Differentiation of β-lactamase-negative ampicillin-resistant Haemophilus influenzae from other H. influenzae strains by a disc method

We established breakpoints for differentiating ampicillin (ABPC)-susceptible strains from resistant strains among Haemophilus influenzae isolates according to susceptibility to various β-lactam antibiotics, using a disc method. Susceptibility testing of isolates for 13 β-lactam agents was followed by analysis of the resistance genes, using a polymerase chain reaction (PCR) to identify the TEM-1 β-lactamase gene (bla) and the ftsI gene encoding penicillin-binding protein (PBP) 3, which affects β-lactam minimum inhibitory concentrations (MICs). A total of 228 H. influenzae isolates were classified into 114 β-lactamase-negative, ABPC-susceptible (BLNAS) strains; 29 β-lactamase-negative, ABPC-resistant (BLNAR) strains; 53 low-BLNAR strains with a low degree of ABPC resistance; 27 TEM-1-producing strains (BLPAR); and 5 strains with ftsI gene mutations in addition to TEM-1 production (BLPACR) according to the PCR results. To identify resistant strains by disc-method susceptibility testing, the zone of inhibition was measured for ABPC (10 μg/disc), cefaclor (30 μg/disc), cefpodoxime (10 μg/disc), and cefdinir (5 μg/ disc) discs. Strains were identified as BLNAS without resistant genes when the diameter was ≥27 mm for the ABPC disc and ≥21 mm for the cefaclor disc. Other strains were identified as BLNAR when the diameter was ≤22 mm for the cefpodoxime disc and ≤17 mm for the cefdinir disc. Remaining strains were identified as low-BLNAR. These criteria differentiated resistance types with high accuracy. A discrepancy was noted between genetic results and disc-testing breakpoints for differentiating resistant from susceptible H. influenzae. A disc-testing breakpoint for cefditoren (5 μg/disc) was proposed, with the susceptibility statistically defined as a diameter of ≥24 mm, which corresponds to the breakpoint (1 μg/ml) of the microdilution method recommended by the Japanese Society of Chemotherapy.