Shining Light on Oral Biofilm Fluorescence In Situ Hybridization (FISH): Probing the Accuracy of In Situ Biogeography Studies.

The oral biofilm has been instrumental in advancing microbial research and enhancing our understanding of oral health and disease. Recent developments in next-generation sequencing have provided detailed insights into the microbial composition of the oral microbiome, enabling species-level analyses of biofilm interactions. Fluorescence in situ hybridization (FISH) has been especially valuable for studying the spatial organization of these microbes, revealing intricate arrangements such as "corncob" structures that highlight close bacterial interactions. As more genetic sequence data become available, the specificity and accuracy of existing FISH probes used in biogeographical studies require reevaluation. This study examines the performance of commonly used species-specific FISH probes, designed to differentiate oral microbes within in situ oral biofilms, when applied in vitro to an expanded set of bacterial strains. Our findings reveal that the specificity of several FISH probes is compromised, with cross-species hybridization being more common than previously assumed. Notably, we demonstrate that biogeographical associations within in situ oral biofilms, particularly involving Streptococcus and Corynebacterium, may need to be reassessed to align with the latest metagenomic data.