Intratumoral microbiota heterogeneity and Bifidobacterium abundance are linked to very early recurrence in hepatocellular carcinoma

Although intratumoral bacteria have been reported in hepatocellular carcinoma (HCC), the compositional differences between HCC and adjacent non-tumor tissues remain controversial, and the specific taxa correlated with very early postoperative recurrence remain unexplored. We collected tumor and paired normal tissues from 131 HCC cases for 16 S ribosomal RNA (rRNA) sequencing. Multiple bioinformatics approaches compared microbial composition, diversity, and enriched taxa between tumor and normal tissues. Intratumoral microbiota was also compared between HCC patients with very early recurrence (VER) and those without VER. HCC tissues with high versus low Bifidobacterium abundance underwent transcriptomic sequencing to identify differentially expressed genes and pathways, with immune cell profiling. Fluorescence in situ hybridization (FISH), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemistry evaluated the association between Bifidobacterium presence/abundance and apoptotic signaling pathways, as well as apoptosis-related marker proteins. Microbial diversity did not differ between HCC and normal tissues, but compositional differences and inter‑individual heterogeneity were evident. Bacterial composition overlapped substantially among tumor tissue, adjacent tissue, and bile across taxonomic levels. Intratumoral Bifidobacterium abundance was significantly lower in VER patients than in non-VER patients. FISH confirmed intratumoral Bifidobacterium . Bifidobacterium enrichment correlated with enhanced apoptotic activity, reduced baculoviral IAP repeat containing 5 (BIRC5) expression, and consistent TUNEL and cleaved caspase‑3 staining. Reduced intratumoral Bifidobacterium abundance is linked to very early postoperative recurrence of HCC, though this finding needs validation in multi‑center cohorts.