|Year : 2013 | Volume
| Issue : 1 | Page : 47-52
A new approach of real time polymerase chain reaction in detection of vancomycin-resistant enterococci and its comparison with other methods
A Tripathi, SK Shukla, A Singh, KN Prasad
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
|Date of Submission||30-May-2012|
|Date of Acceptance||06-Feb-2013|
|Date of Web Publication||15-Mar-2013|
K N Prasad
Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow
Source of Support: Indian Council of medical research,, Conflict of Interest: None
Background: Vancomycin-resistant enterococci (VRE) are third leading cause of nosocomial infection. Therefore, an effective, accurate and early detection of VRE along with their minimum inhibitory concentrations (MICs) is required to initiate appropriate therapy and thus better patient outcome. Objective: To detect VRE by real time quantitative polymerase chain reaction (Q-PCR) and to compare the results with chrom ID (C-ID) VRE and PCR. Further the study also determined the fold change of vanA gene by Q-PCR in different groups of VRE isolates classified on the basis of glycopeptides MIC range. Subjects and Methods: A total of 145 (80 VRE and 65 vancomycin-susceptible enterococci) clinical isolates were included in the study. After the screening of VRE isolates MICs were determined by E-test and agar dilution method. Further VRE was confirmed by vanA and vanB specific PCR and Q-PCR. Results: The sensitivity and specificity of C-ID VRE was 100% and 95.38%. However, sensitivity and specificity of conventional and Q-PCR were found to be 100%. Conventional and Q-PCR confirmed that our all isolates were vanA type. Mean R value was significantly higher ( P < 0.001) in group I (MIC > 1024 μg/ml) when compared to group II (MIC 512-1024 μg/ml) and group III (MIC < 512 μg/ml) isolates. The mean R was also significantly higher in group II when compared to group III isolates ( P = 0.038). Conclusion: Q-PCR is a rapid technique to detect vanA in enterococci along with their MIC range, thus it might be helpful to decide the treatment modalities of infections caused by VRE.
Keywords: Minimum inhibitory concentrations, real-time polymerase chain reaction, vancomycin-resistant enterococci
|How to cite this article:|
Tripathi A, Shukla S K, Singh A, Prasad K N. A new approach of real time polymerase chain reaction in detection of vancomycin-resistant enterococci and its comparison with other methods. Indian J Med Microbiol 2013;31:47-52
|How to cite this URL:|
Tripathi A, Shukla S K, Singh A, Prasad K N. A new approach of real time polymerase chain reaction in detection of vancomycin-resistant enterococci and its comparison with other methods. Indian J Med Microbiol [serial online] 2013 [cited 2020 Apr 1];31:47-52. Available from: http://www.ijmm.org/text.asp?2013/31/1/47/108721
| ~ Introduction|| |
Enterococci are among the common microbiota of the gastrointestinal tract of humans and animals. The members of genus Enterococcus are also opportunistic pathogenic bacteria and have emerged as major cause of nosocomial infections worldwide. , Two species responsible for majority of human infections are Enterococcus faecalis and Enterococcus faecium.  The main reason for survival of these organisms in the environment is their intrinsic resistance to several commonly used antimicrobials and ability to acquire resistance against all currently available antimicrobials including glycopeptides either by mutation or transfer of plasmids and transposons through horizontal gene transfer mechanism via conjugation process.  Vancomycin, a glycopeptide antibiotic, once considered the preferred treatment regime of enterococcal infections may not always be effective due to emergence of resistance. Glycopeptide resistance is mediated by six different vancomycin resistance (van) gene operons. vanA and vanB remain the most clinically relevant of the van genes as they are associated with transposons and easily transferred from one to other organisms.  Phenotypically, the vanA gene mediates inducible, high-level resistance to vancomycin (minimum inhibitory concentrations [MICs], 64 to >1024 μg/ml) and teicoplanin (MICs, 16-512 μg/ml) while the vanB gene confers low to moderate-level resistance to vancomycin (MICs, 32-64 mg/ml). 
Many reports are available in the literature regarding the identification of vancomycin-resistant enterococci (VRE) using conventional microbiological methods, which require time, resources, and space. ,, These standard methods are labour-intensive and require 48-72 h to give the result. Therefore, management of VRE infection relies on rapid and sensitive detection.
Chrom ID (C-ID) VRE is a selective chromogenic medium developed for the detection and identification of vancomycin-resistant E. faecium and E. faecalis. Previously it has been found that C-ID VRE culture media is better than other culture based chromogenic media for the detection of VRE. , Conventional polymerase chain reaction (PCR) is also used for detection of VRE, although an improvement over conventional microbiological tests, lack absolute quantitation and requires time-consuming post-PCR analysis. , Real time quantitative polymerase chain reaction (Q-PCR) can quantify the presence of microorganisms in clinical specimens and improve the precision and sensitivity of conventional PCR by addition of a fluorescently labelled probe so that the target gene can be detected and quantified without post-reaction analysis. Since all these methods (C-ID VRE, PCR and Q-PCR) are more reliable than other conventional methods. Therefore, there is need to study which technique is better among these detection methods. Therefore, in the current study, we compared the performance of C-ID VRE medium (bioMe´ rieux, Nu¨rtingen, Germany) and conventional PCR with real time Q-PCR for the detection of VRE. This study also validated efficacy of new culture dependent Q-PCR for rapid detection of vancomycin resistance in clinical isolates of enterococci and evaluate the relation between fold changes of vanA with the level of resistance.
| ~ Subjects and Methods|| |
A total of 145 strains of enterococci (VRE = 80; E. faecalis = 60 and E. faecium = 20) and 65 vancomycin sensitive enterococci (VSE; E. faecalis = 35 and E. faecium = 30) isolated from different clinical specimens at a tertiary care hospital in India were included in the study. The study period was April 2009 to December 2010. The ethics committee of the institute granted approval for the study. All the strains were cultured in brain-heart infusion broth and stored in cryo vials with 12% glycerol at −80°C.
Characterization of clinical isolates
The strains were identified to the genus level by cultural characteristics, Gram's stain, bile esculin, potassium tellurite agar and salt tolerance tests. Vancomycin resistance in all isolates of enterococci was determined by disc diffusion and VRE-agar screen method (containing 6 μg/ml of vancomycin) following Clinical and Laboratory Standard Institute guidelines; MIC values were determined by E-test and agar dilution method. 
Plasmid deoxyribonucleic acid (DNA) extraction
Plasmid deoxyribonucleic acid (DNA) from all enterococcal strains was isolated by a modified alkaline lysis method. In brief cell pellets were initially incubated with lysozyme (20 mg/ml) in 25 mM Tris/10 mM Ethylenediaminetetraacetic acid (EDTA, pH 8.0), containing 50 mM glucose at 37°C for 1 h. After phenol-chloroform extraction, plasmid DNA was precipitated by addition of isopropanol and pelleted by centrifugation. The isopropanol was gently removed, and 70% ethanol was added to the DNA pellet. The plasmid DNA was again pelleted by centrifugation, the supernatant was removed, and the pellet was resuspended in 100 μl of 10 mM Tris-HCl buffer, pH 8.5.
DNA was extracted by boiling from all enterococcal strains. In brief, a sterile transfer device was used to pick four bacterial colonies from a culture plate. The colonies were resuspended in 200 μl of sterile water and boiled for 10 min. Extracted bacterial DNA was stored at -20°C.
Detection of VRE by chromogenic media
C-ID media plates were incubated aerobically at 37°C and examined after 48 h of incubation. Growth of enterococci on these media plates showed vancomycin resistance.
Vancomycin resistance was identified by following vanA and vanB gene specific primers: VanA forward 5'-ggg aaa acg aca att gc-3', vanA reverse 5'-gta caa tgc ggc cgt ta-3' and vanB forward 5' -acg gaa tgg gaa gcc ga-3', vanB reverse 5' -tgc acc cga ttt cgt tc-3' which were taken from previous published article  and synthesized by Europhine MWG Operon, Banglore, India. The amplicon size of vanA and vanB gene was 732 bp and 647 bp respectively. PCR amplification for vanA and vanB were performed in a final volume of 50 μL containing 3 μL of purified plasmid DNA, 1 × PCR buffer (20 mM Tris_HCl/50 mM KCl, pH 8.4), 1.5 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, 0.5 nM each primer, and 2.5 units of Taq polymerase (Bangalore Geni, Bangalore, India). PCR was carried out in a DNA thermal cycler (PTC-200; MJ Research, Cambridge, MA), with an initial denaturation step (94°C, 3 min), 30 cycles of denaturation (94°C, 1 min), annealing (54°C, 1 min), and extension (72°C, 1 min 30 s), followed by a final extension step (72°C, 10 min). E. faecium ATCC 51559 and E. faecalis ATCC 51299 was used as positive controls for vanA and vanB gene respectively. The negative control was performed for each set of PCRs containing all reagents but no DNA template. PCR amplification products were analyzed by agarose gel electrophoresis in 1.5% agarose and stained with ethidium bromide.
Q-PCR was performed by targeting the vanA that codes for vancomycin resistance by using Corbett Research 6000 real-time PCR instrument and Rotor gene 6000 software. Primer and probe sequences were taken from Spartan Biosciences ( http://www.idtdna.com/analyzer/Applications/OligoAnalyzer ) and synthesized from Eurofine MWG Operon, Banglore, India. The sequences are as follows: VanA forward 5' -tat gat ggc cgc tgc agg ta-3', vanA reverse 5' -cgg tga aat tat ccc aag tgg c-3', probe 5' -6FAM tgc act tcc cga act g-TAMRA-3' and vanB forward 5' -ggg aag atg gca gta tcc aag g - 3', vanB reverse 5' -caa gcg att tcg ggc tgt ga-3', probe 5' -6FAM tga gcc acg gta tct tc TAMRA-3'. Each 25 μl reaction contained 1 × TaqMan universal master mix (QIAGEN, Hilden, Germany), forward and reverse primer (20 nmol each), and TaqMan probe (10 nmol) and bacterial DNA 5 μl (1 μg). The reaction was performed with preliminary denaturation for 10 min at 95°C, followed by 50 amplification cycles of denaturation at 95°C for 16 s, and combined annealing/extension steps at 50°C for 50 s. To check for amplicon contamination, every run contained at least two 'no template' controls in which nuclease free H 2 O was substituted for template. The reporter dye 6-fluorescein amidite (6FAM) signal was measured against the internal reference dye 6-Carboxyl-X-Rhodamine (ROX) signal to normalize the signals for non-PCR-related fluorescence fluctuations that occurred from well to well.
Construction of standards and calculation of fold change of vanA
For calculation of threshold cycle (Ct) value, a standard curve was generated using 10-fold dilutions of vanA positive E. faecium ATCC 51559 DNA varying from 10 5 -10 1 copies. These curve were considered acceptable if a difference of 3.3 ± 0.3 cycles was demonstrated between each of the 10-fold dilutions, and if the correlation coefficient was at least 0.99 [Figure 1]. Ct results for experimental samples were extrapolated from the standard curve. Q-PCR assay targeting enterococci 23S ribosomalRNA gene was used as housekeeping gene and control for efficacy of DNA extraction and normalization for the number of cells amplified per reaction.  The relative fold change of vanA was analyzed using "2−(ΔCt)" mathematical equation. In brief the relative amount of vanA was calculated as follows: ΔCt = Ct (experimental) − Ct (housekeeping); Ratio (R) =2 -(ΔCt) .
|Figure 1: Construction of standards.(a) Bold line (-) shows the result of experiment in which serial dilutions (105-101) containing known quantities of the E. faecium ATCC 51559 strain plasmid deoxyribonucleic acid (DNA) containing vanA fragment were subjected to real time quantitative polymerase chain reaction (Q-PCR) while dotted line (….) shows the results of three represented clinical isolates. The amplifi cation curve shifted to right as the quantity of DNA was reduced. The system was sensitive enough to detect as few as 10 copies of vanA DNA. (b) Plot of threshold cycle (ct) against the target quantity with the later plotted on a common logarithm scale. The linearity of graph demonstrated the large dynamic range and the accuracy of Q-PCR assay|
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Data were analyzed using SPSS software (version 12.0, SPSS, Chicago, IL., USA). Mean R value of vanA in relation to MIC range was assessed by Student t-test. All the P values were two sided and considered significant when less than 0.05.
| ~ Results|| |
Bacterial strains and level of vancomycin resistance
Total 145 strains were included in this study. Of these, 80 were VRE and 65 were VSE strains which were confirmed by disc diffusion and VRE-agar screen methods. Out of 145 isolates, 62 E. faecalis and 21 E. faecium were detected as VRE by C-ID detection method. E. faecium was stained purple, and E. faecalis was stained blue or blue-green color.
On the basis of MIC to vancomycin, all VRE strains were grouped into following categories: Group I (MIC >1024 μg/ml), group II (MIC 512-1024 μg/ml) and group III (MIC < 512 μg/ml) including 55, 13 and 12 isolates in each group respectively. Likewise for teicoplanin, strains were also grouped in to two categories: Group I (>512) and group II (128-256) [Table 1].
|Table 1: Determination of genotypes and in vitro activity of glycopeptides against clinical isolates of enterococci|
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Sensitivity and specificity of different methods
The sensitivity and specificity of different methods are presented in [Table 2]. Both the molecular methods such as PCR and Q-PCR were found to be 100% sensitive and specific. PCR and Q-PCR confirmed that all VRE isolates were vanA type. None, of our isolates could amplify vanB either by PCR or by Q-PCR. Furthermore, all VSE isolates were found to be negative by both vanA and vanB genotypes.
|Table 2: Comparison of different diagnostic methods for detecting vancomycin-resistant enterococci in clinical isolates of enterococci|
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All 80 VRE isolates confirmed by phenotypic and genotypic methods were also positive for vanA Q-PCR. There was difference between ct values of vanA among all three VRE groups [Figure 2]a. The mean ct value of group I (25.05 ± 3.95) was significantly lower compared to group II (32.12 ± 4.45, P < 0.001) and in group II than group III (35.16 ± 3.91, P < 0.05). The mean ct value was also lower in group II compared to group III and data was close to significant (P =0.077). The mean fold change ratio (R) value of vanA gradually decreased from group I to group III [Figure 2]b. The mean R value of vanA in group I (10.62 ± 14.57) was significantly higher than group II (3.07 ± 5.25, P =0.011) and group III (0.76 ± 1.22, P <.001). The mean R value was also higher in group II compared to group III (P =.038). However, there was no significant difference was found in R value of vanA, when data were compared between teicoplanin MIC range (group I vs. group II; 7.49 ± 6.88 vs. 6.60 ± 12.46, P = 0.803). Further, our vanB Q-PCR assay failed to detect vanB gene in our isolates.
|Figure 2: Calculation of threshold cycle (Ct) and mean fold change ratio (r) (2−(ΔCt)) in vancomycin-resistant enterococci (VRE) groups (a) The graph displays a scatter diagram of differences in Ct values between all three VRE groups. The horizontal line represents the geometric mean while error bar represents the® SD (b) The relative fold change of vanA target genes compared to those of a housekeeping gene (23S rRNA) was calculated as follows; ΔnCt (experimental)− Ct (housekeeping), Ratio (r) 2−(ΔCt). The data shows the means and standard deviations of mean R value in all three VRE groups **P<0.001, *P<0.05|
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| ~ Discussion|| |
There are various laboratory and molecular methods for the identification of VRE which can differ in speed, sensitivity, and specificity. The results of the present study suggested that the Q-PCR detection assay is considerably good in comparison to C-ID and conventional PCR for VRE detection isolated from different clinical specimens. Furthermore, this assay also determined the vancomycin MIC range with detection of VRE isolates.
Previous studies suggested that C-ID detection method is better than other chromogenic media and conventional methods for detection of VRE. , Delmas et al. reported the sensitivity of C-ID VRE without enrichment was 92% but the enrichment step was significantly increased the rate of VRE detection from fecal samples and rectal swabs.  In our study, when enrichment was not performed for detection VRE, this method was 100% sensitive and gave three false-positive isolates of van-negative E. faecalis or E. faecium (from 145 isolates). Therefore, C-ID VRE needs confirmatory molecular or phenotypic testing to reach any conclusion.
Although among molecular methods, conventional PCR and Q-PCR both methods gave equal response in term of sensitivity and specificity. However, conventional PCR method is qualitative rather than quantitative. A study has already reported that Q-PCR is more rapid and sensitive technique than the other conventional methods for the detection of VRE.  Real-time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods required agarose gels for detection of PCR amplification which is the time consuming post PCR reaction step.
vanA and vanB types of VRE have plasmid encoded transferable vancomycin resistant gene and both are the highly prevalent genotypes in patients with nosocomial infection. Previous studies suggested that in vitro or in vivo transfer potential of vanA is higher than vanB, which indicates that nosocomial spread of vanA gene in health-care settings is a major problem.  The supremacy of vanA type VRE in our population demands the rapid and sensitive identification of such VRE strains.
Susceptibility and breakpoint measurements are determined based on the MIC that guides the treatment protocol of patients. Previously it had been reported that the culture based conventional methods required 48 h for VRE confirmation.  However, this study did not quantitate the level of resistance along with MIC. Previously it has been described that the copy number of Extended spectrum beta lactamases (ESBL) genes or their hosting plasmids in clinical isolates correlates with the MICs of beta-lactams.  The present study introduced first time a new application of Q-PCR that can detect VRE along with predicted MIC range. We found that the fold change, the indicator of vanA copy numbers had direct correlation with MIC values. Moreover, this technique is also has the advantage to differentiate between VSE and VRE. However, further studies are required to apply this method in the detection of low level inducible resistance to vancomycin which might be helpful in the early diagnosis of VRE.
In summary, the results of the present study suggest that vanA specific Q-PCR is accurate and more rapid technique than the standard culture dependent methods for detection VRE. The difference in fold change of vanA detected by Q-PCR also indicates the level of resistance to glycopeptide by enterococci. In the era of emerging drug resistance with limited treatment options Q-PCR appears to be a valuable method not only for the detection of VRE but also guiding appropriate therapy in order to provide better patient care in future.
| ~ Acknowledgment|| |
Aparna Tripathi acknowledge the financial assistance received from Indian Council of Medical Research (ICMR) Government of India, New Delhi through Senior Research fellowship grant no. 80/625/2009-ECD-I. Sanket Shukla also acknowledge the Department of Biotechnology (DBT), Govt. of India through Senior Research fellowship grant no. (DBT-JRF)/09-10/634.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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