a Institute of Microbiology, Semmelweis University, PO Box 370, H-1445 Budapest; b Laboratory of Bacteriology, Nógrád County Institute of the National Service for Public Health and Health Officers, Salgótarján, Hungary
Sir,
The glycopeptide vancomycin is the first-choice alternative to a penicillinaminoglycoside combination for treatment of systemic enterococcal infections. The frequency of vancomycin-resistant enterococcus (VRE) isolates has increased worldwide following reports from France in 19881 and the UK.2 Vancomycin resistance has been most common in Enterococcus faecium, but vancomycin-resistant Enterococcus faecalis strains have also been reported. The frequency of VRE isolates among nosocomial infections in the USA was only 1% in 1989, but had increased to 7.9% by 1993.3
Here we report a PCR study showing that an E. faecalis strain, isolated from a toe ulcer, carries the vanA gene. This is the first report of this gene in a Hungarian clinical isolate of E. faecalis.
A vancomycin- and teicoplanin-resistant strain of E. faecalis was isolated from a toe ulcer of a 64 year old diabetic male patient admitted to the surgical ward of the Nógrád County Hospital, Salgótarján, Hungary. The strain was identified both by classical methods and by the Rapid ID 32 Strep kit (bioMérieux, Lyon, France). Drug susceptibility was tested by an ATB STREP panel (bioMérieux). The vancomycin and teicoplanin MICs were >256 and 96 mg/L, respectively, as determined by Etest (AB Biodisk, Solna, Sweden).
The patient was discharged from hospital 14 days after admission, following amputation of the toe. The isolate was transferred to the Institute of Microbiology of the Semmelweis University, Budapest, Hungary for further studies. It was re-identified with a Rapid ID 32 Strep panel
(bioMérieux) and the vancomycin MIC was retested by a broth microdilution method, as described by the NCCLS. These tests confirmed the results obtained at the Salgótarján laboratory.
With the ATB panel, the strain appeared to be susceptible only to ampicillin/amoxycillin, sulphamethoxazole trimethoprim combination and rifampicin.
Results of phenotypic examination suggested that the strain carried the vanA gene, so DNA was extracted for PCR amplification to confirm this. Bacterial cells from an overnight broth culture were subjected to lysostaphinproteinase K digestion. The oligonucleotide primers designed to amplify the above gene were as follows: Van A1, 5'-GGGAAAACGACAATTGC-3'; Van A2, 5'-GTACAATGCCGTTA-3'. The PCR reaction consisted of denaturation at 92°C for 3 min, followed by 30 cycles of heating to 92°C for 1 min, cooling to 56°C for 1 min, then heating to 72°C for l min. Vials were then kept at 72°C for 3 min. Two standard laboratory strain (ATCC 51559 vanA positive, ATCC 29212 vanA negative) were used as positive, as well as negative controls. The amplified product, which was 732 bp long,4 was subjected to ethidium bromide agarose gel electrophoresis (Figure).
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Acknowledgments
This work was supported by grant T 021251 from the National Research Fund, Hungary (OTKA).
Notes
J Antimicrob Chemother 2000; 46: 325327
* Corresponding author. Tel/Fax: +36-1-210-2959; E-mail: rozfer{at}net.sote.hu
References
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2 . Uttley, A. H., George, R. C., Naidoo, J., Woodford, N., Johnson, A. P. et al. (1989). High-level vancomycin-resistant enterococci causing hospital infection. Epidemiology and Infection 103, 17381.[ISI][Medline]
3 . Centers for Disease Control and Prevention. (1993). Nosocomial enterococci resistant to vancomycinUnited States, 19891993. Morbidity and Mortality Weekly Report 42, 5979.[Medline]
4 . 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, 1434.