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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPACT OF DIET AND GUT MICROBIAL ECOLOGY ON FOODBORNE BACTERIAL PATHOGENS AND ANTIMICROBIAL RESISTANCE IN FARM ANIMALS
2008 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
Despite multiple personnel vacancies, substantial progress has been made on these diverse projects by making modifications in milestone timing, personnel re-assignments, and streamlining specific milestones. CAMPYLOBACTER ANTAGONISTS: Over 1200 strains of anaerobic bacteria were cultured from the intestinal tracts of swine and turkeys. These strains were tested in cross hatch inhibition assays for antagonizing C. jejuni. None were found to inhibit C. jejuni growth. The milestones of the research have been met. Unfortunately, the results have been unpromising and consequently future milestones associated with this line of research have been discontinued. PROBIOTIC APPROACH TO REDUCING ANTIBIOTIC RESISTANCE IN SWINE: Previous research from other investigators and from our lab at the National Animal Disease Center implicated commensal, normal intestinal bacteria in healthy swine both as reservoirs of antibiotic resistance genes and as sites for the evolution of those resistance genes. In FY 2008 we tested an important hypothesis - that antibiotic sensitive commensal bacteria could block, compete with, or displace antibiotic resistant bacteria of the same species, thereby reducing the incidence of antibiotic resistance in swine. We administered a cocktail mix of five antibiotic sensitive (swine) strains of the commensal bacterium Megasphaera elsdenii to neonatal swine. The piglets were given multiple doses both before and after weaning. At ten days after weaning, only antibiotic sensitive M. elsdenii strains were recovered from the dosed piglets. At 25 and 39 days after weaning, however, multiply antibiotic resistant M. elsdenii strains from the mother sow colonized and eventually predominated in the dosed piglets. Control piglets (not dosed) were colonized with antibiotic resistant sow strains earlier. One explanation for these results (our current working hypothesis) is that Megasphaera elsdenii strains colonizing swine exhibit specificity for the host swine genotype. If this 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. CARBADOX-INDUCED GENE TRANSFER AGENT VSH-1: We published our findings that the antibiotic carbadox stimulates production of a novel gene transfer mechanism, VSH-1, and transfer of a tylosin-resistance gene between cells of the swine pathogen Brachyspira hyodysenteriae. In FY 2008, by using a new DNA (pyro-) sequencing technology recently available at the National Animal Disease Center, we sequenced the B. hyodysenteriae genome in two days and discovered that VSH-1, remarkably, has a "split genome". Its gene clusters appear to be independently regulated but through a common control mechanism. This observation, if further confirmed, will have implications for understanding the functions and evolution of other gene transfer agents now being discovered throughout the bacterial world. Research in this project addresses National Program 108 Food Safety Action Plan Section 1.1.5 -Antimicrobial resistance, (identification of factors responsible for mediating AR).


4.Accomplishments
1. Probiotic Approach to Reducing Antibiotic Resistance in Swine. Two goals of our research are to ameliorate antibiotic use and to reduce antibiotic resistant bacteria in swine. Previous research from other investigators and from our lab at the NADC implicated commensal (normal) intestinal bacteria in healthy swine both as reservoirs of antibiotic resistance genes and as sites for the evolution of those resistance genes. In FY 2008 we tested an important hypothesis - that antibiotic sensitive commensal bacteria could block sow-to-piglet transmission of antibiotic resistant bacteria of the same species, thereby reducing the incidence of antibiotic resistance in the piglets. We administered a cocktail mix of five antibiotic-sensitive (swine) strains of the commensal anaerobic bacterium Megasphaera elsdenii to neonatal swine to block the horizontal flow of antibiotic-resistant M. elsdenii strains from the sow. The piglets were given multiple doses both before and after weaning. At ten days after weaning, only antibiotic sensitive M. elsdenii strains were recovered from the dosed piglets. At 25 and 39 days after weaning, however, multiply antibiotic resistant M. elsdenii strains from the mother sow colonized and eventually predominated in the dosed piglets. Control piglets (not dosed) were colonized with antibiotic resistant sow strains earlier. One explanation for these results (our current working hypothesis) is that Megasphaera elsdenii strains colonizing swine exhibit specificity for the host swine genotype. Thus exogenous dosed strains cannot prevent sow-to-piglet transmission. If this is more broadly true for intestinal commensal bacteria, then probiotic applications requiring colonization of animal or human by dosing non-specific probiotic bacteria are unlikely to be uniformly successful. This work aligns with Component 1.1 of the National Program 108 Food Safety Action Plan and addresses Problem Statement 1.1.5 (Antibiotic Resistance).


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

None

Review Publications
Anderson, R.C., Krueger, N.A., Stanton, T.B., Callaway, T.R., Edrington, T.S., Harvey, R.B., Jung, Y.S., Nisbet, D.J. 2008. Effects of select nitrocompounds on in vitro ruminal fermentation during conditions of limiting or excess added reductant. Bioresource Technology. 99(18)8655-8661.

Stanton, T.B., Humphrey, S.B., Sharma, V.K., Zuerner, R.L. 2008. Collateral effects of antibiotics - carbadox and metronidazole induce VSH-1 and facilitate gene transfer among Brachyspira hyodysenteriae strains. Applied and Environmental Microbiology. 74(10):2950-2956.

Last Modified: 10/24/2014
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