A molecular analysis of quinolone-resistant Haemophilus influenzae: validation of the mutations in Quinolone Resistance-Determining Regions

Abstract

The mechanism of quinolone-resistance is considered to be amino acid mutations in the type II topoisomerase. We validated the genetic mechanisms of quinolone resistance in Haemophilus influenzae. We obtained 29 H. influenzae strains from a nationwide surveillance program in Japan (including 11 quinolone-resistant strains [moxifloxacin: MFLX or levofloxacin MIC ≥2 μg/ml]). We analyzed the sequences of the Quinolone Resistance-Determining Regions (QRDRs) in GyrA, GyrB, ParC and ParE. Furthermore, we induced resistance in susceptible strains by exposing them to quinolone, and investigated the relationship between mutations in the QRDRs and the MICs. Five amino acid substitutions in GyrA (at Ser84 and Asp88) and ParC (at Gly82, Ser84 and Glu88) were found to be closely related to the MICs. The strains with a MFLX MIC of 0.125-1 and 2-4 μg/ml had one and two mutations, respectively. The strains with a MFLX MIC of ≥8 μg/ml had three or more mutations. The strains with induced resistance with MFLX MICs of 0.5-1 and ≥2 μg/ml also had one and two mutations, respectively. We confirmed that these five mutations strongly contribute to quinolone resistance and found that the degree of resistance is related to the number of the mutations. In addition, the three strains of 18 susceptible strains (16.7%) also had a single mutation. These strains may therefore be in the initial stage of quinolone resistance. Currently, the frequency of quinolone-resistant H. influenzae is still low. However, as has occurred with β-lactams, an increase in quinolone use may lead to more quinolone-resistant strains.

Keywords: GyrA; Haemophilus influenzae; Moxifloxacin; ParC; Quinolone Resistance-Determining Regions; Quinolone-resistant.