Contribution of PBP3 Substitutions and TEM-1,TEM-15,and ROB-1 Beta-Lactamases to Cefotaxime Resistance in Haemophilus influenzae and Haemophilus parainfluenzae

Abstract

Aims: To investigate the relative contributions of naturally occurring penicillin-binding protein 3 (PBP3) substitutions, and TEM-1, TEM-15, and ROB-1 beta-lactamases on resistance to a third-generation cephalosporin in Haemophilus influenzae and Haemophilus parainfluenzae. Results: The minimum inhibitory concentration (MIC) of cefotaxime (CTX) was assessed after transformation with PCR-amplified ftsI genes expressing altered PBP3 and/or small plasmids encoding beta-lactamases into an isogenic environment of H. influenzae and H. parainfluenzae. Group III PBP3, comprising substitutions N526K, S385T, and L389F, conferred CTX resistance to H. influenzae according to EUCAST interpretative criteria. Group III-like PBP3, comprising substitutions N526H and S385T, increased the CTX MIC of H. parainfluenzae ninefold, but the level did not transgress the resistance breakpoint. Production of TEM-15 beta-lactamase conferred CTX resistance on both H. influenzae and H. parainfluenzae. A nitrocefin hydrolysis assay showed TEM-15 to be a less efficient enzyme compared to TEM-1. Conclusions: TEM-15 and PBP3 substitutions impose an additive effect on resistance to third-generation cephalosporins in both H. influenzae and H. parainfluenzae. The effect of PBP3 substitutions on beta-lactam resistance in H. parainfluenzae can be addressed by transfer of ftsI genes in vitro.