In vitro Derivation of Fluoroquinolone-resistant Mutants from Multiple Lineages of Haemophilus influenzae and Identification of Mutations Associated with Fluoroquinolone Resistance.

Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of p-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to p-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzoe isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and Delta Glu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae. In conclusion, H. influenzoe clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than beta-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.