Simple SummaryA growing body of research has shown the connection between gut microbiota and colorectal cancer. However, most studies analyze fecal microbiota, which do not reliably represent the bacterial populations associated with colon mucosa. We analyzed the microbiota and metabolome directly collected from the surface of colon polyps, and showed different bacterial and metabolite signatures that discriminate between patients with low- and high-grade dysplastic polyps. We identified bacterial genera and species that are enriched in the early stages of tumor development and may act as drivers of carcinogenesis. Moreover, we revealed that differences in metabolite profiles accompanied the changes in bacterial composition associated with tumor stage, and that gut bacteria are involved in the production and consumption of significantly altered metabolites. Our findings pave the way for future mechanistic investigations to elucidate the role of specific bacteria in colon carcinogenesis and to design preventative measures based on microbiota modulation.AbstractAccording to the driver–passenger model for colorectal cancer (CRC), the tumor-associated microbiota is a dynamic ecosystem of bacterial species where bacteria with carcinogenic features linked to CRC initiation are defined as “drivers”, while opportunistic bacteria colonizing more advanced tumor stages are known as “passengers”. We reasoned that also gut microbiota-associated metabolites may be differentially enriched according to tumor stage, and be potential determinants of CRC development. Thus, we characterized the mucosa- and lumen-associated microbiota (MAM and LAM, respectively) and mucosa-associated metabolites in low- vs. high-grade dysplastic colon polyps from 78 patients. We show that MAM, obtained with a new biopsy-preserving approach, and LAM differ in composition and α/β-diversity. By stratifying patients for polyp histology, we found that bacteria proposed as passengers by previous studies colonized high-grade dysplastic adenomas, whereas driver taxa were enriched in low-grade polyps. Furthermore, we report altered “mucosa-associated metabolite” levels in low- vs. high-grade groups. Integrated microbiota-metabolome analysis suggests the involvement of the gut microbiota in the production and consumption of these metabolites. Altogether, our findings support the involvement of bacterial species and associated metabolites in CRC mucosal homeostasis in a tumor-stage-specific manner. These distinct signatures may be used to distinguish low-grade from high-grade dysplastic polyps.