JOURNAL OF CLINICAL MICROBIOLOGY, June 2005, p. 2920–2922 0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.6.2920–2922.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 6
Evaluation of a New Molecular System for Simultaneous Identification of Four Enterococcus Species and Their Glycopeptide Resistance Genotypes U. Eigner,1 A. Fahr,1* M. Weizenegger,1 and W. Witte2 Department of Microbiology and Hygiene, Laboratory Group Limbach, Heidelberg, Germany,1 and Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany2 Received 11 February 2005/Accepted 12 February 2005
We compared the performances of a new DNA-based strip assay and the VITEK 2 system for identification of 105 enterococcal strains and differentiation of their van gene-associated resistance levels. Both methods provided excellent results. The molecular assay showed advantages in time to result for identification of van-associated genes. many; RKI Wernigerode, Germany; and bioMe´rieux, France). The following species were tested: 37 E. faecalis, 38 E. faecium, 20 E. gallinarum, and 9 E. casseliflavus strains and 1 E. flavescens strain. Thirty-one isolates contained the vanA genotype, 24 the vanB, 20 the vanC1, and 10 the vanC2/3 genotype, and 20 isolates were van gene negative. All tests were performed according to the manufacturers’ instructions. For ID of the enterococcal species, the ID-GPC card was used, and for susceptibility testing, the AST P 535 card was used. AST results were interpreted following NCCLS standards (12). The accuracy of the VITEK 2 system for the detection of the VanA, -B, and -C phenotypes was determined by means of vancomycin and teicoplanin susceptibility test results. The MICs of both antibiotics were compared to the ranges recommended for the different genotypes (2). VanC strains may show MICs interpreted as sensitive by NCCLS standards; therefore, the need for additional ID was taken into account. For the GenoType Enterococcus assay, up to five colonies of an overnight culture were suspended in 150 l distilled water. Bacterial DNA was released by a 15-min heating step at 95°C in a dry incubator and 15 min of sonication. For amplification, 5 l of the DNA solution was added to 45 l of a multiplexed amplification mix containing biotinylated primers, deoxynucleoside triphosphates, 2.5 mM MgCl2, and 1 U HotStarTaq polymerase (QIAGEN, Hilden, Germany). The amplification protocol was as follows: 15 min at 95°C; 10 cycles of 95°C for 30 s and 58°C for 120 s; 20 cycles of 95°C for 25 s, 53°C for 40 s, and 70°C for 40 s; and a final extension at 70°C for 8 min. Hybridization and detection were carried out in an automated washing and shaking device (Profiblot; Tecan, Maennedorf, Switzerland). For colorimetric detection of hybridized amplicons, streptavidin-conjugated alkaline phosphatase and the appropriate substrate were added. Of the 105 tested Enterococcus species, 99% were correctly identified with the molecular method (Table 1). One E. flavescens strain tested showed a positive signal with the E. casseliflavus probe due to the close relationship of these two species. All 85 van gene-containing isolates were correctly identified, and 20 van-negative E. faecalis isolates were reported negative by the GenoType assay. For 105 Enterococcus species tested, 97% were correctly identified by the VITEK 2 system. One
Enterococci play an important role in nosocomial infections, e.g., urinary tract infections, endocarditis, sepsis, and wound infections (10, 20). During recent years, an increasing prevalence of vancomycin-resistant enterococci (VRE), from 25 to 50%, has become a reason for concern in clinical settings, especially in the United States (1, 6). The prevalence of VRE in Europe remains low, i.e., 2 to 5% in most countries (18). Prevalences of more than 10% and dissemination in intensive care units have also been reported (7, 19). Susceptibility testing of VRE using molecular analysis of the van gene cluster is feasible (5), and various methods based on PCR have been developed (4, 9, 11, 13, 14, 15). Amplification methods show advantages with regard to time, cost, and accuracy (8, 13, 17). Fast detection of VRE is necessary to prevent its spread in the hospital setting (2). We evaluated the efficiency and reliability of a DNA strip assay (GenoType Enterococcus; Hain Lifescience GmbH, Nehren, Germany) designed for the simultaneous detection of the most frequently encountered enterococcal species, Enterococcus faecium, E. faecalis, E. gallinarum, E. casseliflavus, and E. flavescens (2), and of the vancomycin resistance genotypes vanA, -B, -C1, and -C2/3. The assay is based on the “reverse hybridization” format. The DNA probes are immobilized on a nitrocellulose strip. This technique allows multiplexed analysis of amplified bacterial DNA similar to that of chip-based systems. The results of the GenoType assay were compared to those of the VITEK 2 system for automated rapid identification (ID) and antimicrobial susceptibility testing (AST). The remaining enterococcal ID discrepancies were resolved by sequencing a 16S rRNA gene fragment for ID. Discrepancies for van gene testing were resolved by Etest and a specific PCR method using published sequences (3, 4, 9). A challenge set of 105 Enterococcus strains from frozen stock cultures was chosen. The isolates were derived from routine samples and from reference strains (American Type Culture Collection, Manassas, Virginia; Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Ger* Corresponding author. Mailing address: c/o Labor Limbach, Im Breitspiel 15, D-69126 Heidelberg, Germany. Phone: 0049-6221-3432192. Fax: 0049-6221-3432-263. E-mail:
[email protected]. 2920
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TABLE 1. Enterococcal species and van genotype detection by VITEK 2 and GenoType Enterococcus tests No. (%) correctly detected by indicated test Species
van genotype
No. of isolates tested
Enterococcal speciesa
van genotypeb
GenoType
VITEK 2
GenoType
VITEK 2
E. faecium
vanA vanB
24 14
24 (100) 14 (100)
23 (96) 14 (100)
24 (100) 14 (100)
23 (96) 14 (100)
E. faecalis
vanA vanB van negative
7 10 20
7 (100) 10 (100) 20 (100)
6 (100) 10 (100) 20 (100)
7 (100) 10 (100) 20 (100)
7 (100) 9 (90) 20 (100)
E. gallinarum E. casseliflavus E. flavescens
vanC1 vanC2/3 vanC2/3
20 9 1
20 (100) 9 (100) 0 (0)
20 (100) 9 (100) 0 (0)
20 (100) 9 (100) 1 (100)
20 (100) 9 (100) 1 (100)
Total a b
105
104 (99)
102 (97)
105 (100)
103 (98)
Discrepant results for ID confirmed by 16S rRNA gene sequencing. Discrepant results confirmed by PCR.
E. faecium, one E. faecalis, and one E. flavescens isolate were misidentified. Additional tests proposed by the system were a pigmentation test to differentiate E. gallinarum and E. casseliflavus and a motility test to differentiate E. faecium and E. gallinarum. Of the 85 van genotypes tested, VITEK 2 identified 98% with the correct MIC; the van-negative isolates were reported as showing sensitivity to vancomycin and teicoplanin. For one E. faecium isolate with vanA and one E. faecalis isolate with vanB, the MICs reported by VITEK 2 were too low for both antibiotics. Phenotypic methods may miss the correct genotype in the case of isolates susceptible to both vancomycin and teicoplanin (10), especially those with VanB and -C phenotypes. Molecular methods, like the GenoType assay, have the advantage of reporting the actual van genotype and are not dependent on phenotypic expression of resistance. The times needed to achieve results by the two systems were also compared. ID results for VITEK 2 were ready in 2 h 45 min, without including the additional time to perform the phenotypic tests proposed by VITEK 2, such as the motility or pigmentation test. Susceptibility testing of vancomycin and teicoplanin was available in 9 h 7 min, so the report for a VRE isolate may not be available in one workday. Species ID and detection of the van genes with the GenoType Enterococcus assay were available in 4 h 30 min from a positive culture to a result. Incubation time (heating, sonication, cycling) and automated hybridization are “walk away” steps and can be used for manual laboratory work. Band patterns on the strip can be analyzed by eye with an interpretation template chart provided with the kit. The test is monitored for quality by an internal amplification and staining control. A uniform hybridization protocol allows the parallel performance of a variety of other GenoType assays, such as those for detection of methicillinresistant Staphylococcus aureus, mycobacteria, or enterohemorrhagic Escherichia coli combined into one test run (16). This combination increases economy for routine lab testing. In conclusion, both systems performed excellently in fast and reliable detection of enterococcal species and glycopeptide resistance. For excellent technical assistance, we thank U. Wild.
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