Hospital wastewater contains abundant antibiotics, antibiotic resistance genes (ARGs), and pathogens. Last-resort antibiotic resistance genes (LARGs) include the New Delhi metallo-β-lactamase gene bla_NDM, mobile colistin resistance gene mcr and tigecycline resistance gene tet(X) which confers resistance to carbapenems, colistin and tigecycline. The presence and significance of LARGs in hospital wastewater treatment systems (HWTS) have not yet been systematically explored. Here, LARG variants were shown to be prevalent both influents and effluents of HWTS. A total of 989 Enterobacteriaceae isolates that confer resistance to last-resort antibiotics were collected from effluents and multiple genetic contexts of LARGs were analyzed. LARGs-carrying plasmids were confirmed to show high multidrug phenotypes and transferability. We also discovered the co-occurrence of plasmids harboring bla_NDM-1 and mcr-1 in single Escherichia coli, as well as E. coli HM016 containing two unique mcr-1-carrying plasmids. This result might accelerate co-dissemination of LARGs under environmental selection pressure. Different core genetic arrangements in these strains suggest several evolutionary pathways in HWTS. The resistance functions of LARGs were confirmed in vitro and in vivo by mass spectrometry. This study provides novel insights into the diversity, genetic context and function of critical ARGs in HWTS. The results raise the concern that LARGs may further spread into the environment, thus, more stringent discharge standards and regulations for hospital wastewater are urgently needed.

医院废水含有丰富的抗生素、抗生素抗性基因（ARG）和病原体。最后手段抗生素抗性基因 (LARG) 包括新德里金属-β-内酰胺酶基因bla _NDM 、移动粘菌素抗性基因mcr和替加环素抗性基因tet (X)，后者赋予碳青霉烯类、粘菌素和替加环素抗性。 LARG 在医院废水处理系统 (HWTS) 中的存在和重要性尚未得到系统探索。在这里，LARG 变体在 HWTS 的进水和出水中都普遍存在。从废水中收集了总共 989 株对最后手段抗生素具有抗性的肠杆菌科菌株，并分析了 LARG 的多种遗传背景。携带 LARGs 的质粒被证实表现出高多药表型和可转移性。我们还在单个大肠杆菌中同时出现了携带bla _NDM-1和mcr-1的质粒，以及包含两个独特的mcr-1携带质粒的大肠杆菌HM016。这一结果可能会加速 LARG 在环境选择压力下的共同传播。这些菌株中不同的核心遗传排列表明了 HWTS 中的几种进化途径。通过质谱法在体外和体内证实了 LARG 的抵抗功能。这项研究为 HWTS 中关键 ARG 的多样性、遗传背景和功能提供了新的见解。研究结果引起人们对LARGs可能进一步扩散到环境中的担忧，因此迫切需要制定更严格的医院废水排放标准和法规。