Detection of N526K-substituted penicillin-binding protein 3 conferring low-level mutational resistance to β-lactam antibiotics in Haemophilus influenzae by disc diffusion testing on Mueller-Hinton agar according to EUCAST guidelines

EUCAST has recently authorized a new disc diffusion test for routine antimicrobial susceptibility testing of Haemophilus influenzae, calibrated to EUCAST MIC breakpoints. We investigated whether disc diffusion testing as recommended by EUCAST could discriminate strains of H. influenzae carrying the N526K substitution in penicillin-binding protein 3 from the wild-type population. A total of 170 recent clinical isolates, genetically characterized for the presence of acquired and mutational resistance mechanisms, were tested by disc diffusion of β-lactam antibiotics on supplemented Mueller-Hinton agar. Tentative epidemiological breakpoint values for the presence of the N526K substitution were suggested for various β-lactams, and the performances were calculated. Epidemiological cut-off values of 19/20 mm for ampicillin (2 μg) and 11/12 mm for benzylpenicillin (1 U) accurately categorized 96% of the study strains, and outperformed cephalosporin-containing discs in the discrimination of mutational resistance in β-lactamase-non-producing isolates. Current EUCAST interpretative criteria for the categorization of clinical resistance showed concordance between resistance rates based on MIC and zone diameter breakpoints for both ampicillin and cefuroxime, but categorization of individual isolates was not consistent. Disc diffusion testing of H. influenzae accurately identified β-lactamase-non-producing isolates with the N526K substitution by use of discs containing low amounts of penicillins. Cephalosporin-containing discs could detect mutational resistance in β-lactamase-producing isolates, but performed with reduced specificity.