BACKGROUND: Data from surveillance on antibiotic resistance have shown an increasing prevalence of non-enzymatic resistance (β-lactamase-negative ampicillin-resistant) to β-lactam antibiotics among H. influenzae strains in the Czech Republic. Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. The phenomenon of non-enzymatic resistance to β-lactams is complicated by the fact that the phenotypic detection of PBP3 with specific amino acid substitutions (rPBP3) is challenging, since rPBP3 isolates have repeatedly been demonstrated to be split by the epidemiological cut-off values (ECOFF) for aminopenicillins defined by EUCAST. OBJECTIVES: We sought to determine whether the penicillin disc has sufficient detection ability to predict the non-enzymatic mechanism; whether other antibiotics can be used for detection; and what is the agreement between the broth microdilution and disc diffusion methods. METHODS: We undertook susceptibility testing of selected antibiotics according to EUCAST of 153 rPBP3 strains, and sequencing of the ftsI gene to determination amino acid substitutions. RESULTS: For a selected set of rPBP strains: (i) the detection capability for penicillin, ampicillin, cefuroxime and amoxicillin/clavulanate was found to be 91.5%, 94.4%, 89.5% and 70.6%, respectively; (ii) the categorical agreement between the disc diffusion method and the MIC for ampicillin and cefuroxime was 71.1% and 83.8%, respectively. CONCLUSIONS: We observed better recognition of rPBP3 strains by the ampicillin disc than by the penicillin disc. There is frequently a discrepancy in the interpretation of susceptibility results between the methods used.

Department of Microbiology 3rd Faculty of Medicine Kralovske Vinohrady University Hospital and National Institute of Public Health Charles University Ruska 87 100 00 Prague 10 Prague The Czech Republic

National Reference Laboratory for Antibiotics Centre for Epidemiology and Microbiology National Institute of Public Health Srobarova 49 48 100 00 Prague 10 Prague The Czech Republic

National Reference Laboratory for Meningococcal Infections Centre for Epidemiology and Microbiology National Institute of Public Health Srobarova 49 48 100 00 Prague 10 Prague The Czech Republic

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