Metagenomic Screening Identifies Bifidobacterium longum as a Potential Therapeutic Target in Head and Neck Squamous Cell Carcinoma

Introduction and aims Head and neck squamous cell carcinoma (HNSCC) is a major oral health challenge, and accumulating evidence suggests that microbial dysbiosis may contribute to disease development. However, most previous studies relied on 16S rRNA sequencing, limiting species-level resolution and providing inconsistent microbial signatures across cohorts. This study aimed to characterize HNSCC-associated microbial alterations using genome-resolved metagenomics and identify candidate microbiota-based interventions. Methods Shotgun metagenomic data from 150 oral and faecal samples spanning an in-house case–control cohort and two public datasets were integrated and analysed using a unified bioinformatics pipeline. Microbial diversity, differential biomarkers, co-occurrence networks, and species-level genome bins were analysed. Candidate probiotic strains were predicted using metaProbiotics and further evaluated in a CAL27 zebrafish xenograft model. Results Faecal communities showed stronger disease-associated restructuring than oral communities, with reduced observed richness and significant Bray–Curtis separation, whereas oral community differences were not significant. Integration of LEfSe and random forest identified eleven faecal consensus markers, including control-enriched Roseburia faecis, Phocaeicola vulgatus , and Bifidobacterium longum , and four cancer-enriched oral markers, including reproducible Capnocytophaga sputigena . Microbial networks were consistently less connected in HNSCC across both niches. Among 990 dereplicated species-level genome bins, Bifidobacterium represented the dominant high-confidence probiotic genus. In vivo validation showed that B. longum and the induction chemotherapy component docetaxel, cisplatin, and 5-fluorouracil (TPF) reduced tumour burden and inflammatory/angiogenic responses, while combination treatment modulated macrophage-associated migration and partially restored microbial network connectivity. Conclusion Genome-resolved metagenomic profiling revealed distinct oral and gut microbial features associated with HNSCC and identified B. longum as a candidate adjunctive probiotic intervention requiring further mammalian and clinical validation. Clinical relevance This study provides microbial biomarkers and candidate probiotic strategies that may support future microbiome-based risk assessment and adjunctive management of HNSCC in oral healthcare settings.