Impact of different amino acid substitutions in penicillin-binding protein 3 on beta-lactam susceptibility in Haemophilus influenzae

Abstract

25Purpose: Beta-lactam antibiotics in combination with a beta-lactamase inhibitor are the first-26 line treatment option for Haemophilus influenzae infections. However, beta-lactamase-27 independent resistance to beta-lactams is increasing. This resistance mechanism has been 28 linked to amino acid substitutions in the penicillin-binding protein 3 (PBP3), but how these 29 substitutions lead to decreased binding affinities to certain beta-lactam antimicrobials 30 remains unknown. 31 Methods:We investigated beta-lactam resistance and amino acid substitutions in PBP3 32 from fifty-three clinical isolates of H. influenzae collected in Switzerland from January to April 33 2016. Identification of key polymorphisms and classification of strains into PBP3 amino acid 34 substitution groups I, II, and M was done as previously described. Based on published PBP3 35 crystal structures, we investigated how the group-specific amino acid substitutions impact the 36 beta-lactam binding site. 37Results: We found that both group I and group II substitutions disrupt the Asn526-Arg517-38Glu324 interaction, which might affect the configuration of the beta-lactam binding site. 39Amino acid substitutions in group M strains are distant from the active site and have most 40 likely no impact on beta-lactam binding. In accordance with this observation, all group M 41 strains showed minimal inhibitory concentrations (MICs) within the susceptible range for all 42 tested antimicrobials and were not significantly different to the wild type (beta-lactamase 43 producers excluded), while group I and group II strains showed significantly higher MICs for 44 beta-lactam antimicrobials. 45Conclusion: Group M strains are phenotypically equal to the wild type, while amino acid 46 substitutions of group I and group II might affect the beta-lactam binding through a common 47 mechanism by disrupting the Asn526-Arg517-Glu324 interaction. 48 49