JAC Advance Access originally published online on October 3, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1264-1267; doi:10.1093/jac/dkl398
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
High-level resistance to moxifloxacin and gatifloxacin associated with a novel mutation in gyrB in toxin-A-negative, toxin-B-positive Clostridium difficile
1 Centre for Food Safety, School of Agriculture Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland 2 Department of Medicine for the Older Person Mater Misericordiae University Hospital, Dublin 7, Ireland 3 UCD Conway Institute of Biomolecular and Biomedical Research University College Dublin, Dublin 4, Ireland
*Corresponding author. Tel: +353-1-716-6268; Fax: +353-1-716-6091; E-mail: denise.drudy{at}ucd.ie
Received 16 June 2006; returned 24 July 2006; revised 22 August 2006; accepted 11 September 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Objectives: To determine the mechanism of high-level resistance to fluoroquinolone antimicrobials in toxin-A-negative, toxin-B-positive (A–B+) Clostridium difficile isolates.
Methods: Following culture 16–23S PCR ribotyping was used to determine genomic relationships between A–B+ C. difficile isolates. Antimicrobial susceptibilities were determined using Etests in the presence and absence of the efflux pump inhibitors reserpine (20 µg/mL), L-phenylalanine-L-arginine-ß-naphthylamide (PAßN; 20 µg/mL) and verapamil (100 µg/mL). Genomic regions including the quinolone-resistance-determining-region (QRDR) of gyrA and gyrB were amplified and characterized.
Results: PCR ribotyping profiles identified one major cluster of A–B+ C. difficile, universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin; MICs > 32 mg/L). All isolates with high-level resistance had a transversion mutation (A
T) resulting in the amino acid substitution Asp-426Val in gyrB. Non-clonal isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3 and 0.4 mg/L, respectively) with reduced susceptibility to levofloxacin (MIC 3 mg/L) consistent with the wild-type genotype. The MICs for resistant isolates were not significantly affected by the addition of any of the efflux pump inhibitors. No amino acid substitutions were identified in the QRDR of gyrA.
Conclusions: High-level resistance to fluoroquinolones in A–B+ C. difficile is associated with a novel transversion mutation in gyrB. The emergence of universal resistance in different C. difficile strain types may be a factor promoting outbreaks in hospitals.
Keywords: fluoroquinolone resistance , A–B+ C. difficile , transversion mutation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Clostridium difficile is a major cause of bacterial diarrhoea in the developed world.1 Patients in hospital receiving antibiotics are the most at risk. Certain classes of antimicrobials have been associated with a high risk of C. difficile-associated disease (CDAD) including clindamycin, cephalosporins and ampicillin/amoxicillin.1 Historically, fluoroquinolone antimicrobials were considered low risk for CDAD, although a number of case reports associated with ciprofloxacin exposure have been published.2 However, recent studies indicate a shift in the risk associated with the use of fluoroquinolone antimicrobials.3–5
The risk of antibiotic-associated CDAD appears to increase if C. difficile is resistant to the administered antibiotic. A number of outbreaks in different states in the United States were associated with a clonal clindamycin-resistant strain, wherein this drug was identified as a specific risk factor.6 More recently, an investigation into an outbreak caused by the hyper-virulent fluoroquinolone-resistant strain (PCR-027/NAP1) in the Quebec region of Canada indicated that fluoroquinolone antimicrobials were more likely to induce CDAD.4 Furthermore, Muto et al.5 reported that exposure to levofloxacin was an independent risk factor for CDAD in a large outbreak in Pittsburgh.
A recent outbreak at one Dublin hospital identified the emergence of a clonal toxin-A-negative, toxin-B-positive (A–B+) C. difficile strain type demonstrating high-level resistance to several fluoroquinolone antibiotics. A second investigation identified these strains in a number of additional healthcare settings in Dublin. The purpose of this study was to determine the contribution of target gene mutations and active efflux to fluoroquinolone resistance in these isolates.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Bacterial strains and culture
Seventy C. difficile strains were studied. These included 30 representative C. difficile strains that were collected during surveillance of a hospital outbreak in 2003, 10 isolates collected from that institution prior to the outbreak and 30 isolates collected from six healthcare settings in Dublin in 2004. C. difficile strains were cultured on cycloserine-cefoxitin-fructose agar (CCFA). Type strains VPI10463 and 1470 along with a non-toxigenic strain (R10567 [GenBank] ) were included as controls.
Antibiotic susceptibility of C. difficile
Fluoroquinolone susceptibility was determined using Etests (AB Biodisk, Solna, Sweden). The MIC was interpreted by the Clinical and Laboratory Standards Institute (CLSI) breakpoints for trovofloxacin (
2 mg/L, susceptible; 4 mg/L, intermediate; 
8 mg/L, resistant).
Molecular analysis of C. difficile strains
Genomic DNA was extracted from broth cultures of C. difficile and PCR ribotyping and PCR–RFLP (toxinotyping) were performed as previously described.
PCR amplification and sequencing of the quinolone-resistance-determining-region (QRDR) of gyrA and gyrB
Primers GyrAF (TTG AAA TAG CGG AAG AAA TGA), GyrAR (TTG CAG CTG TAG GGA AAT C), GyrBF (GAA GGT CAA ACT AAA ACA AA) and GyrBR (GGG CTC CAT CTA CAT CAG) were designed from the sequence of the C. difficile 630 genome and were used to amplify 633 and 514 bp amplicons from gyrA and gyrB, respectively (http://www.sanger.ac.uk/Projects/C_difficile/blast_server.shtml). Amplicons were purified using a QIAquick PCR purification kit (Qiagen, GmbH, Germany) and sequenced commercially by Qiagen. Clustal W amino acid sequence alignments were produced for comparison.
Nucleotide sequence accession numbers
The nucleotide sequence data for partial sequences of the gyrB gene were submitted to GenBank and were assigned the accession numbers DQ642011 [GenBank] , DQ642012 [GenBank] and DQ642013 [GenBank] , respectively.
Efflux contribution
Ten representative isolates (Table 1) were chosen for efflux studies. The MICs of all five antibiotics were determined using Etest strips, in the presence and absence of the following efflux pump inhibitors: reserpine (20 µg/mL); L-phenylalanine-L-arginine-ß-naphthylamide (20 µg/mL); and verapamil (100 µg/mL). Concentrations of PAßN and reserpine up to 80 µg/mL, and verapamil up to 800 µg/mL, were used to determine the effect (if any) of these inhibitors on bacterial growth.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
Antimicrobial susceptibility patterns and efflux contribution
Sixty C. difficile were universally resistant to all five fluoroquinolones tested (MICs > 32 mg/L). Control isolates VPI10463, 1470 and R10567 [GenBank] and 10 clinical isolates were susceptible to moxifloxacin and gatifloxacin (MICs of 0.3 and 0.4 mg/L, respectively) and showed reduced susceptibility to levofloxacin (MIC 3 mg/L). These strains were resistant to ciprofloxacin and ofloxacin. The MICs for representative isolates are shown in Table 1. The addition of efflux pump inhibitors had no effect on MICs or on bacterial growth of any of the isolates investigated (data not shown).
16–23S PCR ribotyping
16–23S ribotyping identified one major cluster containing 60 A–B+ isolates. Toxinotyping confirmed that these isolates were toxinotype VIII (PCR-017) (data not shown). The remaining ten clinical isolates belonged to two different ribotype groups (data not shown).
Amplification and sequence analysis of gyrA and gyrB
Amplicons from gyrA and gyrB were sequenced for 10 representative isolates (Table 1). Sequence analysis of the QRDR of gyrA revealed a number of silent mutations. Deduced sequence analysis of the QRDR of gyrB revealed an Asp-426Val amino acid substitution at codon 426 in all resistant isolates. A Ser-366Ala substitution was found in all A–B+ C. difficile (data not shown). As this substitution was found in clinical strains resistant to moxifloxacin, gatifloxacin and levofloxacin and in the 1470 type strain that was susceptible to moxifloxacin and gatifloxacin, it is unlikely to contribute to the fluoroquinolone-resistant phenotype. No amino acid substitutions were identified in any of the susceptible A+B+ clinical isolates or control strains R10567
[GenBank]
and VPI10463.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
This study identified an A–B+ clone of C. difficile resistant to five fluoroquinolone antimicrobials. The ß-subunit of DNA gyrase is the primary target wherein a single nucleotide transversion resulted in a novel Asp
Val substitution at codon 426. This is the first report of gyrB mutations in this strain type.
Fluoroquinolone resistance associated with chromosomal mutations in gyrA and gyrB has been previously described in C. difficile. Ackermann et al.7 described two substitutions in gyrA corresponding to codon 83 in Escherichia coli. Thirteen of 18 isolates studied had a substitution corresponding to Thr-83Ile while one strain had a Thr-83Val substitution. Dridi et al.8 described mutations in gyrA and gyrB associated with fluoroquinolone resistance in several serogroups of C. difficile. The substitutions in gyrA included Thr-83Ile in six strains (serogroups H1, A9 and 1C), an Asp-71Val substitution in one strain (serogroup H) and an Ala-118Thr substitution in one serogroup D strain. Two substitutions in gyrB, including an Arg-447Leu substitution in a single non-typeable isolate and an Asp-426Asn substitution in five isolates (4 serogroup C and 1 serogroup K), were described.
Codon Asp-426 has been highlighted as a critical region in gyrB and mutations corresponding to this codon have been associated with fluoroquinolone resistance in E. coli, Staphylococcus aureus and Streptococcus pneumoniae.9 In C. difficile, Dridi et al.8 described an Asp-426Asn substitution in five isolates with MICs of moxifloxacin of 8 or 16 mg/L. Substitution of Asp with Asn results in the replacement of a negatively charged polar hydrophilic amino acid with an uncharged polar residue. In this study, Asp is substituted with valine at position 426 in fluoroquinolone-resistant A–B+ C. difficile isolates. This results in the introduction of a non-polar side chain and the loss of negative charge. Moreover, as valine is a branched chain amino acid it can add bulk to the protein backbone thereby restricting conformational flexibility. It is tempting to speculate that Val-426 may alter the shape of the drug-binding pocket more significantly than Asn thereby producing a more extreme phenotype, as measured by MIC. This may explain the high-level resistance encountered in the A–B+ C. difficile strains in this study.
Increased fluoroquinolone resistance associated with newer groups of fluoroquinolones with increased anti-anaerobic activity has been described.9 Ackerman et al.10,11 reported C. difficile moxifloxacin resistance rates of 12% and 50% in two studies where the majority of resistant isolates clustered into two clonal groups. None of the patients with moxifloxacin-resistant C. difficile strains had received this antibiotic. Similarly, in this study neither moxifloxacin nor gatifloxacin was used clinically. This highlights the possibility that resistance to the newer antimicrobials may result from mechanisms that were acquired or evolved following exposure to older fluoroquinolone antimicrobials.
Isolates demonstrating high-level resistance to fluoroquinolones in this study were clonal. These A–B+ strains were isolated from patients from three different university hospitals along with three community-acquired specimens indicating the emergence and dissemination of this strain type throughout the greater Dublin area. Wilcox et al.12 have previously highlighted the importance of typing isolates when describing rates of resistance. It is possible that acquired antimicrobial resistance may be at least one mechanism that contributed to the selection and proliferation of this strain type in an environment where fluoroquinolones are frequently used.
In conclusion, we report a novel transversion mutation in gyrB associated with high-level fluoroquinolone resistance in C. difficile PCR-017. Efflux pump activity does not appear to contribute to the resistance phenotype. Emergence of resistance to fluoroquinolones in C. difficile strains may be a factor that contributes to the persistence and dissemination of strain types in the hospital environment. Fluoroquinolone use is now a recognized high risk of CDAD; therefore careful use of this antibiotic class needs to be encouraged as part of infection control policies to reduce C. difficile disease.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
The cooperation of our colleagues in the Medical Microbiology Departments of the Mater Misericordiae University Hospital, St Vincents' University Hospital and St James University Hospital with regard to sample collection is gratefully acknowledged. We acknowledge the financial support provided by The Health Research Board, Ireland (RP/2005/72), The Mater Foundation and The Newman Scholarship Programme—University College Dublin.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
|---|
1 Kyne L, Farrell RJ, Kelly CP. (2001) Clostridium difficile. Gastroenterol Clin North Am 30:753–77.[CrossRef][Web of Science][Medline]
2 Cain DB and O'Connor ME. (1990) Pseudomembranous colitis associated with ciprofloxacin. Lancet 336:946.[Web of Science][Medline]
3 McCusker ME, Harris AD, Perencevich E, et al. (2003) Fluoroquinolone use and Clostridium difficile-associated diarrhea. Emerg Infect Dis 9:730–3.[Web of Science][Medline]
4 Pepin J, Saheb N, Coulombe MA, et al. (2005) Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 41:1254–60.[CrossRef][Web of Science][Medline]
5 Muto CA, Pokrywka M, Shutt K, et al. (2005) A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol 26:273–80.[CrossRef][Web of Science][Medline]
6
Johnson S, Samore MH, Farrow KA, et al. (1999) Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. N Engl J Med 341:1645–51.
7
Ackermann G, Tang YJ, Kueper R, et al. (2001) Resistance to moxifloxacin in toxigenic Clostridium difficile isolates is associated with mutations in gyrA. Antimicrob Agents Chemother 45:2348–53.
8
Dridi L, Tankovic J, Burghoffer B, et al. (2002) gyrA and gyrB mutations are implicated in cross-resistance to ciprofloxacin and moxifloxacin in Clostridium difficile. Antimicrob Agents Chemother 46:3418–21.
9 Hooper DC. (1999) Mechanisms of fluoroquinolone resistance. Drug Resist Updat 2:38–55.[CrossRef][Web of Science][Medline]
10
Ackermann G, Degner A, Cohen SH, et al. (2003) Prevalence and association of macrolide-lincosamide-streptogramin B (MLSB) resistance with resistance to moxifloxacin in Clostridium difficile. J Antimicrob Chemother 51:599–603.
11 Ackermann G, Tang-Feldman YJ, Schaumann R, et al. (2003) Antecedent use of fluoroquinolones is associated with resistance to moxifloxacin in Clostridium difficile. Clin Microbiol Infect 9:526–30.[CrossRef][Web of Science][Medline]
12
Wilcox MH, Fawley W, Freeman J, et al. (2000) In vitro activity of new generation fluoroquinolones against genotypically distinct and indistinguishable Clostridium difficile isolates. J Antimicrob Chemother 46:551–6.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Spigaglia, F. Barbanti, T. Louie, F. Barbut, and P. Mastrantonio Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2463 - 2468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. Critchley, L. S. Green, C. L. Young, J. M. Bullard, R. J. Evans, M. Price, T. C. Jarvis, J. W. Guiles, N. Janjic, and U. A. Ochsner Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections J. Antimicrob. Chemother., May 1, 2009; 63(5): 954 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Saxton, S. D. Baines, J. Freeman, R. O'Connor, and M. H. Wilcox Effects of Exposure of Clostridium difficile PCR Ribotypes 027 and 001 to Fluoroquinolones in a Human Gut Model Antimicrob. Agents Chemother., February 1, 2009; 53(2): 412 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wright, D. Drudy, L. Kyne, K. Brown, and N. F. Fairweather Immunoreactive cell wall proteins of Clostridium difficile identified by human sera J. Med. Microbiol., June 1, 2008; 57(6): 750 - 756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Spigaglia, F. Barbanti, P. Mastrantonio, J. S. Brazier, F. Barbut, M. Delmee, E. Kuijper, I. R. Poxton, and on behalf of the European Study Group on Clostridi Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe J. Med. Microbiol., June 1, 2008; 57(6): 784 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kim, T. V. Riley, M. Kim, C. K. Kim, D. Yong, K. Lee, Y. Chong, and J.-W. Park Increasing Prevalence of Toxin A-Negative, Toxin B-Positive Isolates of Clostridium difficile in Korea: Impact on Laboratory Diagnosis J. Clin. Microbiol., March 1, 2008; 46(3): 1116 - 1117. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J Mehlhorn and D. A Brown Safety Concerns with Fluoroquinolones Ann. Pharmacother., November 1, 2007; 41(11): 1859 - 1866. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||