1 Antibiotic Resistance Monitoring and Reference Laboratory and 3 Laboratory of HealthCare Associated Infection, Specialist and Reference Microbiology Division, Health Protection AgencyColindale, London NW9 5HT; 2 British Society for Antimicrobial Chemotherapy, Birmingham B1 2JS, UK
Received 29 May 2003; returned 30 June 2003; revised 3 July 2003; accepted 9 July 2003
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Abstract |
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Keywords: E. faecalis, epidemic strains, resistance
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Introduction |
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The purpose of this work was to investigate the basis of a strong association observed between HLGR and high-level ciprofloxacin resistance (MICs 32 mg/L) in isolates of Enterococcus faecalis collected in the UK and Ireland as part of the BSAC Bacteraemia Resistance Surveillance Programme, 2001. We sought to ascertain whether this relationship reflected clonal spread or, considered less likely, the emergence of linked transferable resistance.
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Materials and methods |
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During 2001, 24 laboratories in the UK and Ireland each collected up to 10 consecutive isolates of enterococci from bacteraemias as part of the BSAC Bacteraemia Resistance Surveillance Programme (http://www.bsacsurv.org). The isolates were all from separate patients. MICs of antibiotics, including gentamicin and ciprofloxacin, were determined centrally by the BSAC agar dilution method,6 and species identification was confirmed by amplification of ddlE.faecalis by PCR.7
A criterion of 512 mg/L was used to define HLGR in this study, as this level of resistance is likely to abrogate synergy between gentamicin and cell-wall-active agents, and usually indicates the presence of an acquired resistance gene (A. Sinclair & N. Woodford, unpublished data).
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA8 was carried out to investigate the relatedness of isolates. Banding patterns were analysed using BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium), with a criterion of 80% similarity to define strains.
Conjugal transfer of HLGR and ciprofloxacin resistance
Conjugative transfer of HLGR was investigated from selected donor isolates to E. faecalis recipient strain JH2-2, using a cross-streak plate method.9 Transconjugants were selected on Brain-Heart Infusion agar (Oxoid, Basingstoke, UK) containing gentamicin (200 mg/L), rifampicin (100 mg/L) and fusidic acid (25 mg/L), and were examined for increased ciprofloxacin MICs by Etest (Cambridge Diagnostics, Cambridge, UK).
Statistical methods
The association between high-level resistances to gentamicin and ciprofloxacin was tested with Fishers exact test (http://www.graphpad.com/quickcalcs/CatMenu.cfm).
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Results and discussion |
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A total of 223 enterococci were collected from the 24 hospitals in the BSAC Bacteraemia Resistance Surveillance Programme. Of these, 152 were identified as E. faecalis, 66 (43%) of them with HLGR (MICs 512 mg/L). Sixty of these 66 (91%) were also highly resistant to ciprofloxacin (MICs
32 mg/L; Table 1). In contrast, only three of 83 (4%) E. faecalis isolates with normal gentamicin susceptibility (MICs
128 mg/L) showed this high level of ciprofloxacin resistance (P < 0.0001). Isolates with high-level resistance to both gentamicin and ciprofloxacin were collected at 18 of the 24 participating laboratories. No such association was seen for isolates of other Enterococcus spp. collected during the survey.
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Thirty-eight representative isolates of E. faecalis, with both HLGR and high-level ciprofloxacin resistance were typed by PFGE. These isolates were from 18 hospitals, and typing revealed two large clusters (Figure 1). Cluster 1 included 14 isolates related at 84.8% similarity and isolated at seven hospitals. Cluster 2 included 10 isolates that were related at 83.3% similarity and isolated at six hospitals. Isolates in clusters 1 and 2 were not closely related (60% similarity). Isolates in both clusters were also resistant to erythromycin (MICs > 256 mg/L). Hospitals submitting isolates belonging to these clusters were scattered throughout the UK, and one cluster 2 isolate was collected in the Republic of Ireland. Only one hospital submitted both cluster 1 and cluster 2 isolates.
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Twenty-one comparator isolates without the double high-level resistance phenotype were also selected for PFGE. These were from 16 hospitals and comprised: six isolates with HLGR, but susceptibility to ciprofloxacin; seven with normal gentamicin susceptibility, but with high-level ciprofloxacin resistance; and eight with neither HLGR nor high-level ciprofloxacin resistance. Eighteen of these comparators belonged neither to cluster 1 nor to cluster 2, whilst three grouped with cluster 2 (Figure 1). One of these latter isolates was highly resistant to ciprofloxacin (MIC, 128 mg/L), but normally susceptible to gentamicin (MIC, 16 mg/L), and was collected at a hospital that submitted doubly-resistant cluster 2 isolates, implying that it might have lost, or not acquired, the gentamicin resistance gene; the two others were from a single hospital, and were highly resistant to ciprofloxacin (MICs, 128 mg/L), but showed gentamicin MICs of 256 mg/L. Although not meeting the criterion for HLGR, such MICs were substantially raised and implied the likely presence of an aminoglycoside-modifying enzyme. Indeed, both isolates contained the gene encoding AAC(6')-APH(2'') (data not shown). Isolates of E. faecalis that failed to match the formal criteria for HLGR despite possessing the gene for this enzyme have been isolated previously in the UK.10
Transfer of HLGR in vitro
HLGR was transferred to E. faecalis JH2-2 from two of four tested isolates in cluster 1, but from none of four tested isolates in cluster 2. None of the transconjugants derived from cluster 1 donors acquired ciprofloxacin resistance.
In conclusion, the association between HLGR and high-level ciprofloxacin resistance in E. faecalis from bacteraemia noted in the 2001 BSAC Bacteraemia Resistance Surveillance Programme reflected the dissemination of two strains with this phenotype, rather than the emergence of linked transferable resistance. One of these resistant strains was recovered at seven of 24 survey hospitals, and the other from six. Limited conjugation studies confirmed that HLGR was not genetically linked to ciprofloxacin resistance in isolates of cluster 1. Although transfer of HLGR from isolates of cluster 2 was not detected in vitro, dissociated resistance to ciprofloxacin and gentamicin was observed in some isolates of this cluster, discounting direct genetic linkage of the resistances.
Further work is needed to characterize these strains of E. faecalis and define more fully their epidemiology. E. faecalis has several defined virulence determinants,11 and their presence in the two strains reported here deserves investigation.
Note added in proof
After submission of this manuscript, similar dissemination of HLGR E. faecalis strains was reported in Sweden by Hällgren et al. 12
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Acknowledgements |
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Footnotes |
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References |
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