Analyzing The Structure Of Oral Biofilms At Micron Level

Biofilms are scientifically and medically important for oral and medical diseases. We established in vitro multispecies biofilms generated from human saliva using SHI medium, and created Combinatorial Labeling and Spectral Imaging-Fluorescent In Situ Hybridization (CLASI-FISH) system which enables detailed analysis of biofilm at micron level. The aim of this study is to use CLASI-FISH to examine oral biofilm structures at micron scale. Salivary microbial biofilms were set up in vitro using SHI medium with different culture conditions, including the presence of arginine, sucrose. Genus-and species-specific probes were developed and tested for simultaneous detection of bacterial strains at multiple spatial scales. Segmentation was carried out in FIJI and quantification carried out using linear dipole analysis with DAIME. Proximity analysis showed strong self-association for all taxa and associations between some taxon pairs. The detection frequency of a particular species is generally consistent with the abundance of this species detected by the temporal sequencing, however, for each view spot, only very limited number of species are dominant at the micron level regardless its abundance in temporal sequencing. Unlike the general concept that bacteria within biofilm are relatively uniformly distributed, our study shows that biofilm is far more complexed: it is a highly diversified ecosystem with many different spatial structures created by local “founding” microbial communities that are less than 100 microns apart. This new discovery may lead to new molecular understanding on the different treatment outcomes of various anti-biofilm reagents.