Fusobacterium lineage profiling facilitates the clarification of the associations between non-nucleatum Fusobacterium and colorectal cancer.

Non-nucleatum Fusobacterium may play a nonnegligible role in colorectal cancer (CRC) and certain Fusobacterium lineages (namely, L1 and L5) have shown specific associations with CRC. We aim to clarify the complex connections between Fusobacterium and CRC. We found that the widely adopted quantitative PCR (qPCR) method could overestimate F. nucleatum abundance and, in fact, reflect L1 levels in clinical samples. A lineage-specific qPCR assay targeting L1/L5 was developed and validated using mock and clinical samples. Its application in independent cohorts confirmed that L1 was overabundant in CRC, whereas L5 correlated with lymphovascular invasion. Importantly, faecal L1 abundance was more predictive of CRC than F. nucleatum, supported also by cross-population metagenomic data. CRC-associated virulence and colonisation genes were found in various L1 species other than F. nucleatum. Our results highlight the clinical importance of L1/L5 in CRC with high-diversity Fusobacterium contexts and suggest that non-nucleatum Fusobacterium may also contribute to CRC.