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

The high bacterial community complexity greatly hinders the monitoring of clinically prevalent extended-spectrum beta-lactam resistant bacteria, which are usually present as rare but key populations involving 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 methodology to profile beta-lactam resistome in a municipal wastewater treatment plant (WWTP) and its receiving river. The results showed that clinically prevalent carbapenemase genes (e.g., NDM and KPC family) and extended-spectrum beta-lactamase genes (e.g., CTX-M, TEM and OXA family) considerably existed in the WWTP and showed prominent potential in horizontal dissemination. Strikingly, carbapenem and polymyxin resistance genes co-occurred in highly virulent Enterobacter kobei (MCR-1 and NDM-6) and Citrobacter freundii (ArnA and NDM-16) genomes. Overall, this study exemplifies phenotypic metagenomics for high-throughput surveillance of targeted fraction of clinically-important resistome and substantially expands current knowledge on extended-spectrum beta-lactam resistance in WWTPs.