Muscle-invasive bladder cancer (MIBC) is a severe type of bladder cancer (BCa), with its pathogenesis and relevant interacting factors still open to investigation. Several studies have recently identified microbiome differences between BCa and healthy bladder tissues, mostly in urine samples and confined to the genus level. This study aims to apply 2bRAD sequencing for Microbiome (2bRAD-M) to explore the microbial environment of the bladder in MIBC patients and compare the microbial differences between tumor and normal bladder tissue.

A total of 15 MIBC patients were recruited. All patients underwent radical cystectomy (RC), with tumor tissue and its surrounding normal tissue taken as controls. Employing 2bRAD-M to characterize the bladder microbiome of MIBC patients and to explore whether microbial differences exist between tumor and normal tissues.

A total of 353 species were detected. Normal tissues showed slightly higher alpha diversity than that of tumor, but no significant differences were shown between the two groups in both alpha and beta diversity. MIBC tissue has a similar microbial composition to normal bladder tissue, with Ralstonia_sp000620465, Ralstonia_pickettii and Cutibacterium_acnes being the most dominant species shared by both groups. Brachybacterium_paraconglomeratum and Haloparvum_sedimenti were found to be significantly overexpressed in tumor tissues, and species such as Anoxybacillus_A_rupiensis and Pseudoxanthomonas_A_sp004284195 were less enriched in MIBC tissues than in the normal group. 29 discriminatory character taxa were also revealed by linear discriminant analysis effect sizea (LEfSe). The results of the random forest analysis indicated Ralstonia_sp000620465 and Ralstonia_pickettii as the two most critical species distinguishing the differences between the two groups. Meanwhile, further prediction of COG annotation and KEGG pathway revealed significant differences in microbial function between tumor and normal tissues.

This is the first study employing 2bRAD-M to explore the tumor microbial environment of MIBC patients. Overall, the microbial composition of bladder tissue in MIBC patients is less variable than in normals, but expression differences exist at the species level, which will help provide more insight into MIBC pathogenesis.