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.

移动遗传元件（MGE）的交换促进了功能性状的传播，包括细菌群落内抗菌素耐药性。目前缺乏在复杂微生物群落中绘制 MGE 空间图并识别其细菌宿主的工具，限制了我们对这一过程的理解。在这里，我们将单分子 DNA 荧光原位杂交 (FISH) 与多重核糖体 RNA-FISH 相结合，以实现 MGE 和细菌分类群的同时可视化。我们对噬菌体和抗菌素耐药性（AMR）质粒进行了空间定位，并在人类口腔生物膜中鉴定了它们的宿主分类群。这揭示了不同的 AMR 质粒和原噬菌体簇，与宿主细菌密集的区域一致。我们的数据表明，细菌类群的空间异质性导致群落内 MGE 分布的异质性，MGE 簇是由水平基因转移热点或 MGE 携带菌株的扩张产生的。我们的方法可以帮助推进生物膜中 AMR 和噬菌体生态学的研究。