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Objectives: Linezolid belongs to the new class of oxazolidinones with a novel mechanism of action. Linezolid inhibits ribosomal protein synthesis at the step of formation of the initiation complex.Linezolid has been approved for the treatment of nosocomial infection with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), mainly Enterococcus faecalis and Enterococcus faecium. Treatment of MRSA and VRE infections can be challenging and great efforts are being made to prevent nosocomial infections with these bacteria.
Until now, linezolid-resistant enterococci (LRE) are rare in Germany but increasing use of linezolid could lead to spread of resistance. The resistance mechanism reported most frequently is a G to T single nucleotide polymorphism (SNP) at bp 2576 (Escherichia coli numbering) of the genes coding for 23S rRNA synthesis.
The aim of this study was to compare broth microdilution and pyrosequencing for the detection of linezolid resistance in clinical VRE isolates.
Material and methods: 55 VRE isolates (53 Enterococcus faecium and 2 Enterococcus faecalis) had been collected at the University Medical Center Hamburg-Eppendorf, Germany between 1995 and 2003.
Antimicrobial susceptibilities to linezolid and vancomycin were determined by broth microdilution according to CLSI guidelines.
Detection of the G2576T SNP and estimation of the percentage of 23S rRNA genes carrying the G2576T SNP were done by the use of pyrosequencing
Results: All strains were resistant to vancomycin (MICs ≥ 8 µg/mL, breakpoint for resistance ≥ 8 µg/mL).Two clinical isolates showed increased MICs of linezolid of 16 µg/mL (breakpoint for resistance ≥ 8 µg/mL) and the G to T mutation at bp 2576 was detected by pyrosequencing in both strains.Estimated from the relative peak heights given by the pyrosequencing software both isolates carried the G2576T SNP in 2 23S rRNA genes.Eleven clinical isolates showed intermediate linezolid MICs (4 µg/mL) but none of these strains carried the G to T SNP at bp 2576.The remaining 42 clinical isolates were susceptible to linezolid
Conclusion: Our study shows that pyrosequencing is a valuable tool to address the question of linezolid resistance in enterococci.In accordance to the broth microdilution MIC results pyrosequencing detected the G2576T SNP as the cause of linezolid resistance in the two isolates.Alternatively, mutations which have been described in in vitro mutants of linezolid-resistant enterococci may be responsible for resistance and the pyrosequencing technology could be adapted.
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