Identification of Haemophilus haemolyticus in clinical samples and characterization of their mechanisms of antimicrobial resistance.

Objectives: The objectives of this study were to establish the frequency of Haemophilus haemolyticus in clinical samples, to determine the antimicrobial resistance rate and to identify the mechanisms of resistance to β-lactams and quinolones.

Methods: An updated database was used to differentiate between MALDI-TOF MS results for Haemophilus influenzae and H. haemolyticus. Antimicrobial susceptibility was studied by microdilution, following EUCAST criteria. The β-lactamase types were identified by PCR analysis of isolates that tested positive for nitrocefin hydrolysis. Mutations in the ftsI gene were identified in isolates with ampicillin MICs ≥0.25 mg/L. Mutations in the quinolone resistance-determining region (QRDR) were identified in isolates with ciprofloxacin MICs ≥0.5 mg/L.

Results: Overall, we identified 69 H. haemolyticus isolates from 1706 clinical isolates of Haemophilus spp. from respiratory, genital, invasive, and other infection sources. The frequency of H. haemolyticus was low in respiratory samples compared with that of H. influenzae, but in genital-related samples, the frequency was similar to that of H. influenzae. We found low antimicrobial resistance rates among H. haemolyticus isolates, with 8.7% for ampicillin, 8.7% for co-trimoxazole, 7.2% for tetracycline and 4.3% for ciprofloxacin. Mutations in the ftsI gene classified the isolates into four groups, including the newly described Group Hhae IV, which presents mutations in the ftsI gene not identified in H. influenzae and H. haemolyticus type strains. Three ciprofloxacin-resistant H. haemolyticus isolates with mutations affecting GyrA and ParC were identified.

Conclusions: The frequency of H. haemolyticus was low, especially in respiratory samples, where H. influenzae is the main pathogen of this genus. Although antimicrobial resistance rates were low, three ciprofloxacin-resistant H. haemolyticus clinical isolates have been identified for the first time.