[System analysis of the ecological distribution of bacteriophages in hospital wastewater].

Phage therapy is one of the most important tools for the treatment of infections with multi-drug resistant bacteria. Such phages are usually isolated from hospital effluents, however, no systematic study on the distribution of phages in hospital effluents has been conducted so far. The aim of this study was to isolate the corresponding phages of common pathogenic bacteria isolated in the clinic as hosts, so as to assess the ecological distribution of phages in hospital wastewater and to provide a reference for the isolation and application of phages of drug-resistant bacteria in the clinic. A cross-sectional study design was used in this study. The 125 pathogenic bacteria (belonging to 16 different strains) isolated from the clinical microbiology laboratory of Qilu Hospital of Shandong University from May to June 2023 were selected as the target strains, and the phages corresponding to these strains were isolated and purified from the hospital wastewater by using the double-layer plate sandwich method. At the same time, the distribution of pathogenic bacteria in the same batch of wastewater was analyzed with the help of mNGS sequencing technology, so as to preliminarily investigate the abundance correspondence between pathogenic bacteria and phages in wastewater. The results showed that a total of 56 phage strains were isolated from 125 clinical pathogens as hosts, corresponding to six pathogens, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. All six pathogenic bacteria contained strains with different degrees of drug resistance, with a higher percentage of multi-drug resistant strains in A. baumannii, Escherichia coli and P. aeruginosa. The phage acquisition rates of these six pathogens were, in descending order, Escherichia coli (80%), Stenotrophomonas maltophilia (75%), Pseudomonas aeruginosa (70%), Klebsiella pneumoniae (66.67%), Acinetobacter baumannii (36.36%) and Staphylococcus aureus (12.5%). Preliminary mNGS sequencing results showed that the pathogenic bacteria with higher abundance in the batch of effluent were Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Klebsiella aerogenes, Klebsiella michiganensis and Pseudomonas aeruginosa. In conclusion, phages of most common clinical Gram-negative pathogens were isolated from hospital wastewater with high isolation rates; however, phages of Gram-positive pathogens were isolated at lower rates, and only phages corresponding to Staphylococcus aureus were isolated in this study. The corresponding mNGS sequencing results showed that the distribution of Gram-negative pathogens in sewage may had a positive correlation with the ecological distribution of phages.噬菌体疗法是治疗多重耐药菌感染的重要手段之一。这类噬菌体通常从医院污水中分离得到，然而迄今为止对医院污水中噬菌体的分布情况尚无系统研究。本研究旨在以临床分离的常见病原菌为宿主，分离其对应的噬菌体，以此评估医院污水中噬菌体的生态分布情况，并为临床耐药菌噬菌体的分离和应用提供参考。本研究采用横断面研究设计，选择2023年5—6月山东大学齐鲁医院临床微生物实验室分离得到的125株病原菌（分属于16个不同的菌种）作为目标菌株，利用双层平板夹心法从医院污水中分离纯化这些菌株对应的噬菌体。同时，借助宏基因组二代测序技术（mNGS）分析同批次污水中病原菌的分布情况，初步探究污水中病原菌与噬菌体之间的丰度对应关系。结果显示，以125株临床病原菌为宿主，共分离获得56株噬菌体，分别对应鲍曼不动杆菌、肺炎克雷伯菌、大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌、嗜麦芽窄食单胞菌等6种病原菌。这6种病原菌均包含了不同耐药程度的菌株，其中鲍曼不动杆菌、大肠埃希菌和铜绿假单胞菌中多重耐药菌株占比较高。这6种病原菌的噬菌体获得率由高到低依次为：大肠埃希菌（80%）、嗜麦芽窄食单胞菌（75%）、铜绿假单胞菌（70%）、肺炎克雷伯菌（66.67%）、鲍曼不动杆菌（36.36%）和金黄色葡萄球菌（12.5%）。初步的mNGS测序结果显示，在该批次污水中，丰度较高的病原菌分别为屎肠球菌、肺炎克雷伯菌、大肠埃希菌、粪肠球菌、鲍曼不动杆菌、产气克雷伯菌、密歇根克雷伯菌和铜绿假单胞菌。综上，医院污水中可分离到大多数临床常见革兰阴性病原菌的噬菌体，且分离率较高；然而革兰阳性病原菌的噬菌体分离率较低，在本研究中仅分离到与金黄色葡萄球菌对应的噬菌体。对应的mNGS测序结果表明革兰阴性病原菌在污水中的分布与噬菌体的生态分布可能具有一定的正相关关系。.