EFFECTS OF DIETS, ANTIMICROBIALS AND MINERALS ON THE PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF FECAL BACTERIA IN FEEDLOT CATTLE by MEGAN E. JACOB B. S., University of Wyoming, 2005 A THESIS submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2007 Approved by: Approved by: Co-Major Professor Co-Major Professor T.G. Nagaraja Sanjeev Narayanan Abstract Antimicrobials are included in finishing cattle diets for growth promotion, feed efficiency, and protection against liver abscesses. The inclusion of in-feed antimicrobials at or below therapeutic concentrations may provide a selective pressure for antimicrobial resistant microorganisms. Additionally, heavy metals such as copper and zinc may be included in cattle diets because of growth-promoting effects. Heavy metal resistance genes are on transferable plasmids that also contain antimicrobial resistance genes. The objectives of this research were to 1) determine the prevalence of food-borne pathogens, Salmonella and E. coli O157, in cattle fed diets with or without monensin and tylosin and 0 or 25% wet corn distillers grains (WDGS), 2) determine the prevalence of food-borne pathogens in cattle fed elevated concentrations of copper and zinc 3) evaluate the effect of antimicrobials on antimicrobial susceptibility of food-borne pathogens and commensal fecal bacteria, and 4) determine a possible association between in-feed antimicrobials and the concentration of antimicrobial resistance genes in the feces of cattle. Inclusion of 25% WDGS was associated with a higher prevalence of E. coli O157 on one of two sample collection days; however, there was no association between the use of monensin and tylosin, or copper and zinc on the prevalence of food-borne pathogens. Including monensin and tylosin in cattle diets was associated with an increased resistance of enterococci to macrolides, but was not related to concentration of the common macrolide resistance gene, ermB. In cattle fed diets with copper and/or zinc, no differences were observed in antimicrobial susceptibility or the concentration of antimicrobial resistance genes. In conclusion, results indicate that including growth-promoting antimicrobials in cattle diets at below therapeutic concentrations only limitedly impacted antimicrobial susceptibility and concentration of fecal antimicrobial resistance genes; however, this research encompassed only a select number of microorganisms. The positive association between WDGS and E. coli O157 prevalence in cattle has important implications for food safety, and warrants further investigation. Table of Contents List of Figures.vi List of Tables.vii Acknowledgements.viii CHAPTER 1 - Literature Review. 1 Introduction. 1 Definition of Antimicrobial Resistance. 4 Mechanisms for Bacterial Antimicrobial Resistance. 6 Transfer of Antimicrobial Resistance. 7 Food-safety Implications . 8 Antimicrobial Susceptibility of E. coli O157. 9 Antimicrobial Susceptibility of Salmonella enterica. 11 Antimicrobial Susceptibility of Campylobacter species . 12 Antimicrobial Susceptibility of Listeria species. 13 Antimicrobial Susceptibility of commensal E. coli. 14 Antimicrobial Susceptibility of Enterococcus species . 16 Susceptibility to Metals . 17 Conclusions. 17 References. 20 CHAPTER 2 - Effects of feeding wet corn distillers grains with solubles with or without monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal food-borne pathogenic and commensal bacteria in feedlot cattle . 31 INTRODUCTION. 31 MATERIALS AND METHODS. 32 Animals, Diets and Sampling Schedule. 32 Bacteriological Procedures . 32 Antimicrobial Susceptibility Testing. 34 Quantification of Antimicrobial Resistance Genes. 35 Bacterial Strains and Plasmids. 35 iiiPrimer Design. 35 PCR Running Conditions. 36 Cloning Techniques. 36 Real-time PCR Standardization. 36 Extraction of DNA from Fecal Samples. 37 Real-Time PCR on Fecal Samples. 37 Statistical Analysis. 37 RESULTS. 38 Prevalence of E. coli O157 and Salmonella. 38 Antimicrobial susceptibility. 39 Quantification of fecal ermB and tetM genes. 40 DISCUSSION. 40 REFERENCES . 51 CHAPTER 3 - Effects of Feeding Elevated Concentrations of Supplemental Copper and Zinc on Antimicrobial Susceptibilities of Fecal Bacteria in Feedlot Cattle. 60 INTRODUCTION. 60 MATERIALS AND METHODS. 61 Study design and sampling . 61 Bacteriological procedures. 62 Antibiotic susceptibility determination. 64 Susceptibility determinations for copper and zinc. 64 Quantification of erm(B) and tet(M) genes in the feces. 65 Bacterial strains and plasmids. 65 Primers and PCR running conditions. 65 Cloning techniques. 66 Real-time PCR standardization. 66 Extraction of DNA and real-time PCR of fecal samples. 66 Statistical analysis. 67 RESULTS. 67 Prevalence of food-borne pathogens in the feces. 68 Antibiotic susceptibili