Epidemiology of the virulence and antimicrobial resistance of Escherichia coli from canine and ...

160 Pages·2014·1.94 MB·English

Epidemiology of the virulence and antimicrobial resistance of Escherichia coli from canine
and feline clinical isolates

by

Kamoltip Thungrat

A dissertation submitted to the Graduate Faculty of
Auburn University
in partial fulfilment of the
requirements for the Degree of
Doctor of Philosophy

Auburn, Alabama
May 10, 2015

Keywords: Escherichia coli, Small Animals,
Antimicrobial resistance, Virulence factors

Copyright 2015 by Kamoltip Thungrat

Approved by

Dawn Boothe, Chair, Professor of Anatomy, Physiology and Pharmacology
Mark Liles, Associate Professor of Biological Science
Stuart Price, Associate Professor of Pathobiology
Nedret Billor, Professor of Mathematics and Statistics
ABSTRACT

Escherichia coli resistance impacts the veterinary professional because of therapeutic
failure and the public health significance. The lack of national monitoring programs in small
animal practices limits evidence-based empirical antimicrobial choices. The objectives of the
resistance studies were to establish the current status of E. coli susceptibility to routinely-selected
antimicrobials and to assess veterinary prescribing behaviors.
Clinical canine or feline E. coli isolates (n=3172) were collected from six geographic
regions in the US between May 2008 and January 2013. Minimum inhibitory concentrations
(MIC) of E. coli isolates were determined for 17 antimicrobials. Population MIC distributions
were bimodal with the second mode above the resistant breakpoint for all drugs except for
gentamicin, amikacin, and meropenem. The highest percentage of isolates were resistant to
doxycyline (100%), and cephalothin (98%). None of isolates was susceptible to all drug tested;
46% were single-drug resistant (SDR), and 52% multi-drug resistant (MDR).
Most antimicrobial prescriptions were amoxicillin-clavulanic acid (25%) at 14.9±5.4
mg/kg q12h and enrofloxacin (22%) at 5.6±2.8 mg/kg q24h used for treatment of urinary tract
infections (UTI). Our findings reported that most antimicrobial prescriptions were inappropriate
for the drug selected, and the dosing regimen.
Uropathogenic E. coli (UPEC) isolates acquire virulence genes that encode virulence
factors (VF) necessary for colonization of the urinary tract. VFs may offer targets to alter a
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method of antimicrobial therapy. The objectives of virulence studies were to investigate the
associations among severity of UTI, antimicrobial resistance, and virulence within UPEC.
Virulence profiles of UPEC differ significantly with severity of infections and resistance
patterns. Most severe disease isolates were phylogroups B2 (50%), and ABU were phylogroup D
(63%). MDR isolates exhibited shifts in phylogenetic distribution (A, B1>D, B2), compared to
SDR isolates (B2, D>B1, A). These results suggest that as the resistance level increases,
virulence decreases.
The expression levels of virulence genes were computed by principal component and
linear discriminant analyses to classify severity of UTI as ABU and non-ABU. The model
estimated the non-ABU with a 19% error rate. These findings suggest that the model can be
useful in practices by identifying those isolates that do not need antimicrobial therapy.
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ACKNOWLEDGMENTS

I would like to express my sincere appreciation to many people who have contributed to
this dissertation, personally and professionally. My major advisor Dr. Dawn Boothe provided the
opportunity and guidance that allowed me to successfully conduct my research. Thanks to my
committee members, Dr. Mark Liles, Dr. Nedert Billor and Dr. Stuart Price for their time and
assistance. Special thanks to Jameson Sofge for managing my funding and handling the ordering
of all of my supplies which without none of this would have been at all possible. Many thanks to
Megan Behringer, Dr. Heather Gossett and Dr. Marike Visser for their emotional support, and
being great friends. Crisanta Cruz-espindola who have helped me and supported me on several
things. Thank you for being my great friends during these years in Auburn.
Most of all I would like to thank my family, my father Prasit, mother Wilai and my sister
Dr. Arada Thungrat who kept me motivated throughout the entire process. Without their love I
would have never had made it this far in my studies and for that I am endlessly grateful.
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TABLE OF CONTENTS

Abstract ......................................................................................................................................... ii
Acknowledgments ....................................................................................................................... vi
List of Tables ................................................................................................................................ x
List of Figures .............................................................................................................................. xi
List of Abbreviations .................................................................................................................. xii
CHAPTER 1 LITERATURE REVIEW ..................................................................................... 1
1.1 The evolution of pathogenic Escherichia coli .......................................................... 1
1.2 Virulence factors and virulence ................................................................................ 2
1.2.1 Phylogenetic distribution of VFs ................................................................. 2
1.2.2 Phylogenetic analysis of E. coli ................................................................... 3
1.2.3 E. coli pathotypes ......................................................................................... 5
1.2.4 Phylogenetic distribution of pathogenic E. coli ........................................... 6
1.2.5 Pathophysiology of urinary tract infections (UTIs) by E. coli..................... 7
1.2.6 Virulence factors of uropathogenic E. coli .................................................. 9
1.3 The link between virulence and resistance ............................................................... 15
1.4 Antimicrobial resistance ......................................................................................... 16
1.4.1 Acquisitions of antimicrobial resistance and multidrug resistance ............ 17
1.4.2 Mechanisms of antimicrobial resistance in E. coli .................................... 17
1.4.3 Definitions of MDR, XDR and PDR ......................................................... 20
1.5 Antimicrobial resistance risk assessment ................................................................ 22
1.5.1 Methodological considerations of ARRA .................................................. 23
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1.5.2 Data for ARRA: requirements and sources ............................................... 25
1.5.3 Epidemiology: a tool for the assessment of risk ........................................ 25
1.5.4 Surveillance programs and the role of diagnostic laboratories .................. 26
1.5.5 Antimicrobial susceptibility testing
and interpretation of results ....................................................................... 27
1.6 Research objectives ................................................................................................. 31
1.7 References ............................................................................................................... 33
CHAPTER 2 ANTIMICROBIAL SUSCEPTIBILITY PATTERNS OF CLINICAL
ESCHERICHIA COLI ISOLATES FROM
DOGS AND CATS IN THE UNITED STATES ..................................................................... 46
2.1 Introduction ............................................................................................................ 46
2.2 Materials and methods ........................................................................................... 47
2.2.1 Source of isolates ....................................................................................... 47
2.2.2 Sample collection and susceptibility testing .............................................. 48
2.2.3 Interpretation of susceptibility testing results ............................................ 49
2.2.4 Data collection ........................................................................................... 50
2.2.5 Statistical analyses ..................................................................................... 50
2.3 Results .................................................................................................................... 52
2.3.1 Isolate description ...................................................................................... 52
2.3.2 Pharmacodynamic data .............................................................................. 55
2.3.3 Percentages of antimicrobial resistance ..................................................... 60
2.3.4 Type of resistance ...................................................................................... 65
2.4 Discussion .............................................................................................................. 67
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2.5 Conclusion ............................................................................................................. 72
2.6 References .............................................................................................................. 73
CHAPTER 3 ANTIMICROBIAL PRESCRIBING PRACTICES FOR TREATMENT OF
ESCHERICHIA COLI INFECTIONS IN DOGS AND CATS
IN UNITED STATES ................................................................................................................. 80
3.1 Introduction ............................................................................................................ 80
3.2 Materials and methods ........................................................................................... 81
3.2.1 Sample collection and susceptibility testing .............................................. 81
3.2.2 Questionnaires............................................................................................ 81
3.2.3 Statistical analyses ..................................................................................... 82
3.3 Results .................................................................................................................... 83
3.3.1 Demographic data ...................................................................................... 83
3.3.2 Antimicrobial prescription behaviors ........................................................ 84
3.3.3 Risks of resistance to an antibiotic after
exposure to that same antimicrobial ......................................................... 94
3.3.4 Success rate of antimicrobial therapy ........................................................ 95
3.4 Discussion .............................................................................................................. 96
3.5 Conclusion ............................................................................................................. 99
3.6 References .............................................................................................................. 99
CHAPTER 4 PHYLOGENETIC ORIGIN AND VIRULENCE GENOTYPE IN RELATION TO
ANTIMICROBIAL RESISTANCE AMONG ESCHERICHIA COLI ISOLATES FROM
CANINE URINARY TRACT INFECTIONS .......................................................................... 107
4.1 Introduction .......................................................................................................... 107
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4.2 Materials and methods ......................................................................................... 109
4.2.1 E. coli isolates .......................................................................................... 109
4.2.2 Antimicrobial susceptibility testing ......................................................... 109
4.2.3 DNA extraction ........................................................................................ 110
4.2.4 Phylogenetic grouping ............................................................................. 110
4.2.5 Virulence genes detection ........................................................................ 110
4.2.6 E. coli ST131 strains detection ................................................................ 111
4.2.7 Statistical analyses ................................................................................... 113
4.3 Results .................................................................................................................. 114
4.3.1 Distribution of severity of clinical signs and phylogenetic groups .......... 114
4.3.2 Relationship between phylogenetic group and phenotypic resistance ..... 115
4.3.3 Distribution of VF genes .......................................................................... 118
4.3.4 Screening of clone O25b:H4-ST131 ........................................................ 122
4.4 Discussion ............................................................................................................ 122
4.5 Conclusion ........................................................................................................... 125
4.6 References ............................................................................................................ 126
CHAPTER 5 PREDICTING ASYMPTOMATIC BACTERIURIA IN CLINICAL
ESCHERICHIA COLI UROPATHOGENS .............................................................................. 132
5.1 Introduction .......................................................................................................... 132
5.2 Materials and methods ......................................................................................... 133
5.2.1 E. coli isolates .......................................................................................... 133
5.2.2 RNA purification and reverse transcription ............................................. 134
5.2.3 qRT-PCR experiment ............................................................................... 134
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5.2.4 Data calculations and interpretation ......................................................... 134
5.2.5 Statistical analyses ................................................................................... 135
5.3 Results .................................................................................................................. 137
5.3.1 Principal component analysis .................................................................. 137
5.3.2 Linear discriminant analysis .................................................................... 140
5.4 Discussion ............................................................................................................ 142
5.5 Conclusion ........................................................................................................... 143
5.6 References ............................................................................................................ 144
CHAPTER 6 GENERAL CONCLUSIONS ............................................................................. 146

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LIST OF TABLES

Table 1.1 Antimicrobial drugs categories and agents used to this study ................................... 22
Table 2.1 Pharmacodynamic data (MIC statistics) .................................................................... 57
Table 2.2 Percent of antimicrobial resistance by geographical region and demographic data ... 61
Table 2.3 Percent of antimicrobial resistance by infection location ........................................... 63
Table 2.4 Odds ratios for the association of percent of antimicrobial
resistance for each of 15 antimicrobials ..................................................................... 64
Table 2.5 Odds ratio for the association of resistant types ........................................................ 66
Table 3.1 A summary of antimicrobial selections and dosing regimen ................................ 88-89
Table 3.2 Odds ratios of antimicrobial selections with variable factors ................................ 91-92
Table 3.3 The likelihood of antimicrobial selection and susceptibility ..................................... 94
Table 4.1 The list of primer of virulence genes ................................................................ 112-113
Table 4.2 The list of antimicrobial resistance phenotypes ....................................................... 116
Table 4.3 The distribution of VF genes categorized by severities ........................................... 119
Table 4.4 The distribution of VF genes categorized by phylogenetic groups .......................... 121
Table 5.1 Summary of the data collected from q-RT-PCR ..................................................... 137
Table 5.2 The results of principal component analysis............................................................. 138
Table 5.3 The results of cross validation of a predictive model for 4 severities ...................... 141
Table 5.4 The results of cross validation of a predictive model ABU vs non-ABU ................ 142
x