Department of Microbiology, Medical School, University of Thessalia, Papakyriazi 22, Larissa, Greece 41222
Keywords: resistance, enterococci, VRE
Sir,
The emergence of vancomycin-resistant enterococci (VRE) has raised major concerns because of the limited therapeutic options for treating infections resulting from these organisms. In the USA, the National Nosocomial Surveillance System has described a 20-fold rise in VRE recovered from bloodstream infections during the past 10 years. The limitation in therapeutic options has resulted in the development of new drugs such as quinupristin/dalfopristin and linezolid.
Although linezolid has been used in clinical practice for a relatively short period of time, there are already several reports of linezolid-resistant enterococci.13 In Greece, this antibiotic is only used for the treatment of infections caused by vancomycin-resistant enterococci and teicoplanin-resistant Staphylococcus epidermidis strains, which are circulating in several Greek hospitals (data not shown). To our knowledge, this is the first report of a linezolid-resistant Enterococcus faecium strain isolated in a tertiary care Greek hospital.
The isolate was recovered from a blood culture of a 20-year-old male patient, who was admitted to the University Hospital of Larissa after a car accident. He was operated on immediately for a fractured femur. During corrective surgery, a fatty embolism occurred and he was immediately transferred to the intensive care unit (ICU) where he was treated for about a month. The patient was empirically treated with aminoglycosides, teicoplanin and piperacillin plus tazobactam. However, the isolation from blood culture on the 14th day of an oxacillin-resistant S. epidermidis strain, resistant to teicoplanin and other antimicrobial agents, was the main cause for the replacement of teicoplanin by linezolid. Four days later, an E. faecium isolate, resistant to glycopeptides and linezolid (MIC 16 mg/L) was recovered from blood culture.
The microorganism was identified to the species level by the automated Vitek System (bioMérieux, Hazelwood, MO, USA). Susceptibility testing to penicillin, ampicillin, ciprofloxacin, gentamicin (high-level), streptomycin (high-level), tetracycline, nitrofurantoin and vancomycin was performed using the Vitek System, and susceptibility to erythromycin, rifampicin, linezolid and quinupristin/dalfopristin was tested by Etest strips (AB Biodisk, Solna, Sweden). MICs of vancomycin, teicoplanin and linezolid were determined by the agar dilution method (NCCLS guidelines).4 To examine the stability of linezolid resistance, the isolate was subcultured on drug-free agar once weekly for 2 months and then re-tested to determine the final linezolid MIC. Enterococcus faecalis ATCC 29212 was used as a control for the estimation of MICs. PCRs for erm(B), vanA/B and esp genes were carried out as described previously.5 To elucidate the mechanism of linezolid resistance, the gene encoding domain V of the 23S rRNA was amplified.3 The G2576T mutation, which is mainly associated with the expression of linezolid resistance in clinical isolates, creates a cutting site for the restriction endonuclease NheI.6 Thus, PCR-RFLP analysis of the 420 bp amplicons was performed to detect this mutation: 10 µL of the PCR products was digested with 20 U of NheI for 4 h at 37°C, and the fragments were separated by electrophoresis through a 3.5% Metaphor agarose gel. In addition, in order to detect the presence of mutations other than G2576T, the 420 bp PCR product was sequenced.13,7 The epidemiological relationship of the isolate to other isolates of VRE from our hospital, including those from stool samples from patients in the ICU, was assessed by PFGE using SmaI digests.1
Following repeated subcultures on drug-free agar, the expression of linezolid resistance remained stable. The isolate was also resistant to various other antimicrobial agents, such as penicillin, ampicillin, tetracycline, erythromycin, vancomycin, teicoplanin, streptomycin, gentamicin, nitrofurantoin, rifampicin and ciprofloxacin, but remained susceptible to quinupristin/dalfopristin. PCRs were positive for vanA and erm(B), but negative for the esp gene. The results of NheI PCR-RFLP analysis of the 420 bp rDNA amplicons showed that the isolate was heterozygous: amplicons were cleaved to give undigested products of 420 bp and digested products of 326 bp and 94 bp. E. faecalis ATCC 29212, which is linezolid-susceptible, gave only the undigested 420 bp band. Analysis of sequencing data failed to detect the G2576T mutation
PFGE analysis revealed that the linezolid-resistant E. faecium strain belonged in a clone that differed markedly from the endemic E. faecium hospital clones (Figure 1). Furthermore, two vancomycin- and linezolid-resistant E. faecium strains, isolated from stool culturesthe first from the studied patient (patient 1) and the second from another ICU patient (patient 2)showed the same SmaI pattern as the blood strain, suggesting a possible transfer of the strain between the two patients (Figure 1).
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Emerging linezolid resistance is a fact. Therefore, one could suggest that clinical laboratories should be routinely testing linezolid susceptibility in all clinically significant enterococci. Worldwide surveillance programmes should closely monitor all linezolid resistance reports in order to trace any trends in the development of resistance.
Footnotes
* Corresponding author. Fax: +30-41-682508; E-mail: anm{at}otenet.gr
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