Antimicrobial resistant determinants (ARDs) can be transmitted from livestock systems through meat products or environmental effluents. The public health risk posed by these two routes is not well understood, particularly in non-pathogenic bacteria. We collected pooled samples from 8 groups of 1741 commercial cattle as they moved through the process of beef production from feedlot entry through slaughter. We recorded antimicrobial drug exposures and interrogated the resistome at points in production when management procedures could potentially influence ARD abundance and/or transmission. Over 300 unique ARDs were identified. Resistome diversity decreased while cattle were in the feedlot, indicating selective pressure. ARDs were not identified in beef products, suggesting that slaughter interventions may reduce the risk of transmission of ARDs to beef consumers. This report highlights the utility and limitations of metagenomics for assessing public health risks regarding antimicrobial resistance, and demonstrates that environmental pathways may represent a greater risk than the food supply.DOI: http://dx.doi.org/10.7554/eLife.13195.001

It has been proposed that livestock production effluents such as wastewater, airborne dust and manure increase the density of antimicrobial resistant bacteria and genes in the environment. The public health risk posed by this proposed outcome has been difficult to quantify using traditional microbiological approaches. We utilized shotgun metagenomics to provide a first description of the resistome of North American dairy and beef production effluents, and identify factors that significantly impact this resistome. We identified 34 mechanisms of antimicrobial drug resistance within 34 soil, manure and wastewater samples from feedlot, ranch and dairy operations. The majority of resistance-associated sequences found in all samples belonged to tetracycline resistance mechanisms. We found that the ranch samples contained significantly fewer resistance mechanisms than dairy and feedlot samples, and that the resistome of dairy operations differed significantly from that of feedlots. The resistome in soil, manure and wastewater differed, suggesting that management of these effluents should be tailored appropriately. By providing a baseline of the cattle production waste resistome, this study represents a solid foundation for future efforts to characterize and quantify the public health risk posed by livestock effluents.

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Foodborne illnesses associated with pathogenic bacteria are a global public health and economic challenge. The diversity of microorganisms (pathogenic and nonpathogenic) that exists within the food and meat industries complicates efforts to understand pathogen ecology. Further, little is known about the interaction of pathogens within the microbiome throughout the meat production chain. Here, a metagenomic approach and shotgun sequencing technology were used as tools to detect pathogenic bacteria in environmental samples collected from the same groups of cattle at different longitudinal processing steps of the beef production chain: cattle entry to feedlot, exit from feedlot, cattle transport trucks, abattoir holding pens, and the end of the fabrication system. The log read counts classified as pathogens per million reads for Salmonella enterica, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Clostridium spp. (C. botulinum and C. perfringens), and Campylobacter spp. (C. jejuni, C. coli, and C. fetus) decreased over subsequential processing steps. Furthermore, the normalized read counts for S. enterica, E. coli, and C. botulinum were greater in the final product than at the feedlots, indicating that the proportion of these bacteria increased (the effect on absolute numbers was unknown) within the remaining microbiome. From an ecological perspective, data indicated that shotgun metagenomics can be used to evaluate not only the microbiome but also shifts in pathogen populations during beef production. Nonetheless, there were several challenges in this analysis approach, one of the main ones being the identification of the specific pathogen from which the sequence reads originated, which makes this approach impractical for use in pathogen identification for regulatory and confirmation purposes.

This study compared the antimicrobial effects of epsilon-polylysine (epsilon-PL) against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in 6 food extracts and in broth. The food extracts (10% (w/w) in distilled water) evaluated were fat-free and whole fat milk, beef, bologna, rice, and vegetables (50:50 ratio of broccoli and cauliflower). epsilon-PL was tested at 0.005% and 0.02% (w/v) against E. coli O157:H7 and L. monocytogenes, and 0.02% and 0.04% (w/v) against S. Typhimurium. The substrates were inoculated (5 log CFU/mL) and periodically analyzed for surviving populations during storage at 12 degrees C for 6 d. In general, all 3 pathogens reached 7 to 9 log CFU/mL within 2 d in control substrates (no epsilon-PL). Immediate bactericidal effects (P < 0.05) following exposure to epsilon-PL were obtained in the rice (all pathogens) and vegetable (E. coli O157:H7 and S. Typhimurium) extracts. During storage, antimicrobial effects of epsilon-PL were more pronounced in the food extracts than in the broth medium. The greatest antimicrobial activity for all 3 pathogens was obtained in the rice and vegetable extracts, where counts were reduced (P < 0.05) to below the detection limit (0.0 log CFU/mL) by one or both epsilon-PL concentrations tested. In the other food extracts (fat-free milk, whole fat milk, beef, and bologna), both epsilon-PL concentrations tested generally resulted in lower (P < 0.05) pathogen levels at the end of storage compared to initial counts, with better bactericidal effects exerted by the higher of the 2 epsilon-PL concentrations. Additional research is needed to explore the potential antimicrobial effects of epsilon-PL in real food systems.