ProFiT-SPEci-FISH: a novel approach for linking plasmids to hosts in complex microbial communities at the single-cell...

Plasmids are influential drivers of bacterial evolution, facilitating horizontal gene transfer and shaping microbial communities. Current knowledge on plasmid persistence and mobilization in natural environments is derived from community-level studies, neglecting the single-cell level, where these dynamic processes unfold. Pinpointing specific plasmids within their natural environments is essential to unravel the dynamics between plasmids and their bacterial hosts.

Here, we overcame the technical hurdle of natural plasmid detectability in single cells by developing SPEci-FISH (Short Probe EffiCIent Fluorescence In Situ Hybridization), a novel molecular method designed to detect and visualize plasmids, regardless of their copy number, directly within bacterial cells, enabling their precise identification at the single-cell level. To complement this method, we created ProFiT (PRObe FInding Tool), a program facilitating the design of sequence-based probes for targeting individual plasmids or plasmid families.

We have successfully applied these methods, combined with high-resolution microscopy, to investigate the dispersal and localization of natural plasmids within a clinical isolate, revealing various plasmid spatial patterns within the same bacterial population. Importantly, bridging the technological gap in linking plasmids to hosts in native complex microbial environments, we demonstrated that our method, when combined with fluorescence-activated cell sorting (FACS), can track plasmid-host dynamics in a human fecal sample. This approach identified multiple potential bacterial hosts for a conjugative plasmid that we assembled from this fecal sample's metagenome. Our integrated approach offers a significant advancement toward understanding plasmid ecology in complex microbiomes. Video Abstract.