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Emergence of linezolid-resistant Enterococcus faecalis in Spain and rapid characterisation by real-time PCR

Abstract number: 1733_387

Cercenado E., Marin M., Cuevas O., Bouza E.

Objectives: Linezolid was introduced in our institution in 2004. From May 2005 to September 2006 we determined the in vitro activity of linezolid against all staphylococci and enterococci isolated in our laboratory. We characterise the first linezolid-resistant (LNZ-R) clinical isolates of Enterococcus faecalis in Spain.

Methods: MICs were determined by the broth microdilution method using commercialised MicroScan panels (Dade-Behring). CLSI guidelines (2006) were followed for determination of breakpoints. LNZ-R isolates were confirmed by E-test. Detection of the G2576T mutation was performed by PCR and sequenciation of the 23S rRNA gene, as well as by real-time PCR (Woodford et al; JCM 2002; 40: 4298–4300) using a fluorescent dye-labeled detection probe.

Results: Over the period of study, we tested 6,214 staphylococci (3,817 S. aureus; 2,407 coagulase-negative staphylococci), and 1,850 enterococci (1,439 E. faecalis; 340 E. faecium; 71 other species). All staphylococci were susceptible to linezolid (MIC ≤ 4 mg/L). Three E. faecalis isolates (0.16%) were LNZ-R (MICs 64, and 128 mg/L; 2 and 1 isolate, respectively. The isolates were recovered in 2006 and corresponded to 3 patients hospitalised in different wards that received linezolid for >15 days previous to the isolation of the LNZ-R isolates. PCR and sequenciation detected the G2576T mutation only in 2 isolates (MICs 64 mg/L). Real-time PCR was positive in all 3 isolates, being one strain (MIC = 128 mg/L) heterozygous, and the other 2 homozygous. Results were obtained in less than 2 h. The G2576T mutation in the heterozygous strain was not detected by PCR amplification and sequencing of the 23S rRNA gene.

Conclusions: We characterise for the first time in Spain the emergence of LNZ-R E. faecalis isolates in patients previously treated with linezolid. Since the G2576T mutation was not detected in the heterozygous isolate by PCR and sequencing, the real-time PCR must be the method of choice for the characterisation of this emergent resistance mechanism.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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