Characterisation of Resistance to ß-Lactam Antibiotics in a Population of Haemophilus Influenzae

Abstract

Increased global β-lactam resistance in H. influenzae is due to the production of plasmid mediated TEM and ROB β-lactamases and/or reduced affinity penicillin binding proteins (PBPs). β-lactamase-negative, ampicillin resistant (BLNAR) isolates have amino acid substitutions in the transpeptidase region of ftsI, the gene encoding PBP3. BLNAR isolates are usually resistant to cefuroxime and other β-lactams. The resistance phenotypes of 250 clinically significant strains were determined using agar dilution minimum inhibitory concentrations (MICs). B-lactamase production was detected phenotypically in 130 strains; three produced ROB-1 and 90 produced an altered TEM enzyme as determined by PCR amplification. Eighteen BLNAR strains, six B-lactamase positive co-amoxiclav resistant (BLPACR) strains and four isolates with reduced susceptibility to cefotaxime were amongst the phenotypes identified. 71.2% of the clinical isolates were resistant to at least one of ampicillin/amoxicillin, co-amoxiclav and cefuroxime. An attempt was made to determine the mechanism of imipenem resistance detected in fifteen isolates (MIC range 8–16 mg/L). β-lactamase mediated resistance was discounted and efflux pump investigations were inconclusive. Sequence analysis of the transpeptidase region of ftsI of selected clinical isolates revealed four amino acid substitutions (D350N, M377I, A502V and N526K) when compared to the fully susceptible Rd strain. Imipenem resistant mutants were selected by serial passage of ATCC 49247 in imipenem-containing broth. Imipenem resistance was transformed into the susceptible Rd recipient strain, using chromosomal DNA or PCR amplimers of ftsI from an imipenem resistant mutant of ATCC 49247. Sequencing of the transpeptidase region of ftsI revealed no differences between the parent, mutant and transformant suggesting that imipenem resistance may be due to amino-acid substitutions outside the PBP3 transpeptidase region. As β-lactam resistance increases, carbapenems are becoming important treatment alternatives for serious H. influenzae infections. The detection of imipenem resistance in clinical isolates is a matter of concern.