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JAC Advance Access originally published online on August 13, 2003
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Journal of Antimicrobial Chemotherapy (2003) 52, 711-714
© 2003 The British Society for Antimicrobial Chemotherapy

Two widely disseminated strains of Enterococcus faecalis highly resistant to gentamicin and ciprofloxacin from bacteraemias in the UK and Ireland

Neil Woodford1,*, Rosy Reynolds2, Jane Turton3, Fiona Scott3, Alistair Sinclair1, Andrea Williams1 and David Livermore1

1 Antibiotic Resistance Monitoring and Reference Laboratory and 3 Laboratory of HealthCare Associated Infection, Specialist and Reference Microbiology Division, Health Protection Agency—Colindale, 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


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
A strong association was observed between high-level resistance to ciprofloxacin and gentamicin for isolates of Enterococcus faecalis collected in the UK and Ireland as part of the BSAC Bacteraemia Resistance Surveillance Programme, 2001. Thus, 60 of 66 E. faecalis isolates with gentamicin MICs >= 512 mg/L were highly resistant to ciprofloxacin (MICs >= 32 mg/L), compared with only three of 83 E. faecalis isolates with normal gentamicin susceptibility (MICs <= 128 mg/L) (P < 0.0001). Pulsed-field gel electrophoresis of SmaI-digested genomic DNA was used to investigate 38 representative E. faecalis isolates with the double resistance from 18 hospitals. Based on the criterion of >=80% banding pattern similarity, two large clusters were observed: cluster 1 included 14 isolates, from seven hospitals, that were related at 84.8% similarity; cluster 2 included 10 isolates, from six hospitals, that were related at 83.3% similarity. Sporadic isolates and small clusters with the double resistance were also observed, but were not closely related to those in clusters 1 and 2. Further work is needed to characterize these two ‘epidemic’ E. faecalis strains and to investigate the presence of virulence genes.

Keywords: E. faecalis, epidemic strains, resistance


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
High-level gentamicin resistance (HLGR) in enterococci, which is defined by the BSAC as MICs >= 1024 mg/L,1 is usually mediated by the bifunctional aminoglycoside-modifying enzyme, AAC(6')-APH(2'').2 The gene encoding this enzyme may be associated with transposable elements located on plasmids, or integrated into the chromosome; in the former case, HLGR is often transferable between strains. In contrast, high-level fluoroquinolone resistance is most often associated with chromosomal mutations in gyrA and/or parC, which encode subunits of DNA gyrase and topoisomerase IV, respectively.3 There are isolated reports of plasmid-mediated fluoroquinolone resistance in Enterobacteriaceae, and of the transformation of pneumococci by resistant gyrA and parC sequences,4 but transferable fluoroquinolone resistance has not been reported in enterococci. However, ‘non-transferable’ chromosomal resistance markers in enterococci can occasionally be mobilized during transposition events, if transposons are inserted into flanking DNA.5

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.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Bacteria

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 Fisher’s exact test (http://www.graphpad.com/quickcalcs/CatMenu.cfm).


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Association between HLGR and ciprofloxacin resistance

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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  		Table 1.. Gentamicin and ciprofloxacin susceptibilities for isolates of E. faecalis collected during the 2001 BSAC Bacteraemia Resistance Surveillance Programme
 
Typing of resistant E. faecalis isolates

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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  		Figure 1. Dendrogram showing the relatedness of E. faecalis isolates from bacteraemias as determined by PFGE. Isolates with high-level resistance to gentamicin (Gent) and ciprofloxacin (Cipro) belonging to cluster 1 and cluster 2 are indicated by the filled grey circle and grey bar and by the black circle and black bar, respectively. Smaller black and white symbols indicate other isolates belonging to smaller clusters, based on >=80% similarity. Brackets show pairs of ciprofloxacin-resistant isolates, from two hospitals, that differed in HLGR status. MICs are shown in mg/L.

 
Isolates with both HLGR and high-level ciprofloxacin resistance did not belong exclusively to clusters 1 and 2. Four other strains were identified, each containing small numbers of isolates related to each other at >=80% similarity. Some sporadic isolates also showed this double resistance phenotype (Figure 1).

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


   Acknowledgements
 
We wish to thank Katharine Grant for her considerable contribution to this work. The project would not have been possible without the BSAC Bacteraemia Resistance Surveillance Programme, supported in 2001 by Merck, Pharmacia and Wyeth. We are grateful to them, to the BSAC, and to the hospitals that contributed isolates. This work was presented at the 13th ECCMID, Glasgow, UK in May 2003.


   Footnotes
 
* Corresponding author. Tel: +44-20-8200-4400; Fax: +44-20-8358-3292; E-mail: neil.woodford{at}hpa.org.uk Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Andrews, J. M. for the BSAC Working Party On Susceptibility Testing. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

2 . Chow, J. W. (2000). Aminoglycoside resistance in enterococci. Clinical Infectious Diseases 31, 586–9.[CrossRef][Web of Science][Medline]

3 . Onodera, Y., Okuda, J., Tanaka, M. et al. (2002). Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV of Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 46, 1800–4.[Abstract/Free Full Text]

4 . Ruiz, J. (2003). Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. Journal of Antimicrobial Chemotherapy 51, 1109–17.[Abstract/Free Full Text]

5 . Carias, L. L., Rudin, S. D., Donskey, C. J. et al. (1998). Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate. Journal of Bacteriology 180, 4426–34.[Abstract/Free Full Text]

6 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract]

7 . Dutka-Malen, S., Evers, S. & Courvalin, P. (1995). Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. Journal of Clinical Microbiology 33, 24–7.[Abstract]

8 . Woodford, N., Morrison, D., Johnson, A. P. et al. (1995). Plasmid-mediated vanB glycopeptide resistance in enterococci. Microbial Drug Resistance 1, 235–40.

9 . Woodford, N., Morrison, D., Cookson, B. et al. (1993). Comparison of high-level gentamicin-resistant Enterococcus faecium isolates from different continents. Antimicrobial Agents and Chemotherapy 37, 681–4.[Abstract/Free Full Text]

10 . Weinbren, M. J., Johnson, A. P. & Woodford, N. (2000). Defining high-level gentamicin resistance in enterococci. Journal of Antimicrobial Chemotherapy 45, 404–5.[Free Full Text]

11 . Eaton, T. J. & Gasson, M. J. (2001). Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Applied and Environmental Microbiology 67, 1628–35.[Abstract/Free Full Text]

12 . Hällgren, A., Saeedi, B., Nilsson, M. et al. (2003). Genetic relatedness among Enterococcus faecalis with transposon-mediated high-level gentamicin resistance in Swedish intensive care units. Journal of Antimicrobial Chemotherapy 52, 162–7.[Abstract/Free Full Text]


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