Integrated Microbial Profiling Identifies Bacterial and Fungal Communities in Pseudomyxoma Peritonei Mucin

Pseudomyxoma peritonei (PMP) is a rare mucinous malignancy in which tumour-associated microbial communities remain poorly characterised, particularly, their fungal component and spatial organisation. We applied an integrated workflow combining 16S and 18S rRNA gene sequencing, microbial culture, histochemical staining, chromogenic RNAscope in situ hybridisation and a patient-derived xenograft model to investigate bacterial and fungal communities in control tissues and low- and high-grade PMP mucin. Bacterial and fungal DNA was detected in PMP-associated mucin, with grade-associated differences in bacterial composition and predicted functional potential. Viable bacteria were recovered from human PMP samples and xenograft-derived mucin. Fungal structures were independently supported by periodic acid-Schiff and Calcofluor White staining, while RNAscope localised bacterial 16S and fungal 18S rRNA signals within epithelial and mucin-rich tumour regions. Comparable microbial signals were observed in the xenograft model. Together, these complementary approaches identify PMP mucin as a multi-kingdom microbial niche and provide the first characterisation of its associated mycobiome. This integrated analytical framework may facilitate the investigation of microorganisms in other low-biomass, mucin-rich tumour environments and provides a basis for future mechanistic studies of microbe-host interactions in PMP.