Abstract The exchange of mobile genetic elements (MGEs) facilitates the spread of functional traits including antimicrobial resistance within bacterial communities. Tools to spatially map MGEs and identify their bacterial hosts in complex microbial communities are currently lacking, limiting our understanding of this process. Here we combined single-molecule DNA fluorescence in situ hybridization (FISH) with multiplexed ribosomal RNA-FISH to enable simultaneous visualization of both MGEs and bacterial taxa. We spatially mapped bacteriophage and antimicrobial resistance (AMR) plasmids and identified their host taxa in human oral biofilms. This revealed distinct clusters of AMR plasmids and prophage, coinciding with densely packed regions of host bacteria. Our data suggest spatial heterogeneity in bacterial taxa results in heterogeneous MGE distribution within the community, with MGE clusters resulting from horizontal gene transfer hotspots or expansion of MGE-carrying strains. Our approach can help advance the study of AMR and phage ecology in biofilms.

Spatial mapping of mobile genetic elements and their bacterial hosts in complex microbiomes