Molecular characterization of hospital vancomycin-resistant Enterococcus faecalis isolated in Slovakia

Helena Bujdáková1,*, Miriam Filipová1, Anna Lísková2 and Hana Drahovská3

1 Comenius University, Faculty of Natural Sciences, Department of Microbiology and Virology, Mlynská dolina B-2, 842 15 Bratislava; 2 Institute of Clinical Microbiology, Hospital Nitra; 3 Comenius University, Faculty of Natural Sciences, Department of Molecular Biology, Bratislava, Slovak Republic

Keywords: Enterococcus, vancomycin, resistance, VRE

Sir,

Vancomycin-resistant enterococci (VRE) represent a reservoir of glycopeptide resistance in hospital patients, especially in high-risk wards.1 In Slovakia, the incidence of VRE has not yet been published, although Bíliková et al.2 reported the participation of enterococci, with susceptibility to vancomycin over 16 mg/L, in bacteraemias in six Slovak hospitals. Thus, monitoring of glycopeptide resistance in enterococci is crucial because of possible transmission of van genes in the enterococcal population.

This report, to our knowledge, is the first molecular characterization of hospital vancomycin-resistant Enterococcus faecalis strain 605 isolated in Slovakia. The strain was obtained from a patient with chronic nephritis after repeated treatment for Escherichia coli infection with a second-generation cephalosporin and with quinolones (ciprofloxacin and pefloxacin). Subsequently, infection appeared with significant bacteriuria caused by enterococci. The patient was treated with vancomycin (1000 mg per day, serum level measured AxSYM, Abbott Laboratories) for 7 days. After this therapy, the patient was found to be colonized with VRE. Resistance to vancomycin was confirmed by WHO reference laboratories (European Antimicrobial Resistance Surveillance–UK National External Quality Assurance Scheme). The MIC range was 32–48 mg/L.

This vancomycin-resistant Enterococcus was species-identified as E. faecalis by the EN-COCCUStest (Pliva-Lachema, Czech Republic), and was confirmed by PCR with species-specific primers for ddlE. faecalis and ddlE. faecium genes (these genes encode D-Ala:D-Ala ligases).3 The presence of vanA or vanB operons was studied with PCR assay using primers and conditions described by Kariyama et al.4 In agreement with other reports on the prevalence of the vanA genotype in VRE in Europe,5 this genotype was the only one found in our strain (Figure 1). In addition to vancomycin resistance, E. faecalis 605 was resistant to teicoplanin, erythromycin, quinupristin/dalfopristin and high levels of gentamicin (MIC > 500 mg/L) as determined by the disc diffusion method,6 as well as to other aminoglycosides: tobramycin (MIC 256 mg/L), netilmicin (MIC 32 mg/L), amikacin (MIC 64 mg/L), isepamicin (MIC 32 mg/L) and kanamycin (MIC > 512 mg/L), tested by the agar dilution method.6 The strain remained susceptible to ampicillin, streptomycin, chloramphenicol, rifampicin, nitrofurantoin and ciprofloxacin. The aminoglycoside-resistance profile suggested the presence of the bifunctional enzyme, aminoglycoside acetyltransferase-6'+aminoglycoside phosphotransferase-2'' [AAC(6')+APH(2'')], which destroys all clinically available aminoglycosides with the exception of streptomycin. Positive PCR7 detection of the genes encoding the enzyme confirmed that resistance to aminoglycosides is mediated mainly by AAC(6')+APH(2''). The genes for aminoglycoside resistance are predominantly harboured at mobile elements, so vancomycin-resistant E. faecalis 605 was conjugated with standard recipient strain Enterococcus faecium BM 4105 rifr. The frequency of transfer was relatively high (5.0 x 10–7). Transconjugants possessed a resistance profile similar to the donor strain, with the exception of resistance to quinupristin/dalfopristin, which is, however, located only chromosomally. The potency to transfer resistance determinants was also proved by isolation of a plasmid (~60 kb) from the donor strain as well as from transconjugants. Moreover, E. faecium BM 4105 transconjugants acquired vanA genes (Figure 1), confirming the movement not only of resistance to erythromycin and aminoglycosides, but also of vancomycin and teicoplanin.



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Figure 1. Detection of vanA genes in donor strain E. faecalis 605 and its transconjugant E. faecium 4105. Lane 1, lambda restricted with enzyme Pst1; lane 2, clinical isolate E. faecalis 605; lane 3, transconjugant E. faecium 4105.

 
In summary, molecular study of vancomycin-resistant E. faecalis 605 describes the mechanisms of resistance and the power of this hospital isolate to disseminate resistance markers including van genes. Analysis such as this is important for the creation of antibiotic policy and our understanding of the epidemiology of resistance markers in hospital wards.

Acknowledgements

This research was supported by grant numbers 1/8221/01 and 1/7239/20 from the Slovak Research Grant Agency.

Footnotes

* Corresponding author. Tel: +421-2-60296-436; Fax: +421-2-60296-686; E-mail: bujdakova{at}fns.uniba.sk Back

References

1 . Waren, D. K., Kollef, M. H., Seiler, S. M. et al. (2003). The epidemiology of vancomycin-resistant Enterococcus colonization in a medical intensive care unit. Infection Control and Hospital Epidemiology 24, 257–63.[ISI][Medline]

2 . Bíliková, E., Hanzen, J., Roidová, A. et al. (2001). Bacteraemia due to vancomycin-resistant enterococci in Slovakia. Journal of Antimicrobial Chemotherapy 47, 357–68.[Free Full Text]

3 . 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]

4 . Kariyama, R., Mitsuhatu, R., Chow, J. W. et al. (2000). Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. Journal of Clinical Microbiology 38, 3092–5.[Abstract/Free Full Text]

5 . Kuriyama, T., Williams, D. W., Patel, M. et al. (2003). Molecular characterization of clinical and environmental isolates of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from a teaching hospital in Wales. Journal of Medical Microbiology 52, 821–7.[Abstract/Free Full Text]

6 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA, USA.

7 . Swenson, J. M., Ferraro, M. J., Sahm, D. F. et al. (1995). Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. Journal of Clinical Microbiology 33, 3008–18.[Abstract]





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