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

A total of 198 nonrepetitive clinical strains of Clostridiumdifficile isolated from different French hospitals in 1991 (n= 100) and 1997 (n = 98) were screened for decreased susceptibilityto fluoroquinolones by plating onto Wilkins-Chalgren agar containing16 µg of ciprofloxacin per ml. The frequency of decreasedsusceptibility was 7% (14 of 198) and was identical for theyears 1991 and 1997. Serogroups C, H, D, A9, and K accountedfor five, four, two, one, and one of the resistant strains,respectively, one strain being nontypeable. Arbitrarily primedPCR typing showed that all resistant strains had unique patternsexcept two serotype C strains, which could not be clearly distinguished.All isolates with decreased susceptibility carried a mutationeither in gyrA (eight mutations, amino acid changes Asp71 $->$ Valin one, Thr82 $->$ Ile in six, and Ala118 $->$ Thr in one) or in gyrB (sixmutations, amino acid changes Asp426 $->$ Asn in five and Arg447 $->$ Leuin one). These changes are similar to those already describedin other species except for Asp71 $->$ Val, which is novel, and Ala118 $->$ Thr,which is exceptional. Attempts to detect the topoisomerase IVparC gene by PCR amplification with universal parC primers orDNA-DNA hybridization under low-stringency conditions were unsuccessful.The susceptibilities of all resistant strains to ciprofloxacinand ethidium bromide were not affected by the addition of reserpineat 20 µg/ml. In conclusion, decreased susceptibility tofluoroquinolones in C. difficile is rare in France and is associatedwith the occurrence of a gyrA or gyrB mutation.

INTRODUCTION

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Introduction

Vancomycin and metronidazole are effective antibiotics for thetreatment of Clostridium difficile-associated diarrhea (19,24). However, at least 15% of patients relapse after discontinuationof treatment for poorly understood reasons (23, 24). Other treatmentsmay be more effective in preventing relapses.

The in vitro activities against C. difficile of older fluoroquinoloneslike ciprofloxacin and ofloxacin are poor, but those of somenew compounds like moxifloxacin are markedly increased (1, 3,17, 25). Thus, these drugs could become therapeutic alternativesfor the treatment of C. difficile-associated diarrhea. Decreasedsusceptibility to fluoroquinolones in C. difficile has alreadybeen described (2, 3, 13, 26), but the data reported are controversialand the genetic mechanisms involved are poorly understood.

In the present work, 198 French clinical strains of C. difficilewere screened for decreased susceptibility to fluoroquinolones,and the genetic basis of this phenomenon was investigated.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains. Reference strain ATCC 9689 and 198 nonrepetitive clinical isolatesobtained in 1991 (n = 100) and 1997 (n = 98) from 36 Frenchhospitals (7) were studied. Serotyping of the strains had beenperformed in a previous study (7) by the method of Delméeet al. (12). Serogroups A, C, D, F, G, H, and K accounted for9, 22, 8, 1, 4, 19, and 14% of the strains, respectively, and23% of the strains were untypeable. Strains were cultured inan anaerobic atmosphere at 37°C for 48 h on TCCA medium(brain heart agar supplemented with 5% defibrinated horse blood,0.1% taurocholate, 250 µg of cycloserine per ml, and 10µg of cefoxitin per ml).

Drug susceptibility testing. We first screened C. difficile strains for decreased susceptibilityto fluoroquinolones. The strains were grown at 37°C for24 h in prereduced Wilkins-Chalgren broth (Oxoid, Dardilly,France), 10⁵ CFU were applied on prereduced Wilkins-Chalgrenagar (Oxoid) containing 16 µg of ciprofloxacin per ml,and the plates were incubated for 48 h at 37°C in an anaerobicatmosphere. The inoculum, medium, and incubation time used werethose recommended for anaerobes by the Comité de l'Antibiogrammeof the Société Française de Microbiologie(11).

For the strains with decreased susceptibility as well as forreference strain ATCC 9689, MICs of ciprofloxacin, moxifloxacin,and certain drugs (ethidium bromide, acridine orange, safranin,and acriflavin) were determined by the agar dilution methodwith the same culture conditions and inoculum that were employedfor the screening. MICs of ciprofloxacin and ethidium bromidewere also determined in the presence of 20 µg of reserpine(ICN Pharmaceuticals, Orsay, France) per ml.

The susceptibilities of these strains to other antibiotics (chloramphenicol,erythromycin, tetracycline, and rifampin) had been determinedin a previous study with the ATB ANA susceptibility test (bioMérieux,Marcy l'Etoile, France) (7). Breakpoints used were those recommendedby the Comité de l'Antibiogramme of the SociétéFrançaise de Microbiologie (11).

DNA amplification by PCR and sequencing. For amplification of the quinolone resistance-determining regionsof the gyrA and gyrB genes, oligonucleotide primers were designedfrom the sequence of the genome of C. difficile strain 630 (http://www.sanger.ac.uk/Projects/C_difficile/blast_server.shtml).Primers 5'-AATGAGTGTTATAGCTGGACG-3' and 5'-TCTTTTAACGACTCATCAAAGTT-3'were used to amplify a 390-bp fragment of gyrA from nucleotidepositions 71 to 460. Primers 5'-AGTTGATGAACTGGGGTCTT-3' and5'-TCAAAATCTTCTCCAATACCA-3' were used to amplify a 390-bp fragmentof gyrB from nucleotide positions 1059 to 1448.

The sequences of both strands were determined by the dideoxychain termination method with the primers used for PCR and theautomatic DNA sequencer ABI Prism 373A (Applied Biosystems,Courtaboeuf, France).

Strain genotyping by AP-PCR. Chromosomal DNA was extracted as described above. Arbitrarilyprimed PCR (AP-PCR) was performed with primer AP3 (5'-TCACGATGCA-3')or AP4 (5'-TCACGCTGCA-3'), as described previously (9).

DNA-DNA hybridization. Genomic DNAs from clinical isolate C. difficile 698 and referencestrain Escherichia coli JM109 were obtained by phenol-chloroformextraction and digested with restriction enzymes HindIII andEcoRI (New England Biolabs, Saint-Quentin en Yvelines, France),respectively. Digested DNAs were transferred after electrophoresisto a nylon membrane and hybridized with [³²P]dCTP-labeled PCR-amplifiedDNA fragments used as probes: a 232-bp fragment of gyrA fromE. coli (4), a 343-bp fragment of parC from E. coli (4), andthe above-described 390-bp fragment of gyrA from C. difficile.

RESULTS AND DISCUSSION

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Results and Discussion

Decreased susceptibility to fluoroquinolones: frequency and typing of the strains. The frequency of decreased susceptibility to fluoroquinolones,as defined by an MIC of ciprofloxacin higher than 16 µg/ml,was 7% (14 of 198) and was identical for the years 1991 and1997. The cutoff value of 16 µg/ml was chosen becausethe MIC that inhibited 50% of strains (MIC₅₀) of ciprofloxacinfor C. difficile is 8 to 16 µg/ml (3, 24, 25). All 14strains also showed decreased susceptibility to moxifloxacin(Table 1). Thus, decreased susceptibility to fluoroquinolonesin C. difficile remains relatively rare in our country (7%),and our results are in agreement with data from Sweden, theNetherlands, and the United Kingdom (13, 17, 26).

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TABLE 1. Phenotypic and genotypic characteristics of the fluoroquinolone-resistant strains of C. difficile studied^a

However, two studies have reported contradictory results. InGermany (2), 26% (19 of 72) decreased susceptibility to moxifloxacin(MIC > 8 µg/ml) was found in isolates recovered frompatients or from the hospital or public environments, but someof the strains were genotypically related. In Spain (3), 30%(34 of 113) of the clinical isolates collected from one hospitalhad decreased susceptibility to ciprofloxacin (MIC > 16 µg/ml).However, no typing of the isolates was performed, and the isolateswere collected over a short period (6 months) and could thuscorrespond to only a few clones.

In our work, 9 of the 14 strains with decreased susceptibilitybelonged to two serotypes, C (five strains) and H (four strains)(Table 1). However, these two serotypes were also the most frequentlyobserved among the 198 study strains. The remaining five strainsbelonged to serotypes D (two) and A9 and K (one each), the lastbeing untypeable. All 14 strains could be clearly distinguishedby AP-PCR except for two serotype C strains, 580 and 602 (datanot shown). The patterns observed for these strains with primersAP3 and AP4 were different by only two bands and identical,respectively.

Detection of gyrA and gyrB mutations. The quinolone resistance-determining regions of gyrA and gyrBwere sequenced from nucleotide codons 40 to 145 and 366 to 473,respectively. A mutation in gyrA was present in eight strains,leading to the substitutions Thr82 $->$ Ile (six strains), Asp71 $->$ Val(one strain), and Ala118 $->$ Thr (one strain). The remaining sixstrains carried a mutation in gyrB leading to the alterationsAsp426 $->$ Asn (five strains) and Arg447 $->$ Leu (one strain) (Table 1).

Thr82 corresponds to Ser83 in E. coli, and that position isknown to play a key role in fluoroquinolone resistance in everyspecies (18). In C. difficile, 14 of the 19 resistant strainsinvestigated by Ackermann et al. (2) carried a mutation at codon82 of gyrA, leading to the same Thr82 $->$ Ile substitution in 13cases and to Thr82 $->$ Val in the last case.

Asp71 $->$ Val corresponds to a change at position 72 of GyrA fromE. coli, which has not yet been associated with fluoroquinoloneresistance in any species. It is located in the quinolone resistance-determiningregion and is very likely to be implicated in resistance, butwe do not have direct proof of that. Ala118 $->$ Thr corresponds toa change at position 119, and other alterations at that positionhave occasionally been reported in both GyrA and ParC: Ala $->$ Valin GyrA from Bacteroides fragilis (5) and Salmonella entericaserovar Typhimurium (16); Ala $->$ Pro or Glu in ParC from Staphylococcusaureus (20) and Streptococcus pneumoniae (8). The role in resistanceof this mutation has been demonstrated in S. aureus by cloningthe mutated parC gene into a shuttle vector and introducingit into a wild-type S. aureus strain (14).

The GyrB changes detected in C. difficile are, compared to thosedescribed in E. coli, either identical (Asp426 $->$ Asn) or locatedat the same position (Asp447 $->$ Lys for E. coli) (18). Their frequentoccurrence is surprising because in other species gyrB mutationshave been observed mainly in laboratory mutants (18). However,four of the five gyrB mutations that we found were identicaland concerned serotype C strains isolated from the same hospitalbetween February and December 1991. As already mentioned, twoof these isolates appeared to be genetically related, but thepatterns obtained by AP-PCR for the other two isolates weredifferent. However, it cannot be excluded that these four mutantsin fact derive from a common ancestor.

Search for other resistance mechanisms. We did not find any gene with homology to known parC genes inthe genome of C. difficile 630. PCR experiments with degenerateprimers deduced from conserved regions of the published parCgenes from other bacterial species (21) were also unsuccessful,as was the case for others (2). Low-stringency DNA-DNA hybridizationwith probes specific for gyrA and parC of E. coli did allowus to detect the gyrA gene of C. difficile but not a putativeparC gene. Taken together, these data suggest that C. difficile,like Mycobacterium tuberculosis (10), Helicobacter pylori (22),Campylobacter jejuni (6), and Treponema pallidum (15), lacksgenes for topoisomerase IV.

We also determined the MICs of certain compounds that can besubstrates for efflux pumps. The MICs of acridine orange, acriflavin,ethidium bromide, and safranin for all strains were similar:256 to 1024, 2 to 8, 8 to 32, and 128 to 256 µg/ml, respectively.Furthermore, the MICs of ciprofloxacin and ethidium bromidefor all strains were either unchanged or decreased only twofoldafter addition of reserpine. Some of our strains were coresistantto chloramphenicol, erythromycin, and tetracyclines (Table 1),but these multiresistant strains have been found, by DNA-DNAhybridization and PCR amplification experiments, to carry resistancegenes which are not implicated in efflux, catD, ermB, and tetM,respectively, (N. Hidri, D. Decré, M. Chosson, B. Burghoffer,F. Barbut, and J.-C. Petit, submitted for publication). Thus,the presence of active efflux seems unlikely in the strainsstudied.

In conclusion, our work shows that decreased susceptibilityto both ciprofloxacin and moxifloxacin already exists in Frenchisolates of C. difficile but remains relatively rare and isstrongly associated with the presence of a gyrA or gyrB mutation.

ACKNOWLEDGMENTS

We thank Guillaume Arlet helpful discussions and VéroniqueBarbu for technical assistance.

This research was supported by grants from INSERM (PARMIFR 9609)and the UPRES EA2392 Research Group on Clostridium difficile.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Bactériologie, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75 571 Paris Cedex 12, France. Phone: 33 (1) 49 28 29 10. Fax: 33 (1) 49 28 24 72. E-mail: jacques.tankovic{at}sat.ap-hop-paris.fr.

REFERENCES

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