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.
This is the final report for project 3625-31320-002-00D terminated in December 2010 and replaced with 3625-31320-003-00D.
We discovered that giving Prevotella bryantii to dairy cattle as a probiotic therapy ameliorated experimental rumen lactic acidosis, by significantly reducing lactic acid production. This treatment offered cattle producers an alternative treatment to antibiotics for acidosis, the digestive disturbance known to affect large numbers of cattle annually.
Swine intestinal Megasphaera elsdenii strains were found to transfer tetracycline resistance genes at high frequency. Tetracycline resistance genes were identified as mosaic genes whose sequences match those of the swine pathogen Streptococcus suis and the foodborne pathogens Campylobacter and Enterococcus faecalis. The findings suggest resistance genes can be exchanged among these bacteria and indicate the importance of considering both nonpathogenic and pathogenic bacteria in efforts to mitigate against antibiotic resistance in the food chain.
Probiotic dosing of newborn piglets before and after weaning with a cocktail of five antibiotic sensitive strains of Megasphaera elsdenii (M. elsdenii) delayed but did not prevent the transmission of antibiotic resistant M. elsdenii strains from sow to offspring. Based on molecular fingerprinting, the sow strains were highly diverse. Additionally, an antibiotic-sensitive M. elsdenii strain introduced into the offspring swine became tetracycline-resistant (in the absence of antibiotic exposure). The results provide an explanation for the persistence of antibiotic-resistant bacteria in animals and humans never treated with antibiotics. These findings are also important because they demonstrate this probiotic approach to reduce antibiotic resistance will not work.
The swine pathogen Brachyspira hyodysenteriae’s (B. hyodysenteriae) genome was sequenced. We found that VSH-1, an antibiotic resistance transfer mechanism inside that bacterial genome, itself has a "split genome". We established that low levels of carbadox, a swine dietary antibiotic, stimulate 100-fold increases in VSH-1 transfer of resistance to the antibiotics tylosin, streptogramin, and lincomycin between strains of B. hyodysenteriae. Carbadox also promotes transduction of genes and stimulates transfer of genetic elements among other intestinal bacterial species, including food-borne pathogens such as Escherichia coli and Salmonella. These results indicate that collateral effects of certain antibiotics (other than resistance) need to be considered so that producers can make prudent choices of antibiotics for animal management.
Through metagenomics analysis we discovered an unexpected increase in Escherichia coli populations in swine fed the commercial antibiotic combination aureomycin-sulfathiazole-penicillin (ASP250). Investigations of antibiotic feeding effects on intestinal microbiota are important for understanding of how growth enhancing antibiotics work and how antibiotic alternatives should behave.