Carbapenem-Nonsusceptible Haemophilus influenzae with Penicillin-Binding Protein 3 Containing an Amino Acid Insertion

The prevalence of beta-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae has become a clinical concern. In BLNAR isolates, amino acid substitutions in penicillin-binding protein 3 (PBP3) are relevant to the beta-lactam resistance. Carbapenem-nonsusceptible H. influenzae isolates have been rarely reported. Through antimicrobial susceptibility testing, nucleotide sequence analysis of ftsl, encoding PBP3, and the utilization of a collection of H. influenzae clinical isolates in our laboratory, we obtained a carbapenem-nonsusceptible clinical isolate (NUBL1772) that possesses an altered PBP3 containing V525_N526insM. The aim of this study was to reveal the effect of altered PBP3 containing V525_N526insM on reduced carbapenem susceptibility. After generating recombinant strains with altered ftsl, we performed antimicrobial susceptibility testing and competitive binding assays with fluorescent penicillin (Bocillin FL) and carbapenems. Elevated carbapenem MICs were found for the recombinant strain harboring the entire ftsl gene of NUBL1772. The recombinant PBP3 of NUBL1772 also exhibited reduced binding to carbapenems. These results demonstrate that altered PBP3 containing V525_N526insM influences the reduced carbapenem susceptibility. The revertant mutant lacking the V525_N526insM exhibited lower MICs for carbapenems than NUBL1772, suggesting that this insertion affects reduced carbapenem susceptibility. The MICs of beta-lactams for NUBL1772 were higher than those for the recombinant possessing ftsl of NUBL1772. NUBL1772 harbored AcrR with early termination, resulting in low-level transcription of acrB and high efflux pump activity. These findings suggest that the disruption of AcrR also contributes to the reduced carbapenem susceptibility found in NUBL1772. Our results provide the first evidence that the altered PBP3 containing V525_N526insM is responsible for the reduced susceptibility to carbapenems in H. influenzae.