products for a long time, i.e., to increase shelf life of the product. He developed vaccines against cholera, anthrax and rabies, which were very deadly diseases at that time. He discovered silkworm disease known as pébrine and isolated the causative microorganisms of this disease. He introduced sterilization techniques and developed steam sterilization method, hot air oven and autoclave. These methods are backbone of the sterilization techniques and are widely used in health care industry, and food and beverage industry. He isolated microorganisms responsible for chicken cholera and rabies. He demonstrated that the causative agents of these diseases were too small to be seen through microscope and these could pass through filters. Later on this filterable agent of disease was known as virus. He suggested methods to control cross infections in hospitals. He introduced attenuated live vaccines for prophylactic use. He discovered microorganisms such as Staphylococcus, Streptococcus and Pneumococcus.

GOLDEN AGE OF MICROBIOLOGY ROBERT KOCH AND PRINCIPLES OF MICROBIOLOGY Robert Koch (Fig. 1.7) was a German physician and is well known for his contributions in the field of medical techniques, and is called Father of Microbial Techniques and Father of Medical Microbiology. He brought perfection in bacteriological techniques, staining methods, pure culture techniques, use of solid media to culture microorganisms. He isolated anthrax bacillus (1876), Mycobacterium tuberculosis (1882) and Vibrio cholerae (1883). As the reports were just pouring about the causative organisms of different diseases by different investigators, it became necessary to introduce tests to prove that the microorganisms isolated were indeed the causative agents of that particular disease. These tests serve now as a guideline to prove a relationship between the disease and the causative organism and these should be fulfilled before the organism can be confirmed as a real cause of disease. These criteria are known as Koch’s postulates and these are considered as principles of microbiology. 8

Koch’s Postulates Three postulates were formulated by Robert Koch and Friedrich Loeffler in 1884. In 1905, EF Smith added fourth postulate. These postulates were applied by Koch to find out the course of diseases like anthrax and tuberculosis (Fig. 1.8). These postulates are applicable for other diseases too.

Fig. 1.7: Robert Koch

CHAPTER 1     History and Principles of Microbiology

y

y

y

There are microorganisms which do not follow Koch’s postulates, e.g., the pathogens like Mycobacterium leprae and spirochetes. He believed that cholera and leprosy were caused by microorganisms but all criteria set according to Koch’s postulates are not fulfilled by these pathogens. Further, he proved that Vibrio cholerae could be isolated from both sick and healthy people, which invalidated postulate 2. The limitations of Koch’s postulates are even more intense in case of viral diseases where it was found that many viruses do not cause illness in every infected individual—it was the violation of postulate 1. Postulates 2 and 3 cannot be fulfilled for viruses that do not replicate in cell culture, or for infections for which a suitable animal species has not been identified like spirochetes or Mycobacterium species.

UNIT I     Introduction

y

Facteria

Fig. 1.8: Koch’s postulates—A summary

Postulate I—Association: The microorganism must be present in the body of suffering animal and should not be found in healthy animal. A particular microorganism should be associated with a particular disease. Postulate II—Isolation: The causative organism must be isolated from the body of diseased animal. This isolated organism must be cultured in laboratory to get a pure culture. Its growth characteristics and biochemical characteristics must be noted down. Postulate III—Inoculation: The isolated organism from pure culture must produce similar disease in a healthy laboratory animal. Postulate IV—Reisolation: The microorganism must be reisolated from the inoculated laboratory animal and identified as causative microorganism. The reisolated microorganism must show exactly the same growth and biochemical characteristics as recorded in second step. Later, a fifth criterion was also introduced, which clarifies that specific antibodies to the microorganism should be demonstrated in the patient’s serum suffering from the disease.

Limitations of Koch’s Postulates Koch’s postulates have severe limitations too, which even Koch realized as:

Modern Concepts of Koch’s Postulates Here are Koch’s postulates for the recent times as suggested by Fredericks and Relman: • The antigen in the form of nucleic acid (from a causative organism) must be present in majority of suffering people having same type of disease. The nucleic acid must be present in diseased organ when anatomically observed. • The nucleic acid of the pathogenic organism must not be present in a healthy person. • Nucleic acid of pathogen must disappear or reduce when the disease resolves. The nucleic acid sequence of a pathogen should: ƒ Establish association with disease. ƒ Be biologically consistent with characteristics of that particular group of organisms. ƒ The results obtained from nucleic acid sequences must be reproducible in other labs.

Contributions of Robert Koch Robert Koch has contributed in various ways in the field of microbiology. Some of these are mentioned here: y Before Koch, it was emphasized that the disease begins spontaneously. With his experiments, he proved the germ theory of disease and explained that disease is not due to curse but it originates from germs. y His four postulates are a guideline when an association between disease and causative microorganism is to be established. y In order to study morphology of an organism, he introduced staining techniques. y Nowadays, we all are familiar with heat fixing of the smear before staining. This was also introduced by Koch. 9

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY y

y y

y

He introduced the dyes for staining microorganisms like aniline dyes which are easy to work with, fast to act and give good results. Technique to obtain pure cultures of microorganism was developed by him. Agar is used in many European dishes as a solidifying agent from a long time. Koch was first to use agar as solidifier in culture media. This facilitated a scientist to observe colonial morphology of an organism when grown in labs. He introduced a skin test for diagnosing tuberculosis which is a hypersensitivity reaction.

A

APPLICATIONS OF MICROBIOLOGY Pasteurization

B

Pasteurization is a process of heating and then rapidly cooling liquids like milk, honey, juices or food items like eggs, butter and cheese in order to kill microorganisms that may spoil them or cause diseases (Figs 1.9A and B). Although every single microorganism in the product is not destroyed, for example bacterial spores. Today, the method is used widely in the dairy industry and other food processing industries to achieve food preservation and food safety. The pasteurization process is based on the use of one of the following time and temperature relationships. y High temperature short treatment (HTST): This process uses higher heat for less time to kill pathogenic bacteria, e.g., milk is pasteurized at 161°F (72°C) for 15 seconds. HTST causes less damage to the nutrient composition and sensory characteristics of food as compared to low temperature long treatment (LTLT). y Low temperature long treatment: This process uses lower temperature for a longer time to kill pathogenic bacteria. For example, milk is pasteurized by this method by exposing it to 145°F (63°C) for 30 minutes.

Figs 1.9A and B: A. Pasteurization–mechanism showing passage; B. Use of high and low temperature in process y

y

Flash pasteurization: In order to pasteurize food, “Flash pasteurization” method is used. It involves HTST in which pourable products like juices are heated for 3–15 seconds at a temperature of 74°C which destroys harmful microorganisms. After heating, the product is cooled and packed. Most drink boxes and pouches are pasteurized in this way because it allows extended unrefrigerated storage. Steam pasteurization: This technology uses heat to control or reduce harmful microorganisms in beef/meat. Beef is exposed to pressurized steam for approximately 6–8 seconds (Fig. 1.10). The steam raises the surface temperature of beef to 191°–200°F (88°–93°C). The carcasses are then cooled with a cold water spray. This process kills microorganisms like Escherichia coli, Salmonella and Listeria.

Fig. 1.10: Steam pasteurization used in meat processing factory

10

CHAPTER 1     History and Principles of Microbiology Irradiation pasteurization: Poultry products, red meat, spices, fruits and vegetables are all subjected to small amounts of gamma rays. This process effectively kills vegetative bacteria and parasitic food-borne pathogens. Shelf life of food items is also increased.

Tyndallization Tyndallization was discovered by John Tyndall (Fig. 1.11), who was a student of Pasteur. It is a process used for sterilizing substances, usually food and is also called fractional sterilization. He concluded that bacteria occur in two forms known as heat labile, which are easily killed when exposed to heat. Another form of bacteria is heat stable, which could not be killed by exposing to high temperature and are heat resistant. They could not be killed by continuous boiling of the broth and grow in the broth after some period of time. He further demonstrated that if such broths are subjected to intermittent (discontinuous) boiling-steaming at 100°C on three consecutive days for 30 minutes of each exposure, these heat resistant forms can be killed in broth and no microbial growth will occur thereafter in such broths as broth becomes germ free. At first exposure, the vegetative cells are killed, but spores may remain. At intervals, spores germinate and grow into vegetative cells which are killed during second exposure. On third exposure, the broth becomes completely free of germs and no growth occurs for prolonged period of time. Tyndallization process is usually effective in practice. But it is not very reliable because there are chances that some of the spores may survive which later germinate. Tyndallization method is mainly used for sterilizing plant seeds these days.

Fig. 1.11: John Tyndall

Fermentation Fermentation is a process where carbohydrates are broken down into alcohols and organic acids during anaerobic respiration. Here, carbon and energy (in the form of ATP) provide an electron donor and an electron acceptor in a series of coupled oxidoreduction reactions. This process leads to the formation of a variety of waste products like ethanol in cultures of yeasts, organic acids and alcohols in cultures of bacteria and a mixture of lactic, acetic, formic and succinic acids by the enterobacteria. Fermentation is accompanied by the production of both acids and gases like carbon dioxide and hydrogen. The fruit juices on incubation, in presence of yeast, give rise to wine and this is the main reaction taking place in wine production, but when this substratum gets contaminated by bacteria the wine gets sour. Pasteur solved this problem and he found that on heating this mixture, bacteria were destroyed and a good quality wine was obtained.

UNIT I     Introduction

y

Note Other applications include Sterilization and Disinfection; Aseptic Techniques; Culture methods; Isolation Techniques; Staining Methods; Use of PPE, etc. All these are discussed at relevant places in this book.

MICROBIOLOGY IN NURSING The nurses have to take care of patients—both indoor and outdoor. They are involved in various other aspects in a hospital such as collection of sample from appropriate sites, report reading, patient care, sterility checks in OTs, sterilization of medical equipment, disinfection of the wards and biomedical waste management. To perform these duties sincerely and accurately, a nurse must be well aware of the microbiological concepts. Some of the duties performed by a nurse in correlation with microbiology are mentioned  here: y Knowledge of microbiological aspects imparts a nurse with knowledge about handling a patient and his/her clinical samples, without infecting oneself. y Water is sufficient to kill microorganisms when boiled to high temperature. This knowledge is applied to sterilize instruments used in hospitals like metal instruments, surgical knives, blades, scissors and needles. y The use of antiseptics to minimize septic conditions caused by growth of microorganisms in the wounds. y Universal hand washing technique followed by all nurses is based on the fundamental principle of microbiology as hand washing reduces/removes the load of microorganisms and prevents transmission of hospital acquired infections too. 11

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY y

y

y

y

y

y

y

Different blood groups are identified in labs by a nurse based on the knowledge of immunology, which is a necessary task before blood transfusion. Hypersensitivity reaction like Mantoux test is used to check tuberculosis. This test is again based on immunological knowledge. The knowledge about reactions between specific antigens and antibodies is fundamental for the tests like radioimmunoassay (RIA), complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA), immunodiffusion and agglutination reactions. These tests are very useful in diagnostic labs. The aseptic procedures followed in OTs and minor OTs are based on the principles of sterilization— both gaseous and liquid states of sterilizing agents are used in this process. In a situation when a patient’s body does not respond to antibiotic treatment, antibiotic sensitivity test is done on the isolated organism from the infected site. It helps in selection of targeted treatment. The mass immunization programs are based on the knowledge of microbiology and immunology. These immunization derives have helped in eradicating dreadful diseases like smallpox and providing protection for diseases like mumps, hepatitis and diphtheria, etc. Biomedical waste management is necessary part of the hospital. Collection, segregation, transportation and disposal of biomedical waste are based on the principles of microbiology.

ANTIMICROBIAL THERAPY Paul Ehrlich Paul Ehrlich (Fig. 1.12A) was one of the earliest pioneers in the field of antimicrobial therapy. He along with Sahachiro Hata discovered Salvarsan to treat syphilis and Trypan red dye

A

B

Figs 1.12A and B: A. Paul Ehrlich; B. Alexander Fleming

12

Fig. 1.13: Action of penicillin—Original culture plate of Alexander Fleming

for treating African sleeping sickness. This was the foundation of chemotherapy. He also developed a method to stain the tubercle bacilli—the causative agent of tuberculosis.

Alexander Fleming Sir Alexander Fleming (Fig. 1.12B) discovered the wonder drug Penicillin from mold Penicillium notatum in 1929. The discovery was accidental. He found that the fungus Penicillium produced a substance that destroyed bacterial growth of Staphylococcus. He named it as penicillin. He also showed that the culture filtrate of mold inhibited the growth of Staphylococcus aureus. He called this substance penicillin (Fig. 1.13), which acted on Gram-positive bacteria. For the discovery of this antibiotic, Fleming, Florey and Chain got Nobel Prize in 1945. Penicillin eventually came into use during World War II as a result of the work of a team of scientists led by Howard Florey of the University of Oxford.

Selman Abraham Waksman Waksman and his coworkers isolated Actinomycin in 1940, Streptothricin in 1942, Streptomycin in 1943, and Neomycin in 1949. Streptomycin is produced by Streptomyces griseus. It is a secondary metabolite produced by Streptomyces griseus which is not required for its growth but helps it to compete with other bacteria for food and space in the environment. Streptomycin is used in the treatment of tuberculosis. For commercial purpose Penicillium chrysogenum is used in place of P. notatum as it gives more yield. This drug was later used in Second World War. The antibiotic era began from here. Later Streptomyces griseus was obtained from Streptomyces by SA Waksman in 1944 and he received Nobel

CHAPTER 1     History and Principles of Microbiology Later many new pathogens emerged like: AIDS (1981), Zika virus (2016), Ebola virus (2014), SARS coronavirus and avian influenza virus (1997). Middle East respiratory syndrome coronavirus (MERS-CoV) reported from Oman and Saudi Arabia on 19th December, 2016 was responsible for outbreak. The most newly emerged viruses are COVID-19 and Omicron. Many new pathogens will keep on introducing themselves in future.

VIROLOGY

IMMUNOLOGY

By the end of nineteenth century, bacterial etiology of a large number of diseases had been confirmed. But there were many diseases, like measles, smallpox, influenza, etc., for which bacterial etiology could not be assigned. The field of virology was introduced with Pasteur’s discovery of rabies. Later Ivanovsky (Fig. 1.14A) in 1892 showed that tobacco mosaic disease was caused by applying filtered juice of diseased plants to healthy plants. These filterable but virulent agents that were alive only in a host’s body were termed virions (poison). Beijerinck in 1898 confirmed previous findings and gave the name “virus” for such filterable infectious agents. The first human disease of viral origin was yellow fever discovered by Sir Walter Reed in 1902. Goodpasture (Fig. 1.14B) in 1930 developed a technique of virus cultivation in chicken eggs. Much development took place in field of virology with the discovery of electron microscope by Ruska (Fig. 1.14C) in 1934. With introduction of tissue culture, cultivation of viruses became easy. The possibility that a viral infection could lead to cancer was first put forth by Ellerman and Bang in 1908. Later in 1980, when human T cell leukemia was isolated, this theory was proved.

The study of host response to infection was also studied in the field of microbiology. In 1888, Nuttall observed that defibrinated blood or serum could kill bacteria and in 1889, Buchner showed that this effect can be removed by heating the sera for 1 hour at 55°C. This factor was given the name of Alexine, which was renamed as complement by Bordet in 1895. Antibody was discovered by von Behring and Kitasato in 1890 (Figs 1.15A and B). Metchnikoff (Fig. 1.15C) discovered phenomenon of phagocytosis and suggested that it was important in the body’s defense mechanism against the microbial invasions of tissues. Later on this led to the idea that cells were important in immunity and cellular concept of immunity came into existence.

A

B

UNIT I     Introduction

Prize for this discovery in 1952. The Streptomycin was used in treatment of tuberculosis. The role of opportunistic pathogens among immunocompromised patients in crowded hospitals was also analyzed and understood in 20th century. Efforts were made to eradicate diseases globally by various eradication programs and one of the dreadful diseases like smallpox was finally eradicated in 1977 worldwide.

Immunization An accidental discovery was made by Pasteur when culture of chicken cholera bacillus was left on the bench for several weeks. He found that this culture lost its ability to cause disease but could still protect against subsequent infection by this particular organism. Later on this process was given the name of attenuation and was used to prepare attenuated vaccines in 1881.

C

Figs 1.14A to C: A. Ivanovsky; B. Goodpasture; C. Ruska

13

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

A

B

Figs 1.15A to C: A. Von Behring; B. Kitasato Shibasaburō; C. Élie Metchnikoff

Fig. 1.16: Lady Mary Montagu

Lady Mary Montagu (Fig. 1.16) used the artificial induction of immunity by immunizing agents. She used the immunization practice for smallpox in Asia. Material from pustule of an infected person was scratched into the skin of a healthy person to be immunized. It resulted in mild form of smallpox and gave lifelong immunity. Edward Jenner in 1796 observed that his milkmaid who suffered from cowpox infection was protected against smallpox when they came in contact with smallpox virus. He introduced the term and technique of vaccination (vacca— cow), and used a similar but attenuated organism in healthy individuals. Later on, Pasteur developed attenuated vaccines against chicken cholera and anthrax in 1880 and against rabies in 1885.

SCOPE OF MEDICAL MICROBIOLOGY Medical microbiology is one of the recent and youngest sciences helpful in the diagnosis of the diseases. The other branches of microbiology like immunology, bacteriology, 14

C

virology and mycology have already emerged as separate independent sciences. Many microorganisms have been studied in detail and still studies are going on. Medical microbiology is mainly concerned with etiology, pathogenesis, laboratory diagnosis and treatment of infection along with the epidemiology and control of infection in the community. In order to manage a bacterial infection in a patient, first step is to establish a clinical diagnosis. Then the causative agent of the disease is to be isolated from the clinical sample. The antibiotic susceptibility test is determined in laboratory to give specific treatment before giving appropriate antibiotic. The whole therapeutic strategy is monitored. If antibiotic therapy is not successful then antimicrobial agents are observed in body fluids to give target treatment. Confirmation of bacterial cure is indicated by disappearing of signs and symptoms of the disease—known as follow-up. Mostly the physicians give treatment based on the signs and symptoms of a disease. But there are many diseases which may have same clinical conditions caused by a large number of organisms. In such situations, laboratory is needed so as to diagnose the organism and target the causative microorganism of the disease. Many times even when the clinical symptoms are evident like in urinary tract infections, it becomes difficult to prescribe an appropriate chemotherapeutic agent because a variety of organisms exist which can cause similar infection. In such cases, the laboratory investigation can provide confirmation about the causative organism. On the basis of investigation, it could be decided which drug is to be given. A clinical cure does not always indicate an absolute eradication of infection therefore a lab’s help is always needed. Major discoveries of bacteria and names of the Nobel Prize winners in microbiology are given in Table 1.1 and Table 1.2, respectively.

CHAPTER 1     History and Principles of Microbiology Year

Name of the scientist

Contributed to the…

1908

Ehrlich and Metchnikoff

Theories of immunity and phagocytosis

1945

Fleming, Chain and Florey

Penicillin

1952

Waksman

Streptomycin

1953

Krebs

Krebs cycle

1954

Enders, Weller and Robbins

Cultured polio virus in cell culture

1958

Lederberg, Beadle and Tatum

Genetic control of biochemical reactions

Organism

Discovered by

Year

Mycobacterium leprae

GH Hansen

1861

Bacillus anthracis

Robert Koch

1876

Micrococcus amorphous

Thomas Burrill

1878

Neisseria gonorrhoeae

Albert Neisser

1879

Malarial parasite

CL Laveran

1880

Staphylococcus

Ogston

1881

Pneumococcus

Pasteur and Sternberg

1881

Mycobacterium tuberculosis

Robert Koch

1882

1962

Watson, Crick and Wilkins

Physical structure of DNA

Bacillus of glanders

Loeffler and Schutz

1882

1965

Jacob, Monod and Lwoff

Corynebacterium diphtheriae

Klebs and Loeffler

1883

Regulation of protein synthesis

1966

Rous

Cancer-causing viruses

Vibrio cholerae

Robert Koch

1883

1969

Streptococcus

Fehleisen

1883

Delbruck, Hershey and Luria

Mechanism of viral infection in bacteria

Clostridium tetani

Nicolaier

1884

1972

Edelman and Porter

Escherichia coli

Escherich

1885

Nature and structure of antibodies

Neisseria meningitidis

Weichselbaum

1887

1980

Paul Berg

Brucella melitensis

Bruce

1887

DNA recombinant technology

1982

Aaron Klug

Structure of TMV

Tobacco mosaic virus

Ivanovsky

1892

1987

Susumu Tonegawa

Clostridium perfringens

Welch and Nuttall

1892

Genetics of antibody production

Babesia microti

Smith and Kilbourne

1893

1989

Cancer-causing genes

Yersinia pestis

Yersin

1894

Michael Bishop and Varmus

Spirillum desulfuricans

Beijerinck

1894

1997

Stanley Prusiner

Clostridium botulinum

Ermengem

1896

Prions and relation to neurological diseases

Shigella

Shiga

1896

2005

Marshall and Warren

Helicobacter pylori causes peptic ulcers

Malarial parasite

Ronald Ross

1899

2008

Discovery of HIV

Viral origin of yellow fever

Walter Reed

1900

Francoise Sinoussi and Montagnier

Leishmania donovani

Leishman

1903

2011

Treponema pallidum

Schaudinn and Hoffmann

1905

Beutler, Jules A Hoffmann and Steinman

Role of dendritic cells in adaptive immunity

2015

Causative organism of Rocky Mountain spotted fever

Ricketts

1909

William C Campbell and Satoshi Ōmura

A novel therapy against infections caused by roundworm parasites

Trypanosoma cruzi

Carlos Chagas

1909

TABLE 1.2: Nobel Prize winners in microbiology

UNIT I     Introduction

TABLE 1.1: Major discoveries of bacteria

Recent Updates • Minal Dakhave Bhosale from Pune created India’s first

Year

Name of the scientist

Contributed to the…

1901

Behring

Diphtheria antitoxins

1902

Ross

Malarial transmission

1905

Koch

Cultured tubercle bacilli

1907

Laveran

Malarial parasite

coronavirus testing kit in 2020.

• Gita Ramjee, world-renowned virologist from India died

after contracting COVID-19, on her return from London in April 2020. • Dr Krishna Ella from Bharat Biotech is behind the manufacture of COVID-19 vaccine.

Contd...

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SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY Timeline of microbiology developments has been shown in Table 1.3. TABLE 1.3: Timeline of microbiology developments Timeline

In Year

Observed “little animals” (Antony Leeuwenhoek)

1677

First scientific Smallpox vaccination (Edward Jenner)

1796

Advocated washing hands to stop the spread of disease (Ignaz Semmelweis)

1850

Disproved spontaneous generation (Louis Pasteur)

1861

Supported Germ Theory of disease (Louis Pasteur)

1862

Practiced antiseptic surgery (Joseph Lister)

1867

First proof of Germ Theory of disease with Bacillus anthracis discovery (Robert Koch)

1876

Growth of bacteria on solid media (Robert Koch)

1881

Outlined Koch’s postulates (Robert Koch)

1882

Developed acid-fast stain (Paul Ehrlich)

1882

Developed Gram stain (Christian Gram)

1884

First Rabies vaccination (Louis Pasteur)

1885

Invented Petri dish (JR Petri)

1887

Discovered viruses (Dmitri Iosifovich Ivanovsky)

1892

Recognized viral dependence on cells for reproduction (Martinus Beijerinck)

1899

Proved mosquitoes carried the yellow fever agent (Walter Reed)

1900

Discovered cure for syphilis (Paul Ehrlich)

1910

Discovered Penicillin (Alexander Fleming)

1928

Developed a method to sequence DNA (W Gilbert and F Sanger)

1977

Polymerase Chain Reaction invented (Kary Mullis)

1983

First microbial genomic sequence published (H Haemophilus) (TIGR)

1995

ASSESS YOURSELF Long Answer Questions 1. Write down the contributions of Koch in microbiology? How has it affected the medical microbiology? 2. Write about the contributions of Louis Pasteur in microbiology. 3. What are the principles of microbiology in nursing?

Short Notes Write notes on: 1. Pasteurization 3. Fermentation 5. Robert Koch and his postulates

2. Tyndallization 4. Edward Jenner

Multiple Choice Questions 1. What is the name of process used in dairy industry? a. Fermentation b. Tyndallization c. Pasteurization d. None of these

16

2. The process by which a bacterial virulence power is reduced is known as: a. Disinfection b. Sterilization c. Attenuation d. None of the above 3. Agar-agar was used in culture media as a solidifying agent by: a. Pasteur b. Joblot c. Neisser d. Koch 4. Discoveries about microorganisms blossomed after the introduction of: a. Staining techniques b. Microscope c. Culture media d. All of these 5. Pasteur performed swan neck experiment to disapprove: a. Theory of spontaneous generation b. Germ theory of disease c. Both (a) and (b) d. None of the above

CHAPTER 1     History and Principles of Microbiology 11. Who introduced vaccination method for prevention of smallpox? a. Edward Jenner b. Joseph Lister c. Robert Koch d. None of the above 12. Who introduced methods of obtaining bacteria in pure cultures using solid media? a. Robert Koch b. Louis Pasteur c. Joseph Lister d. Paul Ehrlich

UNIT I     Introduction

6. Who gave the terms—aerobic and anaerobic for organisms growing in presence and absence of oxygen, respectively? a. Koch b. Leeuwenhoek c. Hansen d. Pasteur 7. The vaccine as a term was used by: a. Pasteur b. Jenner c. Landsteiner d. None of these 8. Discoverer of penicillin was: a. Pasteur b. Leeuwenhoek c. Fleming d. Jenner 9. Who is called as father of antiseptic surgery: a. Pasteur b. Loeffler c. Lister d. Fraenkel 10. Who is known as the Father of Microbiology? a. Paul Ehrlich b. Joseph Lister c. Louis Pasteur d. None of these

Answer Key Multiple Choice Questions 1. c 8. c

2. c 9. c

3. d 10. c

4. b 11. a

5. a 12. a

6. d

7. b

17

Importance and Relevance of Microbiology in Nursing Chapter Outline

Every day nurses encounter microbial world while working in medical health care settings as they take care of various groups of patients, including those with contagious infections, therefore it is necessary for them to have good knowledge of microorganisms.

IMPORTANCE OF MICROBIOLOGY FOR A NURSE

y

y

y

Role of Microbiology in Nursing In Diagnostic Labs

y

y

The diagnostic microbiology helps in identifying the causative organism and further helps in targeted treatment of the disease. It helps to recognize the importance of proper collection of specimens for bacteriological examination. It further helps to get accurate reports from the laboratory. Antibiotic susceptibility test helps in treatment of disease with suitable antibiotics.

With knowledge about the mode of spread of infection, it becomes easy to control spread of the diseases.

The knowledge about portals of entry and exit of a disease producing organism helps a nurse to use preventive measures to save community and hospital spread of infection, e.g., in tuberculosis case, a negative pressure room works in a positive way for patient health. Nurses play an important role in immunization drives to control threats of various diseases like diphtheria or MMR, etc. The knowledge of immunization schedules and the cold chain that is used to deliver vaccines helps in safe delivery of vaccines and sera.

In Maintaining Sterile Field y

y

y

In Community Health y

CHAPTER

• Importance of Microbiology for a Nurse

INTRODUCTION

y

2

y

Creating and maintaining a sterile field in the hospitals is also based on the knowledge of microbiology, for example, creating sterile field in operation theater (OT)—principles of asepsis. The knowledge of microbiology enables a nurse to recognise the importance of using sterile equipment in a health care setting to prevent hospital acquired infections. The knowledge of sterilization techniques is mandatory for a nurse to prevent cross infections by maintaining supply of sterile equipment and tools. This simple hygienic act of hand washing prevents or reduces the chances of spreading infections.

CHAPTER 2     Importance and Relevance of Microbiology in Nursing UNIT I     Introduction

2

HAPTER

Fig. 2.3: Fumigation

Fig. 2.1: Generation of biomedical waste in a hospital

In Management of Biomedical Waste y

The proper disposal of biomedical waste is equally important and knowledge of microbiology helps in this field also. The segregation of waste in different bins according to their origin is very important step before sending for disposal in order to avoid environmental pollution (Fig. 2.1). It is discussed separately and a separate chapter in this book.

While Working at Different Places in a Hospital Some of the places in health care settings are discussed here to understand the importance of microbiology in patient care. Infectious Disease or Communicable Disease Wards Nurse uses aseptic methods while giving nursing care in communicable diseases’ wards, following precautions to not to transmit the infection to other patients. Burns’ Unit The nurse takes all precautions while caring burn patients— following aseptic techniques and using sterile equipment (Fig. 2.2).

Fig. 2.2: Sterile surgical equipment

Operation Theater Before operation, it is duty of a nurse to ensure that the atmosphere of the OT is free of microorganisms—for this fumigation (Fig. 2.3) of OT from time to time is done. The sterility of an OT is checked by culturing swabs taken from different sites of an OT. There should not be growth of any o organism on culture plates after overnight incubation at 37 C. Obstetric Unit The wound infection of birth canal after child birth or abortion is a major cause of maternal mortality rate causing peritonitis or phlebitis. The nurse plays a positive role during antenatal care, during delivery or after giving birth for 6 weeks’ period called puerperium by following aseptic techniques herself and teaching same to the new mother. Intensive Care Nursery In intensive care nurseries (Fig. 2.4), especially experienced staff is employed. Nurses keep a check to maintain sterile atmosphere in these intensive care nurseries.

Fig. 2.4: Intensive care nursery

19

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY Central Sterilization Unit A nurse must have a sound knowledge about the sterilization methods and controls of sterilization followed in central sterilization unit (CSU), so that good quality could be maintained while performing nursing care (Fig. 2.5). Note The article on Role of Microbiology in Nursing is published in “Clinical Microbiology: Open Access” 7th Annual Summit on Microbiology: Education, R&D and Market, September 28–29, 2018; San Antonio, USA. You can follow the link to have an access to this article as follows: https://www.longdom.org/proceedings/role-of-microbiologyin-nursing-44045.html

Fig. 2.5: Central sterilization unit in a hospital

ASSESS YOURSELF Long Answer Questions 1. What is the importance of microbiology in the field of nursing? 2. What is the role of microbiology in a health care setting?

Short Notes Write notes on: 1. Relevance of microbiology in nursing 2. Role of nurse in operation theater

Multiple Choice Questions 1. Asepsis is necessary in: a. Operation theater b. Homes c. Markets d. Schools 2. Biomedical waste treatment is needed for: a. Recycling of the waste b. Proper disposal of the pathogenic waste c. Both (a) and (b) d. None of the above

20

3. The process to make equipment completely free from microorganism is: a. Disinfection b. Sterilization c. Both (a) and (b) d. None of the above 4. Immunization schedules are based on the knowledge about: a. Etiology of the disease b. Immunology c. Mode of transmission of the disease d. All of the above 5. Opportunistic pathogens can cause disease and they reside in: a. Surroundings b. In the body c. On the body d. All of the above Answer Key Multiple Choice Questions 1. a

2. b

3. b

4. b

5. d

UNIT

II

General Characteristics of Microbes The microorganisms were noticed and reported a long before Antonie van Leeuwenhoek’s reports about little animalcules. After Lord Mahavira, it was stated by Abu Ali ibn Sina in 1020 that there are certain bad elements which contaminate the body and cause disease. He indicated in “The Canon of Medicine” about certain diseases that could be contagious like tuberculosis. In 14th century Ibn Khatima also said that plague was contacted by people when some contagious minute particles enter the body. Although, all these statements were based on the hypothetical indications and the world of microbiology came into existence with Leeuwenhoek when he studied microorganisms with his self-made microscope. Slowly these single-celled organisms were recognized by scientists all over the world. These single-celled wonders were the first forms of life that appeared on this earth about 4 billion years ago. At that time all forms of life were microscopic. Microorganisms are studied by various other disciplines of science and technology too like biochemistry, molecular biology, genetic engineering or industrial microbiology to name a few. But the microbiological study for a student of health science turns more precious as one has to deal with pathogenic organisms. These microorganisms range from mild to severe nature of pathogens and some of them cause diseases, which can take precious human life. Therefore, for a nursing student who is closely concerned with human life, the knowledge of microorganisms is unavoidable– it must be attained.

LEARNING OBJECTIVES After going through this unit, you will be able to:  Describe the structure, classification, morphology and growth of bacteria.  Identify microorganisms.

UNIT OUTLINE The chapters included in this unit are as follows: Chapter 3: Structure and Classification of Microbes Chapter 4: Morphology of Bacteria Chapter 5: Colonization Chapter 6: Growth and Nutrition of Microorganisms Chapter 7: Blood and Body Fluids Chapter 8: Laboratory Methods for Identification of Microorganisms Chapter 9: Detection of Motility and Staining Techniques Chapter 10: Culture and Media Preparation

3

Structure and Classification of Microbes Chapter Outline

• Methods Used in Classification • Tools of Identification

INTRODUCTION Classification is a way of arranging a wide variety of organisms in nature. It is not possible to study the characteristics of each and every microorganism, therefore, we study the characteristics of a population of different groups of microorganisms by classifying them. The requisites for classification are: y Stability: Every attempt should be made to devise classifications that need only minor changes as new information becomes available. y Predictability: By knowing the characteristics of one member of a taxonomic group, it is possible to assume that the other members of the same group probably have similar characteristics.

METHODS USED IN CLASSIFICATION Generally, three methods are used for arranging bacteria into Texas, which are as follows: 1. Intuitive method: It is based on the properties of the organisms. 2. Numerical taxonomy: This method of classification has great practical usefulness and is unbiased also. Equal weightage is given to each characteristic of microorganism.

CHAPTER

• Classification

By using the computational methods, one can calculate the similarity percentage of each strain. For any two strains, %S = NS/(NS + ND) (For 2 strains) Here, NS is the number of characteristics that are similar, ND is the number of characteristics that are different, %S is percentage of similar characters. 3. Genetic relatedness: It depends on the genetic relatedness between organisms. This method is stable and more predictable. It is most objective and is based on the hereditary material, i.e., DNA. Table 3.1 summarizes different criteria used in classification of bacteria.

TOOLS OF IDENTIFICATION Biochemical testing: It is based on: y The nutrient requirements and ‘metabolic byproducts’ of a particular microorganism. y The physiological behavior of the organism under observation. Dichotomous keys series: The tests are done in a logical order and each test result indicates next test to be done. The collective results of multiple tests create a profile allowing identification of microorganisms (Fig. 3.1).

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY TABLE 3.1: Different criteria used in the classification of bacteria Norms of classification

Examples

Benefits

Genetical Make up

% of G and C ratio along with DNA hybridization

It determines the relatedness within genera and families

Physiological behavior

pH, temperature, concentration of salts required to grow, osmotic pressure, light energy, sensitivity toward antibiotics, etc.

Helps in distinguishing species, genera or higher orders

Biochemistry

Nature of cellular components like RNA, inclusion bodies, different pigments, nuclear material and cell wall

Helps in distinguishing species, genera and higher groups of bacteria

Serology

Fluorescent antibody technique and slide agglutination

Distinguishes strains and species

Sequence of bases in tRNA

tRNA sequencing

Shows relatedness among all living things

Phage typing

Susceptibility of cells to a group of bacteriophages

Distinguishing and identification of strains

Protein profiles

Separation of proteins by electrophoresis

Distinguishes different strains

Morphology

Size and shape of cells, arrangement, presence or absence of flagella, pili, capsules and endospores

First level of separation into genera and species

Growth

Characteristics of growth in solid and liquid media

Distinguishes into species, genera and higher groups of organisms

Staining

Gram staining, acid-fast staining or other differential staining techniques

Separate bacteria into main divisions

Nutrition

Autotrophs or heterotrophs and the kind of source energy is used by them like carbon, nitrogen or sulphur, etc.

Distinguishes species, genera and higher groups of organisms

Abbreviations: C, cytosine; DNA, deoxyribonucleic acid; G, guanine; tRNA, transfer ribonucleic acid

Fig. 3.1: Dichotomous key

24

CHAPTER 3     Structure and Classification of Microbes

Enterotube: In order to identify microorganisms, one of the commercial devices for rapid identification is Enterotube II (Fig. 3.2). It performs multiple tests simultaneously. Results are obtained within 24 hours and the identity of microorganism is revealed. y Serology: Differences in antibody reactivity toward an antigen also reveal different bacterial strains. y Phage typing: When different test phage samples are applied (in dots) to the surface of bacteria (to be tested) grown on agar plates, clear zones appear where bacteria have been infected and killed after 24 hours. Profile of phage sensitivity reveals the identification of bacteria. y DNA base composition: Members of the same genera or species have nearly identical DNA sequences, and hence, the same proportions of G/C base pairs and A/T base pairs because the base pair, G = C and A = T. G/C + A/T = 100% (e.g., if G/C = 40%, then A/T = 60%) By determining the G/C content of the DNA from a test organism and comparing this to known values, is a quick way to identify microorganisms. ƒ If %G/C is different, the microorganism cannot be a match. ƒ If %G/C is same, there might be a match, but additional testing is necessary to confirm the identity. y DNA hybridization: It is done on the basis of capability of separated DNA strand to make another complementary strand according to the base pairs. With enough heat, DNA strands are separated. It is cooled down and it allows complementary strands to make base pairs. This y

y

UNIT II     General Characteristics of Microbes

Fig. 3.2: Enterotube II

technique is used in a variety of ways to see if DNA from two different sources are similar or not. Usually, the DNA from one source is immobilized, the other is labeled to allow detection of complementary base pairs. Ribosomal RNA comparison: Prokaryotic ribosomes contain three different rRNA molecules—large subunit contains 23S and 5S rRNA, and small subunit contains 16S rRNA sequence which is typically used for ribotyping. The sequence on 16 rRNA is highly specific, therefore, a degree of difference reflects that the organism is evolutionary apart. This method is primarily used for classifying prokaryotic microorganisms.

CLASSIFICATION So far 1.7 million organisms have been identified on the earth. All cellular organisms evolved from a common ancestor and it is suggested because of similar plasma membrane, using adenosine triphosphate (ATP) for energy and using DNA for genetic storage. They later evolved in different domains due to random mutation and natural selection (Fig. 3.3). Gene sequencing now allows for more accurate and precise placement of organisms in the taxonomic pyramid of relatedness. The rRNA sequences show three distinct groups of life: 1. Bacteria 2. Archaea 3. Eukarya—protists, fungi, animals and plants (Prior to sequencing, Bacteria and Archaea had been grouped together in the kingdom Monera).

25

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

Fig. 3.3: Schematic representation of three domains of life: Bacteria, Archae and Eukarya

Bacteria Bacteria are single-celled, microscopic living organisms. The bacterial classification not only helps in studying them in a systematic way but also helps in getting hold of treatment of a particular disease because mostly, the bacteria showing similar characteristics are sensitive to same type of chemotherapeutic drugs. Bacterial classification based on different factors is discussed here as follows.

Classification Based on Staining This is based on the difference lying in the structure of bacterial cell wall (Table 3.2). The bacterial cell wall is composed of lipopolysaccharide (lipid), peptidoglycan

(protein) and carbohydrates. Difference lies in the different ratio of lipopolysaccharide and peptidoglycan among different bacteria. Gram staining helps in categorizing bacteria in two broad classes as Gram-positive and Gram-negative. 1. Gram-positive bacteria, have a thick layer of peptidoglycan. The Gram-positive bacteria do not let the primary stain to be removed from their cell wall when decolorized and appear as violet colored organisms under the microscope. 2. Gram-negative bacteria, have less amount of peptidoglycan and more lipopolysaccharide in cell wall. They get decolorized and primary stain is not retained by their cell walls and appear as pink organisms due to the color of counter stain, Safranin.

TABLE 3.2: Different microorganisms and staining methods for microscopic study

26

Microorganisms

Methods of observation under microscope

Reasons

Coliform bacilli and cocci

Gram staining method

Difference in cell wall makes them fall into two main categories: Gram-positive and Gram-negative

Treponema pallidum

Fluorescent antibody technique or dark field microscopy

Too thin and delicate to make smears as is done in Gram staining

Mycobacteria: M. leprae and M. tuberculosis

Acid fast staining

Due to high content of fatty acids in cell wall simple dyes cannot penetrate

Mycoplasma pneumoniae

No staining technique

Cell wall is absent so staining cannot be done as it depends on presence of cell wall in an organism

Rickettsiae

Tissue stains like Giemsa staining

Very small intracellular microorganisms

Chlamydiae trachomatis

Inclusion bodies are observed under the microscope

Very small intracellular microorganisms

Legionella pneumophila

Difficult intake of safranin as counterstain

Prolonged period of time is required to counterstain

CHAPTER 3     Structure and Classification of Microbes

Classification Based on Morphology Cohn classified the bacteria in 1872, in following categories. y Cocci (In Greek ‘kokkos’ means berry): Spheres or oval in shape. ƒ Monococci: Existing as single cells, e.g., Monococcus flavus. ƒ Diplococci: When the cell divides in such a way that after division the cells do not separate from each other, e.g., Diplococcus pneumoniae.

Staphylococci: Cell division occurs in three planes and cells do not separate from one another, instead they arrange themselves as bunches of grapes, e.g., Staph. aureus. ƒ Streptococci: Cells do not separate from each other and form a chain, like Streptococcus pyogenes. ƒ Tetracocci: When the cells are arranged in groups of four due to cell division taking place at right angles, e.g., Gaffkya tetragena. ƒ Sarcina: Cell division is in three planes and it arranges the cells in the form of a cube, e.g., Sarcina lutea. Bacilli (rod-shaped): These are straight rod-shaped microorganisms. ƒ Diplobacilli: When these exist as two cells. ƒ Palisades: When arranged in groups of four or more. ƒ Streptobacilli: In chains. Vibrio: Comma-shaped and curved bacteria, e.g., Vibrio cholerae. Spirilla: These are helically curved rigid rods, e.g., Spirillum minus. ƒ

y

y y

UNIT II     General Characteristics of Microbes

3. Mycoplasma are neither Gram-positive nor Gram-negative bacteria because they lack true cell wall. Mycobacteria have rigid cell wall and can be stained with acid fast staining. They can be stained by acid fast staining technique due to the presence of mycolic acid in their cell wall. 4. Chlamydiae and Rickettsiae are Gram-negative but due to their very small size and their existence as intracellular parasite, cannot be observed under microscope. 5. Legionella needs special induced application of safranin dye to show characteristics of Gram-negative bacteria. Figure 3.4 shows the arrangements and shapes of bacteria.

Fig. 3.4: Arrangements and shapes of bacteria

27

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

Fig. 3.5: Types of bacteria based on number and position of flagella y y y y y

Spirochetes: These are the rods with highly flexible forms, e.g., Treponema pallidum. Coccobacilli: These bacteria are intermediate between cocci and bacilli, e.g., Brucella. Actinomycetes: These are bacteria having branched filaments, e.g., Actinomycetes meyeri. Mycoplasma: These are wall-less organisms and have no fixed shape, e.g., M. pneumoniae. Chlamydia: These organisms resemble viruses in size and reproduce only inside the cells. They are obligate intracellular parasites, filterable and fail to grow in cell-free media. They possess both DNA and RNA, e.g., Chlamydia trachomatis.

Classification Based on Presence of Flagella The flagella are used for movement by the bacteria and are rooted in the cell membrane. On the basis of position of flagella, the bacteria can be classified as: (Fig. 3.5) y Atrichous: When the bacteria do not possess flagella and are, therefore, non-motile. y Monotrichous: When the bacteria have a single flagellum, e.g., V. cholerae. y Amphitrichous: Bacteria have flagella situated at two ends of the cell, e.g., Alcaligenes faecalis. y Peritrichous: Which has the flagella arranged on all over the cell, e.g., S. typhi. y Lophotrichous: When the flagella form a tuft at one particular point. y Polytrichous: Bacteria have a number of flagella at different locations on the cell.

28

Fig. 3.6: Classification based on nutrient requirements

Classification Based on Nutrient Requirements (Fig. 3.6) (Given in detail in Chapter 6)

Classification Based on Existence at Different Temperature (Given in detail in Chapter 6)

Classification Based on Oxygen Requirement (Given in detail in Chapter 6)

Classification Based on Hydrogen Ion Concentration (Given in detail in Chapter 6)

Classification Based on Medical Importance The bacteria can be classified as extracellular and facultative parasites or as obligate parasites (Fig. 3.7). The facultative pathogens can be Gram-positive such as cocci like, Staphylococci and enterococci, or bacilli

CHAPTER 3     Structure and Classification of Microbes UNIT II     General Characteristics of Microbes

Fig. 3.7: Medically important bacteria

like, Corynebacterium. They can be Gram-negative such as cocci like Brucella and Bordetella, and bacilli like, Vibrio and Pseudomonas. The spirochetes like, Borrelia, Treponema and Mycobacterium tuberculosis are obligate human pathogens. • Prokaryotic species: It is defined as a population of cells

with similar characteristics (no sexual reproduction).

• Pure culture: These are clones/populations derived from a

single cell that are genetically identical.

• Strains: Each culture or group that is slightly different is

called a strain. For example, Escherichia coli (E. coli)—normal intestinal flora E. coli 0157:H7 that produces a toxin which all other stains do not produce and is a deadly pathogen of humans.

Archaea These include all prokaryotes with walls that do not have peptidoglycan. They often carry out unusual metabolism and live in extreme environmental conditions. There are no kingdoms to which it belongs.

Eukarya Domain eukarya has four kingdoms: 1. Kingdom Protista (unicellular eukaryotes): These are simple eukaryotes which do not fit elsewhere and

are nutritionally diverse: Autotrophs, heterotrophs, intracellular parasites. For example, algae and protozoa. 2. Kingdom Fungi: They absorb organic material through the plasma membrane and are mostly saprophytic, e.g., yeasts, molds and mushrooms. 3. Kingdom Animalia: They are multicellular animals and ingest organic food through the mouth. The cells are organized into tissues. 4. Kingdom Plantae: The multicellular plants are included in this kingdom. They undergo photosynthesis to convert CO2 + H2O into organic molecules. The cells are organized into tissues.

Viruses The viruses do not fit the domain system as they are acellular and usually classified by Family and Genus. They are usually referred by common name, e.g., human immunodeficiency virus (HIV), Genus—Lentivirus, Family—Retroviridae. Viral species: It is defined as a population of viruses with similar characteristics (including morphology, genes and enzymes) that occupy a particular ecological niche. Viruses have developed themselves to stay as intracellular parasites. They usually only infect one type of cells, which are the one that best supports the viral replication. The viruses tend to be very specific about their preference site of infection, e.g., HIV infects only human T helper cells. 29

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

ASSESS YOURSELF Long Answer Questions 1. What are the different criteria used for classification of bacteria? Discuss the different tools of classification. 2. What are the different ways of classifying bacteria?

Short Notes Write notes on: 1. Classification based on pathogenicity 2. Classification based on morphology

Multiple Choice Questions 1. The organism’s name is in: a. English b. Latin c. French d. None of these 2. Name the apparatus used to put test in one place to identify an unknown organism is: a. Enterotube II b. Enterotoxic tube c. Both (a) and (b) d. None of these 3. Bacilli are: a. Rods b. Cocci c. Coccobacilli d. Spirilla 4. The name of organism has a specific pattern: a. First genus and then species b. First species and then genus c. Both (a) and (b) d. None of the above

30

5. On the basis of rRNA, the three domains of life are: a. Bacilli, cocci and spirilla b. Bacteria, archaea and eukarya c. Bacteria, algae and fungi d. None of the above 6. % Similarity (%S) of each strain to every other strain is calculated by which method? a. Intuitive method b. Numerical taxonomy c. Genetic relatedness d. DNA homology experiments 7. Two organisms which are very closely related to each other have which of the following property? a. Similar mol% G+C values b. Different mol% G+C values c. Similar mol% G+C values and heteroduplexes are formed d. Different mol% G+C values and heteroduplexes are not formed 8. Which among the following kingdoms were proposed by Whittaker? a. Monera b. Protista, Fungi c. Plantae, Animalia d. Monera, Protista, Fungi, Plantae, Animalia Answer Key Multiple Choice Questions 1. b 8. d

2. a

3. a

4. a

5. b

6. b

7. c

4

Morphology of Bacteria

Chapter Outline

• Size and Form of Bacteria • Bacterial Cell Structure • Motility

CHAPTER

• Bacterial Reproduction/Multiplication • Bacterial Spores

INTRODUCTION

Characters

Prokaryotes

Eukaryotes

In 1866, Haeckel proposed a third kingdom Protista, which is further divided into two groups: 1. Prokaryotes (with primitive nucleus)—Bacteria and blue green algae. 2. Eukaryotes (with true nucleus)—Fungi, algae, slime molds and protozoa. The differences between prokaryotic and eukaryotic cells are tabulated as follows (Table 4.1):

• Ribosomes • Mesosomes • Contractile vacuole • Cytoplasmic streaming

70S Present Present

80S Absent Absent

Absent

Present

Cell wall • Sterols • Muramic acid

Present Absent Present

Absent Present Absent

TABLE 4.1: Differences between prokaryotes and eukaryotes

Nucleus • Location

Free in cytoplasm Absent

Almost central in cytoplasm Present

Absent

Present

DNA • Chromosome • Replication

One, circular Binary fission

• Metabolism

Variety of pathways

Multiple and linear By mitosis and meiosis Common metabolic pathway

Characters

Prokaryotes

Eukaryotes

Structure • Size • Cellular organization

Small in size (≤5 μm) Unicellular

Large Multicellular

Cytoplasm • Mitochondria • Golgi apparatus • Endoplasmic reticulum

Not present Not present Not present

• Nuclear membrane • Nucleolus

Present Present Present Contd...

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

SIZE AND FORM OF BACTERIA

Structures External to the Cell Wall

Bacteria are very small and most of them are approximately 0.5–5.0 μm in length and 0.2 μm in diameter. The smallest known bacterium is Phytoplasma, which infects plants just like mycoplasma infects animals. Very large rounded bacterium is Achromatium oxaliferum, which is about 100 μm × 45 μm and Epulopiscium fishelsoni, which lives in the gut of a brown surgeonfish of Red Sea and has dimensions of 80 μm in thickness and is more than 600 μm in length. Comparative sizes of the microorganisms are shown in Figure 4.1.

Flagella

Morphological Types The shape of bacteria is due to its rigid cell wall. (The classification based on morphology of bacteria is given in Chapter 3.)

BACTERIAL CELL STRUCTURE Bacteria are unicellular living organisms without chlorophyll and have both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Bacteria are capable of performing all essential processes of life like growth, metabolism and reproduction. They have rigid cell walls containing muramic acid. When the bacterial cell is observed under the microscope, various structural components can be seen as discussed ahead.

Bacterial flagella are hair-like, helical appendages that protrude through the cell wall and are responsible for screwlike propulsive movements. They act as organs of locomotion. The flagellum is a long, thin filament, twisted spirally in an open, regular waveform and is about 0.02 μm thick (Figs 4.2A and B). The number of flagella and their position on the bacterial cell depends on the species of particular bacteria. (The bacterial classification based on the numbers of flagella is given in Chapter 3.) A flagellum is composed of three parts namely (Fig. 4.3): y Basal body: It is associated with the cytoplasmic membrane and cell wall. It is the extreme basal part of the flagellum. y Hook: It is the shorter, broader and thicker part that passes through the cell wall. y Filament: It is the thinner, elongated, terminal part and is usually several times as long as the cell. Some Gram-negative bacteria have a sheath surrounding the flagellum, which is continuous with the cell wall. The chemical composition of the basal body is unknown, but the hook and filament are composed of protein subunits flagellim, arranged in a helical fashion. There may be two or more sets of encircled rings too. In Gram-negative bacteria, four types of rings, i.e., M, S, P, and L are observed.

Fig. 4.1: Comparative sizes of the microorganisms

32

CHAPTER 4     Morphology of Bacteria UNIT II     General Characteristics of Microbes

A

B Figs 4.2A and B: Parts and attachment of flagellum. A. Gram-negative; B. Gram-positive

Ring M attaches to the cell membrane, ring S is located just above it, P ring attaches to peptidoglycan and L to the outer lipopolysaccharide membrane. The outermost rings P and L are absent in Gram-positive bacteria.

Fig. 4.3: Structure of bacterial flagellum

Fimbriae and Pili These appendages are hollow, non-helical and filamentous (Fig. 4.4). Fimbriae are bristle like, thinner, shorter and more numerous than flagella. Pili are hair like outgrowths on surface of bacteria. Gram-negative bacilli including saprophytic intestinal commensals and pathogenic species of family Enterobacteriaceae, possess pili. They may occur in motile and non-motile strains. Under electron microscope reveals them as almost straight appendages. Pili do not play role in motility but F pili or sex pili serve as the fertility factor of maleness and are involved in transfer of genetic material during bacterial conjugation. They also act as receptor sites for certain bacteriophages. Some pili play a major role in human infection by allowing

Fig. 4.4: Fimbriae and Pili in Escherichia coli cells

33

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY pathogenic bacteria to attach to epithelial cell lining of the respiratory, intestinal or genitourinary tracts. This further prevents the bacteria from washing away with the flow of mucus or body fluids and permits them to cause infection.

Capsules, Microcapsules and Loose Slime Some bacterial cells are surrounded by a viscous substance forming a covering layer or envelope around the cell wall. In wet state, it is wide enough (0.2 μm or >) to be seen under light microscope and is called a capsule. When it is thin, it may be termed as microcapsule. If it is abundant, many cells are embedded in a common matrix, the material is called slime layer. Most bacterial capsules are composed of polysaccharide, e.g., Klebsiella pneumoniae. The capsule of B. anthracis is composed of glutamic acid—a peptide. Advantages The capsules help the bacteria in various ways: y They inhibit the engulfment of pathogenic bacteria by WBCs and thus contribute to invasive or infective ability known as virulence. y They may block attachment of bacteriophages. y They may promote attachment of bacteria to smooth surfaces, e.g., Streptococcus mutans, which causes dental caries, attaches itself to the smooth surface of teeth because of water insoluble capsular glucan. y They may promote the stability of bacterial suspension by preventing the cells from aggregating and settling out. y Capsule protects the bacteria against ingestion by phagocytes of the host, e.g., capsule of Streptococcus pyogenes is composed of hyaluronic acid and is antiphagocytic.

A

Cell Wall Beneath the external structures like capsules, sheaths and flagella but external to the cytoplasmic membrane, is the cell wall, which is very rigid structure and gives shape to the bacterial cell. Its main function is to prevent the cell from shocking, expanding and bursting in unfavorable conditions. Cell wall is mainly composed of peptidoglycan, which is insoluble, porous, cross–linked polymer of enormous strength and rigidity. Peptidoglycan differs in composition and structure from one species to another, but it is basically a polymer of N-acetylglucosamine, N-acetylmuramic acid, L-alanine, D-alanine, D-glutamate and diamino acid.

Cell Wall of Gram-positive Bacteria Gram-positive bacteria have much higher amount of peptidoglycan in their cell walls than Gram-negative bacteria. Polysaccharide that is covalently linked to the peptidoglycan is present in Gram-positive bacteria. Teichoic acid which is an acidic polymer of ribitol phosphate or glycerol phosphate is linked covalently to peptidoglycan. Teichoic acid not only contributes to the Gram positivity but also protects the bacteria from thermal injury by providing cationic pool to the cytoplasmic membrane for stabilization (Fig. 4.5A).

Cell Wall of Gram-negative Bacteria The walls of Gram-negative bacteria are more complex as compared to Gram-positive bacteria. There is an outer membrane that surrounds the thin underlying layer of peptidoglycan, rich in lipids. This outer membrane is an impermeable barrier and prevents the external harmful chemicals to enter the cell. The outer membrane is joined to the underlying peptidoglycan layer by Braun’s lipoproteins.

B Figs 4.5A and B: Diagrammatic representation of cell wall. A. Gram-positive; B. Gram-negative

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CHAPTER 4     Morphology of Bacteria

Structures Internal to the Cell Wall Cytoplasmic Membrane The bacterial protoplasm is limited externally by a thin, elastic membrane, which is 5–10 nm thick. It covers the bacterial protoplasm and the components completely. It consists of lipoproteins. The prokaryotic cells have higher content of protein in their cytoplasmic membrane as compared to eukaryotes. The membrane in cross section appears as phospholipid bilayer under electron microscope. The phospholipids have lipophilic fatty acid tail and hydrophilic head (Fig. 4.6). The phospholipids are arranged in such a way that their hydrophilic polar regions are externally aligned and in contact with a layer of protein at each surface. It does not contain sterols like cholesterol, which is present in eukaryotic cells, but molecules like sterols are present in its membrane known as hopanoids, which are called membrane rafts. These hopanoids make the membrane very active and provide fluidity to it. The proteins are either loosely held in the membrane or are integrated with membrane. The membrane serves as a hydrophobic barrier but at the same time, it is semipermeable. The cytoplasmic

membrane also contains various enzymes involved in respiratory metabolism and synthesis of capsular and cell wall components. As ATP is generated in membrane, certain nutrient transport systems and flagellar motility are also associated with it. Any damage to the cytoplasmic membrane can cause death of the cell.

Cytoplasm of Bacterial Cell The cytoplasm of the bacterial cell is a viscous watery solution or soft gel, containing a variety of organic and inorganic solutes and numerous small granules called ribosomes. The cytoplasm of bacteria differs from that of the higher eukaryotic organisms as it does not contain endoplasmic reticulum, mitochondria and does not show cytoplasmic streaming movement. There is no formation, migration and disappearance of vacuoles in the bacterial cytoplasm. Ameboid movement is also not present. The various cytoplasmic inclusions observed in cytoplasm are discussed as follows (Fig. 4.7).

UNIT II     General Characteristics of Microbes

The membrane is a bilayered structure consisting mainly of phospholipids, proteins and lipopolysaccharides (LPS). This LPS is toxic in nature and is known as endotoxin and occurs in the outer layer of the membrane. The outer membrane allows selective molecules to pass through the channels known as porins and may also serve as receptors for attachment of bacteriophages and bacteriocins (Fig. 4.5B). (The classification based on Gram staining is given in Chapter 3.)

Ribosomes Bacterial ribosomes are smaller than eukaryotic cells and have a sedimentation constant of 70S composed of 30S and 50S subunits. They are threaded together on strands of mRNA to form polysomes and it is at this site that the code of the mRNA is translated into peptide sequences. The difference of bacterial RNA from eukaryotic RNA makes it possible to use antibacterial agents like streptomycin, which interferes with bacterial metabolism at the ribosomal level without affecting human ribosomal function.

Fig. 4.6: Bacterial cytoplasmic membrane

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Facteria Metabolic products – exotoxins versus endotoxins The bacteria produce poisonous products as a result of metabolism and it is known as toxins. They play an important role in pathogenicity of an organism and can be differentiated as exotoxins and endotoxins. Some of the differences between the two are given in Table 4.2. TABLE 4.2: Differences between exotoxins and endotoxins Exotoxins

Endotoxins

 Exotoxin and endotoxin produced by bacteria Secreted by an organism

Integral part of cell wall

Produced by both Gram-positive and Gram-negative bacteria

Produced by Gram-negative bacteria only

It is polypeptide

It is lipopolysaccharide

Heat labile and turn nontoxic when heated at 60°C

Heat stable and can tolerate a temperature of 60°C and more

Highly antigenic

Weakly antigenic

Can be converted into toxoids by treating with formalin

Cannot be converted into toxoids

Highly toxic and is fatal in very small quantities

Weakly toxic

Very specific in action and binds to specific receptors

Non-specific in action

They are non-pyrogenic

Pyrogenic—produce fever in the host by interleukin 1 induction. Shock is also produced

The toxin is located on extra chromosomal gene or plasmid

Located on chromosomal gene

They are filterable

They are not filterable

No enzymatic activity

They are enzymatic

They have high molecular weight—about 10 KDa

They have low molecular weight – about 50–1000 KDa

Can be detected by tests like neutralization and precipitation, etc.

Can be detected by a specific test—Limulus lysate assay

For example, S. aureus, Streptococcus pyogenes, Bacillus cereus and Bacillus anthracis.

For example. E. coli, Vibrio cholerae, Shigella and Salmonella typhi

Cytoplasmic Inclusion Granules Various types of inclusion bodies are found in bacterial cell like volutin, polysaccharide, lipids and crystals. They are present in the largest amount when the bacteria have access to an abundance of energy yielding nutrients, and these diminish or disappear under starvation. They impart specific character to a particular bacterial species, like volutin granules or metachromatic or BabesEarnest granules that can be visualized by special staining techniques in Corynebacterium diphtheriae. y Volutin granules are composed of polyphosphate and store energy. 36

y

y

Lipid granules can be stained with fat soluble dyes like Sudan black. These granules are composed of β-hydroxy butyric acid and act as a carbon and energy storage product. Lipid granules can be seen in Bacillus genus. Polysaccharide granules of starch or glycogen can be seen in the cytoplasm of certain bacteria. They serve as storage products.

Mesosomes (Chondroids) Mesosomes are convoluted membranous bodies that develop by complex invaginations of the cytoplasmic membrane into the cytoplasm and often form the site of cross wall formation in

CHAPTER 4     Morphology of Bacteria UNIT II     General Characteristics of Microbes

Fig. 4.7: Bacterial cell showing complete structure

Gram-positive bacteria. Mesosomes are the principal sites of respiratory enzymes in bacteria.

Bacterial Nucleus The genetic information of a bacterial cell is contained in a single long molecule of double-stranded DNA, which occurs in the form of a closed circle. The bacterial nucleus has no nuclear membrane or nucleolus. Sometimes a smaller extra chromosomal piece of DNA is present in addition to nucleoid called plasmid. The differences between bacterial nucleus and eukaryotic nucleus are given in Table 4.3. TABLE 4.3: Differences between prokaryotic and eukaryotic nucleus Characters

Prokaryotic nucleus

Eukaryotic nucleus

Location

Free in cytoplasm Absent Absent Absent One, circular

Almost central in cytoplasm Present Present Present Multiple and linear

Nuclear membrane Nucleolus Deoxyribonucleoprotein Chromosome

MOTILITY The presence of flagella is commonly inferred from the observation of motility either microscopically or by noting the occurrence of spreading growth in semisolid agar medium. On microscopic observation of wet films by hanging drop method, motile bacteria are seen swimming in different directions across the field with a drifting, wriggling or tumbling movement (Fig. 4.8).

Motility is important for cell survival. The ultimate benefit of motility is that it allows a cell to fetch essential resources more efficiently in a competitive environment. Spirochetes move due to specialized internal structures known as axial filament. There is fast spiral rotation on the long axis with slow progression in the axial line. Gliding movement is observed in Cytophaga species, whereas most bacteria exhibit capability of swimming toward or away from various chemical compounds, i.e., positive chemotaxis or negative chemotaxis. For this purpose, bacteria possess chemoreceptors on the cytoplasmic membrane. To observe the motility of a microorganism, hanging drop method is adapted in laboratories.

BACTERIAL REPRODUCTION/ MULTIPLICATION Binary Fission Multiplication takes place by simple binary fission in true bacteria (Fig. 4.9). The cell grows in size and the protoplasm divides into two, approximately equal halves, by the ingrowth of a transverse septum from the plasma membrane and cell wall.

Conidia Formation In mycelial bacteria, growth takes place by extension of the vegetative filaments, which later produce conidia by transverse division. These conidiospores germinate under favorable conditions and give rise to fresh mycelia (Fig. 4.10A). 37

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Fig. 4.8: Motility of bacteria

A

Fig. 4.9: Binary fission

38

B

Figs 4.10A and B: A. Conidia formation in streptomyces; B. Budding in Rhodomicrobium

Budding

Conjugation

The protoplasm along with cell wall pinches on one end of the cell, known as bud. Later on this bud develops into a new bacterial cell (Fig. 4.10B).

In some species of bacteria, a crude type of conjugation can also be observed where two cells come in contact with each other and a conjugation canal is formed in between (Fig. 4.11).

CHAPTER 4     Morphology of Bacteria

Certain bacterial species, like Bacillus and Clostridium produce spores to survive in a dormant state through a long period of starvation or other adverse environmental conditions. Later the spores, which are metabolically dormant, can undergo germination and form a vegetative cell.

Sporulation

Fig. 4.11: Conjugation

Then the nuclear material from one cell is transferred to another. The one cell which donates the genetic material is called male (F+) and the recipient is called female (F–) strain of bacteria. Mostly it provides extra genetic material to the recipient cell and brings mutations.

Sporulation occurs as a response to starvation or, at least, the exhaustion of a nutrient in the medium (Fig. 4.12). It does not take place as long as conditions continue to favor the vegetative growth. It mostly occurs when multiplication is being arrested at the end of the log phase and in the early stationary phase of artificial culture. The spore is formed inside the parent vegetative cell— endospore. The spore is a metabolically dormant form which under appropriate conditions can undergo germination and grow to form a vegetative cell. Note that the sporulation in bacteria is not a method of reproduction, but is a means of survival.

UNIT II     General Characteristics of Microbes

BACTERIAL SPORES

Fig. 4.12: Sporulation in bacterial cell

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Fig. 4.13: Bacterial spore

Endospores are extremely resistant to desiccation, staining, disinfection and heat. The spores of Clostridium botulinum type A can resist boiling for several hours. All endospores contain large amount of dipicolinic acid in combination with calcium, located in the central part of the spore and contribute to resistance of spores. The impermeability of spore cortex, low water content, very low metabolic and enzymatic activity also contribute to the resistance. The bacterial spores of all medically important species are destroyed by autoclaving at 120°C for 15 minutes.

Fig. 4.14: Germination of bacterial spore

Spore The cell membrane of the bacterial cell grows inward and forms a spore wall around a small part of the protoplasm having nuclear material at one end of the cell, known as forespore. The nuclear material lies in core material and is surrounded by inner membrane. The germ cell wall lies outside and acquires a thick covering layer called cortex, and a thin but tough outer spore coat consisting of several layers. Spores of some species have an additional loose covering known as exosporium having ridges and folds. The location of the mature spore varies according to species, like spherical, subterminal or central position. The remainder of the parent cell disintegrates and the spore is freed (Fig. 4.13).

Germination of Spore Germination occurs when the external conditions are favorable for growth (Fig. 4.14). The spore swells, cortex disintegrates, the coat breaks and a single vegetative cell 40

emerges. The spore is activated by hydrogen ion concentration, temperature variation and abrasions. Then the autolysin named nisin and hydrolytic enzymes degrade the spore coat. With the disintegration of spore coat, the spore swells and a single germ cell emerges from it, which later on changes into a vegetative cell.

Facteria Spores of Bacillus stearothermophilus are used as indicator to check the sterilization process because they are destroyed in the autoclave at a temperature of 121oC within 15 minutes when the pressure is 15 pounds per square inch. Absence of growth, when the spores are allowed to grow in culture media after autoclaving, indicates proper sterilization.

Differences between Gram-positive and Gram-negative bacteria are enlisted in Table 4.4.

CHAPTER 4     Morphology of Bacteria

Gram-positive bacteria

Gram-negative bacteria

Staining characteristics Retain crystal violet even after washing with alcohol or acetone

Do not retain the stain when washed with alcohol or acetone

Outer membrane Outer membrane is absent

Outer membrane is present

Cell wall • The wall is smooth • • • • • • • • • • •

The lipid content in the wall is very low Cell wall contains Teichoic acid One layered Thick (20–80 nm) Chemically composed of Peptidoglycan, teichoic acid and lipoteichoic acid Periplasmic space is present in some Less amount of lipids Porin proteins are absent More permeable More Peptidoglycan No toxic property of cell wall

• Wall is wavy and comes in contact with plasma membrane only at some loci • The lipid content in the wall is 20–30% • Teichoic acid is absent • Two layered • Thin (8–10 nm) • Chemically composed of Lipopolysaccharide, lipoproteins and peptidoglycan • Periplasmic space is present in all • More amount of lipids • Porin proteins are present • Less permeable • Less Peptidoglycan • Cell wall contains LPS which are endotoxins

Flagellum Basal body of the flagellum contains two rings

Basal body of the flagellum has four rings

Mesosomes Quite prominent

Less prominent

Medical Importance A few pathogenic bacteria belong to Gram-positive group

Most of the pathogenic bacteria belong to Gram-negative group

UNIT II     General Characteristics of Microbes

TABLE 4.4: Differences between Gram-positive and Gram-negative bacteria

ASSESS YOURSELF Long Answer Questions 1. Discuss the various cellular components of the bacterial cell. Draw a well-labeled diagram in support of it. 2. What is the difference between a spore and inclusion granules? Discuss it. 3. Write down the differences between Gram-positive and Gramnegative bacteria. 4. What is the difference between prokaryotes and eukaryotes?

Short Notes Write notes on: 1. Bacterial cell wall 2. Flagella 3. Germination of spores

Multiple Choice Questions 1. Spores are: a. Metabolically inert b. Dormant c. Resistant to environmental changes d. All of the above

2. Extra chromosomal material of DNA in the bacteria is known as: a. Bacteriophage b. Spores c. Plasmid d. Cosmid 3. Fimbriae help the bacteria in: a. Motility b. Adherence to surfaces c. Sexual reproduction d. All of the above 4. Which structure is involved in rudimentary sexual conjugation? a. Fimbriae b. Pili c. Flagella d. None of the above 5. The capsule helps the bacteria to come over: a. Phagocytosis b. Action of antimicrobial agents c. Engulfment by bacteriophage d. All of the above

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42

6. Outer membrane is present in cell wall of: a. Gram-positive bacteria b. Gram-negative bacteria c. Both (a) and (b) d. None of the above 7. Endospore has a rich content of: a. Dipicolinic acid and iron b. Dipicolinic acid and calcium c. Dipicolinic acid and magnesium d. None of the above 8. Which of the following bacteria lack a cell wall and are therefore resistant to penicillin? a. Mycoplasma b. Cyanobacteria c. Bdellovibrios d. Spirochetes

9. Which of the following contain fimbriae? a. Gram-positive b. Gram-negative c. Both (a) and (b) d. None of the above 10. The cell wall of many Gram-positive bacteria can be easily destroyed by enzyme known as: a. Lysozyme b. Lipase c. Pectinase d. Peroxidase Answer Key Multiple Choice Questions 1. d 8. a

2. c 9. b

3. b 10. a.

4. b

5. d

6. b

7. b

5

CHAPTER

Colonization

Chapter Outline

• Specific Adherence of Bacteria to Cells and Tissue Surfaces

INTRODUCTION Colonization is the presence of microorganisms on or inside the body, the organism grows and multiplies but there is no interaction between host and organism. Pathogenic bacteria have evolved themselves by having various means of pathogenic capabilities like fimbriae or toxins, to establish themselves in the selected tissue to be colonized by them—virulence factors.

• Pathogenicity

plaque. Corynebacterium diphtheriae colonizes exclusively in the throat.

Species Specificity Microorganisms show species specificity. Some pathogenic bacteria infect only certain species of animals, e.g.,

SPECIFIC ADHERENCE OF BACTERIA TO CELLS AND TISSUE SURFACES The pathogenic bacteria mostly prefer the sites on host’s body that are in close contact with the environment to cause infection (Fig. 5.1). The virulence factors that help the bacteria in colonizing are tabulated in Table 5.1.

Tissue Tropism Particular bacteria are known to have a preference for certain tissues over others, e.g., S. mutans is abundant in dental plaque but does not occur on epithelial surfaces of the tongue; the reverse is true for S. salivarius, which is attached in huge amount to epithelial cells of the tongue but is absent in dental

Fig. 5.1: Colonization by bacteria

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY TABLE 5.1: Various virulence factors of microorganisms Virulence factors

Description

S-layer

Proteins that form the outermost cell envelope of a broad spectrum of bacteria, enabling them to adhere to host cell membranes

Capsule

A detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may mediate specific or non-specific attachment

Lipopolysaccharide (LPS)

A distinct cell wall component of the outer membrane of Gram-negative bacteria to mediate specific adherence

Teichoic acids and lipoteichoic acids (LTA)

Cell wall components of Gram-positive bacteria that are involved in non-specific or specific adherence

Biofilm

Exopolysaccharide or slime produced by bacteria attaches embedded cells to a surface

Common pili

Pili are thought to play a role in adherence and biofilm formation

Fimbriae

Fimbriae help to bind on eukaryotic cell surfaces

Sex pilus

Binds mating prokaryotes together for the purpose of DNA transfer

Mucous

Mucopolysaccharide layer of glycosaminoglycans covers cell’s mucosal surfaces

Receptor

A macromolecular binding site on a eukaryotic cell surface that binds specific adhesins

Adhesin

Surface structure or macromolecule that binds a bacterium to a specific surface

N. gonorrhoeae and Bordetella pertussis infections are limited to humans; enteropathogenic E. coli K-88 infections are limited to pigs; E. coli CFA I and CFA II infect humans; E. coli K-99 strains infect calves; Group A streptococcal infections occur only in humans. In addition, certain native species and symbionts are quite specific in their associations with specific animal hosts.

Genetic Specificity within a Species Certain strains or races within a species may be genetically immune to a pathogen, e.g., males are not susceptible to mastitis; females are not susceptible to orchitis; a percentage of females are not susceptible to urinary tract infection (UTI) caused by E. coli.

Mechanisms of Adherence to Cell or Tissue Surfaces by Pathogens The mechanisms for adherence may involve two steps: 1. Non-specific adherence: It is the reversible attachment of the bacterium to the eukaryotic surface (also called “docking”). It involves non-specific attractive forces which allow affinity of the bacterium to the eukaryotic cell surface like hydrophobic interactions, electrostatic attractions, atomic and molecular vibrations, Brownian movement, recruitment and trapping by biofilm with the help of bacterial capsule. 2. Specific adherence: This is the irreversible permanent attachment of the microorganism to the host cell surface (also called ‘anchoring’) (Fig. 5.2). It involves the 44

Fig. 5.2: Specific adherence of bacteria

permanent formation of many specific locks and key bonds between complementary molecules on each cell surface. The adhesins situated on the capsule, cell wall or fimbriae attach themselves to the host cell membrane and form a covalent bond. For attachment, the host cell membrane has glycolipid receptors on the surface of the cells. Once the bonds are formed, the attachment under physiological conditions becomes virtually irreversible.

PATHOGENICITY The pathogenicity of microorganisms is the mechanism adopted by them to cause disease in a specific host. The pathogenic bacteria must have two integral properties to be pathogenic: toxigenesis and invasiveness.

Toxigenesis The bacteria produce toxins, which help in pathogenicity and the ability to produce toxins is known as toxigenesis.

CHAPTER 5     Colonization

Invasiveness

y

y

y

It is the capability of a pathogen to invade the host’s tissue. It involves mainly three processes: 1. Way of adherence and initial multiplication of bacteria that is a method of colonization. 2. Production of substances like hyaluronidase, which is an enzyme secreted by bacteria that facilitates its invasion in the host’s tissue. 3. The ability of a pathogenic microorganism to overcome the defence mechanism of host-like production of toxins or capsule formation.

Factors that Help in Invasion The invasion of a host by a pathogen may be aided by the production of bacterial extracellular substances, which act against the host by breaking down primary or secondary defences of the body called invasins. Most invasins are proteins (enzymes) that act locally to damage host cells and/or have the immediate effect of facilitating the growth and spread of a pathogen. The damage to the host as a result of this invasive activity may become part of the pathology of an infectious disease.

Spreading Factors

y

y y

y y

y

The spreading factors affect the physical properties of tissue matrices and intercellular spaces, thereby promoting the spread of the pathogen. Some of these are:

Hyaluronidase is the original spreading factor, produced by Streptococci, Staphylococci and Clostridia. The enzyme attacks the interstitial cement (ground substance) of connective tissue by depolymerizing hyaluronic acid. Collagenase is produced by Clostridium histolyticum and Clostridium perfringens. It breaks down collagen, the framework of muscles. Neuraminidase is produced by intestinal pathogens such as Vibrio cholerae and Shigella dysenteriae. It degrades neuraminic acid (also called sialic acid), an intercellular cement of the epithelial cells of the intestinal mucosa. Streptokinase and staphylokinase are produced by streptococci and staphylococci, respectively. Kinase enzymes convert inactive plasminogen to plasmin which digests fibrin and prevents clotting of the blood. The relative absence of fibrin in spreading bacterial lesions allows more rapid diffusion of the infectious bacteria. Streptolysin produced by streptococci specifically lyses phagocytes and their granules. Phospholipases, produced by Clostridium perfringens (i.e., alpha toxin), hydrolyze phospholipids in cell membranes by removal of polar head groups. Lecithinases produced by Clostridium perfringens, destroy lecithin (phosphatidylcholine) in cell membranes. Hemolysins are produced by Staphylococci (i.e., alpha toxin), Streptococci (i.e., Streptolysin) and various clostridia, may be channel-forming proteins or phospholipases or lecithinases that destroy red blood cells and other cells (i.e., phagocytes) by lysis. Coagulase, formed by Staphylococcus aureus, is a cellassociated and diffusible enzyme that converts fibrinogen to fibrin, which causes clotting. Coagulase activity is almost always associated with pathogenic Staph aureus.

UNIT II     General Characteristics of Microbes

The toxins may be associated with cell wall and are released after cell lysis—endotoxins, or they may be secreted by bacteria like exotoxins. The toxins get transported in the body via blood or lymph from the original site of microbial invasion to produce toxemia. (The differences between exotoxins and endotoxins are given in Chapter 4.)

ASSESS YOURSELF Long Answer Questions

Multiple Choice Questions

1. What do you understand by colonization? Discuss it. 2. Enumerate the factors responsible for pathogenicity in detail.

1. What is the name given to the ability of a bacteria to stay in the body and cause disease? a. Colonization b. Establishment c. Foundation d. None of these 2. The pathogenicity depends on: a. Invasiveness b. Toxigenicity c. Both (a) and (b) d. None of these

Short Note 1. Write a note on invasiveness of bacteria.

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46

3. Invasins are: a. Lipids b. Proteins c. Carbohydrates d. Amino acids 4. Spreading factor promotes: a. Spread of metabolic products b. Spread of infection c. Both (a) and (b) d. None of the above 5. The bacteria adhere to the host cells by: a. Specific factors of adhesion b. Non-specific factors of adhesions c. Both (a) and (b) d. None of the above 6. The role of bacterial capsules as virulence factors is usually related to their ability to interfere with: a. Antibody binding b. B lymphocyte activation c. Antibacterial penetration of bacterial cells d. Phagocytosis e. The release of interferon-gamma and other macrophage activating cytokines

7. A patient develops explosive, watery diarrhea 24 hours after eating seafood. What bacterium is most likely involved? a. Campylobacter fetus b. Salmonella typhimurium c. Shigella flexneri d. Vibrio cholerae e. Vibrio parahaemolyticus 8. The survival of Mycobacteria after ingestion by macrophages is attributed to: a.  Bacterial inhibition of complement activation via the alternative pathway. b.  Bacterial inhibition of phagolysosome formation and interference with endosomal acidification c. The poor immunogenicity of the cell wall glycolipids. d. The bacterium’s rapid escape from the endosome into the cytoplasm of infected cells. e. The bacterium’s resistance to oxygen-active radicals released into the phagolysomsome. Answer Key Multiple Choice Questions

1. a 2. c 3. b 4. b 5. c 6. d 7. e 8. b

Growth and Nutrition of Microorganisms

Chapter Outline

• Bacterial Growth Curve • Bacterial Mode of Nutrition

INTRODUCTION The growth involves an orderly increase in all the components of an organism and is associated with multiplication. The method of division in the majority of bacteria is by binary fission. There is a logarithmic increase in number of bacteria. The time required for a bacterium to give rise to two daughter cells under favorable conditions is called generation time or population doubling time. In case of E. coli, there is generation time of 20 minutes. There are many bacteria which are slow growing and may take long time to grow, for example, Mycobacterium tuberculosis takes nearly 20 hours, whereas Mycobacterium leprae takes 20 days.

CHAPTER

• Factors Influencing Bacterial Growth

of cells. If the number of cells present at different times after inoculation in a medium is measured, and the number is plotted in relation to time, the resultant plot is referred to as growth curve. Increase in total number of cells can be observed by bacterial counts.

BACTERIAL GROWTH CURVE In order to observe bacterial growth in labs, a liquid medium is used but the multiplication of the cells is arrested after a few cell divisions because of the depletion of nutrients or accumulation of toxic substances in the medium. To overcome this difficulty, a device called a chemostat (Fig. 6.1) is used, which has a continuous supply of nutrients with continuous removal of grown bacteria. Bacterial growth could be observed in two ways—increase in size of the individual cell and increase in the total number

6

Fig. 6.1: Chemostat

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY a time when the microorganisms try to adapt themselves to grow in the fresh medium. The cells in an inoculum may be striving for enzymes, metabolic intermediates and other factors, therefore, some time is required for these materials to build up to their optimal levels. In another situation, when composition of medium is totally different from the previous one, entirely new enzymes may be synthesized by the process of induction or by selection of mutants.

Logarithmic or Log Phase Fig. 6.2: Total and viable count growth curve

Two types of growth curves can be drawn (Fig. 6.2): 1. Total count: This is based on the number of cells present irrespective of whether they are living or dead. 2. Viable count: This measures only those cells that are capable of growing and producing a colony on a suitable growth medium.

Phases of Growth Curve The bacterial growth curve can be explained in four main phases having a transition phase in between each of these phases (Fig. 6.3).

Lag Phase In this phase, there is no appreciable multiplication of cells although they may increase in size and show metabolic activity also. The duration of this phase varies according to the condition and amount of inoculum. This phase represents

It is also known as exponential phase. In this period, the cells divide by binary fission at a constant rate and there is a linear relationship between time and the number of cells. The bacterial cells divide exponentially in culture media in vitro although rate of division is much slower in vivo. When infectious organisms overpower the body’s defence mechanism and get out of control, it becomes very difficult to eradicate these organisms as time goes on. The actual rate of growth is related to the generation time, i.e., the time taken between two divisions under particular environmental conditions. To grow at such a fast rate, the bacterial cells have high metabolic activity.

Stationary Phase In the meantime, when the exponential growth is no longer possible and the rate of multiplication decreases until it ceases altogether, and the cells pass into the stationary phase. Theoretically, an equilibrium is attained where the rate of growth and death of the cells is the same. The normal metabolism of cells is associated with biosynthesis and when

Fig. 6.3: Bacterial growth curve

48

CHAPTER 6     Growth and Nutrition of Microorganisms

Effects of Stationary Phase y

y

y

The cells in stationary phase have increased storage products like polysaccharides and lipids. Moreover, the Gram-positive cells in stationary phase may become Gram-negative, therefore, for morphological studies, it is important to choose a young culture. Many bacterial species produce secondary metabolites (that is a product made when the cells have stopped dividing) in stationary phase which are very diverse in nature, like antibiotics and exotoxins. In spore-forming species of bacteria, the initiation of sporulation begins at the end of exponential phase or early in the stationary phase.

Death or Decline Phase After a period of time, the cells in the culture start dying and they turn incapable of multiplication even when transferred to a fresh medium. There are various causes of death or loss of viability in this phase. There may be depletion of a particular nutrient or accumulation of toxic products in the medium. There is a rapid fall in the viable count as well as total count because microorganisms undergo autolysis. The rapidity of the onset of death phase is an important factor that may have an impact on the spread of infection.

Bacteria can be classified into two groups based on the fact that whether they produce their own food or take readymade food—autotrophic and heterotrophic bacteria.

Autotrophic Bacteria Autotrophic bacteria make their own food from outside sources of energy. They are further divided into chemoautotrophs and photoautotrophs.

Chemoautotrophic Bacteria Chemoautotrophic bacteria utilize energy liberated from the oxidation of inorganic raw materials in their medium of growth. They make their energy from oxidation of chemical reactions involved in medium. This energy is used in carbon assimilation. The examples of nitrifying bacteria, sulfur oxidizing and iron bacteria come under this category and are named so according to the source of energy utilized by them, e.g., nitrifying bacteria like Nitrosomonas, and sulfur bacteria like Thiothrix. y Nitrifying bacteria make energy by oxidation reaction by the nitrification reaction. y Sulfur oxidizing bacteria oxidize sulfur compound hydrogen bisulfide to sulfur. This oxidation process releases energy. y An iron oxidizing bacterium liberates energy by involving ferrous and ferric compounds. Some chemosynthetic bacteria depend upon liberation of carbon dioxide and water for energy. For example, Gallionella ferruginea (Fig. 6.4) is a chemoautotroph which is found in different aquatic habitats and plays an important role in oxidizing iron and fixing it in the environment.

UNIT II     General Characteristics of Microbes

growth no longer exists, still a slow metabolism keeps on going in the cells which is enough to maintain life of a cell. There may be depletion of one or more nutrients in the medium along with accumulation of waste products. For example, during fermentation, organic acids are the common end products but they may accumulate and lower the pH of the medium which are enough to cease the growth.

BACTERIAL MODE OF NUTRITION In order to grow, microorganisms depend on an adequate supply of different types of nutritional sources. Mainly oxygen, hydrogen, nitrogen and carbon are mandatory elements. Phosphorus and sulfur are needed in less quantity and some elements are vital but only in trace amounts like iron, magnesium, manganese, sodium and potassium. Bacterial mode of nutrition is varied and is classified into nutritional groups on the basis of four major criteria: 1. The kind of energy used for growth 2. The source of carbon 3. The electron donors used for growth 4. A criterion for respiratory microorganisms is whether they can grow in absence or presence of oxygen, i.e., aerobic or anaerobic bacteria.

Fig. 6.4: Chemoautotroph—Gallionella ferruginea

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Textbook of APPLIED MICROBIOLOGY

A

B

Figs 6.5A to C: A. Bacteriochlorophyll; B. Thiocystis spp of bacteria; C. Typical Photoautotrophic bacteria

Photoautotrophic Bacteria The bacteria like Cyanobacteria, utilize the solar energy to synthesize their own food with the help of photosynthetic pigments present in their cell membranes. They have photosynthetic pigments which are bacteriochlorophyll and bacteriopheophytin (Figs 6.5A to C). These pigments occur in thylakoids. The process of energy generation is almost anaerobic type and source of reducing power used is water present in organic and inorganic compounds. Because these bacteria do not require oxygen, they can survive in areas having low oxygen density.

Heterotrophic Bacteria Those bacteria which are unable to synthesize their own food and depend on preformed organic matter present in their medium for survival are called heterotrophic bacteria. They are further of three types.

Saprophytes These microorganisms depend upon organic matter present in the medium for their food. Their presence is beneficial

Fig. 6.6: Streptococcus thermophiles

50

C

as well as harmful for human interests. These bacteria lead to fermentation, (which is anaerobic breakdown of carbohydrates), putrefaction (that is anaerobic breakdown of proteins) and decay (that is anaerobic breakdown of organic compounds). Saprophytic bacteria—Streptococcus thermophilus helps in making yoghurt (Fig. 6.6). They serve to clean the environment by degrading dead and decaying matter. Fungi also play an important role in it. They dispose of the organic remains and are referred to as nature’s scavengers. They are used in sewage treatment. The methanogens, which help in sewage disposal are a good example of it. These bacteria are also used in curing of tea, coffee and tobacco. They may cause food poisoning. Spoilage of food along with destruction of common household articles is also due to saprophytic microorganisms.

Symbiotic Bacteria These bacteria live in mutual coordination with other organisms. They live in cordial environment, which is suitable to both the organisms. The commensal bacteria living in and on the human body play many roles like (Fig. 6.7):

Fig. 6.7: Symbiotic bacteria and their benefits

CHAPTER 6     Growth and Nutrition of Microorganisms

y

y y y

They help in synthesizing vitamins, like B12 and K in our intestine. When present on skin or other mucosal linings of the organs, they serve as barriers and do not let the pathogenic bacteria to colonize. The bile juices secreted in excess are also metabolized by symbiotic bacteria. Short chain fatty acids are produced by these bacteria in human body. Some bacteria also help in reducing hydrogen ion concentration in intestine and make it less acidic.

Parasitic Bacteria These bacteria live in or on the body of other living beings. They depend on their host for survival and derive food from them. They cause disease or are pathogenic microorganisms which are responsible for producing disease by attacking host cells or releasing toxins such as Salmonella typhi.

FACTORS INFLUENCING BACTERIAL GROWTH Under optimal conditions, bacteria can grow and divide extremely rapidly, and bacterial populations can double as quickly as every 10 minutes. The various physical conditions that could affect the growth of bacteria are discussed as follows.

Influence of Carbon Dioxide All bacteria require the presence of carbon dioxide for growth and this amount is provided by the atmospheric carbon dioxide or by the cellular metabolism endogenously. The bacteria like Brucella abortus, when first isolated from the body require a much higher concentration of carbon dioxide, i.e., 5–10%, and this is provided in the environment with the help of candle jar used for culture. Such bacteria are known as capnophilic.

Influence of Temperature There is a particular temperature range for each species within which it grows (Figs 6.8A to D). In the laboratory, bacteria are grown at an optimum temperature in a thermostatically controlled incubator. There are bacteria that grow between 25°C and 45°C and are known as mesophiles, e.g., Pseudomonas aeruginosa. Temperature is the most important factor that determines the rate of growth, multiplication, survival, and death of all living organisms. y High temperatures damage microbes by denaturing enzymes, transport carriers, and other proteins. Microbial membranes are disrupted by extreme temperature. y At very low temperatures membranes also solidify and enzymes also do not function properly.

UNIT II     General Characteristics of Microbes

y

Influence of Oxygen It is necessary to provide oxygen for a strict aerobe and to remove it completely from the environment of a strict anaerobe. There are pathogenic bacteria like Vibrio cholerae, which are obligate aerobes. Mostly, the medically important microorganisms are facultative anaerobes. They can grow as aerobic bacteria under normal conditions and can survive and multiply in absence of oxygen too, for example, coliform bacteria. y

y

Anaerobic bacteria grow in absence of oxygen and obligate anaerobes may even die in presence of oxygen like Clostridium tetani. The reason for such a behavior lies in the fact that the necessary enzymes needed for survival and growth are active only in absence of oxygen. The microaerophilic bacteria require oxygen to survive, but the level of oxygen should be less than that present in the atmosphere, e.g., Campylobacter coli.

A  Psychrophile – Mold

C  Mesophile – E. coli

B  Psychrotroph – Vibrio

D  Thermophile – Archaea

Figs 6.8A to D: Different types of bacteria as per the different conditions of the temperature they live in

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Types of Temperature Favoring Bacterial Growth y

y y

Minimum growth temperature: The lowest temperature at which organisms grow is the minimum growth temperature. Optimum growth temperature: The temperature at which the most rapid rate of multiplication occurs. Maximum growth temperature: The highest temperature at which growth occurs. A temperature only slightly above this point frequently kills the microorganisms by inactivating critical enzymes.

Types of Bacteria According to Temperature The microorganisms are susceptible to heat; especially moist heat can cause coagulation and denaturation of proteins: y Psychrophiles/cryophiles: The term psychrophile was first used by S. Schmidt-Nelson. Psychrophilic bacteria can grow best at a temperature range of –5°–30°C. These are cold‐tolerant bacteria and have optimal and maximal growth temperatures above 15°C and 20°C, respectively. Importance of psychrophiles lies in their ability to cause spoilage of refrigerated and frozen food. Examples: Pseudomonas, Aeromonas, Bacillus, Clostridium, etc.

Applied Microbiology • In order to inhibit bacterial contamination—generally, clinical

samples are stored at low temperature before processing.

• Vaccines are transported as well as stored by using ‘cold

chain’.

y

y

y

y

52

Extremophilic organisms are capable of growth and reproduction in cold temperatures. Temperature range is −20°C to +10°C. Examples: Oscillatoria, Chlamydomonas nivalis, Methanogenium, etc. Thermophiles are another group of nonpathogenic bacteria and the word is derived from Greek words ‘thermotita’ meaning heat and ‘philia’ meaning love. They prefer to grow at temperature of 25°–80°C. Examples are Thermus aquaticus, Geogemma barossii, etc. These organisms are important as they cause spoilage in underprocessed canned foods, since most of them form highly heat resistant spores. Mesophiles: They grow best in moderate temperatures of 20°–45°C. Examples are Escherichia coli, Streptococcus pneumoniae, etc. Under moist heat conditions most vegetative mesophilic bacteria have a thermal death point between 50°C and 65°C, whereas spores have thermal death point between 100°C and 120°C. Hyperthermophile: These thrive in extremely hot environments at temperature range of 80°–113°C. They

were first discovered by Thomas D. Brock in 1965, in hot springs in Yellowstone National Park, Wyoming. The cell membranes of hyperthermophiles contain high levels of saturated fatty acids to retain its shape at high temperatures. Examples are Sulfolobus, Methanococcus jannaschii, Thermotoga, etc.

Applied Microbiology Effect of temperature on patient care There is a set indoor temperature in the hospitals which is ideal for all. Infants and older adults may need their rooms warmer than usual due to their poor temperature regulation. For all, the most suitable indoor temperature is that which is warm enough to prevent feeling chilly, yet not too warm so as to cause perspiration. Keep the temperature between 68° and 74°F or 20° and 23°C. Operating rooms and critical care areas are kept slightly cooler to reduce the body’s metabolic demands.

Influence of Moisture About four-fifth by weight of the bacterial cell consists of water and is absolutely necessary for growth and survival. Drying in air kills bacterial cells under natural conditions. Drying and desiccation reduces water content and makes it less available for microbes to survive. Treponema pallidum, Gonococcus and common cold virus die quickly when exposed to drying, whereas tubercle bacilli, S. aureus and smallpox virus may survive for months. The non-sporing microorganisms may survive drying for a period of years if they are desiccated rapidly and completely, preferably frozen, and maintained in a high vacuum (0.01 mm Hg), in a sealed condition and stored at room temperature in dark. This process is known as lyophilization or freeze drying and is used for preserving bacterial cultures in the laboratories.

Effect of Moisture on Growth of Microorganisms Different types of fungi or bacteria require different amounts of water (in vapor form) to reproduce and grow. Majority of the microorganism require relative humidity (RH) of

Applied Microbiology Effect of moisture in patient care: Humidity is the amount of moisture in the air. Humidity in the range of 30% to 50% is normally comfortable. Very low humidity will dry skin and respiratory passages. Most hospitals maintain a low humidity setting to discourage the growth of microorganisms. Vaporizers or humidifiers may be ordered for a patient with a respiratory condition, when more humidity is required.

CHAPTER 6     Growth and Nutrition of Microorganisms

Influence of Light Spectrum Visible light is beneficial for bacterial growth because it is the source of energy for autotrophs. Darkness provides a favorable condition for growth of microorganisms. Infrared is the major source of Earth’s heat. Ionizing rays can produce mutations which may result in death of microorganisms. Ultraviolet rays and radiations present in environment are bactericidal in nature. Cultures die when exposed to sunlight. Even diffused daylight shortens the survival of microorganisms and are beneficial from hygienic point of view. Gamma rays, X-rays and infrared rays have bactericidal effect on the microorganisms (Fig. 6.9).

Applied Microbiology Effect of light on patient care: The amount of light is an important factor in providing comfort. It is provided either by natural or by artificial source. Adequate light or a sunny room can help to improve patients’ emotional state. The light should be bright enough to see without glare, to avoid eye strain and be soft and diffuse, to prevent sharp shadows.

Fig. 6.10: Effect of hydrogen ion concentration on bacterial cell

The bacteria can be classified on the basis of pH of the surrounding as: y Osmophilic bacteria - survive in high osmotic environment. y Osmoduric bacteria - resist changes in osmotic environment. y Halophilic bacteria - survive in environment with high salt concentration. y Acidophilic bacteria - capable of growing at low hydrogen ion concentration or in acidic environment.

UNIT II     General Characteristics of Microbes

60% or more, although they can survive and multiply in >20% RH. Therefore, decreasing temperature and moisture (relative humidity), creates a less favorable environment for microorganisms to grow.

Applied Microbiology Influence of Hydrogen Ion Concentration pH refers to negative logarithm of hydrogen ion concentration. Microbial growth is strongly affected by the pH of the medium. Drastic variations in cytoplasmic pH disrupt the plasma membrane or inhibit the activity of enzymes and membrane transport proteins (Fig. 6.10).

Fig. 6.9: Effect of visible light on bacteria

Effect of Hydrogen Ion A suitable pH is an essential factor in microbial metabolism and growth (Fig. 6.10). Most of the commensal and pathogenic bacteria grow at a neutral or slightly alkaline pH of 7.2–7.6.

Influence of Osmotic Pressure (Solutes and Water Activity) Due to the presence of semipermeable cytoplasmic membrane (SPM), bacteria also undergo osmotic effect, though they can withstand varying contents of salt, sugar and other similar solutes. Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a SPM. For most species of bacteria, the maximum concentration of sodium chloride is 5–12%. The bacteria undergo plasmolysis—shrinkage of the protoplast and its retraction from the cell wall due to the osmotic withdrawal of water. Plasmolysis occurs most readily in Gram-negative bacteria. When bacterial cells are transferred from a concentrated to a weak solution or to distilled water, it causes plasmoptysis and the cell bursts due to cell wall rupture (Fig. 6.11). 53

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Fig. 6.12: Ultrasonic vibrations are used in dentistry to kill oral bacteria Fig. 6.11: Effect of osmotic pressure on bacterial cell

Classification of Bacteria According to Osmotic Pressure y

y

y

Osmotolerant are those microorganisms which can grow at relatively high salt concentration. Examples: Aeromonas spp., Staphylococcus spp, etc. Halophiles grow in the presence of salt at concentration above 0.2–0.6. These bacteria are sensitive to acid, but tolerant to alkali - pH 7.5–14 and are called halophiles or alkalophiles. Examples: Halobacterium halobium, Vibrio cholera, Thermococcus alcaliphilus, etc. Acidophiles are some bacteria which can grow at acidic pH. They grow between pH 0 and 5.5. Examples: Lactobacillus, Ferroplasma, Thiobacillus thiooxidans, Sulfolobus acidocaldarius, etc.

y

Neutrophiles: Grow between pH 5.5 and 8.0; Examples: Lactobacillus acidophillus, E. coli, Pseudomonas aeruginosa, etc. Bacteria prefer media of pH near neutrality, and usually cannot tolerate pH values below 4–5.

Note that strong solutions of acids or alkali like 5% hydrochloric acid or sodium hydroxide respectively are lethal to most of the bacteria except Mycobacterium tuberculosis.

Influence of Mechanical and Sonic Stresses The cell walls of bacteria have enough strength and elasticity, still it is possible to rupture and kill the bacteria when exposed to mechanical stress. When the bacterial suspension is subjected to vigorous shaking with glass beads, or to supersonic or ultrasonic vibrations (9,000–200,000 and over 200,000 cycles per second respectively), the cells rupture due to separation of large molecular components of the cell (Fig. 6.12).

ASSESS YOURSELF Long Answer Questions 1. Enumerate the physical factors affecting growth of bacteria. Explain any two of them. 2. Explain bacterial growth curve. 3. Classify bacteria and add a note on the factors affecting bacterial growth.

Short Notes Write notes on: 1. Effect of temperature on bacterial growth 2. Effect of drying and desiccation on bacteria

Multiple Choice Questions 1. In which phase of bacterial growth curve there is no increase in number of cells but increase in volume? a. Log phase b. Lag phase c. Stationary phase d. Death phase

54

2. Bacteria which need 5–10% CO2 for growth are: a. Halophilic b. Acidophilic c. Capnophilic d. None of these 3. The microorganisms which spoil food stored in refrigerators are: a. Thermophiles b. Psychrophiles c. Both (a) and (b) d. None of the above 4. Medically important bacteria are: a. Aerobes b. Anaerobes c. Facultative anaerobes d. Obligate anaerobes 5. The bacterial multiplication is maximum in: a. Lag phase b. Log phase c. Stationary phase d. None of these Answer Key Multiple Choice Questions

1. b 2. c 3. b 4. c 5. b

Blood and Body Fluids

Chapter Outline

• Body Fluids • Blood

INTRODUCTION Every living organism needs nourishment to stay alive. The unicellular microorganisms derive nutrition directly from the surroundings by diffusion. In case of multicellular organisms like humans, the mechanism of obtaining nutrients and throwing out waste from the body is not simple. The body fluids present in the human body serve as a medium for the exchange of nutrients and waste products. The body fluids constitute nearly one-fourth of body weight in humans.

7

CHAPTER

• Hematological Disorders

saliva, tears, gastric juices, peritoneal and pleural fluids, sebaceous gland secretions, urine and sweat.

Composition The composition of the body fluids (Table 7.1) depends on the exchange of metabolic products between the cells in the biological tissue and the blood. The body fluid acts as a medium for the exchange of materials like glucose, oxygen, ions, carbon dioxide, etc.

BODY FLUIDS The dominating content of body fluids is water as intracellular (60–65%) and extracellular fluid (30–40%). The fluid components outside the cells include the fluid between the cells like—blood, lymph and interstitial fluid. There is nearly 6–10 L of lymph and 3.5–5 L of blood in the body. The body fluid which fills the spaces between the cells and surrounds the cells is known as interstitial fluid and the fluid present inside the cells is known as intracellular body fluid (Fig. 7.1). The body fluids present in a human body include: vitreous humor and aqueous humor, mucus (including phlegm and nasal secretions), bile juices, blood, CSF, earwax, perilymph and endolymph, female and male secretions, breast milk,

Fig. 7.1: Breakdown of total body mass into anhydrous and fluid compartments

SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY TABLE 7.1: Composition of extracellular and intracellular body fluids Extracellular fluid (plasma) Na+

142

mmol/L

K

4

mmol/L

+

Ca

2

mmol/L

Mg2+

1

mmol/L

Cl

105

mmol/L

27

mmol/L

Phosphates

1

mmol/L

Protein

70

g/L

Osmolarity

290

mosm/L

Na+

10

mmol/L

K

160

mmol/L

5 mm in length when a loop is used to stretch the colony on an agar plate (Fig. 16.4B). y Biochemical tests: Oxidase –ve, indol –ve, H2S –ve and citrate +ve

B Figs 16.4A and B: A. White capsules (Glycocalyx) surrounding purple cells; B. String test

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TREATMENT AND PREVENTION Antibiotics: Infections caused by KPC-producing bacteria can be difficult to treat because fewer antibiotics are effective against them. In such cases, a microbiology laboratory must run tests to determine which antibiotics will treat the infection. The drugs used to treat include: Aminoglycosides, Polymyxins, Tigecycline, Fosfomycin, Temocillin.

HAEMOPHILUS INFLUENZAE INTRODUCTION Haemophilus influenzae can cause infections ranging from mild to severe in people of all age groups. Before vaccine was introduced, meningitis frequently occurred in children younger than 5 years of age.

to nitrate and is catalase and oxidase positive. It can ferment glucose and galactose.

Satellite Growth

Small, slender, non-motile, nonacid fast, non-sporing Gramnegative bacilli. At times, it may occur as coccobacillus. Some strains may also produce capsule (Fig. 16.5).

Staphylococcus aureus is streaked across a blood agar culture plate of sample having H. influenzae. The plate is incubated overnight. It is observed that colonies of H. influenzae are larger near the streak of S. aureus and are smaller when they are away from the streak. This is known as satellitism. This is due to the X and V factors secreted by S. aureus. The organism H. influenzae depends on S. aureus to obtain these growth factors. This method is useful in isolating H. influenzae from clinical specimens.

Growth Features

Virulence Factors

The organisms require enriched medium to grow and it is called fastidious. The organism requires X and V factors as necessary growth factors. These growth factors are available in blood. These growth factors can be made available to H. influenzae when heated blood is added to medium because the growth factors are released from RBCs on heating at a temperature of 80°–90°C. The organism can reduce nitrite

Outer membrane protein (OMP), lipo-oligosaccharide (LOS) and capsular polysaccharide give virulence power to this organism. On the basis of capsular polysaccharide, it is further classified into serotypes. The polyribosyl ribitol phosphate (PRP), an antigen of Hib or H. influenzae type B is capable of inducing antibodies [Immunoglobulin (IgA, IgG and IgM)] formation, which are protective and bactericidal. It is a delicate organism and is destroyed by heating at 55oC for 30 minutes. It cannot withstand drying, refrigeration or disinfectants. It dies in 1–48 hours in dried secretions and droplets. The organism normally lives in nose and throat. Young children and elder people are at risk of getting H. influenzae infection. People having special medical conditions are also at risk, like immunocompromised individuals having HIV infection, people with sickle cell disease, asplenic condition, autoimmune diseases, and patients on chemotherapy or radiation therapy. It is capable of causing different types of infections and these are of two types—invasive and noninvasive.

GENERAL CHARACTERISTICS Morphology

Fig. 16.5: Stained preparation of H. Influenzae

164

Some Klebsiella bacteria have become highly resistant to antibiotics. Klebsiella pneumoniae produces an enzyme known as a carbapenemase (referred to as KPC-producing organisms). Therefore, the class of antibiotics called carbapenems will not work to kill the bacteria and treat the infection. Klebsiella infections that are not drug-resistant can be treated with antibiotics. Prevention: The infection can be prevented by observing safety precautions in healthcare facilities.

CHAPTER 16     Gram-Negative Bacilli (Part B) y

Invasive Infection The organism acts as primary pathogen and invades those parts of the body, which are normally free from microorganisms like blood or spinal fluid. Mostly Hib is involved in such infections. The bacteria spreads through blood. Capsule protects it from the action of phagocytes. Invasive type of disease is severe and can be fatal. The most common health problems are­— meningitis, pneumonia, bacteremia, endocarditis, pericarditis, cellulitis, epiglottitis and arthritis. These complications may be fatal. Disease could be fatal. Meningitis: It is a serious disease which is very common in children between 2 months and 3 years of age. Through blood stream, bacteria reach meninges and cause inflammation. Fatality rate is high (90%). Laryngo-epiglottitis: Hib produces inflammation of the larynx and epiglottis and it is also known as croup. The disease is most often seen in children over 2 years of age. There is obstructive edema in the larynx and to save the life, tracheostomy along with antimicrobial drugs and supportive care have to be given. Pneumonia: Occurs in infants and is accompanied with empyema and meningitis at times. Septic arthritis is due to hematogenous spread of the organism in children younger than 2 years of age. The symptoms include: Headache, malaise, pain in the chest, and fever with chills, cough and difficulty in breathing.

Noninvasive Infection The organism does not invade the tissue and causes additive infections or secondary infections. It mostly causes noninvasive secondary infection of the respiratory tract. For example, otitis media and bronchitis. Suppurative lesions like otitis media occur when bacteria directly spreads from the nasopharynx to ears. The organism causes acute bronchitis when it associates with Streptococcus pneumoniae.

LAB DIAGNOSIS y

Samples are collected from blood, throat swab, sputum, cerebrospinal fluid (CSF) and pus depending on the site of infection. The suspected samples are not subjected to refrigeration because the organism dies on refrigeration. Therefore, it is immediately seeded on the transport medium and sent to lab.

y

y

The sample is observed with the help of Gram staining method—Gram-negative coccobacilli. The organism can be demonstrated by immunofluorescence. Capsule is observable by Quellung reaction. In order to isolate this organism, culture is done on chocolate agar or blood agar and is incubated with 5–10% CO2. Serological tests can detect capsular antigen and the tests are: agglutination test, counterimmunoelectrophoresis and coagglutination.

TREATMENT AND PREVENTION

UNIT III     Pathogenic Organisms

PATHOGENICITY

The infection is treated with antibiotics like co-trimoxazole, clavulanic acid and ampicillin. Prevention: Avoiding direct contact with infected person is helpful as the disease is contagious and spreads from person to person. Vaccine is available for Hib, but it is type specific and cannot provide immunity against other diseasecausing strains. The vaccine is recommended for young children below 5 years of age. Vaccine is prepared from purified platelet-rich plasma (PRP), but it is poorly immunogenic. The immunogenicity is improved by combining it with diphtheria or tetanus toxoid, which acts as protein carriers.

Nursing Interventions In order to relieve, the patient is advised: • Coughing exercise which is effective and helps to improve airway passage. • Chest physiotherapy helps in loosening and mobilizing the secretions. • Adequate hydration (2–3 L) loosens the secretions in the lungs by thinning it. • Humidification also helps in loosening pulmonary secretions and improves airway. • The patient is asked to take bed rest and take pain relievers to reduce chest pain. • Fluid intake with electrolytes must be increased to at least 2 L/day so as to compensate unavoidable fluid loss from body. It will provide electrolytes and calories. • The nutrition-rich beverages like shakes and drinks help to restore proper nutrition. • Instruct the patient and his family about the causes, signs, symptoms and management of pneumonia. Ask the patient to come for follow-up.

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PSEUDOMONAS AERUGINOSA INTRODUCTION Pseudomonas are involved in hospital-acquired infections. These organisms live freely in soil, water, plants and air. Generally, it does not cause disease in a healthy person or if it causes, it is of very mild nature. But people having weak immune system may suffer because these bacteria turn pathogenic and cause diseases which could be fatal for patients who have been staying in hospital for a long time.

GENERAL CHARACTERISTICS Morphology These are Gram-negative bacilli and are slender in structure. The organism possesses a polar flagellum and is actively motile. The organism has pili, when freshly isolated from clinical samples (Fig. 16.6).

Growth Features It is an aerobe and when grown on nutrient agar, produces large translucent colonies with irregular margins. A typical fruity odor is emitted by it due to production of tryptophan from aminoacetophenone. y On blood agar, it shows β-hemolysis and on DCA and MacConkey’s medium, it produces non-lactose fermenting colonies. In liquid medium, it forms surface pellicle. y Pseudomonas aeruginosa produces pigments like— pyocyanin, which is most important and is bluish in color. The organism can be isolated on its basis as other species

y

do not produce this pigment. Pyoverdine gives yellowish shade to culture and can be detected when culture plate is examined under ultraviolet light. Pyorubin is bright red, water-soluble pigment and pyomelanin, which is brown to black in color and is not produced commonly. It gets killed when heated to 55°C for an hour. The organism shows resistance to commonly used disinfectants like Dettol or cetrimide. In fact these are incorporated in culture medium to isolate P. aeruginosa from clinical samples. It gets killed by strong phenolic agents, acids and glutaraldehyde. It is susceptible to silver sulfonamide and this compound is used in ointments for topical application in burns.

Virulence Factors Various toxins and enzymes contribute in virulence of the organism and some of these are—exotoxins and endotoxins which are pyrogenic and pyocyanin. These help the organism to colonize in the nasal mucosa and resist the normal defense mechanism of host. The extracellular enzymes and hemolysins contribute in formation of local lesions. The organism is found in soil, water, sewage and gut of mammals as normal fecal flora. Pseudomonas infections are considered opportunistic infections (Figs 16.7A to C).

PATHOGENICITY HAIs: The organism is resistant to normally used disinfectants used in health-care settings, therefore, they play an important part in causing nosocomial infections. The equipment used in hospital like endoscopes, various articles like bedpans or medicines including lotions, eye and ear drops, ointments, distilled water get contaminated with Pseudomonas and serve as potential source of infection in hospitals. People on prolonged stay in hospital are at risk of getting Pseudomonas infection. The infection is spread via unhygienic practices followed by health care workers. The other situations when this organism can cause infections include—patients on chemotherapy, burn wounds, cystic fibrosis cases, HIV patients and persons undergoing surgery or on artificial ventilators.

In healthy persons, it causes mild infections like skin rashes Fig. 16.6: Gram staining of P. aeruginosa

166

or ear or nail infections. It causes both localized and generalized infections:

CHAPTER 16     Gram-Negative Bacilli (Part B)

B

C

Figs 16.7A to C: Infections of P. aeruginosa. A. Nail infection; B. Hot tub rash; C. Folliculitis y

Localized infections like bedsores, infections of wounds and eyes, urinary tract infections after catheterization are most commonly seen in patients having prolonged stay in hospital. The skin rashes are mostly seen after getting hot bath or inadequately chlorinated swimming pools— known as hot tub rash or folliculitis when hair follicles are infected. Contact lenses contaminated with this organism cause eye infections. External ear canal infection called swimmer’s ear is also caused by Pseudomonas.

y

UNIT III     Pathogenic Organisms

A

Generalized infections can be caused by dissemination of organism in body through blood. The infections are of more severe nature when it is in blood or lungs. It can cause pneumonia in weak people. The organism causes bacteremia and patient complains fever with chills, fatigue and myalgia. Bacteremia is the reason for hemodynamic shock (low blood pressure) and can lead to failure of other organs such as liver, kidneys and heart.

Case Scenario A 52-year-old female was hospitalized in semiconscious state. Her bladder control was lost. She had fever with chills on 3rd day. She was catheterized for her bladder management. On 4th day, she started having pain in abdomen and loose motions. Her condition improved when ampicillin was withdrawn. What do you interpret? Microbiological Discussion/Laboratory Diagnosis Sample: Stool sample was taken • Culture: Cultured on blood agar or MacConkey agar. The organism produces bluish pigment. • Microscopy: Gram –ve rods • Biochemical test: IMVIC = ---+

Interpretation Based on the test results the organism was identified as Pseudomonas spp. As her condition improved on stopping antibiotic, so it was a case of antibiotic associated diarrhea or Pseudomonas colitis.

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TREATMENT AND PREVENTION y

Prevention: The infection can be prevented by following universal safety precautions in health-care settings. After swimming one must take off the swimming costume and shower properly. Ears should be dried carefully to prevent swimmer’s ear. Undue stay in hospitals must be avoided.

y

Antibiotics: Pseudomonas infections are becoming more and more difficult to treat because of antibiotic resistance that has been adapted by the organism in due course of time. Resistance to various antibiotics may be developed during the course of treatment. The antibiotics mainly used are gentamycin and third-generation chemotherapeutic agents like: Ceftazidime, ciprofloxacin, aztreonam, carbapenems and ureidopenicillins.

Nursing Interventions • Maintain a clear airway with the help of suctioning, whenever

Facteria

•

Multi drug resistant (MDR) Pseudomonas aeruginosa has been isolated from immunocompromised or patients having neoplastic disease, cystic fibrosis or patients staying in ICU for a long time. As the organism shows multi drug resistance, new drug of choice is doripenem which has shown promising results in vitro. P. aeruginosa produces metallo-beta-lactamase (MBL) which makes it resistant to antibiotics and poses a threat in ICUs, especially.

• • • • • •

necessary in case of respiratory infection. Deliver oxygen, if needed. Immunocompromised patients must be protected from being exposed to infection. Keep a record of wound exudates and sputum. Strictly follow the sterile techniques while changing dressing on infected wounds. Enquire about the history of allergies especially penicillin. Keep a watch on the patient’s renal function when treating with aminoglycosides. Make sure that the antibiotic course is completed successfully. The use of humidifiers in the patient’s room must be avoided.

BORDETELLA PERTUSSIS INTRODUCTION The disease whooping cough is caused by Bordetella pertussis. It is an acute infectious disease. In 20th century, this disease was one of the main causes of child mortality. After the vaccine became available for this disease in 1940s, the incidence was decreased.

medium with 50% blood. It grows slowly and after 72 hours of incubation on Bordet-Gengou medium, small, greyish white, dome-shaped, smooth colonies appear. The colonies have specific type of shiny surface resembling cut mercury drops on charcoal agar. The colonies show a blurred hemolytic zone.

GENERAL CHARACTERISTICS Morphology These are small Gram-negative coccobacilli arranged singly, in pairs and in small groups. In subcultures they become pleomorphic. The organism may possess a feeble capsule when freshly isolated (Fig. 16.8).

Growth Features The organism needs charcoal, blood and ion exchange resins in the medium for isolation. One of the classic mediums used to isolate this organism is glycerol-potato-extract agar

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Fig. 16.8:  Bordetella pertussis (Gram staining)

CHAPTER 16     Gram-Negative Bacilli (Part B)

y

LAB DIAGNOSIS The samples are taken with nasopharyngeal swabs and for isolation, these are inoculated on BG medium with and without antibiotics, separately. The culture plates are incubated at 37oC for 7 days. The colonies appear with characteristic features and are later tested by biochemical tests. The bacteria can be identified by Gram staining method and direct fluorescent antibody test. To observe the presence of antibodies in serum of patient, PCR, IHA, ELISA and CFT can be used.

y

Fig. 16.9: Some virulence factors of B. pertussis

Virulence Factors Pertussis toxin, which possesses tracheal cytotoxin, is mainly responsible for symptoms of the disease (Fig. 16.9). Other antigenic components that contribute in pathogenesis are Lipopolysaccharides endotoxins, Adenylate cyclase, Hemolysin, Agglutinogens, Pertactin and Filamentous hemagglutinin. These antigenic components are responsible for clinical symptoms of the disease and immune response produced in the body. The immunity produced by infection is long lasting.

PATHOGENESIS Reservoir of infection is human being for this disease. The disease is more prevalent in children under 2 years of the age. The organism is an obligate parasite. The disease is toxin mediated and infects the respiratory tract. For the sake of study, the disease can be divided into three stages: 1. Prodromal stage: The infection is acquired when a healthy person inhales contaminated droplets, which are formed when a patient sneezes or coughs. After an incubation period of 5–10 days, there are mild flu like symptoms with fever and irritating cough. 2. Paroxysmal stage: The bacteria attach to the ciliated cells of respiratory tract and while growing, produce toxins. The toxins damage the ciliated cells and irritation is caused with induced secretion of mucus. It leads to bronchospasm and coughing, which is so rapid that it makes the breathing difficult. Due to these circumstances, patient breathes through strained glottis and a whooping sound is heard. This stage continues for 6–8 weeks. 3. Convalescent stage: The stage continues for another 2–4 weeks and severity of the disease decreases later. The bacteria do not invade the blood and some of the complications include—otitis media, conjunctivitis,

y y

UNIT III     Pathogenic Organisms

secondary infections and hemorrhages due to pressure effect of coughing. Decreased supply of oxygen to the brain, leads to neurological involvement too. The patient may die because of secondary bacterial infection that causes pneumonia.

TREATMENT AND PREVENTION The treatment is provided according to symptoms. Vaccination is done as a part of normal immunization schedule. Inactivated bacteria are combined with diphtheria and tetanus vaccine and administered as DPT. Three injections are given through intramuscular route at an interval of 4 weeks, starting at the age of one and half month. There may be minor adverse reactions like—local swelling, erythema and fever. To overcome the adverse reactions, acellular pertussis vaccine prepared from antigenic components is preferred. Avoiding contact with infectious person is helpful in preventing infection. Research has shown that this organism is continuously undergoing mutations. Therefore, due to declining immunity from vaccine and organism’s tendency to change at genetic level, there may be rise in disease incidence at some point of time in future.

Nursing Interventions • Try to ease respiratory effort by positioning and supply of • • • • • •

oxygen. Patient should be observed for severity of cough. Limit paroxysm. Observe vital signs and oxygen saturation. Promote fluid intake, good nutrition and sufficient hydration. Family must be taught about vaccination. Infection control measures must be observed.

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SECTION A     Applied Microbiology

Textbook of APPLIED MICROBIOLOGY

CAMPYLOBACTER AND HELICOBACTER INTRODUCTION Campylobacter and Helicobacter are Gram-negative microaerophilic bacteria. They are widely distributed in the animal kingdom. They are animal pathogens. As they are fastidious and slow-growing in culture, they can cause human gastrointestinal problems. They may cause diarrheal illnesses, chronic superficial gastritis, systemic infection, peptic ulcer disease, and can even lead to gastric carcinoma. A

CAMPYLOBACTER JEJUNI Campylobacters cause important zoonotic infections. First isolated as Vibrio fetus in 1909 from spontaneous abortions in livestock. Campylobacter enteritis was not recognized until the mid-1970s when selective isolation media were developed for culturing campylobacters from human feces. It is most common form of bacteria responsible for acute infectious diarrhea in developed countries. They have higher incidence when compared with Salmonella and Shigella combined. These are Gram-negative organisms with helical (spiral or curved) morphology and tend to be pleomorphic. Characteristics that facilitate penetration and colonization of mucosal environments are motility by polar flagella. These are microaerophilic, become coccoid when exposed to oxygen or upon prolonged culture and neither ferment nor oxidize carbohydrates. This bacteria is commonly found in animal feces. It is one of the most common causes of human gastroenteritis in the world. Food poisoning caused by Campylobacter species can be severely debilitating, but is rarely life-threatening. It has been linked with subsequent development of Guillain-Barré syndrome (GBS), which usually develops two to three weeks after the initial illness.

General Characteristics Morphology Small, thin (0.2–0.5 µm × 0.5–5.0 µm), helical (spiral or curved) cells with typical Gram-negative cell; “Gullwinged” appearance. They have tendency to form coccoid and elongated forms on prolonged culture or when exposed to O2.

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B Figs 16.10A and B: Darting movement

Motility Distinctive rapid darting motility (Figs 16.10A and B): y Long sheathed polar flagellum at one (polar) or both (bipolar) ends of the cell. y Motility slows quickly in wet mount preparation

Growth Features y y y

Microaerophilic and capnophilic 5% O2, 10% CO2, 85% N2 Thermophilic (42°–43°C) (except C. fetus)—Body temperature of natural avian reservoir May become nonculturable in nature

Pathogenicity Zoonotic: Campylobacter remains the leading cause worldwide of zoonotic disease in humans. Often causing the same symptoms as Salmonella, such as mild to severe diarrhea, Campylobacter infections can also be an infectious trigger for the more serious Guillain-Barré syndrome. It is often isolated from healthy cattle, chickens, birds and even flies. It is sometimes present in nonchlorinated water sources such as streams and ponds. Damage is done (ulcerated, edematous and bloody) to the mucosal surfaces of the jejunum, ileum, colon. C. jejuni infections may also produce serious bacteremia in individuals with AIDS. Guillain-Barré syndrome (GBS), a demyelinating disorder resulting in acute neuromuscular paralysis, is a serious sequelae of Campylobacter infection. Up to 40% of patients with the syndrome have evidence of recent

CHAPTER 16     Gram-Negative Bacilli (Part B)

Lab Diagnosis Feces is refrigerated and examined within few hours. Rectal swabs are preserved in semisolid transport medium. Blood sample is taken for suspected case of C. fetus. Care is taken to avoid oxygen exposure. Selective isolation by filtration of stool specimen as it can pass through 0.45 μm filters. The identification tests are summarized as follows.

Fig. 16.11: H. pylori – A. Morphology

General Characteristics Morphology

Tests used

Characteristics to identify

Motility

Darting motility in fresh stool

Microscopy

Gull-wing appearance in gram stain

Gram-negative, microaerophilic, and spiral in structure bacterium. Usually it is found in the stomach. The helical shape helps it in penetrating the mucoid lining of the stomach to establish infection (Fig. 16.11).

Culture

Growth at 25°, 37°, or 42°–43°C—with watery, spreading and round, convex

Growth Features

Susceptibility

Susceptible to malidixic acid and cephalothin

y

Biochemical

• • • •

y

Hippurate hydrolysis (C. jejuni is positive) Catalase + Oxidase + Strong producer of urease

UNIT III     Pathogenic Organisms

Campylobacter infection. Approximately, 20% of patients with GBS are left with some disability, and approximately 5% die despite advances in respiratory care. Campylobacteriosis is also associated with Reiter syndrome, a reactive arthropathy. Multiple joints can be affected, particularly the knee joint. Pain and incapacitation may continue for months.

H. pylori grows on Skirrow’s medium with—Vancomycin, Polymyxin and Trimethoprim (Fig. 16.12). Grows in 3–6 days at 37°C. The colonies appear translucent (12 mm in diameter) and optimal growth occurs in microaerophic environment.

Treatment and Prevention

Pathogenicity

Antibiotics: Gastroenteritis is self-limiting; fluids and electrolytes help in it. Antibiotic treatment can shorten the excretion period. Erythromycin is drug of choice for severe or complicated enteritis and bacteremia. Fluoroquinolones are highly active (e.g., ciprofloxacin was becoming drug of choice) but organism has developed resistance. Azithromycin is effective in recent human clinical trials.

H. pylori is believed to be transmitted orally due to contaminated food or water. H. pylori is found in the deep

Prevention: Control should be directed at domestic animal reservoirs and interrupting transmission to humans.

HELICOBACTER PYLORI Researchers believe that Helicobacer pylori (H. pylori) is responsible for the majority of peptic ulcers. H. pylori infection is common in the United States. Most infected people, however, do not develop ulcers. Most likely, infection depends on characteristics of the infected person, the type of H. pylori, and other factors yet to be discovered.

Fig. 16.12: On Skirrow’s medium

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Textbook of APPLIED MICROBIOLOGY SECTION A     Applied Microbiology

epithelial surface. H. pylori overlies the gastric type but not intestinal epithelial cells. The symptoms are nausea, pain and fever. Acute symptoms last for