Prevalence of β-lactamase-negative ampicillin-resistant Haemophilus influenzae (BLNAR) has become a clinical concern. In BLNAR, amino acid substitutions in penicillin-binding protein 3 (PBP3) are relevant to the β-lactam resistance. Carbapenem-non-susceptible H. influenzae isolates have been rarely reported. Through antimicrobial susceptibility testing and nucleotide sequence analysis of ftsI, encoding PBP3, and utilizing a collection of H. influenzae clinical isolates in our laboratory, we obtained a carbapenem-non-susceptible clinical isolate (NUBL1772) that possessed 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 ftsI, 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 ftsI gene of NUBL1772. The recombinant PBP3 of NUBL1772 also exhibited reduced binding to carbapenem. These results demonstrate that altered PBP3 containing V525_N526insM influences reduced carbapenem susceptibility. The revertant mutant lacking the V525_N526insM exhibited lower MICs of carbapenem than NUBL1772, suggesting that this insertion affects reduced carbapenem susceptibility. MICs of β-lactam for NUBL1772 was higher than those for the recombinant possessing ftsI 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 reduced susceptibility to carbapenem in H. influenzae.
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