Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis resistance to fluoroquinolones were revealed.KEYWORDS Moraxella catarrhalis, fluoroquinolone, gyr, par, quinolone resistancedetermining region M oraxella catarrhalis is a Gram-negative aerobic diplococcus that causes upper and lower respiratory tract infections (1). M. catarrhalis is known to be resistant to penicillin and first-generation cephalosporins, owing to its ability to produce BRO -lactamase; however, it typically remains susceptible to other antibiotics, including fluoroquinolones (2-4). Fluoroquinolones act primarily by inhibiting DNA gyrase and topoisomerase IV. These two enzymes work together during DNA replication, transcription, recombination, and repair, which are essential for bacterial growth. Both DNA gyrase and topoisomerase IV are large, complex enzymes composed of two pairs of subunits. DNA gyrase comprises the GyrA and GyrB subunits, encoded by the gyrA and gyrB genes, respectively, while topoisomerase IV is composed of ParC and ParE, encoded by the parC and parE genes, respectively (5, 6).In Gram-negative cocci, such as Neisseria gonorrhoeae and N. meningitidis, the mechanism of fluoroquinolone resistance involves amino acid substitutions in the quinolone resistance-determining region (QRDR) of GyrA and ParC and reduced fluoroquinolone accumulation in the cells (7-9). In a recent study, we showed that T80I substitution in GyrA is involved in low-level fluoroquinolone resistance in M. catarrhalis clinical isolates (10). However, the molecular mechanism of high-level fluoroquinolone resistance in M. catarrhalis is unknown. Therefore, we selected fluoroquinolone-resistant M. catarrhalis strains in vitro and identified the mechanism of fluoroquinolone resistance.To obtain fluoroquinolone-resistant M. catarrhalis strains, we performed stepwise selection in brain heart infusion (BHI; BD, Tokyo, Japan) agar plates, as previously described, with modifications (11). The ATCC 49143 reference strain was used as the parental strain (P1) and cultured on BHI agar plates containing levofloxacin at half of the previously determined MIC. The agar plates were then incubated at 35°C for 10 h
