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United States Department of Agriculture

Agricultural Research Service

Research Project: Animal Intestinal Microbiomes, Foodborne Pathogens, and Antimicrobials
2013 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:
The diversity of bacterial viruses in the swine intestinal tract was analyzed. In support of Objective 4 we applied statistics to mathematically discount the rare observations that are common to these types of data, and published a description of an accessible program (CatchAll version 3.0) that implements the statistical procedures. CatchAll was used to analyze data from swine bacterial viruses, showing higher estimations of phage diversity than previously realized. Viruses of bacteria are important drivers of bacterial evolution and of ecosystem functions, so discovering that the swine gut harbors thousands of different phages is important for understanding the ecology of bacteria, their viruses, and how they change under different conditions. Further investigations into these viruses might affect our strategies to modulate gut bacteria and improve animal health and food safety.

Bacteria likely to be keystone members of the intestinal microbial communities of healthy swine were isolated in pure culture and characterized. In support of Objective 1 eleven isolates were found to produce butyrate, an important nutrient for swine intestinal tissue health. Six were strains of Megasphaera elsdenii. Ten to twelve diverse bacterial species (e.g. Cloacibacillus porcorum) intimately associated with the swine intestinal tissues (notably ileum) were also isolated. Molecular assays are being developed for these bacterial subpopulations and will be used along with immunological and metagenomic tools to measure the impacts of heat stress and dietary additives on the swine. The research will facilitate the validation, discovery, and development of alternatives to antibiotics for improving swine health and reducing foodborne pathogens. This research and our previous research on dietary antibiotic effects on swine gut microbes led to a cooperative research agreement and invitations to publish three journal articles.


4.Accomplishments
1. Developed software for measuring viral diversity in the intestinal tract. ARS researchers in Ames, Iowa, together with collaborators at Cornell University and the University of Idaho modified and developed a computer program, CatchAll, to estimate the total number of bacterial viral (phage) species in Deoxyribonucleic acid (DNA) samples (metagenome) made from feces and other environmental samples. No previously published programs were adequate. The researchers will apply CatchAll to new swine fecal datasets to learn how the numbers of different gut bacteriophages change over time and with antibiotic treatment. This program and its proven usefulness will benefit virologists, bacteriologists, ecologists, and environmental scientists studying viruses of bacteria and how they interact with their hosts and the environment.


Review Publications
Stanton, T.B. 2013. A call for antibiotic alternatives research. Trends in Microbiology. 21(3):111-113.

Levine, U.Y., Looft, T.P., Allen, H.K., Stanton, T.B. 2013. Butyrate-producing bacteria, including mucin degraders, from the swine intestinal tract. Applied and Environmental Microbiology. 79(12):3879-3881. Available: http://aem.asm.org/content/79/12/3879.long.

Allen, H.K., Bunge, J., Foster, J.A., Bayles, D.O., Stanton, T.B. 2013. Estimation of viral richness from shotgun metagenomes using a frequency count approach. BMC Microbiome. 1(5). Available: http://www.microbiomejournal.com/content/1/1/5.

Looft, T.P., Levine, U.Y., Stanton, T.B. 2012. Cloacibacillus porcorum sp. nov., a mucin-degrading bacterium from the swine intestinal tract and emended description of the genus Cloacibacillus. International Journal of Systematic and Evolutionary Microbiology. 63:1960-1966.

Looft, T.P., Allen, H.K. 2012. Collateral effects of antibiotics on mammalian gut microbiomes. Gut Microbes. 3(5):463-467.

Allen, H.K., Levine, U.Y., Looft, T.P., Bandrick, M.M., Casey, T. 2013. Treatment, promotion, commotion: Antibiotic alternatives in food-producing animals. Trends in Microbiology. 21(3):114-119.

Last Modified: 10/24/2014
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