Laboratory Detection of Haemophilus influenzae with Decreased Susceptibility to Nalidixic Acid, Ciprofloxacin, Levofloxacin, and Moxifloxacin Due to gyrA and parC Mutations

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Abstract
The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of Haemophilus influenzae to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were ≥0.12 μg/ml (range, 0.12 to 32 μg/ml), and the ciprofloxacin MICs for 28 matched control isolates were ≤0.06 μg/ml. In addition, we sequenced the quinolone resistance-determining regions (QRDRs) of gyrA and parC of all strains. The log 2 MICs of all quinolones were plotted against the inhibition zone diameters. The MICs and inhibition zone diameters selected to screen for the resistance mechanism were based on the susceptibility distribution data and the presence or absence of amino acid changes in the QRDRs of GyrA and ParC. Strains for which ciprofloxacin MICs were ≤0.06 μg/ml, levofloxacin and moxifloxacin MICs were ≤0.03 μg/ml, and nalidixic acid MICs were ≤2.0 μg/ml lacked modifications in the QRDR of GyrA. In contrast, all strains for which ciprofloxacin, levofloxacin, and moxifloxacin MICs were ≥0.5 μg/ml and the vast majority of those for which nalidixic acid MICs were ≥32 μg/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen H. influenzae isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).

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