Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle

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Introduction Bovine respiratory disease (BRD) is one of the most important animal health problems in the beef industry. While bacterial culture and antimicrobial susceptibility testing have been used for diagnostic testing, the common practice of examining one isolate per species does not fully reflect the bacterial population in the sample. In contrast, a recent study with metagenomic sequencing of nasal swabs from feedlot cattle is promising in terms of bacterial pathogen identification and detection of antimicrobial resistance genes (ARGs). However, the sensitivity of metagenomic sequencing was impeded by the high proportion of host biomass in the nasal swab samples. Methods This pilot study employed a non-selective bacterial enrichment step before nucleic acid extraction to increase the relative proportion of bacterial DNA for sequencing. Results Non-selective bacterial enrichment increased the proportion of bacteria relative to host sequence data, allowing increased detection of BRD pathogens compared with unenriched samples. This process also allowed for enhanced detection of ARGs with species-level resolution, including detection of ARGs for bacterial species of interest that were not targeted for culture and susceptibility testing. The long-read sequencing approach enabled ARG detection on individual bacterial reads without the need for assembly. Metagenomics following non-selective bacterial enrichment resulted in substantial agreement for four of six comparisons with culture for respiratory bacteria and substantial or better correlation with qPCR. Comparison between isolate susceptibility results and detection of ARGs was best for macrolide ARGs in Mannheimia haemolytica reads but was also substantial for sulfonamide ARGs within M. haemolytica and Pasteurella multocida reads and tetracycline ARGs in Histophilus somni reads. Discussion By increasing the proportion of bacterial DNA relative to host DNA through non-selective enrichment, we demonstrated a corresponding increase in the proportion of sequencing data identifying BRD-associated pathogens and ARGs in deep nasopharyngeal swabs from feedlot cattle using long-read metagenomic sequencing. This method shows promise as a detection strategy for BRD pathogens and ARGs and strikes a balance between processing time, input costs, and generation of on-target data. This approach could serve as a valuable tool to inform antimicrobial management for BRD and support antimicrobial stewardship.

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