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.

In this study, we determined the prevalence of bovine respiratory disease (BRD)-associated viral and bacterial pathogens in cattle and characterized the genetic profiles, antimicrobial susceptibilities, and nature of antimicrobial resistance determinants in collected bacteria. Nasopharyngeal swab and lung tissue samples from 68 BRD mortalities in Alberta, Canada (n= 42), Texas (n= 6), and Nebraska (n= 20) were screened using PCR for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus, bovine herpesvirus 1, parainfluenza type 3 virus,Mycoplasma bovis,Mannheimia haemolytica,Pasteurella multocida, andHistophilus somni. Excepting bovine herpesvirus 1, all agents were detected.M. haemolytica(91%) and BVDV (69%) were the most prevalent, with cooccurrence in 63% of the cattle. Isolates ofM. haemolytica(n= 55),P. multocida(n= 8), andH. somni(n= 10) from lungs were also collected. AmongM. haemolyticaisolates, a clonal subpopulation (n= 8) was obtained from a Nebraskan feedlot. All three bacterial pathogens exhibited a high rate of antimicrobial resistance, with 45% exhibiting resistance to three or more antimicrobials.M. haemolytica(n= 18),P. multocida(n= 3), andH. somni(n= 3) from Texas and Nebraska possessed integrative conjugative elements (ICE) that conferred resistance for up to seven different antimicrobial classes. ICE were shown to be transferred via conjugation fromP. multocidatoEscherichia coliand fromM. haemolyticaandH. somnitoP. multocida. ICE-mediated multidrug-resistant profiles of bacterial BRD pathogens could be a major detriment to many of the therapeutic antimicrobial strategies currently used to control BRD.

Ensiling of forages was recognized as a microbial-driven process as early as the late 1800s, when it was associated with the production of "sweet" or "sour" silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage.

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti . In total, specific inducers were identified for 76 transport systems, amounting to ≈47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti . Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.

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.

In proteobacteria, genes whose expression is modulated in response to the external concentration of inorganic phosphate are often regulated by the PhoB protein which binds to a conserved motif (Pho box) within their promoter regions. Using a position weight matrix algorithm derived from known Pho box sequences, we identified 96 putative Pho regulon members whose promoter regions contained one or more Pho boxs in the Sinorhizobium meliloti genome. Expression of these genes was examined through assays of reporter gene fusions and through comparison with published microarray data. Of 96 genes, 31 were induced and 3 were repressed by Pi starvation in a PhoB dependent manner. Novel Pho regulon members included several genes of unknown function. Comparative analysis across 12 proteobacterial genomes revealed highly conserved Pho regulon members including genes involved in Pi metabolism (pstS, phnC and ppdK). Genes with no obvious association with Pi metabolism were predicted to be Pho regulon members in S.meliloti and multiple organisms. These included smc01605 and smc04317 which are annotated as substrate binding proteins of iron transporters and katA encoding catalase. This data suggests that the Pho regulon overlaps and interacts with several other control circuits, such as the oxidative stress response and iron homeostasis.