High-Throughput\nSingle-Cell Metabolic Labeling, Sorting,\nand Sequencing of Active Antibiotic-Resistant Bacteria in the Environment

Active antibiotic-resistant bacteria\n(ARB) play a major role in\nspreading antimicrobial resistance (AMR) in the environment; however,\nthey have remained largely unexplored. Herein, we coupled bio-orthogonal\nnoncanonical amino acid tagging with high-throughput fluorescence-activated\nsingle-cell sorting (FACS) and sequencing to characterize the phenome\nand genome of active ARB in complex environmental matrices. Active\nARB, conferring resistance to six antibiotics throughout wastewater\ntreatment, were distinguished and quantified. The percentage and concentration\nof active ARB ranged from 0.28% to 45.3% and from 1.1 × 10<sup>4</sup> to 2.09 × 10<sup>7</sup> cells/mL, respectively. Notably,\nthe final effluents retained up to 4.79 × 10<sup>4</sup> cells/mL\nof active ARB. Targeted FACS and genomic sequencing revealed a distinct\ntaxonomic composition of active ARB compared with that of the overall\npopulation. The coexistence of antibiotic resistome and mobilome in\nactive ARB was also identified, including three high-quality metagenomic\nassembly genomes assigned to pathogenic bacteria, highlighting the\nsubstantial health risks due to their activity, phenotypic resistance,\nmobility, and pathogenicity. This study advances our understanding\nof previously overlooked active ARB in the environment by linking\ntheir resistance phenotype to their genotype. This high-throughput\nmethod will enable efficient quantitative surveillance of active AMR,\nproviding valuable insights into risk control and management.