Low -lactamase negative ampicillin resistant Haemophilus influenzae are best detected by testing amoxicillin susceptibility by the broth microdilution method

Antibiotic Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; Consejo Superior de Investigaciones Científicas, Madrid, Spain

^* To whom correspondence should be addressed. Email: jcampos{at}isciii.es.

	Abstract

Ampicillin resistance in Haemophilus influenzae due to alterations in penicillin-binding proteins (BLNAR) is acquiring increasing clinical and epidemiological importance. BLNAR strains with low ampicillin MICs (0.5 to 4 µg/ml) represent the majority of this population in Europe and the United States, but separating them from susceptible isolates is challenging. To investigate the best method to identify low-BLNAR strains, we studied the antibiotic susceptibility of 94 clinical isolates of H. influenzae by microdilution, E-test and disk diffusion: 25 had no resistance mechanisms (gBLNAS); 34 had mutations in the ftsI gene only (gBLNAR); 20 were -lactamase producers only (gBLPAR); and 15 showed -lactamase production and mutations in the ftsI gene (gBLPACR). By current CLSI breakpoints, most gBLNAR isolates were ampicillin susceptible by microdilution (76.5%) or by E-test (88.2%). Most gBLNAR strains (79.4%) were non-susceptible to amoxicillin (the most widely used community antibiotic in the US and Europe) when tested by microdilution. By E-test, 15% of -lactamase positive isolates were non-resistant to ampicillin or amoxicillin. The poorest agreement between E-test and microdilution was for the gBLPAR isolates (25% for ampicillin, 15% for amoxicillin, and 10% for cefaclor). Low-strength disks of ampicillin and amoxicillin-clavulanic acid poorly identified low-BLNAR isolates and are not recommended as a screening method. We suggest new amoxicillin breakpoints for BLNAR isolates as follows: susceptible, MIC 0.5 µg/ml (no resistance mechanisms, PK/PD data favourable); intermediate, MICs of 1 to 2 µg/ml (resistance mechanisms present but PK/PD data favourable), and resistant, MICs 4 µg/ml (resistance mechanisms present and PK/PD data unfavourable).