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-log₁₀ 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.

碳青霉烯耐药性，尤其是肠杆菌科是对全球公共卫生的迫切威胁。废水处理厂是将抗菌素耐药性传播到环境中的关键控制点，然而，部分由于缺乏适当的方法，对废水中的碳青霉烯类耐药菌的发生，鉴定和清除尚无很好的特征。该项目旨在提供一种定量分析原污水和处理过的废水中的耐碳青霉烯（CR）革兰氏阴性细菌（GNB）的方法。建立了使用膜过滤和选择性介质的两步程序，并分别添加了四个碳青霉烯（多柔比南，美洛培南，亚胺培南和厄他培南），用于定量测定废水基质中的CR GNB。还使用两种单独的方法在单个细菌菌落上评估了碳青霉烯酶的生产。使用Vitek®2抗菌药敏试验和纸片扩散试验来验证补充培养基试验的结果并提供分类学鉴定。使用补充介质程序分析了二级和三级废水处理厂的已处理和未处理废水样品中的CR细菌。从补充有碳青霉烯的培养基中选出的所有分离株中，有98％以上被确认为CR GNB。在80％的分离物中观察到碳青霉烯酶的产生，而88％的细菌具有多重耐药性。所有 使用补充介质程序分析了二级和三级废水处理厂的已处理和未处理废水样品中的CR细菌。从补充有碳青霉烯的培养基中选出的所有分离株中，有98％以上被确认为CR GNB。在80％的分离物中观察到碳青霉烯酶的产生，而88％的细菌具有多重耐药性。所有 使用补充培养基程序分析了二级和三级废水处理厂的已处理和未处理废水样品中的CR细菌。从补充有碳青霉烯的培养基中选出的所有分离株中，有98％以上被确认为CR GNB。在80％的分离物中观察到碳青霉烯酶的产生，而88％的细菌具有多重耐药性。所有从补充培养基中分离出的肠杆菌科细菌被确认为CR，且97％的碳青霉烯酶生产呈阳性。使用补充了厄他培南的培养基观察到了废水中CR GNB的最高浓度。多瑞培南补充的培养基对产生碳青霉烯酶的CRE表现出最大的特异性和选择性。总体而言，第二阶段和第三阶段的污水处理厂分别将废水中的累积CR GNB分别降低了约三和五对数₁₀。这项研究建立了一种表征废水基质中可行的CR GNB的方法，并证明了当前的废水处理技术可以有效地减少废水中的CR细菌，包括CRE。