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Frequency of Tetracycline Resistance Genes in Bacterial Genomic DNA of Swine Feces

Frequency of Tetracycline Resistance Genes in Bacterial Genomic DNA of Swine Feces. Sharise Redmond & Jeannette Nguyen Under the Direction of Candace Glendening. Antibiotics. ABX Agents made by bacteria/mold to inhibit bacterial growth Do NOT kill viruses Use

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Frequency of Tetracycline Resistance Genes in Bacterial Genomic DNA of Swine Feces

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  1. Frequency of Tetracycline Resistance Genes in Bacterial Genomic DNA of Swine Feces Sharise Redmond & Jeannette Nguyen Under the Direction of Candace Glendening

  2. Antibiotics • ABX • Agents made by bacteria/mold to inhibit bacterial growth • Do NOT kill viruses • Use • treat infections in humans and animals • growth promotion in animals • Bacteria • Pathogenic - harmful/disease causing • e.g. Salmonella, Escheria coli O157:H7 • Beneficial – non harmful • Commensal - symbiotic relationship which benefits one species while the other is unaffected • lots of commensal bacteria in gut (E. coli spp.)

  3. History of Antibiotics • 1800’s:“germ theory” • Began search for ABX • 1929:Fleming discovered Penicillin • 1942 1st large scale use of Penicillin • Used largely during WWII • 1946: Penicillin widely available clinically • Obtainable OTC by the public until the mid ‘50s • Dev’t of more ABX over next few decades • 1970’s: Antibiotic Resistance (AR) recognized as a real threat • Meningitis & gonorrhea strains resistant to penicillin

  4. AB Resistance • Bacteria’s ability to produce a protein that: • disables an ABX or • prevents transport of the ABX into the cell • Main hypothesis of AR: Genetic mutations + antibiotics in environment = antibiotic resistance • N. gonorrhoeae (gonorrhea)resistance: penicillin tetracycline SOON cephalosporins flouroquinolones

  5. ABX Use in Animals Does the use growth promotional levels of ABX in food animals lead to AR bacteria in our food? • Therapeutic – treatment of bacterial infection • Sub-therapeutic levels – prevention of disease & growth promotion • Antibiotics used for growth promotion  pigs gain weight: • 3.3-8.8% increased weight • 2.5-7.0% feed efficiency • Food Research Institute, Doyle, 1998 • ABX use by farmers is not regulated • ~25 million pounds annually used

  6. History of Tetracycline • 1948: introduction of tetracycline • Made by Streptomyces bacterium in soil • Chemical structure: • “broad spectrum” • low toxicity • 1953: Shigella dysenteriae dev. resistance to tetracycline • Today 2nd to penicillin in the world in production and use • Treat: Respiratory tract infections, typhus, cholera, brucellosis, anthrax, syphilis, Chlamydia, acne • Also used widely for growth promotion in animals.

  7. Tet Mechanisms of Action • Tetracycline inhibits bacterial growth by inhibiting translation. • It binds to the ribosomal subunit and prevents the amino-acyl tRNA from binding to the A site of the ribosome. Inhibition of Protein Synthesis by Tetracycline

  8. Tetracycline Efflux Pump Inactivation Enzyme plasmid Mechanisms of Tet Resistance Ribosomal Protection Protein

  9. Mechanism of resistance for characterized tet and otr genes

  10. Growth Promotion • There has been a lack of serious studies in the amounts of antibiotics given to livestock and its link to the increasing rates of resistance genes.

  11. Previously Done Studies

  12. Central Question • Does the use of tetracycline as a growth promotant affect tetracycline resistance in swine fecal flora? Central Hypothesis • The use of tetracycline as a growth promotant will  frequency of detecting Tet Resistance Genes in swine fecal flora.

  13. Effects of Growth Promotional use of Chlorotetracycline (CTC) • Large-scale, multi-year study led by Julie Funk @ Ohio State Univ. (OSU) • Epidemiological approach to studying the use of CTC as a growth promotant for swine • Looked for AR bacteria

  14. CDC Year 1 Study Design Temporally matched Barn Pair Control (no antibiotics in the feed) Treatment (50g CTC/ton of feed) • Treatment from 10 weeks (50 lbs) until 6 months old (250 lbs). • Pigs sampled pre-slaughter • 14 barn pairs total • 96 pigs per barn • 2688 total pigs sampled

  15. Isolated 100 different Gram Negative bacteria (usually E. coli) from each fecal sample Studied resistance to 4 antibiotics Found phenotypic (tet res) difference between these 2 groups Selected CDC Year 1 Results Gram Negative Fecal Flora Isolates 1.0 .90 .80 .70 .60 Proportion Resistant to CTC .50 .40 .30 .20 .10 0 No CTC CTC Treatment n=268,800 isolates

  16. Objectives • To study the distribution of tetracycline resistance genes found in the fecal flora of pigs • + CTC diet in their finishing phase. • Ctrl: NO growth promotional use of ABX • Our Study Population • 10 barn pairs • 48 pigs per barn • 480 total pigs sampled • Recall there are at least 38 tet resistance genes • Are certain genes found more often under the selective pressure of tetracycline?

  17. 200 mg poop (frozen quickly) Qiagen Stool DNA Extraction Kit (Bacterial Genomic DNA) 200 l genomic DNA (from bacterial population) 1 l 1 l 1 l 1 l Multiplex PCR Group 1 Group 3 Group 2 Group 4 ExperimentalDesign

  18. E-gel Marker Methods: Multiplex PCR • 2 (or more) sets of primers in same tube • Ex: Group 1: • tet(B) 659 bp • tet(C) 418 bp • tet(D) 787 bp • Run each sample through four separate Multiplex PCR reactions. 480 samples x 4 groups = 1920 rxns! B C D B/C/D 2000 800 400 200 100 Ng et al., 2001

  19. Genes Studied

  20. 667 515 406 267 169 Sample Gel (Group 3) (+) Controls _ Individual Pigs from Farm Bailey 3 _ K L M O S (-) Individual Pigs from Farm Bailey 3 _

  21. Sample Gel (Group 4) (+) Controls Individual Pigs from Farm Bailey 3 _ _ ? Q X (-) 904 468 ___ _ Individual Pigs from Farm Bailey 3 _______

  22. Results

  23. Results

  24. Results .3 .33 .49 5 E-10 1 E-4

  25. Results 6 E-4 2 E-5 3 E-7

  26. Results .008 .03 3 E-7

  27. 81% 81% .94 Central Hypothesis • The use of tetracycline as a growth promotant will  frequency of detecting Tet Resistance Genes in swine fecal flora.

  28. Discussion • At least one tet res gene in 81% of both treatment groups • 5/8 tet res genes showed no statistical diff. btw treatment groups or were not high in frequency • tet(C), (L), (M) similar high frequency in both swine groups • tet(B), (D), (K) min. to 0 frequency in both swine groups • tet(S) mostly found in ctrl samples • tet(O) mostly found in CTC samples • Group 4 AR genes in both treatment groups • 3/3 tet res genes statistically diff. btw treatment groups • Most tet genes found in CTC groups • Group 2 data in progress

  29. Future Work • Complete sample processing • 4 more barn pairs • Look at more tet resistance genes • Try to quantitate the amount of each tet gene present in the sample

  30. References • Aminov, R. I.; Chee-Sanford, J. C.; Garrigues, N.; Teferedegne, B.; Krapac, I. J.;. White, B. A.; Mackie, R. I. (2002) Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative Bacteria. Applied and Environmental Microbiology, 68(4), 1786-1793. • Aminov, R. I; Garrigues-Jean, N; Mackie, R. I. (2000) Molecular Ecology of Tetracycline Resistance: Development and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins. Applied and Environmental Microbiology 67 (1), 22-32. • Billington, S. J.; Songer, J. G.; Jost, B. H. (2002) Widespread Distribution of a Tet W Determinant among Tetracycline-Resistant Isolates of the Animal Pathogen Acranobacterium pyogenes. Antimicrobial Agents and Chemotherapy, 1281-1287. • Bryan, A.; Shapir, N.; Sadowsky, M.J. (2004) Frequency and Distribution of Tetracycline Resistance Genes in Genetically Diverse, Nonselected, and Nonclinical Escherichia coli Strains Isolated from Diverse Human and Animal Sources • Chee-Sanford, J. C.; Aminov, R. I.; Krapac, I. J.; Garrigues-JeanJean, N.; Mackie, R. I. (2001) Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities. Applied and Environmental Microbiology, 67(4), 1494-1502. • Chopra, I.; Roberts, M. (2001) Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews, 65(2), 232-260. • Doyle, M. E. (2001) Alternatives to Antibiotic Use for Growth Promotion in Animal Husbandry. Food Research Institute: Briefings, University of Wisconsin-Madison 1-17. • Gevers, D.; Danielsen, M.; Huys, G.; Swings, J. (2002) Molecular Characterization of tet(M) Genes in Lactobacillus Isolates from Different Types of Fermented Dry Sausage. Applied and Environmental Microbiology, 69(2), 1270-1275. • http://dictionary.reference.com • http://en.wikipedia.org/wiki/Tetracycline • Lefers, Mark and Holmgren Lab (2004) http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/Def-A/antibiotic_resistance.html • Levy, M.D., Stuart B. (2002). The Antibiotic Paradox. Cambridge, MA: Perseus Publishing • Mathews, K. H. (2001) Antibiotic Drug Use and Veterinary Costs in U.S. Livestock Production. United States Department of Agriculture Economic Research Service, Agriculture Information Bulletin 766. • Ng, L.-K.; Martin, I.; Alfa, M.; Mulvey, M. (2001) Multiplex PCR for the detection of tetracycline resistant genes. Molecular and Cellular Probes, 15, 209-215. • Rubkin, Roberts, Institute of Medicine (1998) Antimicrobial Resistance: Issues and Options. Washington, DC: Harrison, P. R. and Lederberg, J. National Academy Press. • Villedieu, A.; Diaz-Torres, M. L.; Hunt, N.; McNab, R.; Spratt, D. A.; Wilson, M.; Mullany, P. (2002) Prevalence of Tetracycline Resistance Genes in Oral Bacteria. Antimicrobial Agents and Chemotherapy, 47(3), 878-882. • White, D.G.; Zhao, S.; Simjee, S.; Wagner, D. D.; McDermott, P. F. (2002)Antimicrobial resistance of foodborne pathogens. Microbes and Infection 4, 405-412.

  31. Acknowledgements: • Grant!!! • Julie Funk, MS, DVM, PhD, Asst. Prof. @ OSU School of Veterinary Medicine • Fecal Extraction Team • Andy Bowman • Luc Hesselschwardt • Andy Mack • Jodi Houser • Jamie Berning • Candace Glendening • Each Other • University of Redlands

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