摘要
      肠杆菌中超广谱β-内酰胺酶（ESBL）的出现，尤其是碳青霉烯酶的出现，导致治疗选择有限。因此，充分了解所有潜在的传播途径至关重要，尤其是在医院废水等高风险来源。本研究旨在量化四种肠道机会性病原体（EOPs），即总大肠杆菌、ESBL和碳青霉烯类耐药大肠杆菌及其相应的耐药基因（两种ESBL和五种碳青霉烯酶基因），并对萨格勒布两家大型医院两个季节的医院废水中的肠道细菌分离株进行鉴定。培养显示，总大肠菌群和耐碳青霉烯类大肠菌群的平均水平相似（3.4×104 CFU/mL），推定ESBL大肠菌群水平低10倍（3×103 CFU/mL）。实时PCR显示，在所检测的所有抗性基因中，EOP中的大肠杆菌水平最高（105个细胞当量/mL），blaKPC基因水平最高（高达10−1个基因拷贝/16S个拷贝）。从医院废水中分离出的69株产ESBL和90株产碳青霉烯酶的肠杆菌（CPE）中，所有菌株都具有多重耐药性，大多数被鉴定为大肠杆菌、柠檬酸杆菌、肠杆菌和克雷伯菌。在ESBL分离株中，blaCTX-M-15是最常见的ESBL基因，而在CPE分离株中中，blaKPC-2和blaNDM-1是最常检测到的CP基因，其次是blaOXA-48。使用PFGE、MLST和全基因组测序（WGS）的分子流行病学显示，临床相关变体，如大肠杆菌ST131（blaCTX-M-15/blaTEM-116）和ST541（blaKPC-2）、肺炎克雷伯菌ST101（blaOXA-48/blaNDM-1）和阴沟肠杆菌复合物ST277（blaKPC-2/blaNDM-1）是最常检测到的克隆类型。WGS还揭示了这些和其他分离株中各种各样的抗性基因和质粒，以及blaCTX-M、blaOXA-48和blaKPC-2基因侧翼区域的转座子和插入序列，表明了动员的潜力。我们得出的结论是，医院废水是临床上重要病原体和耐药基因的潜在二级库，因此在排入城市下水道系统之前需要进行有效的预处理。
Abstract
The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

https://www.sciencedirect.com/science/article/abs/pii/S0048969723004205