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Lakshmikantha H.C 1 , Bh. Subramanyam 1 , and L. Zurek 2

Antibiotic resistant and virulent enterococci in animal feed. Lakshmikantha H.C 1 , Bh. Subramanyam 1 , and L. Zurek 2. 1 Department of Grain Science and Industry 2 Department of Entomology Kansas State University. Introduction. animal feed: antibiotics: enterococci.

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Lakshmikantha H.C 1 , Bh. Subramanyam 1 , and L. Zurek 2

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  1. Antibiotic resistant and virulent enterococci in animal feed Lakshmikantha H.C1, Bh. Subramanyam1, and L. Zurek2 1Department of Grain Science and Industry 2Department of Entomology Kansas State University

  2. Introduction animal feed: antibiotics: enterococci

  3. Antimicrobials in animal feed • In the US, 3,000 feed mills produce 121 million tons of feed for various animal species (Feedstuffs 2003) • Antibiotic use in feed as a : ¤ Growth promotion ¤ Prophylactic ¤ Therapeutic • >70% of antibiotics (>22 million pounds) produced in USA are used by the feed industry (Walker et al. 2005) • Selection pressure Antibiotic resistant bacteria

  4. Importance of enterococci • Enterococci are normal fauna of the gastrointestinal tract of humans and animals • Enterococci are not typical food-borne pathogens • Reservoirs for drug-resistant genes and mobile genetic elements Enterococcus faecalis

  5. Antimicrobial resistance and public health • 76 million cases of food borne illness reported annually in the United States (CDC) • >70% bacterial infections in the world are resistant to at least one antimicrobial (CDC) • Vancomycin-resistant enterococci infections, MRSA have become common in U.S. hospitals (National Institute of Allergy and Infectious Diseases)

  6. Research questions • Does animal feed carry enterococci? • Do enterococci harbor drug resistant & virulence genes? • Are these virulence genes expressed? • Is there any gene transfer from Enterococcus spp. to other species viahorizontal gene transfer (HGT)

  7. Horizontal transmission of resistance genes among species Levy and Miller, (1989). Gene transfer in the environment. Science. 247: 350-351

  8. Hypothesis Animal feed carry enterococci with antibiotic resistant and virulent genes

  9. Research objectives • Isolation, quantification, and species level identification of enterococci isolated from animal feed using 16S rRNA gene • Molecular characterization of antibiotic resistance and virulence genes assessed by phenotypic and genotypic assays • Conjugation ability and transfer of virulent genes from Enterococcus spp. to other species via horizontal gene transfer

  10. Materials & Methods

  11. Collection of samples ● ● ● ● • Feed samples: 89 • No. enterococcal isolates: 208

  12. Materials and Methods Feed Isolation Quantification Antibiotic resistance Specific primers; 16S rRNA gene sequences Multiplex PCR Species level identification

  13. TET VAN CIP ERY CHL AMP Antibiotics used in this study • Tetracycline 30µg (TET) • Ampicillin 10µg (AMP) • Erythromycin 15µg (ERY) • Vancomycin 30µg (VAN) • Chloramphenicol 30µg (CHL) • Ciprofloxacin 5µg (CIP) • Gentamicin 120 µg (GEN) • Kanamycin 20µ g (KAN) • Streptomycin 20µg (STR) Diffusion disk assay Followed the guidelines of the Clinical and Laboratory Standards Institute(formerly NCCLS)

  14. Results

  15. Prevalence and distribution of enterococci in animal feed

  16. Enterococcal species distribution in animal feeds

  17. Antibiotic resistance profiles of enterococci isolated from feed environments (n=208) Feed (208) % prevalence Antibiotics

  18. Tetracycline resistance profiles of enterococci isolated from animal feeds Feed (208) (n=208) % prevalence Tetracycline resistance genes

  19. Virulence determinants of enterococci isolated from animal feed Genotypic assay Phenotypic assay Feed (n=208) % prevalence % prevalence Virulence factor Virulence factor

  20. Distribution of virulence determinants in Enterococcus species isolated from animal feed Feed ( n = 208)

  21. Highlights and outcomes of research-to-date • Majority of the isolates showed resistance to tetracycline (48 %), erythromycin (14.4 %), and streptomycin (13 %) • 58 % of isolates were resistant to at least one antimicrobial agent • 26 % of isolates were resistant to two or more antimicrobial agents • 20.6 % of isolates harbors at least one virulence gene and resistance to at least one antimicrobial agent

  22. Conclusions • Animal feed collected from feed mills and livestock facilities harbored antibiotic-resistant and potentially virulent enterococci • Source of enterococcal contamination in animal feed is yet to be determined • Can these drug-resistant genes be transferred to other enteric pathogens via horizontal gene transfer (HGT)?

  23. Acknowledgements Dr Leland McKinney Dr Aqeel Ahmad Research reported here was funded by USDA/CSREES under agreement number 2002-3438-112187

  24. Thank you Questions?

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