Advanced BONCAT-FACs to study the active antibiotic resistome in the environments

Herein, bio-orthogonal non-canonical amino acid tagging (BONCAT), in combination with fluorescent-activated cell sorting (FACs), were developed to distinguish active antibiotic resistant bacteria (ARB) in wastewater treatment plant (WWTP) in a high-throughput and culture-independent way. Active ARB against six types of antibiotics in WWTP were identified, and their abundance were successfully revealed. The further application of FACs followed by 16S rRNA and metagenomic sequencing revealed the active antibiotic resistant communities and genetic basis underlying the phenotypic resistance. Particularly, some active pathogenic ARB including Aeromonas hydrophila and Klebsiella pneumoniae were detected. Moreover, co-existence of the antibiotic resistance genes (ARGs), virulence factor genes (VFGs), and mobile genetic elements (MGEs) were found in the sorted active ARB, suggesting their high risks in spreading AMR. This study unravels both the phenotype and genotype of active ARB in complex environments, and advances our understanding of their health risks.