Sulfamethoxazole-Trimethoprim Resistance in <i>Haemophilus influenzae</i> Clinical Isolates in Japan: Role of FolA and Horizontal Transfer

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 <i>Haemophilus influenzae</i> clinical isolates from Japan, as information on such resistance remains limited. A total of 79 <i>H. influenzae</i> 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 <i>folA</i> fragments, indicating that FolA mediates SXT resistance in <i>H. influenzae</i>. 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 <i>H. influenzae</i> 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.