Vancomycin-resistant enterococci screening

Since the first cases of human infection with vancomycin-resistant enterococci (VRE) were reported in the 1980s, the occurrence of VRE has progressively increased globally. In order to reduce the spread of these pathogens and lower health care-associated infections, identifying colonised individuals is of great importance.(1-4) So far, nine genotypes of glycopeptide resistance, which differ in the level and range of resistance to glycopeptides and in transferability, have been described for enterococci. Six of the van genes are acquired (vanA, vanB, vanD, vanE vanG, vanL) and three (vanC1, vanC2, vanC3) are intrinsic.(4–6) Of these, vanA and vanB are the most common types. VRE isolates are predominantly found in Enterococcus faecium (E. faecium) followed by E. faecalis, the enterococcal species responsible for most infections in humans.  Detection of VRE is traditionally by culture-based methods, with confirmation of species and van genotypes based on molecular methods. Culture-based screening methods for VRE are time consuming and can take generally from one to five days to complete.(7) To shorten the detection time and improve the sensitivity in VRE diagnosis, in the last decade, real-time polymerase chain reaction (PCR) methods and chromogenic selective media have been widely applied in screening methods for VRE.

The target genes in the PCR assay for VRE screening are commonly vanA and vanB. The BD GeneOhmTM VanR assay, Cepheid GeneXpert vanA/vanB assay, and Roche LightCycler VRE detection kit are examples of the commercially available assays for VRE screening. The GeneOhmTM VanR assay and the GeneXpert vanA/vanB assay, combining integrated nucleic acid extraction and automated PCR for the detection of vanA and vanB gene sequences, have been marketed for the direct screening of VRE from perianal or rectal swabs. The LightCycler VRE detection kit provides the primers and the hybridisation probes for the amplification and detection of vanA, vanB1 and vanB2/3 genes. To be compatible with the existing extraction platform for an integrated laboratory workflow in microbiology laboratories, the laboratory-developed real-time PCR assay has also been reported.(8) It is important to note that these PCR assays detect van genes rather than VRE per se.  A positive vanB result by PCR is poorly predictive for VRE owing to the presence of vanB in non-enterococcal species. The specificity of the PCR assays was limited largely due to false-positives in the vanB portion of the assay. PCR assay is therefore a simple, rapid and acceptable method for screening for VRE in a variety of populations in which vanA is the predominant genotype. Samples positive for the vanB genotype should be confirmed by culture owing to the apparent high number of false-positive results. However, the VRE strains harbouring van genes other than vanA or vanB could be missed by the vanA/vanB-based PCR assay. 

Chromogenic selective media commonly used for VRE screening are BBL EnterococcoselTM agar (Bile Esculin Azide Agar, BD Diagnostics) with 6–8mg/L vancomycin and chromIDTM VRE agar (bioMérieux). Enterococcosel agar is aimed at differential isolation and presumptive identification of enterococci. chromID incorporates chromogens targeted by enzymes specific for E. faecium and E. faecalis, leading to the two species as violet and bluish-green coloured colonies, respectively.(9,10) An adequate suppression of the abundant stool flora is a major concern while developing media for VRE. Diluting samples in sterile saline or pre-incubation in enrichment broth before plating can help circumvent this problem. False-positive colonies on chromID agar are most likely to be Candida spp. and gram-negative rods. On the Enterococcosel agar, Enterococcus spp. other than E. faecium or E. faecalis, gram-positive rods, and Streptococcus spp. are observed more commonly. 

Studies have shown that inclusion of broth enrichment provides a better performance for both PCR assay and chromogenic selective agar for the detection of VRE.(6,9,10) There is commercially available VRE broth such as bile-esculin-azide broth with vancomycin (Hardy Diagnostics) but broth made in-house is also used routinely in microbiology laboratories. Besides vancomycin, aztreonam is usually included in the in-house broth so as to suppress the growth of gram-negative bacteria. Although clinical laboratories can detect high-level resistance to vancomycin reliably, there are reports of difficulties in the detection of low-level inducible resistance to vancomycin.(11,12) VRE with low-level inducible resistance can be missed by routine screening methods. VRE strains with the VanB phenotype were originally described as having inducible low-level resistance to vancomycin. Better identification and screening methods for the detection of low-level vancomycin resistance are needed to improve surveillance and prevent transmission of VRE. Studies showed that reducing the vancomycin concentration in screening media substantially increased the sensitivity for detection of VRE.(8,12)  The VRE broth, at the author’s laboratory, was composed of esculin-bile-azide broth (Biolife, Milano, Italy), 60mg/l aztreonam (Sigma-Aldrich, St. Louis, MO, USA) and 4mg/l vancomycin (Hospira, Illinois, USA). Our study showed that both VanA-type and VanB-type VRE could be detected, after overnight cultivation, with an initial inoculum as low as 1–10cfu/ml in the broth.(8)

E. faecium and E. faecalis are the two predominant enterococcal species associated with VRE. VanA and VanB are the most common types of acquired glycopeptide resistance(4,5). The acquisition of the vanA- or vanB‑mediated glycopeptide resistance in enterococcal species other than E. faecalis or E. faecium has been reported.(13,14) Motile enterococcal species, such as E. gallinarum and E. casseliflavus, usually carry the vanC1 and vanC2/3 gene, respectively, and exhibit low-level intrinsic resistance to vancomycin. The presence of vanC genes, which confer low-level vancomycin resistance, is not considered an indication for strict patient isolation precautions. However, coexistence of the vanC genotype with the vanA or vanB genotype may represent an additional threat for the control of enterococcal infections. Although reported sporadically, clinicians and microbiologists should be aware of their occurrence and of the need to detect and characterise these microorganisms promptly.

The ability of motile enterococcal species to acquire glycopeptide resistance determinants warrants further attention for these microorganisms, and the resistance genotypes in enterococci of these species should be closely monitored.

VanA phenotype VRE is defined as having high-level resistance to vancomycin and teicoplanin, whereas the VanB phenotype has various levels of vancomycin resistance and is susceptible to teicoplanin. In recent years, the teicoplanin-susceptible vanA-genotype VRE strains have been increasingly reported and named VanB phenotype-vanA genotype VRE. The mechanism for this phenomenon is still uncertain. The possible mechanisms might be point mutations in the putative sensor domain of vanS, and impairment or rearrangement of vanA gene cluster.(13,15)  Enterococcal resistance to glycopeptides is therefore phenotypically and genotypically heterogeneous, which emphasises the necessity of utilising both phenotypic and genotypic methods to characterise VRE.

Rapid diagnosis is critical for preventing and treating VRE infections. PCR-based molecular diagnostics and the development of chromogenic media have allowed not only a clinically relevant turnaround time for a diagnostic result, but also an increased sensitivity and specificity in screening for VRE. Nevertheless, there is as yet no single stand-alone rapid assay that can recover all VRE strains. A screening assay combining broth enrichment, real-time PCR and chromogenic selective agar might be an efficient strategy in the detection of VRE.  The choice of a screening method, however, should also take into account the local epidemiological status and the cost of the assays. Long-term efficient surveillance requires reliable and rapid laboratory testing at low cost.

1. Boyle JF et al. Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycin-resistant enterococci. J Clin Microbiol 1993;31(5):1280–85.
2. Low DE et al. Clinical prevalence, antimicrobial susceptibility, and geographic resistance patterns of enterococci: results from the SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis 2001;32 Suppl 2:S133–45.
3. Ostrowsky BE et al. Control of vancomycin-resistant enterococcus in health care facilities in a region. N Engl J Med 2011;344 (19):1427–33.
4. Courvalin P. Vancomycin resistance in gram-positive cocci. Clin Infect Dis 2006;42 Suppl 1:S25–34.
5. Boyd DA et al. Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel D-Ala-D-Ser gene cluster, vanL. Antimicrob Agents Chemother 2008;52(7):2667–72.
6. Malhotra-Kumar S et al. Current trends in rapid diagnostics for methicillin-resistant Staphylococcus aureus and glycopeptide-resistant enterococcus species. J Clin Microbiol 2008;46 (5):1577–87.
7. Palladino S et al. Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay. J Clin Microbiol 2003;41(6):2483–86.
8. Fang H et al. Screening for vancomycin-resistant enterococci: an efficient and economical laboratory-developed test. Eur J Clin Microbiol Infect Dis 2011;31:261–65.
9. Kuch A et al. New selective and differential chromogenic agar medium, chromID VRE, for screening vancomycin-resistant Enterococcus species. J Microbiol Methods 2009;77(1):124–26.
10. Stamper PD et al. Comparison of the BD GeneOhm VanR assay to culture for identification of vancomycin-resistant enterococci in rectal and stool specimens. J Clin Microbiol 2007;45(10):3360–65.
11. Woodford N et al. Two distinct forms of vancomycin resistance amongst enterococci in the UK. Lancet 1990;335(8683):226.
12. Pendle S et al. Difficulties in detection and identification of Enterococcus faecium with low-level inducible resistance to vancomycin, during a hospital outbreak. Clin Microbiol Infect 2008;14 (9):853–7.
13. Biavasco F et al. Recovery from a single blood culture of two enterococcus gallinarum isolates carrying both vanC-1 and vanA cluster genes and differing in glycopeptide susceptibility. Eur J Clin Microbiol Infect Dis 2001;20(5):309–14.
14. Mammina C et al. VanB-VanC1 Enterococcus gallinarum, Italy. Emerg Infect Dis 2005;11(9):1491–92.
15. Qu TT et al. Heteroresistance to teicoplanin in Enterococcus faecium harboring the vanA gene. J Clin Microbiol 2009;47(12):4194–96.