Using Culture-Enriched Phenotypic Metagenomics for Targeted High-Throughput Monitoring of the Clinically Important Fraction of the β-Lactam Resistome

High bacterial community diversity and complexity greatly challenge the cost-efficient monitoring of clinically prevalent antibiotic-resistant bacteria, which are usually present as rare and important populations involved in the environmental dissemination of clinical resistance. Here, we introduce culture-enriched phenotypic metagenomics that integrates culture enrichment, phenotypic screening, and metagenomic analyses as an emerging standardized methodology for targeted resistome monitoring and apply it to decipher the extended-spectrum β-lactam resistome in a municipal wastewater treatment plant (WWTP) and its receiving river. The results showed that clinically prevalent carbapenemase genes (e.g., the NDM and KPC families) and extended-spectrum β-lactamase genes (e.g., the CTX-M, TEM, and OXA families) were prevalent in the WWTP and showed prominent potential in horizontal dissemination. Strikingly, carbapenem and polymyxin resistance genes co-occurred in the highly virulent nosocomial pathogens Enterobacter kobei and Citrobacter freundii. Overall, this study exemplifies phenotypic metagenomics for high-throughput surveillance of a targeted clinically important fraction of antibiotic resistomes and substantially expands current knowledge on extended-spectrum β-lactam resistance in WWTPs.

细菌群落的高度多样性和复杂性极大地挑战了对临床常见的耐药细菌进行具有成本效益的监测，这些细菌通常作为罕见但重要的种群存在，参与了临床耐药性在环境中的传播。在此，我们引入了富集培养的表型宏基因组学方法，它将富集培养、表型筛选和宏基因组分析相结合，作为一种新兴的标准化方法用于有针对性的耐药组监测，并将其应用于解读城市污水处理厂（WWTP）及其受纳河流中的超广谱β -内酰胺耐药组。结果表明，临床常见的碳青霉烯酶基因（例如NDM和KPC家族）以及超广谱β -内酰胺酶基因（例如CTX - M、TEM和OXA家族）在污水处理厂中普遍存在，并在水平传播方面显示出显著的潜力。令人惊讶的是，碳青霉烯和多粘菌素耐药基因共同出现在高致病性的医院病原体克氏肠杆菌和弗氏柠檬酸杆菌中。总体而言，这项研究举例说明了表型宏基因组学可用于对具有临床重要性的抗生素耐药组的特定部分进行高通量监测，并极大地扩展了当前关于污水处理厂中超广谱β -内酰胺耐药性的知识。