摘要
      环境抗生素耐药性微生物的人类健康风险评估不仅需要量化环境基质中抗生素耐药性基因的丰度，还需要了解其宿主和遗传背景。此外，区分细胞内和细胞外DNA（iDNA和eDNA）组分中的ARG可能有助于完善我们对ARG可转移性的理解。本研究的目的是了解沿土地利用梯度的细胞外、细胞内和总ARG的（O1）丰度和多样性，以及（O2）生物信息学管道对不同DNA组分中观察到的ARG的假定宿主分配的影响。沿着美国新泽西州拉瑞坦河的土地利用梯度收集沉积物样本。提取DNA以分离eDNA和iDNA，并对选定的ARGs和16S rRNA基因进行qPCR。对不同DNA组分的DNA提取物进行霰弹枪宏基因组测序。ARG主机通过两种不同的生物信息学管道分配：原始读取的网络分析与组装。比较了这两条管道的结果，以评估它们在链接的数量和多样性以及计算机矩阵尖峰主机分配的准确性方面的性能。在DNA组分之间未观察到16S rRNA基因标准化的sul1浓度的差异。与eDNA相比，iDNA和总DNA的总体微生物群落结构更相似，并且通常按采样点聚类。下游位点的iDNA中与可移动遗传元件相关的ARGs增加。关于主机分配，原始读取管道通过网络分析确定了247个ARG主机，而组装管道确定了53个主机。对管道之间进行了其他比较，包括将ARG分配给含有水传播病原体的分类群，以及对处理时间的实际考虑。
Abstract
Human health risk assessment for environmental antibiotic resistant microbes requires not only quantifying the abundance of antibiotic resistance genes (ARGs) in environmental matrices, but also understanding their hosts and genetic context. Further, differentiating ARGs in intracellular and extracellular DNA (iDNA and eDNA) fractions may help refine our understanding of ARG transferability. The objectives of this study were to understand the (O1) abundance and diversity of extracellular, intracellular, and total ARGs along a land use gradient and (O2) impact of bioinformatics pipeline on the assignment of putative hosts for the ARGs observed in the different DNA fractions. Sediment samples were collected along a land use gradient in the Raritan River, New Jersey, USA. DNA was extracted to separate eDNA and iDNA and qPCR was performed for select ARGs and the 16S rRNA gene. Shotgun metagenomic sequencing was performed on DNA extracts for the different DNA fractions. ARG hosts were assigned via two different bioinformatic pipelines: network analysis of raw reads versus assembly. Results of the two pipelines were compared to evaluate their performance in terms of number and diversity of linkages and accuracy of in silico matrix spike host assignments. No differences were observed in the 16S rRNA gene normalized sul1 concentrations between the DNA fractions. The overall microbial community structure was more similar for iDNA and total DNA compared to eDNA and generally clustered by sampling site. ARGs associated with mobile genetic elements increased in iDNA for the downstream sites. Regarding host assignment, the raw reads pipeline via network analysis identified 247 ARG hosts as compared to 53 hosts identified by assembly pipeline. Other comparisons between the pipelines were made including ARG assignment to taxa containing waterborne pathogens and practical considerations regarding processing time.

https://www.sciencedirect.com/science/article/abs/pii/S0043135422003256