Antimicrobial resistance (AMR) in soils represents a serious risk to human health through the food chain and human–nature contact. However, the active antibiotic-resistant bacteria (ARB) residing in soils that primarily drive AMR dissemination are poorly explored. Here, single-cell Raman-D₂O coupled with targeted metagenomics is developed as a culture-independent approach to phenotypically and genotypically profiling active ARB against clinical antibiotics in a wide range of soils. This method quantifies the prevalence (contamination degree) and activity (spread potential) of soil ARB and reveals a clear elevation with increasing anthropogenic activities such as farming and the creation of pollution, thereby constituting a factor that is critical for the assessment of AMR risks. Further targeted sorting and metagenomic sequencing of the most active soil ARB uncover several uncultured genera and a pathogenic strain. Furthermore, the underlying resistance genes, virulence factor genes, and associated mobile genetic elements (including plasmids, insertion sequences, and prophages) are fully deciphered at the single-cell level. This study advances our understanding of the soil active AMR repertoire by linking the resistant phenome to the genome. It will aid in the risk assessment of environmental AMR and guide the combat under the One Health framework.

Single-cell capturing and sequencing of active ARB in soils. (A) Workflow for capturing highly active ARB from soils via targeted single-cell sorting. (B) Active ARB with normalized C-D ratios > 1.6 (top 2%, red dots) were selected for sorting (Left). Representative single-cell Raman spectra of highly active bacteria (red line) and less active or dormant bacteria (green line) (Right). (C) Relative abundance of dominant phyla in bacterial communities of bulk soil (Left), the captured highly active ARB from farmland soils (Middle) and MFS (Right).