Molecular characterization of cefepime and aztreonam nonsusceptibility in Haemophilus influenzae

Background: Cefepime and aztreonam are highly efficacious against H. influenzae, and resistant strains are rare. In this study, we isolated cefepime- and aztreonam-nonsusceptible H. influenzae strains and addressed the molecular basis of their resistance to cefepime and aztreonam. Methods: Two hundred and 28 specimens containing H. influenzae were screened, of which 32 isolates were enrolled and applied to antimicrobial susceptibility testing and whole-genome sequencing. Genetic variations that were detected in all nonsusceptible isolates with statistical significance by Fisher's exact tests were identified as cefepime or aztreonam nonsusceptibility related. Functional complementation assays were conducted to assess the in vitro effects of proteins with sequence substitutions on drug susceptibility. Results: Three H. influenzae isolates were nonsusceptible to cefepime, one of which was also nonsusceptible to aztreonam. Genes encoding TEM, SHV and CTX-M extended-spectrum β-lactamases were not detected in the cefepime- and aztreonam-nonsusceptible isolates. Five genetic variations in four genes and 10 genetic variations in five genes were associated with cefepime and aztreonam nonsusceptibility, respectively. Phylogenetic analyses revealed that changes in FtsI were correlated strongly with the MIC of cefepime and moderately with aztreonam. FtsI Thr532Ser-Tyr557His cosubstitution linked to cefepime nonsusceptibility and Asn305Lys-Ser385Asn-Glu416Asp cosubstitution to aztreonam nonsusceptibility. Functional complementation assays revealed that these cosubstitutions increased MICs of cefepime and aztreonam in susceptible H. influenzae isolates, respectively. Conclusions: Genetic variations relevant to resistant phenotypes of cefepime and aztreonam nonsusceptibility in H. influenzae were identified. Moreover, the effects of FtsI cosubstitutions on increasing MICs of cefepime and aztreonam in H. influenzae were demonstrated.