Sulfamethoxazole-trimethoprim (SXT) is an important empirical treatment agent against various bacterial infections. In this study, we aimed to elucidate the mechanisms underlying SXT resistance in Haemophilus influenzae clinical isolates from Japan, as information on such resistance remains limited. A total of 79 H. influenzae clinical isolates collected in 2018 and 2022 were analyzed. The SXT resistance rates were 38.7% in 2018 and 35.3% in 2022. Multilocus sequence typing analysis revealed that ST422 was the most common sequence type (36.7%), followed by ST107 (26.7%). Horizontal transfer assays using the genomic DNA or PCR-amplified fragments revealed that SXT resistance was transferred to the susceptible isolates via genomic DNA and PCR-amplified folA fragments, indicating that FolA mediates SXT resistance in H. influenzae. Site-directed mutagenesis revealed that the substitution of isoleucine at position 95 in FolA was associated with SXT resistance. All SXT-resistant isolates had an amino acid substitution at position 95 in FolA: leucine in 26 of the 30 strains, valine in 3 strains, and glycine in 1 strain. Our findings demonstrate that SXT resistance in H. influenzae was prevalent and can spread via horizontal transfer. Furthermore, an amino acid substitution at position 95 of FolA played a key role in conferring resistance.
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