ANTIBIOTIC SENSITIVITY OF ESCHERICHIA COLI ISOLATED FROM ANIMALS AND HUMAN

112 Pages·2007·0.88 MB·English

ESCHERICHIA COLI
ANTIBIOTIC SENSITIVITY OF
ISOLATED FROM ANIMALS AND HUMAN SOURCES

By

MOHAMMED KHIDER ABD ALRAHIM AHMED

B. V. Sc. University of Khartoum

2001

Supervisor:

Dr. Suliman Mohammed El Hassan

A thesis submitted in partial fulfillment of the requirements
for the degree of Master of
Veterinary Medicine

Department of Microbiology

Faculty of Veterinary Medicine
University of Khartoum

September 2006
PREFACE

This work has been carried out at the Department of Microbiology,

Faculty of Veterinary Medicine, University of Khartoum, under the

supervision of Dr. Suliman Mohammed El Hassan.
DDDDEEEEDDDDIIIICCCCAAAATTTTIIIIOOOONNNN

To my parents
With thankful
To my Brothers and Sister
With love
To my colleagues and friends
With wishes
ACKNOWLEDGEMENT

First I would like to thank god for giving me strength, confidence and
patience to complete this study.
I whish to express my sincere gratitude to my supervisor Dr. Suliman
Mohammed El Hassan for patience supervision, guidance, encouragement,
advice, and support.
I am also gratefull to the members of department of Microbiology for
their unlimited assistance and advice.
I am deeply indebted to my family for their patience, continued
encouragement and enthusing throughout this work.
Finally I appreciate the support of all people who helped me in any
way to finish this work successfully.
LIST OF CONTENTS

Page
PREFACE -------------------------------------------------------------------- i
DEDICATION------------------------------------------------------------------- ii
ACKNOWLEDGEMENT ---------------------------------------------------- iii
LIST OF CONTENTS --------------------------------------------------------- iv
LIST OF FIGURES ------------------------------------------------------------ xi
LIST OF TABLES ------------------------------------------------------------- xii
ENGLISH ABSTRACT ------------------------------------------------------- viii
ARABIC ABSTRACT --------------------------------------------------------- xv
INTRODUCTION -------------------------------------------------------------- xvii
1. CHAPTER ONE: LITERATURE REVIEW ------------------------ 1
1.1 Historical background ------------------------------------------------- 1
1.2 Classification of E.coli ------------------------------------------------ 2
1.3 Definition of E.coli ---------------------------------------------------- 3
1.4 Normal habitat of E.coli ---------------------------------------------- 4
1.5 Characteristics of E.coli ---------------------------------------------- 5
1.5.1 Morphology ------------------------------------------------------------ 5
1.5.2 Capsules ---------------------------------------------------------------- 5
1.5.3 Cultural characteristics ----------------------------------------------- 6
1.6 Isolation and cultural characterization of E.coli ------------------- 6
1.6.1 Media for isolation of E.coli ---------------------------------------- 6
1.6.1.1 Differential or selective media ------------------------------------- 6
1.6.1.2 Enrich media ---------------------------------------------------------- 7
1.6.1.3 Basic media ----------------------------------------------------------- 7
1.6.1.4 Maintenance and preservation of E.coli --------------------------- 7
1.7 Resistance to physical agents ----------------------------------------- 8
1.8 Biochemical tests ------------------------------------------------------- 8
1.9 Antigens and toxins ---------------------------------------------------- 9
1.9.1 O antigens -------------------------------------------------------------- 9
1.9.2 K antigens -------------------------------------------------------------- 9
1.9.3 H antigens -------------------------------------------------------------- 9
1.9.4 Fimbrial antigens ------------------------------------------------------ 9
1.9.5 Common antigens (CA) ---------------------------------------------- 10
1.10 Classification of pathogenic E.coli ---------------------------------- 10
1.10.1 Entero- toxigenic E.coli (ETEC) --------------------------------- 10
1.10.1.1 Prevalence of the disease caused by (ETEC) -------------------- 10
1.10.2 Enteroinvasive E.coli (EIEC) ---------------------------------------- 10
1.10.2.1 Virulence factors of EIEC ----------------------------------------- 11
1.10.3 Enteropathogenic E.coli (EPEC) ---------------------------------- 11
1.10.3.1 Virulence factors of EPEC ------------------------------------------ 11
1.10.4 Enterohaemorrhagic or verotoxigenic E.coli (EHEC) ---------- 11
1.10.4.1 Virulence factors of EHEC ----------------------------------------- 11
1.10.5 Enteroaggregative E.coli (EAEC) ---------------------------------- 11
1.10.5.1 Virulence factors of EAEC ----------------------------------------- 12
1.10.6 Diffusely adhering E.coli (DAEC) --------------------------------- 12
1.10.6.1 Virulence factors of DAEC ----------------------------------------- 12
1.10.7 Uropathogenic E.coli (UPEC) --------------------------------------- 12
1.10.7.1 Virulence factors of UPEC ------------------------------------------ 12
1.10.8 E.coli that causes neonatal sepsis and meningitis ---------------- 12
1.10.8.1 Virulence factors ----------------------------------------------------- 12
1.11 Enterotoxin ------------------------------------------------------------- 13
1.12 Mode of transmission ------------------------------------------------- 13
1.13 Epidemiology and pathology ---------------------------------------- 13
1.14 E.coli infection in animals and man -------------------------------- 13
1.14.1 Cattle ------------------------------------------------------------------- 14
1.14.1.1 Colibacillosis --------------------------------------------------------- 14
1.14.1.2 Mastitis ---------------------------------------------------------------- 14
1.14.2 Sheep ------------------------------------------------------------------- 15
1.14.3 Goats ------------------------------------------------------------------- 16
1.14.4 Camels ----------------------------------------------------------------- 16
1.14.5 Horses ------------------------------------------------------------------ 16
1.14.6 Pigs --------------------------------------------------------------------- 16
1.14.7 Dogs -------------------------------------------------------------------- 17
1.14.8 Cats --------------------------------------------------------------------- 17
1.14.9 Avian pathogenic E.coli (APEC) ---------------------------------- 17
1.14.9.1 Acute septicemia ----------------------------------------------------- 18
1.14.9.2 Embryo and early chick mortality --------------------------------- 18
1.14.9.3 Respiratory tract infection (air sac disease) ---------------------- 19
1.14.9.4 Synovitis -------------------------------------------------------------- 19
1.14.9.5 Panophthalmitis ------------------------------------------------------ 19
1.14.9.6 Coligranuloma (Hjarre’s disease) ---------------------------------- 19
1.14.9.7 Enteritis ----------------------------------------------------------------19
1.14.9.8 Pericarditis ------------------------------------------------------------ 20
1.14.10 Human ------------------------------------------------------------------ 20
1.14.10.1 Childhood Diarrhea ------------------------------------------------ 20
1.14.10.2 E.coli O157:H7 ----------------------------------------------------- 20
1.14.10.3Urinary tract infection (UTI) ------------------------------------- 21
1.15 Antibiotics ------------------------------------------------------------- 21
1.15.1 Definition -------------------------------------------------------------- 21
1.15.2 Classification and mechanism of action ----------------------------21
1.15.2.1 Nucleic acid inhibitors ---------------------------------------------- 22
1.15.2.2 Protein synthesis inhibitors ----------------------------------------- 23
1.15.2.3 Cell wall synthesis inhibitors --------------------------------------- 24
1.15.2.4 Cell membrane function inhibitors -------------------------------- 25
1.15.3 Toxicity and side effects --------------------------------------------- 25
1.15.4 Antibiotic spectrum --------------------------------------------------- 26
1.15.4.1 Broad spectrum antibiotics ------------------------------------------ 26
1.15.4.2 Narrow spectrum antibiotics ---------------------------------------- 26
1.15.5 Type of action ----------------------------------------------------------26
1.15.6 Clinical use of antibiotics -------------------------------------------- 26
1.16 Drug resistant ---------------------------------------------------------- 27
1.16.1 Multidrug resistance -------------------------------------------------- 27
1.16.2 Mechanism of antibiotics resistance -------------------------------- 28
1.16.2.1 Biologic vs. clinical resistance -------------------------------------- 29
1.16.2.2 Environmentally mediated antimicrobial resistance --------------29
1.16.2.3 Microorganism- mediated antimicrobial resistance ---------------29
1.16.2.3.1 Intrinsic resistance ---------------------------------------------------29
1.16.2.3.2 Acquired resistance --------------------------------------------------30
1.16.3 Common pathways for antimicrobial resistance ------------------31
1.16.3.1 Resistance to β- lactam antibiotic ----------------------------------31
1.16.3.2 Resistance to aminoglycosides ------------------------------------ 31
1.16.3.3 Resistance to fluroquinolones --------------------------------------32
1.16.3.4 Resistance to glycopeptides ---------------------------------------- 33
1.16.3.5 Resistance to tetracyclines ------------------------------------------33
1.16.3.6 Resistance to trimethoprim -----------------------------------------34
1.16.3.7 Resistance to chloramphenicol ------------------------------------ 34
1.17 Antibiotic sensitivity ------------------------------------------------- 35
2. CHAPTER TWO: MATERIALS AND METHOD ------------------ 42
2.1 Sterilization ------------------------------------------------------------- 42
2.1.1 Flaming ----------------------------------------------------------------- 42
2.1.2 Red heat ----------------------------------------------------------------- 42
2.1.3 Hot air oven ------------------------------------------------------------ 42
2.1.4 Steaming at 100ºC ----------------------------------------------------- 42
2.1.5 Moist heat (Autoclave) ---------------------------------------------- 42
2.2 Reagents and indicators ----------------------------------------------- 43
2.2.1 Reagents ---------------------------------------------------------------- 43
2.2.1.1 Alpha- naphthol solution --------------------------------------------- 43
2.2.1.2 Potasium hydroxide --------------------------------------------------- 43
2.2.1.3 Hydrogen peroxide ---------------------------------------------------- 43
2.2.1.4 Methyl red -------------------------------------------------------------- 43
2.2.1.5 Tetramethyl- p- phenylenediamine dihydrochloride --------------43
2.2.1.6 Kovac’s reagent --------------------------------------------------------43
2.2.2 Indicators ----------------------------------------------------------------44
2.2.2.1 Andrade’s indicator ----------------------------------------------------44
2.2.2.2 Bromothymol blue -----------------------------------------------------44
2.2.2.3 Phenol red ---------------------------------------------------------------44
2.2.3 Preparation of media ----------------------------------------------------44
2.2.3.1 Nutrient broth -----------------------------------------------------------44
2.2.3.2 Peptone water -----------------------------------------------------------44
2.2.3.3 Peptone water sugars ---------------------------------------------------44
2.2.3.4 Glucose phosphate medium (MR- VP test medium) -------------- 45
2.2.3.5 Nutrient agar ------------------------------------------------------------ 45
2.2.3.6 Diagnostic sensitivity test agar --------------------------------------- 45
2.2.3.7 MacConkey’s agar -----------------------------------------------------46
2.2.3.8 Motility medium- Gragie tube medium ---------------------------- 46
2.2.3.9 Hugh and Leifson’s (O/F) medium -----------------------------------46
2.2.3.10 Urea agar medium -----------------------------------------------------46
2.2.3.11 Simmon’s citrate medium --------------------------------------------47
2.2.4 Collection of samples ---------------------------------------------------47
2.2.5 Transportation of samples --------------------------------------------- 47
2.2.6 Culture of specimens --------------------------------------------------- 48
2.2.7 Purification ---------------------------------------------------------------48
2.2.8 Microscopic examination -----------------------------------------------48
2.2.9 Identification of bacteria ----------------------------------------------- 48
2.2.10 Biochemical methods -------------------------------------------------- 48
2.2.10.1 Catalase test ------------------------------------------------------------ 48
2.2.10.2 Oxidase test ------------------------------------------------------------ 49
2.2.10.3 Oxidation- fermentation test ----------------------------------------- 49
2.2.10.4 Sugar fermentation test ---------------------------------------------- 49
2.2.10.5 Citrate utilization ----------------------------------------------------- 50
2.2.10.6 Urease activity -------------------------------------------------------- 50
2.2.10.7 Indole production test ----------------------------------------------- 50
2.2.10.8 Methyle red test ------------------------------------------------------ 50
2.2.10.9 Voges- Proskauer test ----------------------------------------------- 50
2.2.10.10 Motility test ---------------------------------------------------------- 51
2.2.10.11 Antibiotic sensitivity ------------------------------------------------ 51
2.2.10.12 Isolation of plasmid DNA ------------------------------------------ 52
2.2.10.13 Detection of plasmid DNA ----------------------------------------- 52
3. CHAPTER THREE: RESULTS ------------------------------------------ 54
3.1 Survey ----------------------------------------------------------------------- 54