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

Abstract

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.

Keywords: antimicrobial resistance; antimicrobial resistance genes; bovine respiratory disease; feedlot cattle; long-read metagenomic sequencing.

Figure 1

Mean qPCR copy numbers (three repeats per n samples—median value and interquartile range) of bacterial Bovine Respiratory Disease pathogens for DNA samples extracted from frozen swabs from 0 h to 16 h of O₂ incubation in BHI broth with 1% glucose (n = 20 samples) for total 16S rRNA gene copies, M. haemolytica (Mh), P. multocida (Pm), and H. somni (Hs) (Nadkarni et al., 2002; Kishimoto et al., 2017). (A) All samples and (B) the results restricted to samples that were culture-positive for M. haemolytica (19 samples), P. multocida (7 samples), and H. somni (4 samples).

Figure 2

Log₁₀-transformed total DNA base pairs and total number of DNA read counts for each enrichment treatment (none, 10 h, and 14 h, n = 16 for each) of frozen swabs to detect bacterial Bovine Respiratory Disease pathogens by metagenomic sequencing. Box and whisker plots: boxes include the median and upper and lower quartiles; whiskers include the minimum and maximum values. *p < 0.01 on Wilcoxon signed-rank test of the total base pairs for enrichment treatment compared with no enrichment.