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United States Department of Agriculture

Agricultural Research Service

Research Project: Animal Intestinal Microbiomes, Foodborne Pathogens, and Antimicrobials
2011 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. IBC-0260 BSL-Exempt; Recertified 08/03/11; IBC-0303 Recertified 6/11/11; IBC-0312 Recertified 11/15/10; IBC-0331 certified 02/16/11.


3.Progress Report
Swine siblings were fed either a commercial diet containing aureomycin, sulfamethazine, and penicillin (ASP250) or the same diet without antibiotics (control group). After two weeks ASP250 fed swine shed increased Escherichia coli populations in their feces. Bacterial genes important for energy production and metabolism in the swine digestive tract increased in the antibiotic treated group, a finding consistent with the animal performance enhancing properties of the ASP250-diet. The ASP250 diet was found, not surprisingly, to raise the fecal levels of genes encoding resistance for the ASP250 antibiotics. Additionally, increases in resistance genes, not in the ASP250 formulation were detected. One example was a gene for aminoglycoside (streptomycin) resistance. A possible explanation is that the ASP250 diet indirectly enriched for aminoglycoside resistance genes because they are carried on the same genetic transfer elements as the genes for aureomycin-, sulfamethazine-, or penicillin-resistance. To our knowledge, this is the first study combining these multiple molecular and culture based technologies to evaluate “collateral” effects of antibiotics. Investigations of antibiotic feeding effects on intestinal microbiota are important for understanding how growth enhancing antibiotics work and how antibiotic alternatives should behave. The genome sequence of the swine commensal (non-pathogenic) anaerobic bacterium Megasphaera elsdenii (M. elsdenii) strain 14-14 was determined, analyzed, and is being assembled. In addition to the previously detected mosaic (hybrid) gene for tetracycline resistance, there were a large number (six or seven) of other antibiotic-resistance genes identified. A second tetracycline resistance gene was discovered in close proximity to the mosaic gene. The M. elsdenii 14-14 genome contains numerous (greater than 40) genes considered or known to be involved in cell-to-cell transfer of antibiotic resistance genes in other bacteria. Consequently, these results indicate that the 14-14 strain of M. elsdenii, a common non-pathogenic bacterium in the intestinal tracts of animals and humans, is multiple drug resistant and appears well-equipped with mobilization mechanisms for transferring the antibiotic resistance genes to other bacteria. These findings are important for two reasons. Information from this analysis will facilitate research to evaluate M. elsdenii’s roles both in the transfer of antibiotic resistance and in the evolution of antibiotic resistance in the swine intestinal tract. Further, these results underscore the importance of considering both nonpathogenic and pathogenic bacteria in strategies to mitigate against antibiotic resistance in the food chain.


4.Accomplishments
1. Dietary antibiotic effects on the swine intestinal microbiome. ARS researchers at Ames, Iowa in collaboration with scientists at Michigan State University examined effects of feeding certain antibiotics to swine. They used a combination of phylotyping (bacterial identification by 16S rRNA gene sequences), metagenomic (sequencing and bioinformatic analysis of bacterial genes), quantitative PCR (qPCR), and culture-based approaches to investigate differences between swine fed a commercial diet containing aureomycin, sulfamethazine, and penicillin (ASP250) and siblings fed the same diet without antibiotics (control group). By comparison with control animals, swine fed ASP250 diets for two weeks: had higher Escherichia coli numbers in their feces; had elevated levels of bacterial genes for energy production and metabolism (consistent with the animal performance enhancing properties of the ASP250-diet); and had higher levels of genes encoding resistance both for the ASP250 antibiotics and other antibiotics in feces. Investigations of dietary antibiotic effects on intestinal microbiomes are important for understanding how certain antibiotics enable animals to grow efficiently with less feed consumption and how antibiotic alternatives should behave. Additionally, feeding subtherapeutic doses of antibiotics to farm animals will likely have collateral effects which should be considered in cost-benefit analyses of antibiotic use. Information is currently being used in the selection of antibiotics and design of antibiotic alternatives.


Review Publications
Levine, U.Y., Teal, T.K., Robertson, G.P., Schmidt, T.M. 2011. Agriculture's impact on microbial diversity and associated fluxes of carbon dioxide and methane. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. Available: http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej201140a.html.

Hamir, A.N., Greenlee, J.J., Stanton, T.B., Smith, J.D., Doucette, S., Kunkle, R.A., Stasko, J.A., Richt, J.A., Kehrli, Jr., M.E. 2011. Experimental inoculation of raccoons (Procyon lotor) with Spiroplasma mirum and transmissible mink encephalopathy (TME). Canadian Journal of Veterinary Research. 75(1):18–24.

Last Modified: 12/21/2014
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