Histological techniques for the visualisation and identification of polymicrobial communities in mucosal tissue

Sequencing studies have generated masses of data linking mucosal bacteria to health and disease, particularly in organs such as the colon, mouth and stomach. However, extracting microbial DNA from mucosal sites with lower bacterial biomass can introduce significant variation given the heterogeneity of bacterial colonisation within pathological microenvironments. Therefore, a histological technique that identifies mucosal samples with high microbial mass samples prior to applying sequencing technologies, would increase reproducibility and reduce issues of low signal: noise ratios commonly observed during 16S rRNA gene sequencing. Using RNAscope technology, we have previously shown that non- H. pylori bacteria invade the gastric lamina propria in H. pylori- infected patients diagnosed with chronic gastritis and gastric intestinal metaplasia. However, RNAscope technology is costly and time consuming. Here, using gastric tissue from patients with Helicobacter pylori -associated precancer and cancer, we applied the use of a modified Gram stain technique to identify gastric tissue with high bacterial biomass. We validated the presence of bacteria using custom-designed RNAscope probes on consecutive tissue sections and then we visualised bacteria within a polymicrobial community at the Phylum, Kingdom and Genus taxonomic levels. This study validates the cost-effective modified Gram stain as an efficient way to select samples with high microbial biomass for downstream applications investigating host-microbiota relationships.