Brumfield Kyle D, Cotruvo Joseph A, Shanks Orin C, Sivaganesan Mano, Hey Jessica, Hasan Nur A, Huq Anwar, Colwell Rita R, Leddy Menu B
Maryland Pathogen Research Institute, University of Maryland, College Park, MD, United States.
University of Maryland Institute for Advanced Computer Studies, University of Maryland, College Park, MD, United States.
Front Water. 2021 Feb;3:626849. doi: 10.3389/frwa.2021.626849. Epub 2021 Feb 15.
Microbial contamination of recreation waters is a major concern globally, with pollutants originating from many sources, including human and other animal wastes often introduced during storm events. Fecal contamination is traditionally monitored by employing culture methods targeting fecal indicator bacteria (FIB), namely . and enterococci, which provides only limited information of a few microbial taxa and no information on their sources. Host-associated qPCR and metagenomic DNA sequencing are complementary methods for FIB monitoring that can provide enhanced understanding of microbial communities and sources of fecal pollution. Whole metagenome sequencing (WMS), quantitative real-time PCR (qPCR), and culture-based FIB tests were performed in an urban watershed before and after a rainfall event to determine the feasibility and application of employing a multi-assay approach for examining microbial content of ambient source waters. Cultivated . and enterococci enumeration confirmed presence of fecal contamination in all samples exceeding local single sample recreational water quality thresholds (. , 410 MPN/100 mL; enterococci, 107 MPN/100 mL) following a rainfall. Test results obtained with qPCR showed concentrations of . , enterococci, and human-associated genetic markers increased after rainfall by 1.52-, 1.26-, and 1.11-fold log copies per 100 mL, respectively. Taxonomic analysis of the surface water microbiome and detection of antibiotic resistance genes, general FIB, and human-associated microorganisms were also employed. Results showed that fecal contamination from multiple sources (human, avian, dog, and ruminant), as well as FIB, enteric microorganisms, and antibiotic resistance genes increased demonstrably after a storm event. In summary, the addition of qPCR and WMS to traditional surrogate techniques may provide enhanced characterization and improved understanding of microbial pollution sources in ambient waters.
娱乐用水的微生物污染是全球主要关注的问题，污染物来源众多，包括在暴雨事件期间经常引入的人类和其他动物粪便。传统上，通过采用针对粪便指示菌（FIB）的培养方法来监测粪便污染，即大肠杆菌和肠球菌，这只能提供少数微生物类群的有限信息，且无法提供其来源信息。宿主相关的定量聚合酶链反应（qPCR）和宏基因组DNA测序是FIB监测的补充方法，可增强对微生物群落和粪便污染来源的理解。在一次降雨事件前后，对一个城市流域进行了全宏基因组测序（WMS）、定量实时PCR（qPCR）和基于培养的FIB测试，以确定采用多分析方法检测环境源水微生物含量的可行性和应用。培养的大肠杆菌和肠球菌计数证实，降雨后所有样本中均存在粪便污染，超过了当地单个样本娱乐用水水质阈值（大肠杆菌，410个MPN/100 mL；肠球菌，107个MPN/100 mL）。qPCR测试结果显示，降雨后大肠杆菌、肠球菌和人类相关基因标记物的浓度分别增加了1.52倍、1.26倍和1.11倍（每100 mL对数拷贝数）。还对地表水微生物组进行了分类分析，并检测了抗生素抗性基因、一般FIB和人类相关微生物。结果表明，暴雨事件后，多种来源（人类、鸟类、狗和反刍动物）的粪便污染以及FIB、肠道微生物和抗生素抗性基因明显增加。总之，在传统替代技术中加入qPCR和WMS可能会增强对环境水体中微生物污染源的表征和理解。
