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United States Department of Agriculture

Agricultural Research Service

Research Project: Animal Intestinal Microbiomes, Foodborne Pathogens, and Antimicrobials
2012 Annual Report


1a.Objectives (from AD-416):
1) Identify and characterize intestinal ecological niches and their impact on foodborne pathogen survival, persistence, colonization, or virulence. In a broader systematic approach, evaluate the interactions among environmental influences (e.g., management, production) and ecological niches on phenotypic and genotypic characteristics and food safety..
2)Evaluate the effects of antimicrobials on intestinal microbiomes, and on the expression and transmission of virulence, fitness, and antimicrobial resistance genes in culture and the host..
3)Develop a functional metagenomic approach to identify gene products that inhibit foodborne pathogen growth, interfere with virulence gene expressions, or reduce antimicrobial resistance (and enhance food safety)..
4) Assess role of commensal intestinal bacteria in evolution, persistence, or transmission of resistance genes. Evaluate novel strategies for reducing antimicrobial resistant organisms and resistance genes..
5)Evaluate the effects of environmental influences (e.g., management, production), ecological niches and vaccine strategies on phenotypic and genotypic characteristics of Campylobacter (specifically in turkeys).


1b.Approach (from AD-416):
Research to control Campylobacter in turkeys will be pursued by subfractionating, identifying and characterizing microbial species in turkey ceca which are potential Campylobacter antagonists. Cecal populations will be subfractionated in vivo by antibiotic applications and dexamethasone induced stress. Potentially useful species will initially be associated with Campylobacter reductions in vivo and confirmed in vitro by using co-cultures. Two other approaches for controlling Campylobacter will be tested: dietary additives (prebiotics and probiotics) and a prime-boost vaccination strategy using C. jejuni surface membrane protein CjaA expressed in a recombinant strain of attenuated Salmonella. The commensal anaerobe Megasphaera elsdenii will be used as an archetype intestinal bacterium to identify common antibiotic resistance genes and transfer mechanisms in the intestinal tract and to test M. elsdenii as a site for tetracycline resistance gene evolution in that ecosystem. In a probiotic type attempt, a mix of five antibiotic sensitive M. elsdenii strains will be used to block the transmission of antibiotic resistant strains from mother sow to offspring pigs. Metagenomic, culture, and PCR methods will be used to assess the effects of dietary antibiotics (ASP250, carbadox, and other antibiotics) on swine and turkey microbiomes. Specific areas of research will include the influence of subinhibitory antibiotics on the production of Salmonella bacteriophages carrying fitness or virulence genes and on phage mediated gene transfer between Salmonella strains (transduction). These studies will be carried out with Salmonella Typhimurium strains in culture and in a mouse model. Functional metagenomics assays involving recombinant E. coli and Salmonella strains containing reporter plasmids will be used to identify gene products which either affect Salmonella virulence or are inhibitory for Salmonella growth.


3.Progress Report:
A) Research on the impact of antibiotics on intestinal microbiomes revealed detectable changes in the bacteriophages (viruses that infect bacteria) due to in-feed antibiotics. By studying a gene that is required for phages to get into a bacterial genome (called an integrase), the results suggested that phage production was stimulated by the in-feed antibiotics carbadox and aureomycin, sulfamethazine, penicillin 250 (ASP250). Antibiotic resistance genes were detected in the phage deoxyribonucleic acid (DNA), although there was no statistically significant difference in the abundance of resistance genes between the medicated and non-medicated animals. Phages are important drivers of bacterial evolution in an ecosystem because they transfer genes between bacteria. The result that in-feed antibiotics increased phage numbers is an indication that antibiotic treatment is providing an evolutionary stimulus to gut microbial communities. B) Megasphaera elsdenii (M. elsdenii) is a common, non-pathogenic bacterium that inhabits the intestinal tracts of animals and humans. It provides nutritional and health benefits to its animal hosts. The genomes (DNAs) of two M. elsdenii parent strains (strain 14-14 and strain 24-50C) and three offspring strains produced from the matings of the parent strains were extensively sequenced and analyzed. The genome sequence comparisons showed M. elsdenii strain 14-14 efficiently transferred resistance to the antibiotics tetracycline, ampicillin, and tylosin to strain 24-50C. There were, however, two different genetic mechanisms for the transfer. Understanding both bacterial reservoirs of antibiotic resistance and the genetic mechanisms for the spread and the evolution of resistance provide tools for rationally assessing the effects of agricultural antibiotic use. This research was recently cited in the Food and Drug Administration’s final Guidance for Industry Number 209 entitled, “The judicious use of medically important antimicrobial drugs in food-producing animals”. This citation demonstrates the importance of this research for regulatory agencies.


4.Accomplishments
1. Functional metagenomics assay for alternatives to antibiotics. ARS researchers in Ames, Iowa developed and successfully tested a high throughput, robotic-based assay to identify ‘toxic’ genes whose products either inhibit Escherichia coli (E. coli) growth or disrupt E. coli bacteria. This ‘functional metagenomic’ assay screens all deoxyribonucleic acids (DNAs) in subfractions of environmental samples in order to find genes toxic for foodborne pathogens, such as E. coli. The researchers plan to add to their collection of inhibitory genes while starting to identify the cloned toxic genes. This research and products benefit animal producers, animal health and food safety industries, commodity groups, and pharmaceutical industries seeking alternatives to existing antibiotics.


Review Publications
Stanton, T.B., Humphrey, S.B., Stoffregen, W.C. 2011. Chlortetracycline - resistant intestinal bacteria in organically-raised and feral swine. Applied and Environmental Microbiology. 77(20):7167-7170.

Bunge, J., Woodard, L., Bohning, D., Foster, J.A., Connolly, S., Allen, H.K. 2012. Estimating population diversity with CatchAll. Bioinformatics. 28(7):1045-1047. Available: http://bioinformatics.oxfordjournals.org/content/28/7/1045.long.

Looft, T.P., Johnson, T., Allen, H.K., Bayles, D.O., Alt, D.P., Cole, J., Hashsham, S., Stedtfeld, R., Stedtfeld, T., Chai, B., Tiedje, J., Stanton, T.B. 2012. In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences. 109(5):1691-1696.

Stanton, T.B., Humphrey, S.B. 2011. Persistence of antibiotic resistance: evaluation of a probiotic approach using antibiotic-sensitive M. elsdenii strains to prevent colonization of swine by antibiotic-resistant strains. Applied and Environmental Microbiology. 77(20):7158-7166.

Allen, H.K., Looft, T.P., Bayles, D.O., Humphrey, S.B., Levine, U.Y., Alt, D.P., Stanton, T.B. 2011. Antibiotics in feed induce prophages in swine fecal microbiomes. mBio [serial online]. 2(6). Available: http://mbio.asm.org/content/2/6/e00260-11.

Bunge, J., Bohning, D., Allen, H.K., Foster, J. 2012. Estimating population diversity with unreliable low frequency counts. Pacific Symposium on Biocomputing (PSB) [serial online]. 17:203-212. Available: http://psb.stanford.edu/psb-online/proceedings/psb12/abstracts/2012_p203.html.

Anderson, R.C., Krueger, N.A., Genovese, K.J., Stanton, T.B., MacKinnon, K.M., Harvey, R.B., Edrington, T.S., Callaway, T.R., Nisbet, D.J. 2012. Effect of thymol or diphenyliodonium chloride on performance, gut fermentation characteristics, and Campylobacter colonization in growing swine. Journal of Food Protection. 75:758-761.

Levine, U.Y., Bearson, S.M., Stanton, T.B. 2012. Mitsuokella jalaludinii inhibits growth of Salmonella enterica serovar Typhimurium. Veterinary Microbiology. 159(1-2):115-122.

Last Modified: 11/27/2014
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