epidemiology, molecular characterization and antimicrobial resistance of escherichia coli isolates

234 Pages·2017·3.56 MB·English

“EPIDEMIOLOGY, MOLECULAR CHARACTERIZATION AND
ANTIMICROBIAL RESISTANCE OF ESCHERICHIA COLI
ISOLATES FROM DIARRHOEIC CALVES”

By
MOTURI SRIVANI
M.V.Sc
RVD/2012-13

Thesis submitted to
P.V. NARASIMHARAO TELANGANA VETERINARY UNIVERSITY
In partial fulfilment of the requirements for the award of the degree of
DOCTOR OF PHILOSOPHY
In the faculty of Veterinary Science
(VETERINARY MICROBIOLOGY)

DEPARTMENT OF VETERINARY MICROBIOLOGY
COLLEGE OF VETERINARY SCIENCE
RAJENDRANAGAR, HYDERABAD-500 030

MARCH, 2017
CERTIFICATE

This is to certify that Mrs. MOTURI SRIVANI has satisfactorily prosecuted
the course of research and that the thesis entitled "Epidemiology, molecular
characterization and antimicrobial resistance of Escherichia coli isolates from
diarrhoeic calves" submitted is the result of original research work and is of
sufficiently high standard to warrant its presentation to the examination. I also certify
that the thesis or part thereof has not been previously submitted by her for a degree of
any University.

Date: , (Dr. Y. NARASIMHA REDDY)
Place: Hyderabad Major Advisor

i
ii

CERTIFICATE

This is to certify that the thesis entitled "Epidemiology, molecular
characterization and antimicrobial resistance of Escherichia coli isolates from
diarrhoeic calves" submitted in partial fulfilment of the requirements for the degree
of the "DOCTOR OF PHILOSOPHY" of P.V. Narasimharao Telangana
Veterinary University, is a record of bonafide research work carried out by Mrs.
Moturi Srivani under our guidance and supervision. The subject of the thesis has
been approved by student's advisory committee.

No part of the thesis has been submitted for any other degree or diploma. The
published part has been fully acknowledged. All the assistance and help received
during the course of the investigation has been duly acknowledged by the author of
the thesis.

(Dr. Y. NARASIMHA REDDY)
Chairman of the Advisory Committee

Thesis approved by the Student Advisory Committee

Chairman: Dr. Y.NARASIMHA REDDY
Professor & University Head ----------------------
Dept. of Veterinary Microbiology
College of Veterinary Science
Rajendranagar, Hyderabad - 500 030

Member: Dr. K.V.SUBRAMANYAM ----------------------
Associate Professor
Dept. of Veterinary Microbiology
NTR College of Veterinary Science
Gannavaram - 521101

Member: Dr. M.LAKSHMAN ----------------------
Professor
Dept. of Veterinary Pathology
Ruska Laboratory, College of Veterinary Science
Rajendranagar, Hyderabad - 500 030

Member: Dr. M.RAMAKOTI REDDY ----------------------
Principal Scientist
Directorate of Poultry Research
Rajendranagar, Hyderabad - 500 030
iii

LIST OF CONTENTS

Chapter Page.
Title
No. No.
INTRODUCTION
I 1-5
REVIEW OF LITERATURE
II
2.1 EPIDEMIOLOGICAL DISTRIBUTION OF E.COLI
7
IN CALVES
2.1.1 Prevalence of E.coli in calves of different areas
7
2.1.2 Prevalence of E.coli in calves of different age groups
9
2.1.3 Prevalence of E.coli pathotypes
10
2.1.3.1 Haemolytic activity of E.coli isolates
12
2.1.4 Prevalence of E.coli serogroups
13
2.2 VIRUELNCE GENES OF E.COLI
15
2.3 ANTIBACTERIAL RESISTENCE IN E.COLI
22
ISOLATES
2.4 ANTIMICROBIAL RESISTANCE GENES
28
2.4.1 ESBL genes
28
2.4.2 Tetracycline resistance genes
29
2.4.3 Sulfamethoxazole resistance genes
30
2.4.4 Sulfonamide and trimethoprim resistance gene
30
2.4.5 Chloramphenicol resistance gene
31
2.4.6 Gentamicin resistance gene
31
2.4.7 Streptomycin resistance gene
32
2.5 ASSOCIATION BETWEEN VIRULENCE AND
32
ANTIMICROBIAL REISITANCE GENES
2.6 ASSOCIATION BETWEEN VIRULENCE GENES
34
AND E.COLI SEROGROUPS
2.7 PLASMID PROFILES
35
MATERIALS AND METHODS
III
3.1 BIOLOGICALS, CHEMICALS AND
38
EQUIPMENT
3.1.1 Bacterial strains and DNA
38
3.1.2 Media, chemicals, buffers and reagents
38
3.1.3 Equipments
39
3.2 COLLECTION OF FAECAL SAMPLES
39
3.3 ISOLATION AND IDENTIFICATION OF E.COLI
41
3.4 BIOCHEMICAL CHARACTERIZATION
41
iv

3.5 MOLECULAR CHARACTERIZATION
42
3.5.1 Template preparation by boiling and snap chilling
42
method
3.5.2 PCR conditions for detection of E.coli
42
3.5.3 Analytical agarose gel electrophoeresis 42

3.6 DETECTION OF VIRULENCE GENES
43
3.6.1 Multiplex PCR for detection of virulence genes (stx
43
1, stx 2, eaeA, hlyA)
3.6.1.2 standardization of multiplex PCR protocol for
44
detection of stx1, stx2, eaeA, hlyA virulence genes
3.6.2 Detection of other virulence genes (STa, LT, cnf2,
45
EAST1 and pic)
3.6.2.1 Standardization of PCR protocols for detection of
45
other virulence genes
3.7 IN VITRO DETECTION OF VIRULENCE
46
FACTORS
3.7.1
Haemolysis assay 46
3.8
SEROGROUPING OF THE E.COLI ISOLATES 46
3.9 DETECTION ANTIBIOTIC RESISTANCE
47
3.10 DETECTION OF ANTIBIOTIC RESISTANCE
48
GENES FOR SELECTED ANTIBIOTICS
3.11 PLASMID PROFILING OF THE BACTERIAL
50
STRAINS
3.12 SEQUENCING ANALYSIS OF PCR PRODUCTS 51

3.13 STATISTICAL ANALYSIS
51
RESULTS
IV
4.1 EPIDEMIOLOGICAL DISTRIBUTION OF
52
E.COLI
4.1.1 Epidemiological distribution of E.coli in diarrhoeic
52
buffalo calves
4.1.2 Epidemiological distribution of E.coli in healthy
53
buffalo calves
4.1.3 Epidemiological distribution of E.coli in diarrhoeic
58
cow calves
4.1.4 Epidemiological distribution of E.coli in healthy cow
58
calves
4.2 MOLECULAR CHARACTERIZATION OF
64
VIRULENCE GENES IN E.COLI ISOLATES
4.2.1 Standardization of multiplex PCR assay targeting
64
stx1 stx2, eaeA and hlyA genes
4.2.2 Standardization of PCR assay targeting LT gene
64
4.2.3 Standardization of PCR assay targeting STa gene
64
4.2.4 Standardization of PCR assay targeting cnf2 gene
64
4.2.5 Standardization of PCR assay targeting EAST1gene
64
v

4.2.6 Standardization of PCR assay targeting pic toxin
69
gene
4.3 DISTRIBUTION OF VIRULENCE GENES
69
4.3.1 Distribution of virulence genes in E.coli isolates
69
from diarrhoeic and healthy buffalo calves
4.3.2 Distribution of virulence genes in E.coli isolates
73
from diarrhoeic and healthy cow calves
4.4 HAEMOLYSIS ON BLOOD AGAR
75
4.5 EPIDEMIOLOGICAL DISTRIBUTION OF
76
E.COLI SEROGROUPS
4.5.1 Distribution of serogroups of E.coli isolates in
76
diarrhoeic buffalo calves
4.5.2 Distribution of serogroups of E.coli isolates in
80
healthy buffalo calves
4.5.3 Distribution of serogroups of E.coli isolates in
82
diarrhoeic cow calves
4.5.4 Distribution of serogroups of E.coli isolates in
84
healthy cow calves
4.6 ANTIBIOTIC RESISTANCE PATTERN OF
88
E.COLI ISOLATES
4.6.1 Antibiotic resistance pattern of E.coli isolates from
88
diarrhoeic and healthy buffalo calves
4.6.2 Antibiotic resistance pattern of E.coli isolated from
89
diarrhoeic and healthy cow calves
4.7 ANTIBIOTIC RESISTANCE PATTERN OF
91
E.COLI PATHOTYPES
4.7.1 Antibiotic resistance pattern of E.coli pathotypes
91
isolated from diarrhoeic buffalo calves
4.7.2 Antibiotic resistance of E.coli pathotypes isolated
94
from healthy buffalo calves
4.7.3 Antibiotic resistance of E.coli pathotypes isolated
95
from diarrhoeic cow calves
4.7.4 Antibiotic resistance of E.coli pathotypes isolated
97
from healthy cow calves
4. 8 PHENOTYPIC ASSOCIATION BETWEEN
ANTIBIOTIC RESISTANCE AND VIRULENCE 99
GENES
4.8.1. Phenotypic association between antibiotic resistance
and virulence genes of E.coli isolates from 99
diarrhoeic buffalo calves
4.8.2 Phenotypic association between antibiotic resistance
and virulence genes of E.coli isolates from healthy 103
buffalo calves
4.8.3 Phenotypic association between antibiotic resistance
and virulence genes of E.coli isolates from 105
diarrhoeic cow calves
4.8.4 Phenotypic association between antibiotic resistance
and virulence genes of E.coli isolates from healthy 107
cow calves
vi

4.9 STANDERDIZATION OF PCR ASSAY FOR THE
DETECTION OF ANTIBIOTIC RESISTANCE 111
GENES
4.10 DISTRIBUTION OF ANTIBIOTIC RESISTANCE
GENES IN E.COLI ISOLATESFROM
117
DIARRHOEIC AND HEALTHY BUFFALO AND
COW CALVES
4.10.1 Distribution of antibiotic resistance genes in E.coli
117
isolates from diarrhoeic and healthy buffalo calves
4.10.2 Distribution of antibiotic resistance genes in E.coli
118
isolates from diarrhoeic and healthy cow calves
4.11 DISTRIBUTION OF ANTIBIOTIC RESISTANCE
123
GENES IN E.COLI ISOLATES
4.11.1 Distribution of antibiotic resistance genes in E.coli
123
pathotypes isolated from diarrhoeic buffalo calves
4.11.2 Distribution of antibiotic resistance genes in E.coli
126
pathotypes isolated from healthy buffalo calves
4.11.3 Distribution of antibiotic resistance genes in E.coli
128
pathotypes isolated from diarrhoeic cow calves
4.11.4 Distribution of antibiotic resistance genes in E.coli
130
pathotypes isolated from healthy cow calves
4.12 ASSOCIATION BETWEEN ANTIBIOTIC
132
RESISTANCE AND VIRULENCE GENES
4.12.1 Association between antibiotic resistance and
virulence genes in E.coli isolates from diarrhoeic 132
buffalo calves
4.12.2 Association between antibiotic resistance and
virulence genes in E.coli isolates from healthy 136
buffalo calves
4.12.3 Association between antibiotic resistance and
virulence genes in E.coli isolates from diarrhoeic 138
cow calves
4.12.4 Association between antibiotic resistance and
virulence genes in E.coli isolates from healthy cow 140
calves
4.13 ASSOCIATION BETWEEN VIRULENCE GENES
142
AND SEROGROUPS
4.13.1 Association between virulence genes and serogroups
142
of E.coli isolates from diarrhoeic buffalo calves
4.13.2 Association between virulence genes and serogroups
145
of E.coli isolates from healthy buffalo calves
4.13.3 Association between virulence genes and serogroups
146
of E.coli isolates from diarrhoeic cow calves
4.13.4 Association between virulence genes and serogroups
147
of E.coli isolates from healthy cow calves
4.14 PLASMID PROFILES OF E.COLI FROM
DIARRHOEIC AND HEALTHY BUFFALO AND 148
COW CALVES
4.15 SEQUENCING OF ANTIBIOTIC RESISTANCE
151
AND VIRULENCE GENES
vii

4.15.1 EAST1 forward sequence
152
4.15.2 EAST1 reverse sequence
152
4.15.3 aac(3)-I reverse sequence
153
DISCUSSION
V
5.1 EPIDEMIOLOGICAL DISTRIBUTION OF
154
E.COLI
5.2 PREVALENCE OF E.COLI IN CALVES OF
156
DIFFERENT AGE GROUPS
5.3 DISTRIBUTION OF VIRULENCE GENES
157
5.4 HAEMOLYSIS ACTIVITY
158
5.5 EPIDEMIOLOGICAL DISTRIBUTION OF
163
E.COLI SEROGROUPS
5.6 DISTRIBUTION OF SEROGROUPS IN
DIARRHOEIC AND HEALTHY BUFFALO AND 165
COW CALVES
5.7 ANTIBIOTIC RESISTANCE PATTERN OF
166
E.COLI ISOLATES
5.8 DISTRIBUTION OF ANTIBIOTIC RESISTANCE
170
GENES
5.9 ASSOCIATION BETWEEN VIRULENCE GENES
174
AND ANTIBIOTIC RESISITANCE GENES
5.10 ASSOCIATION BETWEEN VIRUELNCE GENES
174
AND E.COLI SEROGROUPS
5.11 PLASMID PROFILES
176
SUMMARY
VI 178
LITERATURE CITED
184
APPENDIX I to

IV
viii

LIST OF TABLES

Table No. Title Page No.
1 Details of faecal samples collected from buffalo and cow calves. 40
2 Identifying characteristics of E.coli. 41
3 Details of the primers used for the detection of stx1, stx2, eaeA and 43
hlyA genes.
4 Details of the primers used for the detection of virulence genes 45
5 Composition of PCR reactions for virulence genes 45
6 PCR conditions for detection of virulence genes 46
7 Interpretation chart for antibiotic resistant/sensitivity patterns 47
8 Primers used for detection of antibiotic resistance genes 48
9 Composition of PCR reactions for antibiotic resistance genes 49
10 PCR conditions for amplifying antibiotic resistance genes 49
11 Epidemiological distribution of Ecoli isolates in diarrhoeic buffalo 54
calves of different age groups in Andhra Pradesh and Telangana States
12 Epidemiological distribution of E.coli isolates in healthy buffalo calves 56
of different age groups in Andhra Pradesh and Telangana States
13 Epidemiological distribution of E.coli isolates in diarrhoeic cow calves 59
of different age groups in Andhra Pradesh and Telangana States
14 Epidemiological distribution of E.coli isolates in healthy cow calves of 61
different age groups in Andhra Pradesh and Telangana States
15 Distribution of virulence genes in E.coli isolates from diarrhoeic and 71
healthy buffalo calves
16 Distribution of virulence genes in E.coli isolates from diarrhoeic and 73
healthy cow calves
17 Distribution of serogroups of E.coli isolates from faeces of diarrhoeic 77
buffalo calves in various districts of Andhra Pradesh and Telangana
States
18 Distribution of serogroups of E.coli isolates from faeces of healthy 82
buffalo calves in various districts of Andhra Pradesh and Telangana
States
19 Distribution of serogroups of E.coli isolates from faeces of diarrhoeic 84
cow calves in various districts of Andhra Pradesh and Telangana States
ix

20 Distribution of serogroups of E.coli isolates from faeces of healthy cow 86
calves in various districts of Andhra Pradesh and Telangana States
20(A) Prevalence (%) of E.coli serogroups in diarrhoeic and healthy buffalo 87
and cow calves.
21 Antibiotic resistance pattern of E.coli isolates from diarrhoeic and 89
healthy buffalo calves
22 Antibiotic resistance pattern of E.coli isolates from diarrhoeic and 90
healthy cow calves
23 Antibiotic resistance of different E.coi pathotypes isolated from 93
diarrhoeic buffalo calves
24 Antibiotic resistance of different E.coli pathotypes isolated from 95
healthy buffalo calves
25 Antibiotic resistance of different E.coli pathotypes isolated from 97
diarrhoeic cow calves
26 Antibiotic resistance of different E.coli pathotypes isolated from 98
healthy cow calves
27 Phenotypic association between antibiotic resistance and virulence 100
genes (STEC) of E.coli isolates from diarrhoeic buffalo calves
28 Phenotypic association between antibiotic resistance and virulence 102
genes of E.coli isolates from diarrhoeic buffalo calves
29 Phenotypic association between antibiotic resistance and virulence 104
genes of E.coli isolates from healthy buffalo calves
30 Phenotypic association between antibiotic resistance and virulence 106
genes of E.coli isolates from diarrhoeic cow calves
31 Phenotypic association between antibiotic resistance and virulence 107
genes of E.coli isolates from healthy cow calves
32 Distribution of antibiotic resistance genes in E.coli isolates from 118
diarrhoeic and healthy buffalo calves
33 Distribution of antibiotic resistance genes in E.coli isolates from 121
diarrhoeic and healthy cow calves
34 Distribution of antibiotic resistance genes in E.coli pathotypes isolated 125
from diarrhoeic buffalo calves
35 Distribution of antibiotic resistance genes in E.coli pathotypes isolated 127
from healthy buffalo calves
36 Distribution of antibiotic resistance genes in E.coli pathotypes isolated 129
from diarrhoeic cow calves
37 Distribution of antibiotic resistance genes in E.coli pathotypes isolated 131
from healthy cowcalves
38 Association between antibiotic resistance and virulence genes in E.coli 135
isolates from diarrheic buffalo calves