A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci

Abstract

BACKGROUNDVancomycin-resistant enterococci (VRE) are resistant to most classes of antibiotics. Diagnosis of VRE using standard methods takes 2 to 5 days. Development of a rapid PCR-assay that detects and identifies resistant genes in bacteria would provide time-critical information on the presence of VRE in clinical samples allowing early treatment and management of infected patients.OBJECTIVESInvestigate the use of high resolution melting analysis (HRMA) and 16S-rRNA-PCR approach for rapid and cost-effective identification of VRE.DESIGNDescriptive antibiotic susceptibility studies.SETTINGManchester Academic Health Sciences Centre and School of Translational Medicine, University of Manchester, UK, and Department of Clinical Laboratory Sciences, Taibah University, Saudi Arabia.MATERIALS AND METHODSPCR-HRMA using 16S-rRNA V1-primers was used to detect and identify VRE. DNA from different strains of vancomycin-resistant and -sensitive Enterococcus faecalis (VSE) and Enterococcus faecium were amplified using V1-primer followed by HRMA in a single run. Differentiation of VRE from VSE was based on curve shapes generated against reference organisms (Bacteroides fragilis).MAIN OUTCOMES MEASURESAmplification curves and difference plots for VRE and VSE.RESULTSDifference plots were generated for all vancomycin-resistant and -sensitive E faecalis and E faecium strains by subtracting their fluorescence melting profile from that of a reference-species B fragilis. A characteristic curve shape was produced by vancomycin-sensitive E faecalis and E faecium. However, vancomycin-resistant strains of these bacteria were associated with a markedly different curve shape facilitating a clear differentiation.CONCLUSIONThe 16S-PCR-HRMA approach has the potential for detecting vancomycin-resistant E faecium and E faecalis. Data with VRE provide the basis for combining VRE identification with pathogens speciation in a rapid, cheap assay able to identify a pathogen as an Enterococcus and whether it is vancomycin-sensitive or -resistant E faecium or E faecalis in a single PCR and HRMA run.LIMITATIONSTested on specific, but not all, reference Enterococcus species and clinical isolates.

Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates

“…This traditional method of identification is time-consuming and labor-intensive. Also, it takes up to 5 days or more [4]. On the other hand, HRMA real-time PCR has the capacity to detect more than one target in a single reaction, thus it saves time and cost.…”

“…Moreover, this technique measures the changes in the fluorescence intensity during melting with higher resolution than conventional melting analysis [3]. Consequently, minor variations in the sequence of PCR amplicons cause slight changes in the melt curves and are detected using a dedicated gene-scanning software [4]. During the HRMA method, this process is monitored using intercalating dyes [3].…”

“…Enterococcus faecalis and Enterococcus faecium were identified as the two most prevalent Enterococcus spp. causing healthcare-associated infections (HAI) [4,6]. E. faecium has been reported as the most common Enterococcus spp.…”

“…The vanA is the most common phenotype of VRE and demonstrates higher level of resistance to vancomycin and teicoplanin. The spread of the vanA gene from Enterococcus to Staphylococcus aureus in the same patient demonstrates the mobility of these resistance genes [4,7]. Moreover, divIVA gene encodes a multifunctional protein, which plays an important role in cell division, biofilm formation, resistance, and viability in E. faecalis.…”

“…Hence, the use of molecular techniques is essential more than ever. Although many experimental studies have been performed on different species of Enterococcus by the HRMA method, limited research has been done on VRE strains [4].…”