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Antimicrobial Agents and Chemotherapy, June 2009, p. 2463-2468, Vol. 53, No. 6
0066-4804/09/$08.00+0 doi:10.1128/AAC.01252-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro
Patrizia Spigaglia,1
Fabrizio Barbanti,1
Thomas Louie,2
Frédéric Barbut,3 and
Paola Mastrantonio1*
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy,1 Department of Medicine, University of Calgary, Calgary, Alberta, Canada,2 National Reference Laboratory for Clostridium difficile, Microbiology Unit, Saint-Antoine Hospital, Assistance-Publique Hôpitaux de Paris, Paris, France3
Received 19 September 2008/ Returned for modification 4 December 2008/ Accepted 27 March 2009
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 x 10–6 to 6.6 x 10–5 for MX and from 1.0 x 10–6 to 2.4 x 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.
* Corresponding author. Mailing address: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39 06 49902335. Fax: 39 06 49387112. E-mail: paola.mastrantonio{at}iss.it
Published ahead of print on 13 April 2009.
Antimicrobial Agents and Chemotherapy, June 2009, p. 2463-2468, Vol. 53, No. 6
0066-4804/09/$08.00+0 doi:10.1128/AAC.01252-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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