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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Illustration of the ProFiT-SPEci-FISH pipeline to explore plasmid dynamics and determine their bacterial host in environmental samples. The sample undergoes sequencing, and plasmids are assembled using SCAPP [4]. ProFiT is then employed to design multiple small, specific digoxigenin-labeled probes, tailored for the plasmids or plasmid genes of interest. These probes are ordered and subsequently hybridized to the target plasmid using the SPEci-FISH method. Probes are then detected by anti-digoxigenin antibodies, conjugated to horseradish peroxidase (HRP). This enzyme catalyzes multiple reactions with tyramide-labeled fluorophores, which serve as the substrate, leading to a localized and amplified fluorescent signal that can be visualized using a fluorescence microscope or analyzed using FACS. Dotted lines represent optional steps in the pipeline

Fig. 2

Microscopic visualization and FACS analysis of SPEci-FISH labeled plasmids. A (i) Microscopy images showing SPEci-FISH labeled E. coli TG1 cells containing no plasmid, high-copy number (HCN) and low-copy number (LCN) plasmids, as well as E. coli E2022 cells containing plasmids pE2022-1 to pE2022-4. Cells were stained with Alexa 647 (red) or 488 (green). (ii) Corresponding FACS analyses for these samples, depicting the proportions of positively labeled cells in each sample. B (i) Microscopy images of E. coli E2022 cells with plasmids pE2022-3 and pE2022-2 simultaneously labeled using SPEci-FISH, stained with Alexa 647 and 488, respectively. (ii) Corresponding FACS analyses showing the proportions of positively labeled cells containing both plasmids, pE2022-3 only, or pE2022-2 only

Fig. 3

E2022 plasmids visualized at the single-cell level using STORM. Images of four plasmids present in the isolate E2022, labeled using SPEci-FISH and acquired by Stochastic Optical Reconstruction Microscopy (STORM), see Methods section. Colors reflect the relative normalized density in each image (white, maximum density; black minimum density), see bar in top right image. Arrows indicate pE20222-2 plasmid accumulation at the cell poles. Scale bars are indicated

Fig. 4

Detecting plasmid mobility in gut environments. A The plasmidome of a healthy human fecal sample was assembled to identify a specific plasmid of interest. Probes were designed using ProFiT, and the plasmid was labeled in-situ with SPEci-FISH. Fluorescent cells were sorted using FACS and their 16S rRNA gene was amplified and sequenced to identify the bacterial hosts. B A boxplot showing the proportions of positively labeled cells harboring pAIZM1 in a fecal sample, analyzed by FACS. The top panel represents the proportions without SPEci-FISH labeling (control), while the bottom panel represents the proportions with SPEci-FISH labeling. C Potential bacterial hosts (strain level) of pAIZM1 were determined by sequencing the cells with and without the plasmid, and comparing the abundances. The bar plot shows abundances significantly higher within the fraction containing the plasmid versus the fraction without the plasmid. Colors represent the different phyla. D Simultaneous labeling of pAIZM1 with SPEci-FISH and the 16S rRNA gene with standard FISH for two of its potential hosts, B. fragilis (top) and R. bromii (bottom). pAIZM1 was labeled with Alexa Fluor 647 (right) and the 16S rRNA gene was labeled with Alexa Fluor 488 (left). FACS analyses in the boxplot on the right depict the proportions of positively labeled cells in each bacterial host. Pie charts summarize the proportion of each bacterial host with and without the plasmid

Fig. 5

SPEci-FISH protocol. The workflow of SPEci-FISH includes fixation, permeabilization, hybridization, and enzymatic signal amplification