Sanitation Districts of Los Angeles County, 1955 Workman Mill Road, Whittier, CA 90601, USA.
Sanitation Districts of Los Angeles County, 1955 Workman Mill Road, Whittier, CA 90601, USA.
J Microbiol Methods. 2020 Dec;179:106070. doi: 10.1016/j.mimet.2020.106070. Epub 2020 Oct 2.
Carbapenem resistance, particularly in Enterobacteriaceae, is an urgent threat to public health worldwide. Wastewater treatment plants are a critical control point for the spread of antimicrobial resistance into the environment yet, due in part to the lack of appropriate methods, the occurrence, identification and removal of carbapenem resistant bacteria has not been well characterized in wastewater matrices. This project was designed to provide a method for quantification of viable carbapenem resistant (CR) gram-negative bacteria (GNB) in raw sewage and treated wastewater effluents. A two-step procedure using membrane filtration and selective media supplemented with each of four carbapenems (doripenem, meropenem, imipenem, and ertapenem) was established for the quantification of CR GNB in wastewater matrices. Carbapenemase production was also assessed on individual bacterial colonies using two separate methods. Vitek®2 antimicrobial susceptibility test and disk diffusion assays were used to verify results from the supplemented media test and provide taxonomic identification. Treated and untreated wastewater samples from secondary and tertiary-stage wastewater treatment plants were analyzed for CR bacteria using the supplemented media procedure. Over 98% of all isolates selected from the carbapenem-supplemented media were verified as CR GNB. Carbapenemase production was observed in 80% of these isolates and 88% were multidrug resistant. All Enterobacteriaceae isolates from the supplemented media were verified as CR and 97% tested positive for carbapenemase production. The highest concentrations of CR GNB in wastewater were observed using the ertapenem-supplemented media. Doripenem-supplemented media showed the greatest specificity and selectivity for carbapenemase-producing CRE. Overall, the cumulative CR GNB in wastewater were reduced by approximately three- and five-log by the secondary and tertiary-stage WWTPs, respectively. This study establishes a method for characterization of viable CR GNB in wastewater matrices and demonstrates that current wastewater treatment technologies effectively reduce CR bacteria, including CRE, in sewage.
碳青霉烯类耐药性，特别是肠杆菌科的耐药性，是全球公共卫生的一个紧迫威胁。污水处理厂是控制抗生素耐药性向环境传播的关键控制点，但由于缺乏适当的方法，碳青霉烯类耐药菌在污水基质中的发生、鉴定和去除尚未得到很好的描述。本项目旨在提供一种定量检测污水和处理后废水中活的碳青霉烯类耐药（CR）革兰氏阴性菌（GNB）的方法。采用两步膜过滤法和选择性培养基，培养基中添加了四种碳青霉烯类药物（多尼培南、美罗培南、亚胺培南和厄他培南），建立了用于定量检测污水基质中 CR GNB 的方法。还使用两种不同的方法对单个细菌菌落的碳青霉烯酶产生情况进行了评估。Vitek®2 抗菌药物敏感性测试和纸片扩散法用于验证补充培养基测试的结果，并提供分类鉴定。使用补充培养基程序对二级和三级污水处理厂的处理和未处理废水样品进行了 CR 细菌分析。从碳青霉烯类药物补充培养基中选择的 98%以上的分离株被确认为 CR GNB。在这些分离株中，80%观察到碳青霉烯酶产生，88%为多药耐药。补充培养基中分离的所有肠杆菌科分离株均被确认为 CR 阳性，97%的分离株对碳青霉烯酶产生检测呈阳性。在污水中，使用厄他培南补充培养基观察到的 CR GNB 浓度最高。多尼培南补充培养基对产碳青霉烯酶 CRE 具有最大的特异性和选择性。总体而言，二级和三级污水处理厂分别使废水中的累积 CR GNB 减少了约三个和五个对数级。本研究建立了一种在污水基质中描述活的 CR GNB 的方法，并证明了当前的污水处理技术可有效减少污水中的 CR 细菌，包括 CRE。
