Abstract

Summary Fusobacterium nucleatum , which has four subspecies ( nucleatum , animalis , vincentii and polymorphum ), plays an important role in promoting colorectal cancer (CRC). However, as there is no efficient method of differentiating these subspecies in the context of a rich gut microbiota, the compositions in CRC remain largely unknown. In this study, a PCR-based differentiation method enabling profiling of F . nucleatum infection in CRC at the subspecies level was developed. Based on the analysis of 53 F . nucleatum genomes, we identified genetic markers specific to each subspecies and designed primers for the conserved sequences of those markers. The PCR performance of the primers was tested with F . nucleatum and non- nucleatum Fusobacterium strains, and complete consistence with taxonomy was achieved. Additionally, no non-specific amplification occurred when using human DNA. The method was then applied to faecal ( n = 58) and fresh-frozen tumour tissue ( n = 100) samples from CRC patients, and wide heterogeneity in F . nucleatum subspecies compositions in the gut microbiota among CRC patients was observed. Single-subspecies colonization was common, whereas coexistence of four subspecies was rare. Subspecies animalis was most prevalent, while nucleatum was not frequently detected. The results of this study contribute to our understanding of the pathogenicity of F . nucleatum at the subspecies level and the method developed has potential for clinical and epidemiological use.