High-resolution bacterial 16S rRNA gene profile meta-analysis and biofilm status reveal common colorectal cancer consortia

Meta-analysis of biofilm prevalence in CRC. a Microbial biofilms in Carnoy’s-fixed tissue were detected by FISH with the universal 16S rRNA gene probe Eub338 (stained in red) and the nucleic acid stain DAPI (blue). White brackets denote the mucus layer, visible via autofluorescence of the tissue. Left panel: a biofilm-positive paired normal tissue from the right colon of a CRC patient with abundant bacteria adjacent to the epithelium (arrow). Right panel: a biofilm-negative paired normal tissue from the left colon of a CRC patient with minimal bacteria (arrow) present only in the outer edge of the mucus. Scale bars represent 100？μm. b The locations of tumors from USA, MAL1, and MAL2 are overlaid on a diagram of the colon. c Percentage of biofilm-positive right and left tumors for the three cohorts. All statistics shown are Fisher’s exact tests. d Total percentage of biofilm-positive right- and left-sided tumors from all three cohorts is depicted with a Fisher’s exact test p-value. e Left-sided and right-sided tumors separated according to tumor stage and biofilm status. Multivariate logistic regression analysis was performed after controlling for tumor side. f, g Biofilm-positive samples were stained with DAPI (blue) and probes against four bacterial membership groups: Fusobacterium (yellow), Bacteroidetes (green), Lachnospiraceae (red), and Proteobacteria (magenta). Scale bars in large images represent 100？μm; scale bars in smaller, inset images represent 5？μm. f Representative polymicrobial biofilm from a tumor (left panel) and its paired normal tissue (right panel), with blooms of Fusobacterium in yellow visible only in the tumor. g Representative Proteobacteria-dominant tumor (left panel) and its paired normal tissue (right panel