Applicability of live-fluorescence in situ hybridization (Live-FISH) on soil microbiomes

Soil microbiomes play pivotal roles in ecosystem functioning and have been a trove of commercially important enzymes and drug leads. In recent years, metagenomic scrutiny has exposed the immense taxonomic and functional diversity of these microbiomes, leading to the realization that most microorganisms remain out of reach by conventional cultivation approaches. Live-Fluorescence in situ Hybridization (Live-FISH) coupled with fluorescence activated cell sorting represents a potential strategy to overcome cultivation barriers by enabling the taxa-specific extraction of viable cells for targeted cultivation efforts. However, it remains unknown to what extent this approach is applicable on soil microbiomes. Here, we evaluated the impact of Live-FISH on the viability of microorganisms extracted from temperate topsoil. Using propidium monoazide (PMA) viability qPCR, we observed a one-order of magnitude reduction in the number of viable cells as a result of the treatment. Through PMA viability sequencing, we observed an overall reduction in viable taxa as function of treatment, yet, 501 ASVs retained their viability and could serve as targets for future cultivation efforts. The effects proved to be taxon-specific, and we observed that the viability of Bacillota and Planctomycetota was retained to a larger extent than that of other dominant phyla like Acidobacteriota, which were reduced by five orders of magnitude throughout the steps of the procedure. Furthermore, planctomycetes were amenable to labelling and distinguishable in subsequent microscopy and flow cytometry analyses, demonstrating the utility of this technique to selectively label and extract viable species of this elusive phylum.