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.

We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Twenty-two white-tailed deer were divided into four groups. One group (n=5) received 10(9) colony-forming units (cfu) BCG via a lipid-formulated oral bait; one group (n=5) received 10(9) cfu BCG in culture directly to the oropharynx, one group (n=6) was vaccinated with 10(6) cfu BCG subcutaneously, and one group served as a control and received culture media directly to the oropharynx (n=6). All animals were challenged 3 mo after vaccination. Five months postchallenge the animals were examined for lesions. Results indicate that both oral forms of BCG and parenterally administered BCG offered significant protection against M. bovis challenge as compared to controls. This study suggests that oral BCG vaccination may be a feasible means of controlling bovine tuberculosis in wild white-tailed deer populations.

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.

The objective was to examine effects of treating commercial beef feedlot cattle with therapeutic doses of tulathromycin, a macrolide antimicrobial drug, on changes in the fecal resistome and microbiome using shotgun metagenomic sequencing. Two pens of cattle were used, with all cattle in one pen receiving metaphylaxis treatment (800 mg subcutaneous tulathromycin) at arrival to the feedlot, and all cattle in the other pen remaining unexposed to parenteral antibiotics throughout the study period. Fecal samples were collected from 15 selected cattle in each group just prior to treatment (Day 1), and again 11 days later (Day 11). Shotgun sequencing was performed on isolated metagenomic DNA, and reads were aligned to a resistance and a taxonomic database to identify alignments to antimicrobial resistance (AMR) gene accessions and microbiome content. Overall, we identified AMR genes accessions encompassing 9 classes of AMR drugs and encoding 24 unique AMR mechanisms. Statistical analysis was used to identify differences in the resistome and microbiome between the untreated and treated groups at both timepoints, as well as over time. Based on composition and ordination analyses, the resistome and microbiome were not significantly different between the two groups on Day 1 or on Day 11. However, both the resistome and microbiome changed significantly between these two sampling dates. These results indicate that the transition into the feedlot—and associated changes in diet, geography, conspecific exposure, and environment—may exert a greater influence over the fecal resistome and microbiome of feedlot cattle than common metaphylactic antimicrobial drug treatment.