Single-cell imaging links antibiotic resistance gene copy number and bacterial host identity

Identifying the in situ microbial hosts of antibiotic resistance genes (ARGs) in complex environments remains a major challenge, as many of these genes are associated with mobile genetic elements. Here, we introduce combined-FISH, which integrates direct-geneFISH for ARG detection with catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) for rRNA labelling, thereby enabling the simultaneous visualization and copy number quantification of ARGs in taxonomically identified single cells within microbial samples. Using the sulfonamide resistance gene sul1 as a model target, we designed a polynucleotide probe set covering all non-redundant database sequences of this gene within 5% sequence mismatch. The method was validated using model organisms harboring plasmids with sul1, Escherichia coli K12 J53 with R388 and Acinetobacter defluvii with pOXA58_010030. Combined-FISH enabled quantitative determination of sul1 copy numbers at single-cell resolution, with E. coli harboring 1-8 copies and A. defluvii 1-4 copies per cell. Following plasmid-mediated transfer of sul1 from E. coli to Pseudomonas alloputida, recipient cells typically contained 2-6 sul1 copies, with up to 14 copies detected in individual cells, demonstrating the applicability of the approach for investigating horizontal gene transfer. The method was successfully applied to river biofilm samples containing sul1-harboring bacteria, enabling simultaneous tracking of the bacterial host and its associated ARG within a complex microbial community. Collectively, these results establish combined-FISH as a powerful tool for quantitatively linking ARGs and microbial hosts in situ, and for investigating plasmid copy number and gene transfer at single-cell resolution in technical and environmental systems.