gyrA and gyrB Mutations Are Implicated in Cross-Resistance to Ciprofloxacin and Moxifloxacin in Clostridium difficile

Abstract

A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n ‫؍‬ 100) and 1997 (n ‫؍‬ 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 g of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp713Val in one, Thr823Ile in six, and Ala1183Thr in one) or in gyrB (six mutations, amino acid changes Asp4263Asn in five and Arg4473Leu in one). These changes are similar to those already described in other species except for Asp713Val, which is novel, and Ala1183Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 g/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.Vancomycin and metronidazole are effective antibiotics for the treatment of Clostridium difficile-associated diarrhea (19,24). However, at least 15% of patients relapse after discontinuation of treatment for poorly understood reasons (23, 24). Other treatments may be more effective in preventing relapses.The in vitro activities against C. difficile of older fluoroquinolones like ciprofloxacin and ofloxacin are poor, but those of some new compounds like moxifloxacin are markedly increased (1,3,17,25). Thus, these drugs could become therapeutic alternatives for the treatment of C. difficile-associated diarrhea. Decreased susceptibility to fluoroquinolones in C. difficile has already been described (2, 3, 13, 26), but the data reported are controversial and the genetic mechanisms involved are poorly understood.In the present work, 198 French clinical strains of C. difficile were screened for decreased susceptibility to fluoroquinolones, and the genetic basis of this phenomenon was investigated. MATERIALS AND METHODSBacterial strains. Reference strain ATCC 9689 and 198 nonrepetitive clinical isolates obtained in 1991 (n ϭ 100) and 1997 (n ϭ 98) from 36 French hospitals (7) were studied. Serotyping of the strains had been performed in a previous study (7) by the method of Delmée et al. (12). Serogroups A, C, D, F, G, H, and K accounted for 9, 22, 8, 1, 4, 19, and 14% of the strains, respectively, and 23% of the strains were untypeable. Strains were cultured in...

Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018

“…In C. difficile, as in many other bacterial species, resistance is determined by amino acid substitutions in the quinolone resistance-determining region (QRDR) of the target enzymes (27). Since this bacterium does not have genes for topoisomerase IV, these alterations are located in the QRDR of either GyrA or GyrB, the DNA gyrase subunits (11,34). Recent studies indicated that the replacement of Thr82 with Ile in GyrA characterized 93% of European toxigenic C. difficile isolates resistant to fluoroquinolones, including the hypervirulent epidemic clone 027/NAP1/III (12,34).…”

Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile

“…The QRDRs of gyrB (nucleotides 1059-1448) and gyrA (nucleotides 71-460) were amplified by PCR as described previously [6]. To investigate the entire gyrBA region, beyond the defined QRDR, primers flanking the entire ORFs of gyrB and gyrA were designed.…”

“…In C. difficile, resistance is linked to a homoplasic Thr82Ile substitution in the quinolone resistance-determining region (QRDR) of the GyrA DNA gyrase subunit, a hallmark of globally disseminated 027/BI/NAP1 strains [5]. Several additional mutations that map to the QRDR of gyrA and gyrB have also been reported in C. difficile, whereas the type IV topoisomerase is absent in C. difficile as well as several other pathogenic species [6]. Molecular characterisation of fluoroquinolone resistance in C. difficile also suggests the existence of undetermined mechanisms among resistant strains lacking QRDR mutations [7,8].…”

Clostridium difficile Infection: Pathogenesis, Diagnosis and Treatment

“…Resistance of C. difficile to tetracycline varies in different countries, ranging from 2.4 to 41.67% [32]. Fry et al reported that although the tetracycline resistance gene tetM is predominant in C. difficile, others such as tetW are found in human and animal strains [33].…”

“…Alterations in the quinolone-resistance determining region (QRDR), which confers resistance to fluoroquinolones, have been identified in C. difficile [32]. Resistance of C. difficile to tetracycline varies in different countries, ranging from 2.4 to 41.67% [32].…”