Haemophilus influenzae dominance in fungal ball microbiome revealed through multi-niche metagenomic sequencing.

OBJECTIVE: This study employed metagenomic sequencing to characterize the sinonasal microbiome in patients with unilateral maxillary sinus fungal ball (MSFB), with specific emphasis on bacterial-fungal interactions and functional pathways implicated in fungal ball pathogenesis. METHODS: The study enrolled 30 MSFB patients and 30 healthy controls. Nasal secretion samples were obtained from three anatomical sites in MSFB cases: fungal ball cavity (FC), affected middle nasal meatus (AM), and contralateral unaffected middle nasal meatus (UM). And in the control group, samples were obtained from the healthy middle nasal meatus (HM). Metagenomic sequencing of microbial DNA was performed using the Illumina Novaseq platform. Taxonomic and functional analyses were conducted using Kraken2, Bracken, and HUMAnN2. RESULTS: Bacteria dominated the microbiome in the FC group (98.53%), with Haemophilus influenzae identified as a key biomarker (LDA score > 5). A negative correlation between H. influenzae and Aspergillus flavus was observed in the FC group (r = -0.46, P = 0.013). Functional pathways enriched in the FC group included amino acid biosynthesis (map00290), lipopolysaccharide biosynthesis (map00540), and fatty acid biosynthesis (map00061), supporting H. influenzae survival and immune modulation. FC microbiota showed reduced diversity and distinct composition compared to other groups (PERMANOVA, P < 0.001). No significant differences were found in the composition of the microbiota between the bilateral middle nasal meatus groups of MSFB. CONCLUSION: This study highlights H. influenzae as a critical bacterial biomarker in MSFB. The inverse relationship between H. influenzae and A. flavus may suggest competitive or immune-mediated interactions. These findings advance understanding of non-invasive fungal sinusitis. Future validation in larger fungal ball cohorts or invasive fungal sinusitis is warranted.