2010 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 Presistence of Antibiotic Resistance in Swine: In FY10, we completed research attempting to block the transmission of antibiotic resistant Megasphaera elsdenii (M. elsdenii) strains from sow to offspring by feeding five antibiotic sensitive strains of M. elsdenii. This approach did not prevent the colonization of baby piglets by sow antibiotic resistant strains of this bacterium which were highly diverse in their antibiotic resistance properties. We have now completed a molecular fingerprint analysis (SNP patterns of two genes) of M. elsdenii strains: a) isolated from the sow, b) fed to the offspring, and c) isolated from the offspring. On the basis of their molecular fingerprints, over 700 M. elsdenii isolates were assigned to 42 distinct fingerprint groups indicating substantial diversity between strains. Only two of the five fed strains detectably colonized the offspring. The findings indicate M. elsdenii in the swine intestinal tract displays broad antibiotic resistance properties and has unexpected strain diversity. The finding of strain diversity provides a reasonable explanation for the inability to displace antibiotic resistant strains with antibiotic sensitive strains i.e., there are abundant M. elsdenii strains with different resistance traits in the swine intestine. Displacing them with a handful of antibiotic sensitive strains in a probiotic type approach is not practical. Discovery that the Swine Intestinal Tract has Diverse Butyrate-Producing Bacteria: Of 950 bacteria isolated from swine feces using seven different culture media, we found that 52 (5%) produced detectable butyrate (5 mM) during growth. Identification of these bacteria from their 16S rRNA gene sequences indicated they are taxonomically diverse and include bacteria known as Veillonellaceae (especially Megasphaera), Clostridium cluster XIV, Bacteroides, Prevotella, Enterobacteria, and previously uncultured. The results indicate butyrate-producing bacteria in swine are more diverse than those reported for humans. Butyrate is considered a key nutrient for healthy intestinal tissues. Identification of intestinal butyrate producing bacteria is the first step towards exploiting those bacteria in strategies to improve swine health and food safety. These results were reported at Gut Microbiology Conference in Aberdeen, Scotland (June, 2010). Bacterial Viruses in Swine Feces are Numerous and Diverse: We developed a method to isolate bacteriophages from swine feces that used blending, differential and density gradient centrifugation, and filtration. Numerous phage morphologies were detected by electron microscopy. Phage preparations will be analyzed by using metagenomics and bioinformatics tools to decipher the role of phage in the swine intestinal tract and to assess the effects of dietary antibiotics on phage production. These studies are important for detecting collateral effects of certain antibiotics fed to swine. These results were reported at the International Society for Microbial Ecology in Seattle, Washington (August 2010). 4.Accomplishments 1. Census of Butyrate-producing Bacteria in the Swine Intestine. Butyrate is considered a key nutrient for maintaining healthy intestinal tissues. A healthy intestinal ecosystem is essential for good animal growth and for providing barriers to intestinal pathogens. ARS researchers at Ames, Iowa took the first census of butyrate producing bacteria in the swine intestinal tract. Surveying cultures of nearly a thousand intestinal bacterial isolates, they found that 5% produce butyrate during growth. The bacterial species were diverse and included species of Megasphaera, Clostridium cluster XIV, Bacteroides, Prevotella, and Enterobacteria. Identification of intestinal butyrate-producing bacteria is a first step towards exploiting those bacteria in strategies for improving swine health and food safety and for discovering alternatives to antimicrobial use. Review Publications Anderson, R.C., Huwe, J.K., Smith, D.J., Stanton, T.B., Krueger, N.A., Callaway, T.R., Edrington, T.S., Harvey, R.B., Nisbet, D.J. 2010. Effect of nitroethane, dimethyl-2-nitroglutarate and 2-nitro-methyl-propionate on ruminal methane production and hydrogen balance in vitro. Bioresource Technology. 101:5345-5349. Schloss, P.D., Allen, H.K., Klimowicz, A.K., Mlot, C., Gross, J.A., Savengsuksa, S., Mcellin, J., Clardy, J., Ruess, R.W., Handelsman, J. 2010. Psychrotrophic Strain of Janthinobacterium lividum from a Cold Alaskan Soil Produces Prodigiosin. DNA and Cell Biology. 29(9):533-541.