Influence of DNA from non-viable sources on the riverine water and biofilm microbiome, resistome, mobilome, and resistance gene host assignments
Influence of DNA from non-viable sources on the riverine water and biofilm microbiome, resistome, mobilome, and resistance gene host assignments
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DOI：
10.1016/j.jhazmat.2023.130743
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发表时间：
2023-01-11
影响因子：
13.6
通讯作者：
Fahrenfeld, N. L.
中科院分区：
文献类型：
--
作者：
Deshpande, A. S.;Fahrenfeld, N. L.
霰弹枪宏基因组学研究揭示了环境基质中抗生素耐药基因（ARG）的多样性，相对丰度和宿主。有动机联合收割机结合这种方法与可行性为基础的技术，以更好地定义ARG危险。本研究的目的是评价细胞外DNA（eDNA）和推定的“非活”细胞DNA分离的不同方法的性能，以了解对ARG-宿主分配的影响。成对的水和生物膜样品收集沿着土地利用梯度。为了研究推定的“活细胞”DNA，将样品用单叠氮丙啶（即，PMA-DNA）。为了研究eDNA，分离细胞内和细胞外DNA。qPCR显示过滤器与离心浓缩样品在水中的总16 S rRNA基因拷贝数存在差异，但除此之外，DNA组分之间的基因拷贝浓度没有差异。接下来，对PMA-DNA和总DNA提取物进行宏基因组测序，揭示了两者在细菌群落结构和ARG谱方面的显著差异。在生物膜和水中鉴定了含有致病性ARG宿主的假定可行分类群。与总DNA提取物相比，去除PMA结合的DNA改善了N50和组装作图。这项研究表明，不同的样品制备方法的影响，告知与河流ARGs在水和生物膜的潜在危害。
Shotgun metagenomic studies have revealed the diversity, relative abundance, and hosts of antibiotic resistance genes (ARGs) across environmental matrices. There is motivation to combine this method with viability-based techniques to better define ARG hazard. The objectives of this study were to evaluate the performance of different methods for extracellular DNA (eDNA) and putative "non-viable" cell DNA separation to understand the influence on ARG-host assignments. Paired water and biofilm samples were collected along a land use gradient. To study putative "viable-cell" DNA, samples were treated with propidium monoazide (i.e., PMA-DNA). To study eDNA, intracellular and extracellular DNA were separated. qPCR revealed differences in total 16S rRNA gene copies in water for filter vs. centrifuge-concentrated samples, but otherwise there were no differences in gene copy concentrations between DNA fractions. Next, metagenomic sequencing was performed on PMA-DNA and total DNA extracts revealing significant differences between the two for bacterial community structure and ARG profiles. Putative viable taxa containing pathogenic ARG hosts were identified in biofilm and water. Removing PMA-bound DNA improved N50 and assembly mapping compared to total DNA extracts. This study demonstrates the impact of different sample preparation methods on informing the hazard potential associated with riverine ARGs in water and biofilm.