Effect of Antimicrobial Use in Conventional Versus Natural Cattle Feedlots on the Microbiome and Resistome.

Antimicrobial use (AMU) in the livestock industry has been associated with increased levels of antimicrobial resistance. Recently, there has been an increase in the number of "natural" feedlots in the beef cattle sector that raise cattle without antibiotics. Shotgun metagenomics was employed to characterize the impact of AMU in feedlot cattle on the microbiome, resistome, and mobilome. Sequenced fecal samples identified a decline (q < 0.01) in the genera Methanobrevibacter and Treponema in the microbiome of naturally vs. conventionally raised feedlot cattle, but this difference was not (q > 0.05) observed in catch basin samples. No differences (q > 0.05) were found in the class-level resistome between feedlot practices. In fecal samples, decreases from conventional to natural (q < 0.05) were noted in reads for the antimicrobial-resistant genes (ARGs) mefA, tet40, tetO, tetQ, and tetW. Plasmid-associated ARGs were more common in feces from conventional than natural feedlot cattle. Interestingly, more chromosomal- than plasmid-associated macrolide resistance genes were observed in both natural and conventional feedlots, suggesting that they were more stably conserved than the predominately plasmid-associated tetracycline resistance genes. This study suggests that generationally selected resistomes through decades of AMU persist even after AMU ceases in natural production systems.

Evaluation of metagenomic assembly methods for the detection and characterization of antimicrobial resistance determinants and associated mobilizable elements.

Antimicrobial resistance genes (ARGs) can be transferred between members of a bacterial population by mobile genetic elements (MGE). Understanding the risk of these transfer events is important in monitoring and predicting antimicrobial resistance (AMR), especially in the context of a One Health Continuum. However, there is no universally accepted method for detection of ARGs and MGEs, and especially for determining their linkages. This study used publicly available shotgun metagenomic DNA short-read (Illumina, 100 bp paired-end) sequence data from samples across the One Health Continuum (including beef cattle composite feces from feedlots, catch basin water at feedlots, agricultural soil from feedlot manured surrounding fields, and urban/municipal sewage influent from two municipal wastewater treatment plants) to develop a workflow to identify and associate ARGs and MGEs. ARG- and MGE-based targeted-assemblies with available short-read data were unable to meet this analysis goal. In contrast, de novo assembly of contigs provided enough sequence context to associate ARGs and MGEs, without compromising discovery rate. However, to estimate the relative abundance of these elements, unassembled sequence data must still be used.

Prevalence and Risk Factors Associated With Antimicrobial Resistance in Bacteria Related to Bovine Respiratory Disease-A Broad Cross-Sectional Study of Beef Cattle at Entry Into Canadian Feedlots.

A broad, cross-sectional study of beef cattle at entry into Canadian feedlots investigated the prevalence and epidemiology of antimicrobial resistance (AMR) in Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, bacterial members of the bovine respiratory disease (BRD) complex. Upon feedlot arrival and before antimicrobials were administered at the feedlot, deep nasopharyngeal swabs were collected from 2,824 feedlot cattle in southern and central Alberta, Canada. Data on the date of feedlot arrival, cattle type (beef, dairy), sex (heifer, bull, steer), weight (kg), age class (calf, yearling), source (ranch direct, auction barn, backgrounding operations), risk of developing BRD (high, low), and weather conditions at arrival (temperature, precipitation, and estimated wind speed) were obtained. Mannheimia haemolytica, P. multocida, and H. somni isolates with multidrug-resistant (MDR) profiles associated with the presence of integrative and conjugative elements were isolated more often from dairy-type than from beef-type cattle. Our results showed that beef-type cattle from backgrounding operations presented higher odds of AMR bacteria as compared to auction-derived calves. Oxytetracycline resistance was the most frequently observed resistance across all Pasteurellaceae species and cattle types. Mycoplasma bovis exhibited high macrolide minimum inhibitory concentrations in both cattle types. Whether these MDR isolates establish and persist within the feedlot environment, requires further evaluation.

A direct qPCR screening approach to improve the efficiency of Mycoplasma bovis isolation in the frame of a broad surveillance study.

Culturing Mycoplasma bovis is laborious and unpredictable with most laboratories relying on molecular methods for its detection and identification. However, bacterial culture is still necessary to relate phenotypic characteristics to genotypic traits within and between individual strains. Thus, the main objective of this study was to develop a procedure that saved time and consumables during the culturing of M. bovis within the scope of a broad antimicrobial resistance surveillance project. Deep nasopharyngeal swabs (DNPS) collected from feedlot cattle upon arrival at 10 Southern Alberta feedlots were enriched in broth and an aliquot of the culture was directly used in a M. bovis-specific quantitative PCR (qPCR) assay. Only qPCR-positive cultures were plated onto agar media for the isolation of M. bovis. The detection of M. bovis from broth culture by direct-culture-qPCR proved to be more sensitive (1.61 × 102 CFU/mL) than using a commercial kit (1.61 × 103 CFU/mL) to extract DNA from pure cultures of M. bovis. When isolation of M. bovis from broth-enriched DNPS (n = 208 samples) was used as the gold standard for diagnostics, the qPCR screening approach showed 100% sensitivity, 87.27% specificity, and a kappa index = 0.87 (strong agreement). In contrast, qPCR of DNPS samples (n = 58) exhibited 100% sensitivity, 42.86% specificity, and a kappa index = 0.49 (weak agreement). The qPCR protocol described here together with a high throughput direct-culture-qPCR approach for sample testing made it possible to reduce the labor and cost of M. bovis isolation by eliminating the need to process 97.3% of M. bovis-negative samples. This was possible through the use of qPCR Ct values as a predictive tool of the likelihood of M. bovis isolation. This new procedure could be evaluated for its use in antimicrobial resistance surveillance programs that focus on Mycoplasma species.