Activity-guided discovery of antibiotic-transforming bacteria from environmental microbiomes using D-amino acid-assisted fluorescence-activated cell sorting.

Efficient discovery of functionally active microorganisms from complex environmental microbiomes remains a major challenge in bioremediation research, as conventional enrichment and cultivation approaches are often low-throughput and poorly suited for capturing rare but metabolically active taxa. In this study, we developed an activity-guided screening strategy that integrates fluorescent D-amino acid metabolic labeling with fluorescence-activated cell sorting (HADA-FACS) to enable targeted isolation of metabolically active bacteria. Using ciprofloxacin (CIP) as a representative selective pressure, HADA-FACS enriched active microbial subpopulations from lake sediment, leading to the isolation of a previously overlooked bacterium, Pandoraea sp. AYGW-1. The isolate exhibited measurable CIP transformation activity. UPLC-MS revealed multiple CIP-derived intermediates, providing supportive evidence for structural transformation involving piperazine-ring oxidation and quinolone-core defluorination. Integrated genomic and transcriptomic analyses further revealed coordinated responses associated with transport, stress adaptation, and redox-related functions during CIP exposure. The isolate showed no hemolytic activity and no atypical antibiotic resistance features. Overall, this work demonstrates that HADA-FACS provides an effective activity-guided framework for accessing metabolically active microorganisms from complex environments and linking single-cell activity to functionally relevant transformation processes.