Biology subjects

Rossiter-Pearson, A. E.

Publications and source records attributed to Rossiter-Pearson, A. E..

3 recordsLinked to original sources

bioRxiv2025

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.

microbiology↗

bioRxiv2024

The gatekeeper to gastric cancer; gastric microbiota invade the lamina propria in Helicobacter pylori-associated gastric carcinogenesis

Stomach cancer is the fourth leading cause of cancer-related deaths worldwide. Helicobacter pylori is the main risk factor for gastric adenocarcinoma (GAC), yet the mechanism underpinning this association remains uncharacterised. Gastric intestinal metaplasia (GIM) represents the pre-cancerous stage and follows H. pylori-associated chronic gastritis (CG). Sequencing studies have revealed fewer H. pylori and more non-H. pylori bacteria in GAC. However, the spatial organisation of the gastric microbiota in health and disease is unknown. Here, we have combined RNA in situ hybridisation and immunohistochemistry to detect H. pylori, non-H. pylori bacteria and host cell markers (E-cadherin, Mucins 5AC and 2) from patients with CG (n=9), GIM (n=12), GAC and normal tissue adjacent to tumours (NATs) (n=3). Quantitative analysis of whole slide scans revealed significant correlations of H. pylori and other bacteria in CG and GIM samples. In contrast to sequencing studies, significantly fewer non-H. pylori bacteria were detected in H. pylori-negative patients. Importantly, whilst H. pylori exclusively colonised the gastric glands, non-H. pylori bacteria invaded the lamina propria in 3/4 CG and 5/6 GIM H. pylori-positive patients. Bacterial invasion was observed in 3/3 GAC samples and at higher levels than matched NATs. We propose that H. pylori holds the keys to disrupt the gastric epithelial barrier, facilitating the opportunistic invasion of non-H. pylori bacteria to the lamina propria. Bacterial invasion could be a significant driver of inflammation in H. pylori-associated carcinogenesis. This proposed mechanism would both explain the synergistic roles of H. pylori and other bacteria and redirect attempts to prevent, diagnose and treat GAC.

microbiology↗

bioRxiv2023

Inhibition of Neisseria gonorrhoeae complement-mediated killing during acute gonorrhoea is dependent upon the IgG2:IgG3 antibody ratio

Excessive binding of antibodies to the bacterial cell surface can paradoxically increase resistance of some Gram-negative pathogens to complement-mediated killing (CMK). We examined CMK of 336 Neisseria gonorrhoeae clinical isolates sampled from participants recruited to a clinical trial. Serum bactericidal assays revealed 3% (9/336) of the autologous participant sera that were tested inhibited CMK. Gonococci isolated from these participants were resistant to the autologous host serum, sensitive to a pool of healthy control sera (HCS) and protected by the host serum in a 1:1 mixture with HCS. Analysis of the clinical metadata showed that there were a significantly higher proportion of inhibitory sera found in participants with urethral infections and from men within the transmission network of men who have sex with women (MSW), when compared to the whole cohort. Following antibody purification from selected participants with inhibitory sera (5/9), IgG and IgM protected the autologous isolates from HCS-mediated killing. Only three of these isolates were protected by purified IgA. A closer examination of IgG subclasses using whole gonococcal cell ELISAs revealed a strong correlation between increased IgG2 binding and decreased IgG3 binding to the bacterial cell surface of isolates that were resistant to CMK. This suggests that IgG2 prevents bactericidal IgG3 from initiating CMK and that the IgG2:IgG3 ratio is important for determining either inhibition or killing of isolates. We therefore reveal a previously unreported mechanism by which inhibitory antibodies prevent CMK of N. gonorrhoeae.

microbiology↗