Herman Emily K, Lacoste Stacey R, Freeman Claire N, Otto Simon J G, McCarthy E Luke, Links Matthew G, Stothard Paul, Waldner Cheryl L
Department of Agricultural, Food, and Nutritional Science, Faculty of Agricultural, Life, and Environmental Sciences, University of Alberta, Edmonton, AB, Canada.
Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
Front Microbiol. 2024 May 8;15:1386319. doi: 10.3389/fmicb.2024.1386319. eCollection 2024.
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.
This pilot study employed a non-selective bacterial enrichment step before nucleic acid extraction to increase the relative proportion of bacterial DNA for sequencing.
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 reads but was also substantial for sulfonamide ARGs within and reads and tetracycline ARGs in reads.
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.
牛呼吸道疾病（BRD）是肉牛产业中最重要的动物健康问题之一。虽然细菌培养和抗菌药物敏感性测试已用于诊断检测，但每个物种仅检测一个分离株的常规做法并不能完全反映样本中的细菌群体。相比之下，最近一项对饲养场牛鼻拭子进行宏基因组测序的研究在细菌病原体鉴定和抗菌耐药基因（ARG）检测方面很有前景。然而，鼻拭子样本中宿主生物量的高比例阻碍了宏基因组测序的灵敏度。
这项初步研究在核酸提取前采用了非选择性细菌富集步骤，以增加用于测序的细菌DNA的相对比例。
非选择性细菌富集增加了细菌相对于宿主序列数据的比例，与未富集样本相比，使得BRD病原体的检测率提高。该过程还能以物种水平分辨率增强对ARGs的检测，包括检测未针对培养和药敏试验的目标细菌物种的ARGs。长读长测序方法无需组装即可在单个细菌读数上检测ARGs。非选择性细菌富集后的宏基因组学在与呼吸道细菌培养的六项比较中有四项达成了实质性一致，并且与定量聚合酶链反应有实质性或更好的相关性。分离株药敏结果与ARGs检测之间的比较在读取的大环内酯类ARGs方面最佳，但在读取的和读取的磺胺类ARGs以及读取的四环素类ARGs方面也很显著。
通过非选择性富集增加细菌DNA相对于宿主DNA的比例，我们证明了使用长读长宏基因组测序在饲养场牛深鼻咽拭子中鉴定与BRD相关的病原体和ARGs的测序数据比例相应增加。这种方法有望作为BRD病原体和ARGs的检测策略，并在处理时间、投入成本和目标数据生成之间取得平衡。这种方法可以作为一个有价值的工具，为BRD的抗菌管理提供信息并支持抗菌药物管理。
