A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility

Abstract

BackgroundIdentification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93–57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined.ResultsSite-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93–57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93–57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump.ConclusionsIn parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1196-6) contains supplementary material, which is available to authorized users.

β-lactam Resistance in Pseudomonas aeruginosa: Current Status, Future Prospects

“…Variants in experimentally-evolved resistant bacteria also cluster around the active site and are often at the same residues as those in clinical isolates [124][125][126][127]148]. Variants around the active sites of PBPs also contribute to β-lactam resistance in other species [131,149,150], and some cases have been shown to reduce the affinity of β-lactams for the PBP [150,151]. It seems likely that sequence variants around the active sites in PBPs cause slight confirmational changes that reduce the ability of β-lactams to bind and the active site and react with the catalytic serine [129,132].…”

Characterization of Haemophilus influenzae Strains with Non-Enzymatic Resistance to β-Lactam Antibiotics Caused by Mutations in the PBP3 Gene in the Czech Republic in 2010–2018

“…Clonal experiments to confirm the contribution of individual AA substitutions to β-lactam resistance have not been performed and may be the subject of further studies. According to Thegerström [33], even artificially cloned mutations, other than those detected in this collection, reduced susceptibility to aminopenicillins. This suggests that the range of mutations leading to resistance is wide, and new AA substitutions may continue to occur.…”

Molecular Epidemiology and Antibiotic Resistance Analysis of Non-Typeable Haemophilus influenzae (NTHi) in Guangzhou: A Representative City of Southern China

“…NTHi isolates with rPBP3 variants are classified into three main groups based on the substitution of two key amino acids occurring near the KTG motif, R517H (clustered as group I), N526K (group II) or S385T (group III or III-like), which includes additional substitutions near the SSN motif. The latter confers a higher level of antimicrobial resistance, including resistance to third-generation cephalosporins [ 23 ]. However, BLNAR isolates without amino acid substitutions in the PBP3 transpeptidase domain or mutations outside the KTG or SSN motifs, termed miscellaneous mutations, were also found [ 15 ].…”