Comparison of Testing Methods for Detection of Decreased Linezolid Susceptibility Due to G2576T Mutation of the 23S rRNA Gene in Enterococcus faecium and Enterococcus faecalis

Abstract

E-test, Vitek 2, MicroScan, agar dilution, and disk diffusion were compared for detection of decreased linezolid susceptibility due to 23S rRNA gene G2576T mutation among 32 clinical Enterococcus strains initially reported as intermediate or resistant by E-test alone or Vitek 2 confirmed by E-test. Agar and broth dilution methods were in concordance with PCR detection of the mutation, and disk diffusion was somewhat less sensitive but equally specific.Linezolid provides high rates of clinical cure and microbiological success in complicated infections due to Enterococcus spp., including vancomycin-resistant Enterococcus faecium (3). However, the emergence of resistance during linezolid treatment has been reported for clinical strains of Enterococcus (1,2,7,9,12,13). Clinical resistance to linezolid is associated with a G2576T mutation in domain V of 23S rRNA genes of Enterococcus, and the level of linezolid resistance is directly related to the number of 23S rRNA genes containing this mutation (11,15). Both laboratory and clinical strains of E. faecium with linezolid MICs of 4 g/ml have been shown to carry the G2576T mutation (10,14). Accurate detection by susceptibility testing methods of decreased susceptibility due to G2576T mutation in one or two genes is necessary since this can be a prelude to higher levels of linezolid resistance associated with extensive use of the antibiotic (14).In this study we compared the performance of five different susceptibility testing methods, E-test, disk diffusion, Vitek 2 system, MicroScan WalkAway broth microdilution, and agar dilution, for the detection of decreased linezolid susceptibility of Enterococcus faecalis and E. faecium due to presence of the G2576T mutation.Strain selection. collected during this period. All strains were identified to the species level by using the Vitek 2 system. When the Vitek 2 system failed to identify the strains, identification was obtained by manual biochemical reactions (8). The strains were recovered from blood, urine, respiratory specimens, and various body fluids and tissues. Three well-characterized strains of linezolid-resistant E. faecium (strains 38-13, 45-24, and 38-42) and one strain of lineozlidresistant E. faecalis (strain 41-31) were kindly provided by Paul Schreckenberger of the Loyola University Medical Center, Maywood, IL.Susceptibility testing. E-test linezolid strips with a concentration gradient corresponding to 0.016 to 256 g/ml were utilized with Mueller-Hinton agar as described by the manufacturer (AB Biodisk, Piscataway, N.J.). E-test MICs were determined as 80% growth inhibition, and measured E-test MICs were rounded up to the next twofold dilution for the categorical interpretation. Disk diffusion testing was performed with 30-g linezolid disks (BBL, Becton Dickinson) using Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) standards (4), and 80% growth inhibition was utilized to measure diameters of growth inhibition zones. Agar dilution testing (0.5...

Accuracy of Six Antimicrobial Susceptibility Methods for Testing Linezolid against Staphylococci and Enterococci

“…This is consistent with the report of Qi et al, who showed a high correlation between the presence or absence of mutations in the 23S rRNA gene and the phenotype reported by the MicroScan system (18). VITEK 2 also had high category agreement, provided that the advanced expert system, which reported all linezolid-nonsusceptible staphylococci as susceptible, was not used.…”

“…In one study, Qi et al noted that the linezolid resistance results reported for enterococci by the MicroScan system (with a PC-21 panel) correlated well with the presence of G2576T mutations in domain V of the 23S rRNA gene of the organisms tested. In that same study, however, the Etest and VITEK 2 methods (with an AST GP-61 card) tended to produce false resistance results when compared by using the results of rRNA sequencing as the "gold standard" (18). Problems with false linezolid resistance results for enterococci were also reported by Scheetz et al (19).…”

“…The resistant strains reported in the literature were detected by either Etest or disk diffusion. However, problems with false linezolid resistance results obtained with Etest and VITEK 2 have been reported (18), prompting several manufacturers of automated susceptibility testing systems to issue alerts regarding the detection of linezolid-resistant organisms. We also noted that several isolates of staphylococci and enterococci that were sent to either the CDC or Project ICARE for confirmation of a linezolid-nonsusceptible phenotype produced indistinct zones of inhibition when tested by both the disk diffusion and Etest methods, making interpretation of the test results difficult.…”

Investigation of Linezolid Resistance in Staphylococci and Enterococci

“…However, Qi et al did evaluate the ability of Etest, agar dilution, broth microdilution, disk diffusion, and Vitek2 to detect decreased linezolid susceptibility in enterococci with G2576T mutations (15). The purpose of the study by Qi et al was to assess the impact of G2576T mutation on phenotypic susceptibility.…”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…However, a few reports documented resistance detection independent from linezolid treatment (Rahim et al, 2003;Bonora et al, 2006). Resistance results from point mutations in 23S rRNA, preferably at position 2576 (G > T) Werner et al, 2004;Qi et al, 2006;Werner et al, 2007a) and the level of resistance is dependent on the number of mutated alleles per genome (Marshall et al, 2002;Lobritz et al, 2003;BourgeoisNicolaos et al, 2007;Boumghar-Bourtchai et al, 2009). Once established, resistance levels quickly arise due to recombinational exchange of mutated 23S rDNA alleles under selective pressure (Willems et al, 2003;Boumghar-Bourtchai et al, 2009).…”