β-lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of β-lactamase-negative high-level ampicillin-resistant H. influenzae (High-BLNAR) isolates has been reported in Japan. However, the reasons for the expansion are unknown. High-BLNAR possesses an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of High-BLNAR and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined.
Sixty percent of H. influenzae clinical isolates (n=62/104) were BLNAR. Among BLNAR, 92% (n=57/62) were High-BLNAR. Most were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicate that the expansion of High-BLNAR isolates was multiclonal and still dominant in Japanese clinical setting. One High-BLNAR harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV).
No quinolone-resistant H. influenzae isolates were identified. The minimum inhibitory concentrations (MICs) of the quinolones [lsqb]moxifloxacin, garenoxacin, and tosufloxacin[rsqb] were similar to that of levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained four H. influenzae isolates showing decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA. Three of these were High-BLNAR.
In summary, this study shows that multiclonal High-BLNAR dominates in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in High-BLNAR strains.
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