摘要
      霰弹枪宏基因组研究揭示了环境基质中抗生素耐药性基因（ARGs）的多样性、相对丰度和宿主。有动机将这种方法与基于生存能力的技术相结合，以更好地定义ARG危害。本研究的目的是评估细胞外DNA（eDNA）和假定的“不可行”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。沿着土地利用梯度收集成对的水和生物膜样本。为了研究推定的“活细胞”DNA，用单叠氮丙啶（即PMA-DNA）处理样品。为了研究eDNA，分离了细胞内和细胞外DNA。qPCR显示，过滤样品与离心浓缩样品在水中的16S rRNA总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图谱方面的显著差异。在生物膜和水中鉴定出含有致病性ARG宿主的推定活类群。与总DNA提取物相比，去除PMA结合的DNA改善了N50和组装图谱。本研究证明了不同样品制备方法对告知水中和生物膜中河流ARGs相关的潜在危害的影响。
Abstract
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.

https://www.sciencedirect.com/science/article/abs/pii/S0304389423000250