Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to β-lactam antibiotics

Abstract

Objectives: EUCAST developed an antibiotic susceptibility testing method for Haemophilus influenzae. We assessed the EUCAST testing method and EUCAST clinical breakpoints and newly proposed epidemiological cut-off values against H. influenzae clinical isolates with known molecular mechanisms of resistance to β-lactam antibiotics.

Methods: In total, 89 clinical isolates were used: 30 were β-lactamase negative with PBP3 mutations (gBLNAR), 20 were β-lactamase positive without PBP3 mutations (gBLPAR), 15 were β-lactamase positive with PBP3 mutations (gBLPACR), and 24 were β-lactamase negative without resistance mechanism (gBLNAS). Twelve different β-lactam antibiotics and disc charges were tested.

Results: None of the discs tested fully separated between gBLNAS and gBLNAR populations. According to EUCAST clinical zone diameter breakpoints, overall the best values of sensitivity and specificity were obtained with cefuroxime 30 μg and amoxicillin/clavulanic acid 2/1 μg discs for detection of gBLNAR and gBLPACR populations, although a previous β-lactamase test was needed. Other antibiotic discs could be suitable for epidemiological purposes, such us penicillin 10 U for separating gBLNAR isolates and cefoxitin 30 μg for detection of gBLPACR isolates. By Etest using the EUCAST method, the EUCAST MIC clinical breakpoints for ampicillin and amoxicillin/clavulanic acid showed high specificity, but low sensitivity, for the detection of genotypes with mutations in PBP3.

Conclusions: The main genotypes of β-lactam-resistant H. influenzae can be separated by using the EUCAST disc diffusion method, although it should be noted that overlapping between populations with and without PBP3 mutations is common.