Comparison of agar dilution, broth microdilution, E-test, disk diffusion, and automated Vitek methods for testing susceptibilities of Enterococcus spp. to vancomycin

Abstract

An evaluation was undertaken to determine the optimal method for testing the susceptibilities of 100 clinical isolates and two reference strains of Enterococcus spp. to vancomycin in vitro. Six testing methods were studied by using the following media and incubation times: agar screen with the Synergy Quad Plate (Remel, Lenexa, Kans.), an in-house-prepared brain heart infusion (BHI) agar plate, and an in-house-prepared Mueller-Hinton (MH) agar plate, all incubated for 24 or 48 h; broth microdilution (Sensititre Just One Strip; AccuMed International, Inc. , West Lake, Ohio) with BHI or cation-adjusted MH broth incubated for 24 or 48 h; agar dilution with BHI or MH agar incubated for 24 or 48 h; epsilometer test (E test; AB BioDisk, Solna, Sweden) with BHI or MH agar incubated for 24 or 48 h; disk diffusion with BHI or MH agar incubated for 24 or 48 h; and the automated Vitek method with the gram-positive susceptibilityStaphylococcus aureus card and R02.03 software (bioMerieux, Inc., Hazelwood, Mo.). Growth failures occurred with MH media (n ‫؍‬ 6) but not with BHI media. One growth failure occurred with the Vitek method. Results for each testing method for each Enterococcus strain were interpreted as susceptible, intermediate, or resistant according to current National Committee for Clinical Laboratory Standards (NCCLS) criteria and compared to the vancomycin resistance genotype (i .e., vanA, vanB, vanC-1, or vanC-2/3). For all methods, extension of the incubation time from 24 h to 48 h either produced no difference in the results or gave poorer results. The following methods produced no very major or major interpretive errors: broth microdilution with BHI media incubated for 24 h, agar dilution with BHI media incubated for 24 or 48 h, and E test with BHI media incubated for 24 or 48 h. Unacceptable frequencies of very major errors (>1%) occurred with all methods for which MH media were used. Minor interpretive errors were frequent with all methods. These minor interpretive errors also occurred most frequently with Enterococcus strains with vanC genes, which encoded low-level vancomycin resistance (MIC < 8 g/ml), as opposed to Enterococcus strains which possessed vanA or vanB genes, which encoded higher-level vancomycin resistance (MIC > 64 g/ml). Modification of NCCLS breakpoints, especially for motile Enterococcus spp. (E. casseliflavus, E. flavescens, and E. gallinarum), may resolve this problem; however, in the current study, one E. faecalis strain and one E. faecium strain carried only the vanC gene. The agar screen method may also require reformulation. The current agar screen plate contains 6 g of vancomycin per ml, which may not detect all low-level resistance associated with vanC genotypes. Nevertheless, the clinical significance of this low-level vancomycin resistance remains unknown.

Determination of 16S rRNA Sequences of Enterococci and Application to Species Identification of Nonmotile Enterococcus gallinarum Isolates

“…There also exists a need to accurately and quickly differentiate enterococci with vanC-associated vancomycin resistance from VanA or VanB VRE. Stool screening for VRE commonly involves the use of media supplemented with vancomycin at concentrations as low as 6 g/ml and may also not accurately differentiate E. gallinarum from VanA or VanB VRE or from vancomycin-susceptible enterococci (14,24,28).…”

Use of Molecular and Reference Susceptibility Testing Methods in a Multicenter Evaluation of MicroScan Dried Overnight Gram-Positive MIC Panels for Detection of Vancomycin and High-Level Aminoglycoside Resistances in Enterococci

“…Therefore, it is critical for clinical microbiology laboratories to provide accurate antimicrobial susceptibility testing results for enterococci, so that effective therapy and infection control measures can be initiated. Recent reports have demonstrated the inability of many clinical laboratories (2,23,34) and commercial susceptibility testing systems (12,23,34) to detect glycopeptide resistance, especially patterns of low-or moderate-level resistance to vancomycin in enterococci (strains with a vanC genotype and certain strains of the vanB type). Enterococci with the vanC genotype, i.e., E. gallinarum and E. casseliflavus, which possess an intrinsic property of constitutive low-level VR (13,19), are infrequently recovered from clinical specimens (11,32).…”

“…Overall, 73.2% of strains tested for the VR genes were negative (range, 61.2 to 94.0%), while 23.5% produced a vanA or vanB PCR product, including four strains from WUSM harboring both genes. The remaining 3.4% of strains produced a band consistent with one of the vanC genotypes, usually associated with Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens (12,18,25,31). The percentages of strains lacking the HLSR or HLGR gene were 54.4% (range, 36.0 to 74.0%) and 77.2% (range, 70.0 to 81.6%), respectively.…”

Genetic Methods for Assessing Antimicrobial Resistance

“…In essence, the RFLP procedure results in "bar codes" which specify distinct genes associated with vancomycin resistance. We have found this technique to be particularly useful for determining vancomycin resistance associated with the vanC gene family as this form of vancomycin resistance is not always detectable by conventional culturebased susceptibility methods (27). However, caution must be taken when identifying vanB and vanC-2 by RFLP analysis, as we have noted significant DNA sequence variation among these genes (47).…”