Molecular detection of linezolid resistance in Enterococcus faecium and Enterococcus faecalis by use of 5' nuclease real-time PCR compared to a modified classical approach

Abstract

A nucleotide transversion from guanine to uracil in the 23S rRNA confers linezolid resistance. We describe a real-time PCR using two Taqman probes that detects a single mutated allele among the genomes of Enterococcus faecium and Enterococcus faecalis. Results were confirmed by a classical approach involving LabChip technology assayed with an Agilent Bioanalyzer 2100.

FIG. 1.

Detection of 23S alleles of linezolid-resistant E. faecium and E. faecalis isolates by NheI digestion of purified pooled PCR fragments and subsequent separation in a LabChip 1000 measured in an Agilent Bioanalyzer 2100. The lowest and highest bands correspond to internal size markers. Lanes: M, external size markers; 1, E. faecium ATCC 19434; 2, E. faecium 3698; 3, E. faecium 3695; 4, E. faecium 3819; 5, E. faecium 3936; 6, E. faecium 3938; 7, E. faecium 3935; 8, E. faecium 3939; 9, E. faecalis V583; 10, E. faecalis 3932; 11, E. faecalis 3697; 12, E. faecalis 3696.

FIG. 2.

Detection of the G or T allele type of 23S ribosomal DNA by real-time PCR using two labeled probes. In vitro gene dosage experiments vary the number of wild-type versus mutated alleles in E. faecium (A) and E. faecalis (B) (see text for details). Only results for the FAM-LIZ-TQ-R probe are shown. For better visibility, only one representative per allele mix is shown. Labels indicate the numbers of mutated alleles relative to the overall number of 23S ribosomal DNA copies per genome (E. faecium, 6; E. faecalis, 4). Delta R_n, difference of fluorescence signals of a given template and the no-template control.