Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10⁻⁶ to 6.6 × 10⁻⁵ for MX and from 1.0 × 10⁻⁶ to 2.4 × 10⁻⁵ for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.Recent outbreaks of Clostridium difficile infections (CDI), with increased severity, high relapse rates, and significant mortality, have been related to the emergence of the hypervirulent C. difficile clone toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 (5, 23, 25-29, 31). Several studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of CDI caused by C. difficile III/027/NAP1 and that the acquisition of resistance to the newer fluoroquinolones could have promoted its wide dissemination (6, 17, 30, 32-34).Fluoroquinolones are a family of broad-spectrum antibiotics extensively used in the treatment of a great variety of human infections. The in vitro activity of the older fluoroquinolones, such as ciprofloxacin, has been reported to be moderate or poor against anaerobes, including C. difficile (3, 8), whereas the third and the fourth generations of fluoroquinolones are characterized by improved activity against gram-positive cocci and anaerobic bacteria (19, 36). Fluoroquinolones act by inhibiting the action of DNA gyrase and topoisomerase IV, which are related but distinct enzymes involved in DNA synthesis (18). The mechanisms of resistance to fluoroquinolones in bacteria are basically two: (i) alterations in the targets of fluoroquinolones and (ii) decreased accumulation inside the bacteria due to impermeability of the membrane and/or an overexpression of efflux pump systems (19, 20, 36). The first mechanism of resistance is widespread in many bacteria, and it is due to amino acid substitutions in the quinolone-resistance determining region (QRDR) of the target enzymes (35). This is the principal mechanism of resistance also in C. difficile, and since, as already observed in other species, this bacterium does not have genes for topoisomerase IV, resistance is determined by alterations in the QRDR of either DNA gyrase subunit GyrA or GyrB (10, 38).Different amino acid substitutions have been identified in GyrA and GyrB in fluoroquinolone-resistant C. difficile strains. The most frequent is the amino acid change Thr82 to Ile in GyrA, which also characterizes the epidemic clone III/027/NAP1 (11, 38). Two other GyrA substitutions, Asp71 to Val and Ala118 to Thr, have been more rarely observed (1, 2, 10, 12, 38). Four different amino acid substitutions have been identified in GyrB: Arg447 to Lys, Arg447 to Leu, Asp426 to Asn, and Asp426 to Val (10, 11, 38). In particular, Asp426 to Val has been described in toxin A-negative/toxin B-positive C. difficile epidemic strains of recent isolation (11).In this study, we evaluated the potential of moxifloxacin (MX) and levofloxacin (LE), recently associated with outbreaks caused by C. difficile III/027/NAP1 (25, 31, 33), for the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations in five different susceptible C. difficile strains. The sequence changes occurring in the QRDR of the derived fluoroquinolone-resistant mutants were analyzed and correlated with the in vitro resistance to MX, LE, and gatifloxacin (GA), another fluoroquinolone recently involved in C. difficile outbreaks (17, 33).