companion animals as reservoir of antibiotic resistant and virulent enterococci

234 Pages·2011·2.31 MB·English

ASSESSMENT OF PET DOGS AS A RESERVOIR OF ANTIBIOTIC RESISTANT
BACTERIA

by

DEEPTI KUTTAN PILLAI

M.V.Sc., Nagpur Veterinary College, Maharashtra, India, 2005

AN ABSTRACT OF A DISSERTATION

submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

Department of Diagnostic Medicine/Pathobiology
College of Veterinary Medicine

KANSAS STATE UNIVERSITY
Manhattan, Kansas

2011
Abstract
Transfer of bacteria, including antibiotic resistant strains between companion animals and
people is likely due to close physical contacts. However, surveillance programs on prevalence of
antibiotic resistance are focused mainly on food animals and very little is known about the role
of companion animals in the development and spread of antibiotic resistant bacteria. For this
study, enterococci were chosen as model organism due to intrinsic and acquired antibiotic
resistance and several virulence traits that make them the 3rd most important nosocomial
pathogens. In addition, increased fecal shedding of antibiotic resistant bacteria from stressed
animals has been reported from studies on food animals. To determine whether the gut
microbiota of pet animals serves as a reservoir of clinically important enterococci, 360
enterococcal isolates from two groups: healthy group and pyoderma (stressed) group with 9 dogs
in each were identified and screened for resistance to 10 antibiotics and 4 virulence traits. The
transferability of resistance determinants and clonality of selected isolates were assessed by
horizontal gene transfer assays and pulsed-field gel electrophoresis, respectively. In addition,
overall diversity of bacteria as well as antibiotic and metal resistance genes in feces of healthy
dogs was assessed by tag-encoded parallel pyrosequencing and microarray analysis, respectively.
The most prevalent enterococcal species identified was E. faecalis: healthy group
(70.5%); pyoderma group (44.0%). In the pyoderma group, antibiotic resistance and virulence
traits (esp, gelE) were more frequent than in the healthy group; however, the overall prevalence
of antibiotic resistant strains was low (< 37%) in both groups. The most prevalent resistance
genes were tet(M)and tet(S). The antibiotic resistance traits were transferable in-vitro in E.
faecalis (tetracycline, erythromycin, doxycycline) and E. faecium (tetracycline). Genotyping
revealed less diverse E. faecalis community in pyoderma infected dogs.
Pyrosequencing (~7,500 sequences per dog) revealed Firmicutes as the dominant phylum
and most common genera included Turicibacter, Lactobacillus, Ruminococcus, Clostridium, and
Fusobacterium. Two phyla Lentisphaerae (<1%) and Fibrobacteres (<1%) are reported for the
first time from healthy dogs. Microarray data revealed the presence of several tetracycline,
erythromycin, aminoglycoside, and copper resistance genes; however, most of these originated
from one animal with history of chronic skin infection two year prior to our sampling.
Higher prevalence of antimicrobial resistance in pyoderma infected dogs may be related
to stress; however, this requires further investigation. In conclusion, based on our data, healthy
and pyoderma infected dogs do not represent an important reservoir of clinically significant
antibiotic resistant microbiota.
ASSESSMENT OF PET DOGS AS A RESERVOIR OF ANTIBIOTIC RESISTANT
BACTERIA

by

DEEPTI KUTTAN PILLAI

M.V.Sc., Nagpur Veterinary College, Maharashtra, India, 2005

A DISSERTATION

submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

Department of Diagnostic Medicine/Pathobiology
College of Veterinary Medicine

KANSAS STATE UNIVERSITY
Manhattan, Kansas

2011

Approved by:

Major Professor
Dr. Ludek Zurek
Copyright

DEEPTI KUTTAN PILLAI
2011
Abstract
Transfer of bacteria, including antibiotic resistant strains between companion animals and
people is likely due to close physical contacts. However, surveillance programs on prevalence of
antibiotic resistance are focused mainly on food animals and very little is known about the role
of companion animals in the development and spread of antibiotic resistant bacteria. For this
study, enterococci were chosen as model organism due to intrinsic and acquired antibiotic
resistance and several virulence traits that make them the 3rd most important nosocomial
pathogens. In addition, increased fecal shedding of antibiotic resistant bacteria from stressed
animals has been reported from studies on food animals. To determine whether the gut
microbiota of pet animals serves as a reservoir of clinically important enterococci, 360
enterococcal isolates from two groups: healthy group and pyoderma (stressed) group with 9 dogs
in each were identified and screened for resistance to 10 antibiotics and 4 virulence traits. The
transferability of resistance determinants and clonality of selected isolates were assessed by
horizontal gene transfer assays and pulsed-field gel electrophoresis, respectively. In addition,
overall diversity of bacteria as well as antibiotic and metal resistance genes in feces of healthy
dogs was assessed by tag-encoded parallel pyrosequencing and microarray analysis, respectively.
The most prevalent enterococcal species identified was E. faecalis: healthy group
(70.5%); pyoderma group (44.0%). In the pyoderma group, antibiotic resistance and virulence
traits (esp, gelE) were more frequent than in the healthy group; however, the overall prevalence
of antibiotic resistant strains was low (< 37%) in both groups. The most prevalent resistance
genes were tet(M)and tet(S). The antibiotic resistance traits were transferable in-vitro in E.
faecalis (tetracycline, erythromycin, doxycycline) and E. faecium (tetracycline). Genotyping
revealed less diverse E. faecalis community in pyoderma infected dogs.
Pyrosequencing (~7,500 (mean) sequences per dog) revealed Firmicutes as the dominant
phylum and most common genera included Turicibacter, Lactobacillus, Ruminococcus,
Clostridium, and Fusobacterium. Two phyla Lentisphaerae (<1%) and Fibrobacteres (<1%) are
reported for the first time from healthy dogs. Microarray data revealed the presence of several
tetracycline, erythromycin, aminoglycoside, and copper resistance genes; however, most of these
originated from one animal with history of chronic skin infection two year prior to our sampling.
Higher prevalence of antimicrobial resistance in pyoderma infected dogs may be related
to stress; however, this requires further investigation. In conclusion, based on our data, healthy
and pyoderma infected dogs do not represent an important reservoir of clinically significant
antibiotic resistant microbiota.
Table of Contents

Table of Contents .................................................................................................................... viii
List of Figures .......................................................................................................................... xii
List of Tables ...........................................................................................................................xiv
Acknowledgements ................................................................................................................... xv
Dedication ................................................................................................................................xvi
Abbreviations ......................................................................................................................... xvii
Chapter 1 Literature review .........................................................................................................1
1.1 Introduction .......................................................................................................................1
1.2 Zoonotic pathogens ...........................................................................................................4
1.3 Antimicrobial resistance ....................................................................................................5
1.4 Antimicrobial resistance in zoonotic pathogens .................................................................7
1.4.1 Food animals ..............................................................................................................8
1.4.2 Companion animals .................................................................................................. 10
1.5 Epidemiology of antimicrobial drug resistant bacteria in dogs ......................................... 12
1.6 Sources for acquisition of antibiotic resistant pathogens .................................................. 13
1.7 Stress ............................................................................................................................... 14
1.7.1 Animal stress and bacteria......................................................................................... 14
1.7.2 Stress and gastrointestinal tract (GIT) ....................................................................... 15
1.7.3 Association between stress and antimicrobial resistance in the gut microbiota........... 18
1.8 Enterococci, the opportunistic pathogen .......................................................................... 20
1.8.1 Antibiotic resistance in enterococci ........................................................................... 21
1.8.2 Virulence factors ....................................................................................................... 24
1.9 Epidemiology of antibiotic resistant (AMR) enterococci in dogs ..................................... 25
1.9.1 United States ............................................................................................................. 26
1.9.2 European countries ................................................................................................... 27
1.10 Summary ....................................................................................................................... 32
1.11 Research objectives ....................................................................................................... 34
viii
References ................................................................................................................................ 35
Chapter 2 Population size, diversity, polyphasic characterization of antimicrobial resistance and
virulence traits, horizontal transfer of resistance genes and clonal structure of enterococci
from the feces of healthy and dogs with pyoderma ............................................................. 59
2.1 Abstract ........................................................................................................................... 59
2.2 Introduction ..................................................................................................................... 61
2.3 Materials and methods ..................................................................................................... 65
2.3.1 Study animals ........................................................................................................... 65
2.3.2 Fecal sampling and processing .................................................................................. 66
2.3.3 Isolation of enterococci ............................................................................................. 66
2.3.4 Species determination ............................................................................................... 67
2.3.5 Antibiotic susceptibility test ...................................................................................... 68
2.3.6 Virulence factors ....................................................................................................... 70
2.3.7 Pulsed-field gel electrophoresis (PFGE) .................................................................... 72
2.3.8 Conjugation assay ..................................................................................................... 74
2.3.9 Statistical analysis ..................................................................................................... 75
2.4 Results ............................................................................................................................ 76
2.4.1 Prevalence of enterococci ......................................................................................... 76
2.4.2 Antibiotic susceptibility test ...................................................................................... 77
2.4.3 Virulence factors ....................................................................................................... 80
2.4.4 Clonal diversity......................................................................................................... 81
2.4.5 Conjugation results ................................................................................................... 82
2.5 Discussion ....................................................................................................................... 83
2.6 Conclusions ..................................................................................................................... 95
2.7 Figures and Tables........................................................................................................... 96
References .............................................................................................................................. 121
Chapter 3 Gut microbial diversity of healthy dogs ................................................................... 138
3.1 Abstract ......................................................................................................................... 138
3.2 Introduction ................................................................................................................... 140
3.3 Materials and methods ................................................................................................... 145
3.3.1 Collection of fecal samples ..................................................................................... 145
ix
3.3.2 Extraction of DNA .................................................................................................. 145
3.3.3 bTEFAP sequencing PCR ....................................................................................... 146
3.3.4 AmBead purification ............................................................................................... 147
3.3.5 bTEFAP FLX massively parallel pyrosequencing ................................................... 147
3.3.6 bTEFAP sequence data analysis .............................................................................. 147
3.3.7 Phylogenetic assignment, alignment and clustering of 16S rRNA gene fragments ... 148
3.3.8 Biodiversity ............................................................................................................ 148
3.4 Results .......................................................................................................................... 148
3.4.1 Characteristics of pyrosequencing data.................................................................... 148
3.4.2 Abundance of microorganisms in gut microbiota of healthy dogs ............................ 149
3.4.3 Biodiversity ............................................................................................................ 149
3.4.4 Bacterial composition in the gut microbiota of healthy dogs .................................... 150
3.4.5 Phyla level .............................................................................................................. 150
3.4.6 Genus level ............................................................................................................. 151
3.5 Discussion ..................................................................................................................... 152
3.6 Conclusion .................................................................................................................... 157
3.7 Figures and Tables......................................................................................................... 159
References .............................................................................................................................. 182
Chapter 4 Analysis of antibiotic and metal resistance genes in diverse bacteria of gastrointestinal
tract of healthy dogs ......................................................................................................... 188
4.1 Abstract ......................................................................................................................... 188
4.2 Introduction ................................................................................................................... 189
4.3 Materials and methods ................................................................................................... 191
4.3.1 Collection of fecal samples ..................................................................................... 191
4.3.2 Template DNA extraction ....................................................................................... 191
4.3.3 Preparation of labeled DNA .................................................................................... 192
4.3.4 Microarray hybridization ........................................................................................ 193
4.3.5 Data analysis ........................................................................................................... 194
4.4 Results .......................................................................................................................... 196
4.4.1 Composition of antibiotic resistance genes in diverse fecal microbiota .................... 196
4.4.2 Composition of metal resistance genes in diverse fecal microbiota .......................... 197
x