selection and co-selection of antimicrobial resistance in gut enterococci of swine and cattle fed

233 Pages·2014·6.37 MB·English

by  Martin Courtois

SELECTION AND CO-SELECTION OF ANTIMICROBIAL
RESISTANCE IN GUT ENTEROCOCCI OF SWINE AND
CATTLE FED DIETS SUPPLEMENTED WITH COPPER,
TYLOSIN, AND CHLORTETRACYCLINE

By

RAGHAVENDRA G. AMACHAWADI

BVSc & AH., University of Agricultural Sciences, Bangalore, India, 2003
MS., Kansas State University, 2010

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

2014
Abstract
Copper, as copper sulfate, is often used as an alternative to in-feed antibiotics for growth
promotion in both swine and cattle diets. Gut bacteria exposed to copper can acquire resistance,
which among enterococci is conferred by a plasmid-borne transferable copper resistance gene
(tcrB). The plasmid also carries tetracycline [tet(M)] and macrolide [erm(B)] resistance genes.
Because of the genetic link between acquired copper (tcrB) and antibiotic resistance in
Enterococcus spp., we hypothesized that copper supplementation may exert selection pressure
for enterococci to become resistant to macrolides and tetracyclines, and possibly to other
antibiotics. We conducted studies in cattle and swine to investigate the relationship between
copper supplementation and the fecal prevalence of tcrB-positive enterococci, as well as its
potential co-selection for macrolide and tetracycline resistance. The prevalence was higher in
animals fed diets supplemented with elevated level of copper compared to normal level (P <
0.05). The tcrB-positive isolates belonged to either E. faecium or E. faecalis; the majority was
E. faecium. All tcrB-positive isolates also contained both erm(B) and tet(M) genes; however,
none of them harbored the vanA gene. Median minimum inhibitory concentrations (MIC) of
copper for tcrB-positive and tcrB-negative enterococci were 22 mM and 4 mM, respectively (P <
0.0001). The overall prevalence of erm(B) and tet(M) genes among enterococcal isolates of
cattle were 46.8 % and 57.5%, respectively; in contrast,100% of the swine isolates were positive
for both erm(B) and tet(M) genes. The transferability of the tcrB gene was demonstrated by
filter mating assay. Multi-locus variable number tandem repeat analysis revealed a genetically
diverse population of enterococci. The finding of a strong association between the copper
resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is
significant because enterococci are potential pathogens and have the propensity to transfer
resistance genes to other bacteria in the gut. The occurrence of vancomycin resistant enterococci
in swine in the US is very rare. Strains of E. faecium positive for vanA, that confers resistance to
vancomycin, were isolated and characterized from swine feces. The swine strains belonged to
clonal complex 17, a well-adapted hospital clone throughout the world.
SELECTION AND CO-SELECTION OF ANTIMICROBIAL
RESISTANCE IN GUT ENTEROCOCCI OF SWINE AND
CATTLE FED DIETS SUPPLEMENTED WITH COPPER,
TYLOSIN, AND CHLORTETRACYCLINE

By

RAGHAVENDRA G. AMACHAWADI

BVSc & AH., University of Agricultural Sciences, Bangalore, India, 2003
MS., Kansas State University, 2010

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

2014

Approved by:
Major Professor
H. Morgan Scott
Copyright
RAGHAVENDRA G. AMACHAWADI
2014
Abstract
Copper, as copper sulfate, is often used as an alternative to in-feed antibiotics for growth
promotion in both swine and cattle diets. Gut bacteria exposed to copper can acquire resistance,
which among enterococci is conferred by a plasmid-borne transferable copper resistance gene
(tcrB). The plasmid also carries tetracycline [tet(M)] and macrolide [erm(B)] resistance genes.
Because of the genetic link between acquired copper (tcrB) and antibiotic resistance in
Enterococcus spp., we hypothesized that copper supplementation may exert selection pressure
for enterococci to become resistant to macrolides and tetracyclines, and possibly to other
antibiotics. We conducted studies in cattle and swine to investigate the relationship between
copper supplementation and the fecal prevalence of tcrB-positive enterococci, as well as its
potential co-selection for macrolide and tetracycline resistance. The prevalence was higher in
animals fed diets supplemented with elevated level of copper compared to normal level (P <
0.05). The tcrB-positive isolates belonged to either E. faecium or E. faecalis; the majority was
E. faecium. All tcrB-positive isolates also contained both erm(B) and tet(M) genes; however,
none of them harbored the vanA gene. Median minimum inhibitory concentrations (MIC) of
copper for tcrB-positive and tcrB-negative enterococci were 22 mM and 4 mM, respectively (P <
0.0001). The overall prevalence of erm(B) and tet(M) genes among enterococcal isolates of
cattle were 46.8 % and 57.5%, respectively; in contrast,100% of the swine isolates were positive
for both erm(B) and tet(M) genes. The transferability of the tcrB gene was demonstrated by
filter mating assay. Multi-locus variable number tandem repeat analysis revealed a genetically
diverse population of enterococci. The finding of a strong association between the copper
resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is
significant because enterococci are potential pathogens and have the propensity to transfer
resistance genes to other bacteria in the gut. The occurrence of vancomycin resistant enterococci
in swine in the US is very rare. Strains of E. faecium positive for vanA, that confers resistance to
vancomycin, were isolated and characterized from swine feces. The swine strains belonged to
clonal complex 17, a well-adapted hospital clone throughout the world.
Table of Contents
List of Figures .............................................................................................................................. xiii
List of Tables ................................................................................................................................ xv
Acknowledgements ..................................................................................................................... xvii
Dedication .................................................................................................................................... xix
CHAPTER 1 - Antimicrobial Agents in Food Animal Agriculture: Use, Resistance, and
Alternatives in Swine and Cattle ............................................................................................. 1
Introduction ................................................................................................................................. 1
Antimicrobials and Antimicrobial Resistance ............................................................................ 2
Antibiotic Growth Promoters ...................................................................................................... 5
Antibiotic Alternatives for Growth Promotion in Animals ........................................................ 6
Heavy metals ............................................................................................................................. 10
Resistance to Heavy Metals .................................................................................................. 11
Copper ................................................................................................................................... 12
Use of Copper as a Growth Promoter ................................................................................... 13
Copper Homeostasis ............................................................................................................. 14
Copper homeostasis in Gram-positive bacteria (Enterococcus hirae).................................. 14
Copper resistance in Gram-positive bacteria ........................................................................ 16
tcrYAZB Operon .................................................................................................................... 17
Antimicrobial Use and Resistance Associated with Swine Production .................................... 20
Antimicrobial Use and Resistance Associated with Beef Cattle Production ............................ 23
CHAPTER 2 - Epidemiology of Antimicrobial Resistant Enterococci, Vancomycin Resistant
Enterococci, and their Virulence Characteristics ................................................................... 28
Enterococcus spp. ..................................................................................................................... 28
Enterococcal infections ............................................................................................................. 29
Virulence factors in Enterococcus spp. .................................................................................... 30
Secreted Proteins ................................................................................................................... 31
Adhesins ................................................................................................................................ 31
Cell wall polymers ................................................................................................................ 33
viii
Vancomycin Resistant Enterococci .......................................................................................... 37
References ................................................................................................................................. 41
Dissertation Outline .................................................................................................................. 66
CHAPTER 3 - Effects of in-feed copper, chlortetracycline, and tylosin on the prevalence of
transferable copper resistance gene, tcrB, among fecal enterococci of weaned piglets ........ 70
Introduction ............................................................................................................................... 70
Materials and Methods .............................................................................................................. 72
Animals, dietary treatments, experimental design, and fecal sampling schedule ................. 72
Isolation and species identification of Enterococcus ............................................................ 73
PCR detection of tcrB, erm(B), and tet(M) genes ................................................................ 74
Copper, tetracycline, tylosin and vancomycin susceptibility determinations ....................... 75
Determinations of antimicrobial susceptibility profile ......................................................... 76
Statistical Analysis ................................................................................................................ 76
Results ....................................................................................................................................... 78
Prevalence of the tcrB, erm(B), and tet(M) genes in fecal enterococci ................................ 78
MICs of copper, tetracycline, tylosin, and vancomycin ....................................................... 79
Susceptibilities to other antimicrobials ................................................................................. 80
Discussion ................................................................................................................................. 81
Acknowledgements ................................................................................................................... 86
References ................................................................................................................................. 93
CHAPTER 4 - Occurrence of the transferable copper resistance gene, tcrB, among fecal
enterococci of U. S. feedlot cattle fed copper-supplemented diets ........................................ 98
Introduction ............................................................................................................................... 98
Materials and Methods ............................................................................................................ 100
Animals, study design, and sampling schedule .................................................................. 100
Isolation and speciation of Enterococcus ........................................................................... 101
PCR detections of tcrB, erm(B), tet(M), vanA, vanB and virulence genes ......................... 102
Copper and antibiotic susceptibility determinations ........................................................... 102
Transferability of tcrB gene ................................................................................................ 103
Southern blot hybridization ................................................................................................. 104
ix
Multiple-locus variable number tandem repeat analysis (MLVA) of tcrB-positive
enterococci .......................................................................................................................... 104
Nucleotide sequence accession number .............................................................................. 105
Statistical analyses .............................................................................................................. 105
Results ..................................................................................................................................... 106
Prevalence of tcrB, erm(B), tet(M), and virulence genes among fecal enterococci ........... 106
Phenotypic susceptibilities to copper, tetracycline, tylosin, and vancomycin .................... 107
Transferability of tcrB gene ................................................................................................ 108
Southern hybridization ........................................................................................................ 108
Multiple-locus variable number tandem repeat analysis (MLVA) ..................................... 108
Discussion ............................................................................................................................... 109
Acknowledgements ................................................................................................................. 114
References ............................................................................................................................... 121
CHAPTER 5 - Effects of in-feed copper and tylosin supplementations on copper and antibiotics
resistance in fecal enterococci of feedlot cattle ................................................................... 127
Introduction ............................................................................................................................. 127
Materials and Methods ............................................................................................................ 129
Study design and sampling schedule .................................................................................. 129
Bacteriological procedures .................................................................................................. 130
PCR detections of tcrB, erm(B), tet(M), and virulence genes ............................................ 130
Susceptibility determinations for copper, tylosin and tetracycline ..................................... 131
Susceptibility determinations to other antibiotics ............................................................... 132
Statistical Analysis .............................................................................................................. 132
Results ..................................................................................................................................... 134
Prevalence of tcrB, erm(B), tet(M), and virulence genes in fecal enterococci ................... 134
Phenotypic susceptibilities to copper, tylosin, and tetracycline.......................................... 135
Susceptibilities of enterococci to other antimicrobials ....................................................... 136
Discussion ............................................................................................................................... 137
Acknowledgements ................................................................................................................. 143
References ............................................................................................................................... 142
x