PURPOSE: β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae is frequently isolated from respiratory samples and is particularly problematic in Japan. The aim of this study was to characterize circulating isolates of H. influenzae genotypically by BLNAR-PCR and multilocus sequence typing (MLST), and to determine any associations between them. METHODS: H. influenzae isolates (n=191) were collected from paediatric patients (1 month to 12 years old) between 2000 and 2011 for three types of infections: pneumonia (n=61), acute otitis media (AOM) (n=68) and meningitis (n=62). All were characterized for capsular type by agglutination tests, and for β-lactam resistance by real-time PCR. The sequence types (STs) determined by MLST were analysed using eburst v3. RESULTS: Eighty-eight out of 191 (46.1 %) H. influenzae isolates were BLNAR by PCR; 37 of 61 (60.7 %) from pneumonia; 33 of 68 (48.5 %) from AOM and 18 of 62 (29.0 %) from meningitis cases. MLST identified 40 and 44 STs among isolates from pneumonia and AOM, respectively. BLNAR were found in singletons such as ST156 in pneumonia, and ST161 and ST396 in AOM. In contrast, eight STs were identified in meningitis, of which seven were genotypically closely related, while ST54 was the most frequent (62.9 %), unlike in the MLST database registrations, where ST6 predominated. CONCLUSION: Non-typeable H. influenzae (NTHi), mostly derived from pneumonia and AOM, were genetically diverse, in contrast to the predominance of H. influenzae type b (Hib) among meningitis cases. The associations between certain STs and β-lactam resistance among NTHi were confirmed.
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