2009 Annual Report 1a.Objectives (from AD-416) 1) Identify commensal sources of tetracycline resistance genes;. 2)Evaluate bacteriophage as agents of gene transfer;. 3)Identify protozoal factors that affect pathogen virulence in the rumen; and. 4)Identify dietary strategies to limit acidosis and pathogen reservoirs. 1b.Approach (from AD-416) Isolate commensal bacteria from swine that share niches and exchange genes with Campylobacter. Classify Campylobacter strains for antibiotic susceptibility and amplify and sequence tet genes. Add carbadox to stimulate phage induced tylosin resistance gene transfer in Brachyspira. Assay degree of phage induction and gene transfer. Harvest protozoa from rumen contents and determine associated bacterial populations using ARISA and BLAST. Culture single species of protozoa and allow them to feed upon specific bacteria tagged with fluorescence. Examine protozoa for uptake and sequestration of tagged bacteria. Identify compounds to defaunate the rumen and verify reservoir hypothesis by loss of bacterial pathogens in ruminants. 3.Progress Report PERSISTENCE OF ANTIBIOTIC RESISTANCE IN SWINE: In FY09 we completed research attempting to use antibiotic sensitive Megasphaera elsdenii strains to prevent transmission of antibiotic resistant strains from sow-to-piglet. The approach was insufficient. We have now established through antibiotic resistance phenotype analysis and SNP (single nucleotide polymorphism) determinations that the sow transmitted numerous, diverse M. elsdenii strains to the piglets. Among 125 M. elsdenii sow strains eight different combinations of resistance for the antibiotics tylosin, ampicillin, and chlortetracycline were found. This strain diversity likely made it difficult for exogenously added antibiotic sensitive strains to outcompete the sow strains and prevent them from colonizing the piglets. Importantly, these findings provide an explanation for the often reported persistence of antibiotic resistance in farm animals (and other ecosystems) in the absence of direct antibiotic selection. If the situation with M. elsdenii is more broadly true for intestinal bacteria, then probiotic applications requiring colonization of animal or human by dosing non-specific probiotic bacteria are unlikely to be uniformly successful. These results were described at an invited presentation at the USDA-NIFSI Antibiotic Resistance Conference (April 09). DISCOVERY OF A NON-CONTIGUOUS GENOME FOR THE GENE TRANSFER AGENT, VSH-1: The novel gene transfer mechanism, VSH-1, has a remarkable "split genome". We used QRTPCR (quantitative real time PCR) analysis of gene transcription to demonstrate the VSH-1 separate gene clusters are transcribed independently. These observations have implications for understanding the functions and evolution of other gene transfer agents and cryptic bacterial viruses. These findings were reported at an invited address at the International Intestinal Spirochete Conference in Spain (June, 09). They were also published (J Bact 191:1719). METAGENOMICS DETECTS ANTIMICROBIAL EFFECTS ON SWINE INTESTINAL MICROBIOME: A preliminary metagenomics analysis of antimicrobial effects on swine either fed or not fed ASP250 (ampicillin, sulfamethazine, and chlortetracycline) was conducted. The study revealed a surprising 5-7-fold increase in enterobacteria representation in all swine fed the performance enhancing antimicrobial mixture. Enterobacteria increased to 20-25% of the total bacteria library. If confirmed, the results suggest that performance enhancing antimicrobials can significantly raise intestinal levels and shedding of E. coli related bacteria. These findings were reported in the Symposium “Animal Microbiomes“, at the annual meeting of the Am Soc Microbiol (May, 09). 4.Accomplishments 1. Surprising Discovery of Novel Non-Contiguous Genome for Gene Transfer Agent VSH-1. Previously we discovered that the novel gene transfer mechanism, VSH-1, has a remarkable "split genome". In FY09, we used QRTPCR quantitative real time PCR (QRTPCR) analysis of gene transcription and confirmed our hypothesis that VSH-1 gene clusters are transcribed independently. These observations have implications for understanding the functions and evolution of other gene transfer agents and bacterial viruses now being discovered throughout the bacterial world. Both GTAs, like VSH-1, and traditional bacterial viruses have been proven capable of transferring antibiotic resistance genes and bacterial virulence determinants. 6.Technology Transfer Number of Web Sites Managed 1 Review Publications Stanton, T.B., Humphrey, S.B., Bayles, D.O., Zuerner, R.L. 2008. Identification of a Divided Genome for VSH-1, the Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae. Journal of Bacteriology. 191(5):1719-1721.