Rapid characterization schemes for surveillance isolates of vancomycin-resistant enterococci

Abstract

Surveillance cultures for vancomycin-resistant enterococci (VRE) and subsequent characterization of the isolates can be extremely burdensome and difficult. Therefore, efficient and reliable schemes for the characterization of surveillance isolates are needed. In this study, a commercial agar (bile esculin azide agar with 6 g of vancomycin per ml [BEAA]; Remel, Lenexa, Kans.) was used in the initial screening step to establish relatively rapid (i.e., in <24 h from the time of isolation) phenotype-based and PCR-based schemes for the detection and characterization of VRE. The phenotype-based scheme included Gram staining of growth on BEAA and subculture of cocci on sheep blood agar plates for vancomycin disk diffusion and pyrazinamidase (PYR) testing. For the PCR scheme, inocula for van gene detection were taken directly from the BEAA plates. The phenotypic approach was applied to 378 surveillance cultures that yielded growth on BEAA. Gram staining quickly eliminated gram-positive bacilli from further testing, and a negative PYR test classified 25 additional isolates as probable pediococci. A positive PYR test reliably identified 121 single-patient VRE isolates that included 83 Enterococcus faecium, 33 E. gallinarum, and 5 E. casseliflavus strains. The vancomycin inhibition zone size clearly distinguished VanA and VanB strains from VanC strains within 24 h of BEAA isolation. All VanA and VanB strains failed to produce zones of >6 mm, while only one VanC strain produced a zone of <15 mm. Challenging this phenotypic scheme with 47 stock VRE isolates produced similar findings. In direct PCR analyses, false-negative vanA and vanB results occurred with 12% of the specimens. Many of the false-negative reactions also failed to produce an internal control product, which underscores the need for including control primers when a PCR scheme is used. In summary, the phenotype-and the PCR-based schemes provide efficient methods for characterizing VRE within 24 h of isolation.

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

“…This is a recent concept made possible by the application of DNA hybridization techniques and PCR (4,13). A PCR assay to detect genotypes of VR and to identify organisms to the species level offers a specific and moderately rapid method for susceptibility testing, in particular for detection of low-level glycopeptide resistance (4,26). Molecular methods have also been utilized in clinical and surveillance studies (11,21), and PCR produced excellent results in the EP of this investigation.…”

“…A laboratory report of a VR isolate will initiate a cascade of infection control events that are both time-consuming and costly (5) and that should be focused on organisms of the vanA or vanB pattern. Thus, an important adjunct to susceptibility testing is the need for accurate identification of enterococcal species to differentiate intrinsic vanC VR from low-level vanB VR in E. faecalis or Enterococcus faecium (4,26).…”

Comparison of Simple and Rapid Methods for Identifying Enterococci Intrinsically Resistant to Vancomycin

“…The identification of hospitalized patients infected with vancomycin-resistant enterococci (VRE) has become an important component of infection control programs aimed at minimizing patient-to-patient transmission of these organisms (4). A variety of vancomycin-containing media have been shown to provide a simple and cost-effective means for differentiating VRE from non-VRE (1,12), and use of these media has been adopted in many clinical laboratories. In addition to organisms possessing high-level, transferable vancomycin resistance (predominantly Enterococcus faecalis and Enterococcus faecium), those enterococci that intrinsically express low-level resistance to glycopeptide antibiotics, namely, Enterococcus casseliflavus and Enterococcus gallinarum, will also grow on vancomycincontaining media.…”

Detection of clinical vancomycin‐resistant enterococci in Denmark by multiplex PCR and sandwich hybridization

“…PCR assays to detect glycopeptide resistance genotypes in enterococci have often been described previously (9,(12)(13)(14)(15). In our PCR setup, 16s rDNA primers were included as a control of the DNA preparation, but also to ensure that template actually had been added to the test tube.…”

“…A negative result was only accepted if 16s rDNA had been amplified. Some investigators use control primers as well, but they do not test for the vanA, vanB, vanC-Z and vanC2/ 3 genes in the same reaction as we do (13,15). Thus, the present assay will be useful for screening of enterococci for vancomycin resistance genes.…”