Genomic Characterization of Blood-Derived Non-typeable Haemophilus influenzae in the Kinki Region of Japan Using Whole-Genome Sequencing

Non-typeable Haemophilus influenzae (NTHi)-related invasive disease and antimicrobial resistance are emerging public health concerns, but genomic data on domestic isolates remain limited. We performed a genomic epidemiological analysis of 15 H. influenzae strains isolated from blood samples (n=15) at seven participating medical institutions in the Kinki region of Japan between 2018 and 2020. Whole-genome sequencing with hybrid assembly showed that all isolates were NTHi. Multilocus sequence typing identified 10 sequence types, including recurrent ST165 and ST107, and core-genome single-nucleotide polymorphism (SNP) analysis revealed three clusters, while unclustered strains showed high genetic diversity. Additional pan-genome and Virulence Factor Database (VFDB)-based analyses showed heterogeneous distribution of putative virulence-associated genes, particularly those related to adhesion, iron/heme acquisition, and lipooligosaccharide or outer-membrane structures. Antimicrobial susceptibility testing showed that 73.3% of isolates were phenotypically classified as β-lactamase-negative ampicillin-resistant (BLNAR), while penicillin-binding protein 3 (PBP3) mutation analysis showed that 73.3% were genotypically classified as high-gBLNAR. One ST422 isolate showed reduced susceptibility to moxifloxacin and carried gyrA S84L and parC S84I substitutions. These findings provide regional baseline data on the genomic diversity, resistance mechanisms, and molecular epidemiology of invasive NTHi in the Kinki region of Japan.