Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/7/2010
Publication Date: 2/1/2011
Citation: Durso, L.M., Harhay, G.P., Bono, J.L., Smith, T.P. 2011. Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach. Journal of Microbiological Methods. 84: 278-282.
Interpretive Summary: U.S. cattle output between 1.9 and 14.2 billion pounds of manure each day. There are many fecal bacteria in this manure, including both harmless and pathogenic microorganisms. We are interested in the ecology of manure microorganisms, and have used a new technique, called metagenomic pyrosequencing, to determine the kinds and amounts of bacteria in feedlot beef cattle feces, and also what kinds of genes are carried by these bacteria – including genes for normal metabolic pathways, and genes that are associated with virulence and antibiotic resistance. We compared the numbers and kinds of virulence genes from cattle feces with other samples such as Sargasso Sea, Antarctic marine derived lake, cattle rumen, chicken cecum, human feces, and farm soil. Results show that older methods of characterizing what bacteria are present in fecal samples may under-represent certain bacterial groups. Over eight percent of the DNA sequences from beef feces were associated with virulence factors, a number lower than that for the Sargasso Sea, chicken cecum, and cow rumen samples, but comparable to the Antarctic marine derived lake, human fecal, and farm soil samples. The results generated are semi-quantitative, and can be used to track relative amounts of antibiotic resistance genes in individual animals, and over time in agriculturally impacted environments.
Technical Abstract: The bovine fecal microbiota impact human food safety as well as animal health and nutrition. Although the bacteria of cattle feces has been well characterized using both culture-based and culture independent methods, techniques have been lacking to correlate total community composition with community function. We used pyrosequencing of total community DNA (metagenomics) to describe community composition while also characterizing the functional genes present in beef cattle feces, including genes associated with antibiotic resistance, and bacterial virulence. Results suggest that traditional 16S sequencing using “universal” primers may under-represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes than found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of the metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats means that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time in agriculturally impacted environments, and to generate sample-specific antibiotic resistance gene profiles or fingerprints that can help us to understand the ecology of the microbial communities in each habitat, and the epidemiology of antibiotic resistant gene transport between and among habitats.