The microbiome of common bedding materials before and after use on commercial dairy farms.

BACKGROUND: Bovine mastitis is one of the most economically important diseases affecting dairy cows. The choice of bedding material has been identified as an important risk factor contributing to the development of mastitis. However, few reports examine both the culturable and nonculturable microbial composition of commonly used bedding materials, i.e., the microbiome. Given the prevalence of nonculturable microbes in most environments, this information could be an important step to understanding whether and how the bedding microbiome acts as a risk factor for mastitis. Therefore, our objective was to characterize the microbiome composition and diversity of bedding material microbiomes, before and after use. METHODS: We collected 88 bedding samples from 44 dairy farms in the U.S. Unused (from storage pile) and used (out of stalls) bedding materials were collected from four bedding types: new sand (NSA), recycled manure solids (RMS), organic non-manure (ON) and recycled sand (RSA). Samples were analyzed using 16S rRNA sequencing of the V3-V4 region. RESULTS: The overall composition as well as the counts of several microbial taxa differed between bedding types, with Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes dominating across all types. Used bedding contained a significantly different microbial composition than unused bedding, but the magnitude of this difference varied by bedding type, with RMS bedding exhibiting the smallest difference. In addition, positive correlations were observed between 16S rRNA sequence counts of potential mastitis pathogens (bacterial genera) and corresponding bedding bacterial culture data. CONCLUSION: Our results strengthen the role of bedding as a potential source of mastitis pathogens. The consistent shift in the microbiome of all bedding types that occurred during use by dairy cows deserves further investigation to understand whether this shift promotes pathogen colonization and/or persistence, or whether it can differentially impact udder health outcomes. Future studies of bedding and udder health may be strengthened by including a microbiome component to the study design.

Mobilization of Antibiotic Resistance: Are Current Approaches for Colocalizing Resistomes and Mobilomes Useful?

Antimicrobial resistance (AMR) poses a global human and animal health threat, and predicting AMR persistence and transmission remains an intractable challenge. Shotgun metagenomic sequencing can help overcome this by enabling characterization of AMR genes within all bacterial taxa, most of which are uncultivatable in laboratory settings. Shotgun sequencing, therefore, provides a more comprehensive glance at AMR "potential" within samples, i.e., the "resistome." However, the risk inherent within a given resistome is predicated on the genomic context of various AMR genes, including their presence within mobile genetic elements (MGEs). Therefore, resistome risk stratification can be advanced if AMR profiles are considered in light of the flanking mobilizable genomic milieu (e.g., plasmids, integrative conjugative elements (ICEs), phages, and other MGEs). Because such mediators of horizontal gene transfer (HGT) are involved in uptake by pathogens, investigators are increasingly interested in characterizing that resistome fraction in genomic proximity to HGT mediators, i.e., the "mobilome"; we term this "colocalization." We explored the utility of common colocalization approaches using alignment- and assembly-based techniques, on clinical (human) and agricultural (cattle) fecal metagenomes, obtained from antimicrobial use trials. Ordination revealed that tulathromycin-treated cattle experienced a shift in ICE and plasmid composition versus untreated animals, though the resistome was unaffected during the monitoring period. Contrarily, the human resistome and mobilome composition both shifted shortly after antimicrobial administration, though this rebounded to pre-treatment status. Bayesian networks revealed statistical AMR-MGE co-occurrence in 19 and 2% of edges from the cattle and human networks, respectively, suggesting a putatively greater mobility potential of AMR in cattle feces. Conversely, using Mobility Index (MI) and overlap analysis, abundance of de novo-assembled contigs supporting resistomes flanked by MGE increased shortly post-exposure within human metagenomes, though > 40 days after peak dose such contigs were rare (∼2%). MI was not substantially altered by antimicrobial exposure across all cattle metagenomes, ranging 0.5-4.0%. We highlight that current alignment- and assembly-based methods estimating resistome mobility yield contradictory and incomplete results, likely constrained by approach-specific data inputs, and bioinformatic limitations. We discuss recent laboratory and computational advancements that may enhance resistome risk analysis in clinical, regulatory, and commercial applications.

MEGARes 2.0: a database for classification of antimicrobial drug, biocide and metal resistance determinants in metagenomic sequence data.

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.

Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period.

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.

Assessment of a carbon fibre MRI flatbed insert for radiotherapy treatment planning.

OBJECTIVE: The purpose of this work was to assess heating and radiofrequency (RF) deposition and image quality effects of a prototype three-section carbon fibre flatbed insert for use in MRI. METHODS: RF deposition was assessed using two different thermometry techniques, infrared thermometry and Bragg-grating thermometry. Image quality effects were assessed with and without the flatbed insert in place by using mineral oil phantoms and a human subject. RESULTS: Neither technique detected heating of the insert in typical MRI examinations. We found that the insert was less suitable for MRI applications owing to severe RF shielding artefact. For spin-echo (SE), turbo spin-echo (TSE) and gradient-echo sequences, the reduction in signal-to-noise ratio (SNR) was as much as 89% when the insert was in place compared with the standard couch, making it less suitable as a patient-support material. Turning on the MultiTransmit switch together with using the scanner's quadrature body coil improved the reduction in SNR from 89% to 39% for the SE sequence and from 82% to 12% for the TSE sequence. CONCLUSION: No evidence was found to support reports in the literature that carbon fibre is an unsuitable material for use in MRI because of heating. ADVANCES IN KNOWLEDGE: This study suggests that carbon fibre is less suitable for large-scale MRI applications owing to it causing severe RF shading. Further research is needed to establish the suitability of the flatbed for treatment planning using alternative sequences or whether an alternative carbon fibre composite for large-scale MRI applications or a design that can minimize shielding can be found.

A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-beta-lactam.

We report the first example of a monoclonal antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with k(cat)=8.7 x 10(-6)s(-1) and K(M)=35 microM. This is the first example of a rat monoclonal catalytic antibody.