Antimicrobial resistance has become an imminent concern for public health. As methods for detection and characterization of antimicrobial resistance move from targeted culture and polymerase chain reaction to high throughput metagenomics, appropriate resources for the analysis of large-scale data are required. Currently, antimicrobial resistance databases are tailored to smaller-scale, functional profiling of genes using highly descriptive annotations. Such characteristics do not facilitate the analysis of large-scale, ecological sequence datasets such as those produced with the use of metagenomics for surveillance. In order to overcome these limitations, we present MEGARes (https://megares.meglab.org), a hand-curated antimicrobial resistance database and annotation structure that provides a foundation for the development of high throughput acyclical classifiers and hierarchical statistical analysis of big data. MEGARes can be browsed as a stand-alone resource through the website or can be easily integrated into sequence analysis pipelines through download. Also via the website, we provide documentation for AmrPlusPlus, a user-friendly Galaxy pipeline for the analysis of high throughput sequencing data that is pre-packaged for use with the MEGARes database.

Antimicrobial resistance (AMR) is a threat to global public health and the identification of genetic determinants of AMR is a critical component to epidemiological investigations. High-throughput sequencing (HTS) provides opportunities for investigation of AMR across all microbial genomes in a sample (i.e. the metagenome). Previously, we presented MEGARes, a hand-curated AMR database and annotation structure developed to facilitate the analysis of AMR within metagenomic samples (i.e. the resistome). Along with MEGARes, we released AmrPlusPlus, a bioinformatics pipeline that interfaces with MEGARes to identify and quantify AMR gene accessions contained within a metagenomic sequence dataset. Here, we present MEGARes 2.0 (https://megares.meglab.org), which incorporates previously published resistance sequences for antimicrobial drugs, while also expanding to include published sequences for metal and biocide resistance determinants. In MEGARes 2.0, the nodes of the acyclic hierarchical ontology include four antimicrobial compound types, 57 classes, 220 mechanisms of resistance, and 1,345 gene groups that classify the 7,868 accessions. In addition, we present an updated version of AmrPlusPlus (AMR ++ version 2.0), which improves accuracy of classifications, as well as expanding scalability and usability.

Developments in high-throughput next generation sequencing (NGS) technology have rapidly advanced the understanding of overall microbial ecology as well as occurrence and diversity of specific genes within diverse environments. In the present study, we compared the ability of varying sequencing depths to generate meaningful information about the taxonomic structure and prevalence of antimicrobial resistance genes (ARGs) in the bovine fecal microbial community. Metagenomic sequencing was conducted on eight composite fecal samples originating from four beef cattle feedlots. Metagenomic DNA was sequenced to various depths, D1, D0.5 and D0.25, with average sample read counts of 117, 59 and 26 million, respectively. A comparative analysis of the relative abundance of reads aligning to different phyla and antimicrobial classes indicated that the relative proportions of read assignments remained fairly constant regardless of depth. However, the number of reads being assigned to ARGs as well as to microbial taxa increased significantly with increasing depth. We found a depth of D0.5 was suitable to describe the microbiome and resistome of cattle fecal samples. This study helps define a balance between cost and required sequencing depth to acquire meaningful results.

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

Forty USDA Select and 40 upper two-thirds USDA Choice beef carcasses were used to determine the effects of postmortem aging on tenderness of 17 individual beef muscles. Biceps femoris-long head, complexus, gluteus medius, infraspinatus, longissimus dorsi, psoas major, rectus femoris, semimembranosus, semitendinosus, serratus ventralis, spinalis dorsi, supraspinatus, tensor fasciae latae, teres major, triceps brachii-long head, vastus lateralis, and vastus medialis muscles were removed from each carcass. Seven steaks (2.54-cm thick) were cut from every muscle, and each steak was assigned to one of the following postmortem aging periods: 2, 4, 6, 10, 14, 21, or 28 d postmortem. After completion of the designated aging period, steaks were removed from storage (2 degrees C, never frozen), cooked to a peak internal temperature of 71 degrees C, and evaluated using Warner-Bratzler shear force (WBSF). Analysis of WBSF revealed a 3-way interaction (P = 0.004) among individual muscle, USDA quality grade, and postmortem aging period. With the exception of the Select teres major, WBSF of all muscles (both quality grades) decreased with increasing time of postmortem storage. Nonlinear regression was used to characterize the extent (aging response) and rate of decrease in WBSF from 2 through 28 d postmortem for each muscle within each quality grade. In general, WBSF of upper two-thirds Choice muscles decreased more rapidly from 2 to 10 d postmortem than did corresponding Select muscles. Muscles that had greater aging responses generally had greater 2-d WBSF values. The upper two-thirds Choice psoas major, serratus ventralis, and vastus lateralis muscles required similar aging times to complete a majority of the aging response (< or =0.1 kg of aging response remaining) compared with analogous Select muscles. The upper two-thirds Choice complexus, gluteus medius, semitendinosus, triceps brachii-long head, and vastus medialis muscles required 4 to 6 d less time to complete a majority of the aging response than did comparable Select muscles. Aging times for Select biceps femoris-long head, infraspinatus, longissimus dorsi, rectus femoris, semimembranosus, spinalis dorsi, supraspinatus, and tensor fasciae latae muscles were > or =7 d longer than those for corresponding upper two-thirds Choice muscles. Results from this study suggest that muscle-to-muscle tenderness differences depend on quality grade and aging time and that postmortem aging should be managed with respect to individual muscle and USDA quality grade.