MEGARes and AMR++, v3.0: an updated comprehensive database of antimicrobial resistance determinants and an improved software pipeline for classification using high-throughput sequencing.

Antimicrobial resistance (AMR) is considered a critical threat to public health, and genomic/metagenomic investigations featuring high-throughput analysis of sequence data are increasingly common and important. We previously introduced MEGARes, a comprehensive AMR database with an acyclic hierarchical annotation structure that facilitates high-throughput computational analysis, as well as AMR++, a customized bioinformatic pipeline specifically designed to use MEGARes in high-throughput analysis for characterizing AMR genes (ARGs) in metagenomic sequence data. Here, we present MEGARes v3.0, a comprehensive database of published ARG sequences for antimicrobial drugs, biocides, and metals, and AMR++ v3.0, an update to our customized bioinformatic pipeline for high-throughput analysis of metagenomic data (available at MEGLab.org). Database annotations have been expanded to include information regarding specific genomic locations for single-nucleotide polymorphisms (SNPs) and insertions and/or deletions (indels) when required by specific ARGs for resistance expression, and the updated AMR++ pipeline uses this information to check for presence of resistance-conferring genetic variants in metagenomic sequenced reads. This new information encompasses 337 ARGs, whose resistance-conferring variants could not previously be confirmed in such a manner. In MEGARes 3.0, the nodes of the acyclic hierarchical ontology include 4 antimicrobial compound types, 59 resistance classes, 233 mechanisms and 1448 gene groups that classify the 8733 accessions.

Using a multi-omics approach to explore potential associations with rumen content and serum of cows with different milk production levels based on genomic predicted transmitting ability for milk and phenotypic milk production.

This study aims to compare rumen microbiome and metabolites between second lactation dairy cows in the 75th percentile (n = 12; 57.2 ± 5.08 kg/d) of production according to genomic predicted transmitting ability for milk (GPTAM) and their counterparts in the 25th percentile (n = 12; 47.2 ± 8.61 kg/d). It was hypothesized that the metagenome and metabolome would differ between production levels. Cows were matched by days in milk (DIM), sire, occurrence of disease, and days open in previous lactation. For an additional comparison, the cows were also divided by phenotype into high (n = 6; 61.3 ± 2.8 kg/d), medium (n = 10; 55 ± 1.2 kg/d), and low (n = 8; 41.9 ± 5.6 kg/d) based on their milk production. Samples were collected 65 ± 14 DIM. Rumen content was collected using an oro-gastric tube and serum samples were collected from the coccygeal vessels. High-resolution liquid chromatography-mass spectrometry (LC-MS) was used for rumen and serum metabolite profiling. Shotgun metagenomics was used for rumen microbiome profiling. Microbiome sample richness and diversity were used to determine alpha and Bray-Curtis dissimilarity index was used to estimate beta diversity. Differences in metabolites were determined using t-tests or ANOVA. Pearson correlations were used to consider associations between serum metabolites and milk production. There was no evidence of a difference in rumen metabolites or microbial communities by GPTAM or phenotype. Cows in the phenotypic low group had greater serum acetate to propionate ratio and acetate proportion compared to the cows in the phenotypic medium group. Likewise, serum propionate proportion was greater in the medium compared to the low phenotypic group. Serum acetate, butyrate, and propionate concentrations had a weak positive correlation with milk production. When investigating associations between rumen environment and milk production, future studies must consider the impact of the ruminal epithelium absorption and post-absorption processes in relation to milk production.

Effect of castration timing and weaning strategy on the taxonomic and functional profile of ruminal bacteria and archaea of beef calves.

BACKGROUND: Beef cattle experience several management challenges across their lifecycle. Castration and weaning, two major interventions in the early life of beef cattle, can have a substantial impact on animal performance. Despite the key role of the rumen microbiome on productive traits of beef cattle, the effect of castration timing and weaning strategy on this microbial community has not been formally described. We assessed the effect of four castration time windows (at birth, turnout, pre-weaning and weaning) and two weaning strategies (fence-line and truck transportation) on the rumen microbiome in a randomized controlled study with 32 male calves across 3 collection days (i.e., time points). Ruminal fluid samples were submitted to shotgun metagenomic sequencing and changes in the taxonomic (microbiota) and functional profile (metagenome) of the rumen microbiome were described. RESULTS: Using a comprehensive yet stringent taxonomic classification approach, we identified 10,238 unique taxa classified under 40 bacterial and 7 archaeal phyla across all samples. Castration timing had a limited long-term impact on the rumen microbiota and was not associated with changes in alpha and beta diversity. The interaction of collection day and weaning strategy was associated with changes in the rumen microbiota, which experienced a significant decrease in alpha diversity and shifts in beta diversity within 48 h post-weaning, especially in calves abruptly weaned by truck transportation. Calves weaned using a fence-line weaning strategy had lower relative abundance of Bacteroides, Lachnospira, Fibrobacter and Ruminococcus genera compared to calves weaned by truck transportation. Some genes involved in the hydrogenotrophic methanogenesis pathway (fwdB and fwdF) had higher relative abundance in fence-line-weaned calves post-weaning. The antimicrobial resistance gene tetW consistently represented more than 50% of the resistome across time, weaning and castration groups, without significant changes in relative abundance. CONCLUSIONS: Within the context of this study, castration timing had limited long-term effects on the rumen microbiota, while weaning strategy had short-term effects on the rumen microbiota and methane-associated metagenome, but not on the rumen resistome.