Evaluation of phenotypic with genotypic methods for species identification and detection of methicillin resistant in Staphylococcus aureus.

BACKGROUND: Phenotypic methods for the detection of methicillin resistance are inadequate, due to presence of hetero-resistant population and dependence of environmental factors that may affect the phenotypic expression of resistance. AIMS: Present study was conducted, to evaluate the efficacy of phenotypic methods for the identification of species and mec-A mediated resistance in S. aureus with polymerase chain reaction (PCR), and to assess the prevalence of the Panton-Valentine leukocidin (pvl) toxin in methicillin resistant S. aureus (MRSA) and overall S.aureus population.
MATERIALS AND METHODS: A total of 200 clinical isolates of Staphylococci were subjected to phenotypic and genotypic methods for the species identification and detection of MRSA.
RESULTS: The specificity and sensitivity of conventional methods in the detection of S.aureus, was found to be 100 and 97.59% respectively. However, the performance of phenotypic methods in the detection of MRSA were: Oxacillin disc diffusion (DD)-sensitivity 70.58%, specificity 75.75%; cefoxitin DD-sensitivity 86.27%, specificity 83.33%; and oxacillin agar dilution-sensitivity 92.15%, specificity 90.90%. PVL gene was detected in all mec-A positive isolates irrespective of their types.
CONCLUSION: Phenotypic methods still preferred for the species identification, but for the reliable detection of MRSA an algorithm should include a combination of tests and apply a genotypic method for confirmation of resistance isolates showing discrepant results. Considering the high prevalence of PVL-MRSA, we recommend PCR as assay, as it has an advantage of simultaneous detection of mec-A and pvl genes by multiplex PCR.

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) strains were first described in England in 1961[1] shortly after methicillin became available for clinical use. They have subsequently spread throughout the world and are an important cause of nosocomial and community associated infections. Therefore, rapid and accurate detection of methicillin resistant strains in staphylococci is very essential in order to choose appropriate therapy, to prevent unnecessary use of glycopeptides antibiotics and to take necessary measures for infection control. The mechanism of resistance is due to acquisition of the mec-A gene, which encode for low-affinity penicillin-binding protein 2a (PBP2a). Therefore, presence and absence of mec-A gene indicates methicillin resistance and methicillin susceptibility in staphylococci respectively. Polymerase chain reaction (PCR) for the amplification of the mec-A is presently considered the gold standard for the detecting methicillin resistance in S. aureus.[2] In spite of the growing consensus in the literatures for this method, it is not yet available in the all clinical laboratories, therefore phenotypic methods still remains a methods of choice in the resource-constraint settings. However, the performance of phenotypic methods in the detection of methicillin resistance is not consistent, time consuming and also encounter difficulties in detecting all the resistant isolates, as many environmental factors,[3] and existence of various types of strains among S. aureus population limit its accuracy.[45]

The strains of S. aureus, which do not possess the usual genetic mechanism for oxacillin resistance, but appear resistance phenotypically are known as borderline oxacillin resistant S.aureus (BORSA) and modified S. aureus (MODSA). The BORSA is a strain of S. aureus, that hyper produce beta-lactamase and appear oxacillin resistant, whereas MODSA possess a modification of existing penicillin binding protein rather than a altered or new PBP2a which is encoded by mec-A gene, which is the mechanism of classical MRSA.[4] In yet another type of resistance, strains have the genetic information for methicillin resistance, but only a small number express the resistance in vitro, due to the presence of two sub-population (one susceptible and the other resistant), that may co-exist within a culture of staphylococci.[5] Such strain is termed as heteroresistance and occurs in staphylococci resistant to penicillinase-stable penicillins, such as oxacillin.

Cells expressing hetero-resistance grow more slowly than oxacillin-susceptible population and may be missed at temperature above 35°C and should incubate for full 24 hrs when isolates being tested against oxacillin.[6]

To overcome these difficulties, many studies have been carried out for the evaluation of phenotypic methods for the accurate detection of methicillin resistance and different recommendations have been made regarding the most reliable method for routine use.[247-10] In comparison to gold standard method of PCR, many authors have recommended that cefoxitin could be a surrogate marker for the detection of methicillin resistance in the settings where PCR is not feasible, as it is a better inducer of mec-A gene and disc diffusion test using cefoxitin give clearer endpoints and are easier to read than test with oxacillin.[48-9]

Besides its comparable accuracy to PCR, it was also found very efficient in the detection of BORSA strains, which can avoid the mis-categorization of Methicillin-sensitive S. aureus (MSSA) into MRSA.[4] PCR also encountered difficulty in discriminating MRSA from methicillin-resistant coagulase negative staphylococci (MR-CoNS), because epidemiological studies revealed that mec-A gene are also widely distributed among coagulase negative staphylococci (CoNS) and associated with methicillin resistance.[11-13] So with the detection that fem-A gene encodes a factor essential for methicillin resistance present universally in all S. aureus isolates, MR-CoNS can be excluded.

Panton-Valentine leukocidin (pvl) is a cytotoxin that causes leucocyte destruction and tissue necrosis, produced by less than 5% of S. aureus strains.[14] It has been preferentially linked to furuncles, cutaneous abscess and severe necrotizing skin infections.[15] Reports suggested the strong association of pvl toxin in Community-associated (CA) S. aureus and CA-MRSA infections.[1416]

The purpose of the present study was to compare the efficacy of phenotypic methods for the identification of S. aureus and detection of MRSA with genotypic method of PCR by direct detection of the S. aureus fem-A gene (serve as internal control) and mec-A gene and secondly to assess the prevalence of the pvl toxin in the overall S. aureus population and MRSA by multiplex PCR with the simultaneous detection of mec-A and pvl genes.

MATERIALS AND METHODS

A total of 200 isolates of Staphylococci, isolated from different clinical samples during the period from September 2009 to March 2011 in the tertiary-care teaching hospitals were studied. All the isolates were identified to the species level by using conventional techniques like colony morphology on 5% sheep blood agar, catalase test, slide and tube coagulase test, DNase test (Hi-Media, Mumbai, India), Phosphatase test (Hi-Media, Mumbai, India) and Modified Hugh-Leifson's test.

Case definition and source of data

Hospital-associated MRSA (HA-MRSA) isolate defined as one cultured from a clinical specimens obtained 72 hrs after patient's hospital admission or whose sources of isolation were associated with risk factors for HA-MRSA infection (e.g., recent hospitalization, recent surgery, residence in a long-term care facility, drug use, etc.,)[1718] within one year of MRSA isolation date. In the CDC definition an infection is considered hospital-associated, if it occurs >48 hrs after admission; yet, we chose >72 hours as a cut-off to more conservatively capture hospital-associated infections, i.e., to minimize the mis-categorization of community-associated infections as hospital-associated infections.[19]

Community-associated MRSA (CA-MRSA) isolate were defined as one cultured during the first 72 hrs of a patient's hospital admission, or from patient's whose sources of isolation were not associated with risk factors for HA-MRSA infection as mentioned above.

The secondary data of the patients were obtained from the laboratory investigation register and from medical record file of patients. Data of the study subjects included basic demographic profiles, status of patients (out-patients/in-patients), ward admitted, specimen type, length of hospital stay, clinical notes, and detailed of risk factors associated with HA-MRSA infections as mentioned above.

Detection of MRSA

Phenotypic methods

Disc diffusion methods

In all confirmed S. aureus isolates, oxacillin and cefoxitin disc diffusion (DD) methods were performed for the identification of methicillin-resistant. Four to five colonies from overnight growth was inoculated into 4 to 5 ml cation-adjusted Mueller-Hinton broth (Hi-Media, Mumbai, India) and incubated at 35°C until turbid to 0.5 McFarland standard, and inoculated on two separate Mueller-Hinton agar (MHA) plates, then 1 μg/ml oxacillin disc (Hi-Media, Mumbai, India) and 30 μg/ml cefoxitin disc (Hi-Media, Mumbai, India) placed aseptically and incubated at ambient air, 33-35°C for 16-18 hrs in cefoxitin and full 24 hrs for oxacillin disc diffusion method. Oxacillin DD test was interpreted as follows: Resistance (≤10 mm), moderately sensitive (11-12 mm) and sensitive (≥13 mm), whereas cefoxitin DD ≥ 22 mm as sensitive and ≤ 21 mm as resistant. Reference strains ATCC 29213 (MSSA) and ATCC 43300 (MRSA) included as a control strains in every batch of testing.[6]

Oxacillin agar dilution method

The preparation of oxacillin (Sigma-Aldrich, St Louis, USA) stock solution and testing conditions for oxacillin agar dilution was done as per Clinical and Laboratory Standards Institute (CLSI) guidelines.[6] The range of dilution used was 0.125 to 512 μg/ml. The preparation of test inoculum was similar to DD. Once it matched with standards, 1 μl of bacterial suspension was spot-inoculated on 2% Nacl MHA with a calibrated loop,[620] with various concentrations of oxacillin, starting with the lowest dilution and incubated at ambient air 33-35°C for 24 hrs. Reference strains MSSA (ATCC 29213) and MRSA (ATCC 43300) were included in each batch of testing along with drug free control plates to check the validity and purity of testing. The test result was interpreted as resistant (MIC of ≥ 4 μg/ml) and sensitive (MIC ≤ 2 μg/ml).

Genotypic methods

DNA isolation

The test inoculum was prepared by inoculating two to three isolated colonies of S. aureus into 3 to 4 ml of Brain-Heart infusion (BHI) broth (Hi-Media, Mumbai, India) and incubated overnight at ambient temperature of 35-37°C. The DNA was extracted by using the HiPurA™ Bacterial and Yeast Genomic DNA Miniprep Purification Spin kit (Hi-Media, Mumbai, India). The DNA concentration was determined as micrograms per millilitre according to A260 values by Nanodrop ND-1000 Spectrophotometer (Welmington, USA)

Monoplex polymerase chain reaction for the detection of fem-A gene

The monoplex PCR was performed for the detection of fem-A gene as an internal control to validate that all the isolates tested were S. aureus. The primers, GFEMAR-1 (5’- AAAAAAGCACATAACAAGCG-3’) and GFEMAR-2 (5’ GATAAAGAAGAAACCAGCAG-3’) with 132 bp amplicon size for the amplification of fem-A gene were taken from published sequence by Mehrotra et al.,[21] and checked for specificity against available S. aureus genomes with the BLAST utility available through the National centre for Biotechnology Information website (www.ncbi.nim.nih.gov) under GenBank (accession No. X17688.1) and commercially obtained from Sigma-Aldrich, Bangalore, India. PCR was performed by using Qiagen Taq PCR Master mix Kit (Hilden, Germany). A 25 μl final reaction volume consisted of 12.5 μl of master mix, 1 μl of each forward and reverse primers (0.4 μM), 7.5 μl of RNase free water and 3 μl of DNA template. PCR was performed in thermocycler (Biometra Goettingen, Germany) with the thermocycling conditions of initial denaturation (95°C, 5 mins), followed by 35 cycles of three step cycling conditions of denaturation (94°C, 30 sec), annealing (57°C, 1 min) and extension (72°C, 30 sec) followed by final extension (72°C, 7 min) and soaked at 4°C. Then 5 μl of amplified products were mixed with 2 μl of ethidium bromide (Fermentas, St. Leon-Rot, Germany) and were loaded on a 2% agarose gel (Amresco, Solon, USA) along with GeneRuler™ 100 bp Plus DNA Ladder (Fermentas, St. Leon-Rot, Germany); electrophoresis was performed at 100 volt for 50-60 min and visualized under UV transilluminator (Bio-Doc analyzer, Biometra, Goettingen, Germany).

Multiplex polymerase chain reaction for the detection of mec-A and pvl gene

The primers for the amplification of mec-A gene GenBank (accession No. Y00688) and pvl gene GenBank (accession No. X72700) were MECAP4 (5’-TCCAGATTACAACTTCACCAGG-3’) and MECAP7 (5’- CCACTTCATATC TTGTAACG-3’) as described by Oliveria et al.[22] and luk-PV-1 (5’- ATCATTAGGTAAAA TGTCTGGACATGATCCA-3’) and luk-PV-2 (5’- GCATCAA GTGTATTGGATAGCAAAAGC-3’) as described by Mclure et al.,[23] respectively. PCR was performed by using Qiagen Multiplex PCR kit (Hilden, Germany) with slight modification. A 25-μl final reaction volume consisted of 12.5 μl mastermix, 2.5 μl primer mix (0.2 μM of each primer), 3 μl of DNA template and 7 μl of RNase free water. DNA samples were subjected to thermocycling conditions with initial inactivation step (95°C, 15 min) with three steps cycling condition of denaturation (94°C, 30 sec), annealing (60°C, 90 sec) and extension (72°C, 90 sec) for 35 cycles with final extension (72°C, 10 min) and soaked at 4°C. Then 5 μl of amplified products were mixed with 2 μl of ethidium bromide (Fermentas, St. Leon-Rot, Germany) and loaded on a 2% agarose gel (Amresco, Solon, USA) along with GeneRuler™ 100 bp Plus DNA Ladder (Fermentas, St. Leon-Rot, Germany); electrophoresis was performed at 100 volt for 50-60 min and visualized under UV transilluminator (Bio-Doc analyzer, Biometra, Goettingen, Germany).

RESULTS

A total of 200 isolates of Staphylococci were tested; 119 were detected as S. aureus and 81 were detected as CoNS. Of the 119 S. aureus, fem-A gene were detected in 117 (98.36%) isolates, whereas none reported from CoNS [Table 1, Figure 1]. The performance of conventional method in the accurate identification of S. aureus was evaluated by keeping the PCR (fem-A) as gold standard. All fem-A positive isolates were also identified as S. aureus by standard methods, however out of 83 fem-A negative isolates, 81 were detected as CoNS and 2 were detected as S. aureus by conventional techniques (sensitivity 100% and specificity 97.59%) [Table 2].

Table 1

Comparison of phenotypic and genotypic methods for the identification of S.aureus

Figure 1

Monoplex PCR for the detection of fem-A (162 bp), Lane 1 to 5 = Positive for fem-A gene, M = (Marker 100 bp)

Table 2

Performance of phenotypic method in the identification of S. aureus as defined by PCR

Of the 117 fem-A positive isolates, 51 (43.58%) were amplified by mec-A (162 bp) and 54 (46.15%) were amplified by pvl (433 bp) genes respectively; whereas the presence mec-A and pvl genes were not shown by 66 (56.41%) and 63 (53.84%) isolates respectively [Figure 2].

Figure 2

Multiplex PCR (mec-A and PVL gene). Lane 1,2,3,5 = Positive mec-A (162 bp) and PVL (433 bp), M = Marker (100 bp DNA ladder), Lane 4 = Negative mec-A (162 bp) and positive PVL (433 bp)

Among 51 mec-A positive isolates, 36 (70.58%), 41 (80.39%) and 47 (92.15%) isolates were identified as MRSA by oxacillin DD, cefoxitin DD and oxacillin agar dilution methods respectively [Table 3]. Whereas, of 66 mec-A negative isolates, 44 (66.66%), 48 (72.72%) and 60 (90.90%) isolates were correctly detected as MSSA by oxacillin DD, cefoxitin DD and oxacillin agar dilution methods respectively. The sensitivity, specificity, positive and negative predictive values for oxacillin DD were 70.58, 75.75, 69.23 and 76.92% respectively; for cefoxitin DD were 86.27%, 83.33%, 80% and 88.70% respectively; and for oxacillin agar dilution were 92.15%, 90.90%, 88.67% and 93.75% respectively [Table 4].

Table 3

Comparison of phenotypic and genotypic methods for the detection of MRSA

Table 4

Performance of phenotypic methods for the detection of MRSA as defined by mec-A gene detection by PCR

Majority of mec-A positive isolates (70.58%) which were resistant to both cefoxitin and oxacillin DD had MIC of ≥16 μg/ml, whereas 15.68% isolates (8/51) which were sensitive to oxacillin but resistant to cefoxitin had MIC value of 8 μg/ml. However, variations in the MIC values were seen among the isolates, which was sensitive to both DD methods. Of the seven sensitive isolates by both DD methods, three and four isolates had MIC value of 4 μg/ml and 1-2 μg/ml respectively [Table 5].

Table 5

Comparison of MIC value of mec-A positive isolates (MRSA) with the results of disc diffusion methods (N=51)

Similarly, among the mec-A negative isolates, the majority of isolates [75.75% (50/66)] had MIC value in the range of 0.125-1 μg/ml, and 7.5% (5/66) isolates which were resistant to oxacillin but sensitive to cefoxitin had MIC of 2 μg/ml. Whereas among the 11 mec-A negative isolates, which were resistant to both DD methods, 5, 3 and 3 isolates had MIC value of 2 μg/ml, 4 μg/ml and 8 μg/ml respectively [Table 6].

Table 6

Comparison of MIC value of mec-A negative isolates (MSSA) with the results of disc diffusion methods (N=66)

Of the 51 mec-A positive (MRSA) isolates, 36 (70.58%) isolates were categorized as CA-MRSA and 15 (29.41%) isolates were categorized as HA-MRSA. Of the 54 pvl positive S. aureus isolates, 51 (94.44%) were reported from MRSA and 3 (5.55%) were reported from MSSA. All CA- and HA-MRSA were found to be harbouring pvl gene.

DISCUSSION

For the past 50 years S. aureus has been a dynamic human pathogen that has gained the deepest respect of clinicians, since the first report of MRSA infection in Boston city hospital in 1961.[1] Since then MRSA has become widespread all over the world. As methicillin resistant strains are widely distributed in S. aureus as well as in CoNS, therefore with the detection of a marker specific to S. aureus along with mec-A gene proved to be a more reliable indicator to identify MRSA by differentiating it from mec-A positive CoNS.[42425] In our study, we have used fem-A gene as a specific marker to S. aureus, as besides being a unique feature to S. aureus,[26] it also act as a regulator gene in the expression of high-level methicillin resistant in S. aureus.[24] So, with the inclusion of fem-A gene in our study, it has added advantage of accurate identification of S. aureus along with the detection of any influence of fem-A gene on methicillin resistant in S. aureus.

In the present study, the detection rate of fem-A gene among S. aureus population was 98.31% (117/119) and none detected in CoNS by monoplex PCR, against detection rate of 89.4% and 97% of fem-A and fem-B genes by Kobayashi et al.[24] with the detection of three genes (fem-A, fem-B and mec-A) in a single run PCR (multiplex). This difference could be due to simultaneous detection of three genes in a single run by multiplex PCR,[24] against one gene (fem-A) in present study by monoplex PCR, that may increase the detection rate of fem-A gene in the present study.

Similarly, on evaluating the efficacy of conventional methods in the detection of S. aureus vs Monoplex PCR (fem-A); 100% fem-A positive and 2.4% fem-A negative isolates of S. aureus were identified by conventional methods (sensitivity 100% and specificity 97.59%) in the present study. This is in contrast to other studies having detected fem-A gene in few CoNS isolates, but similar in having good co-relation of standards methods in the detection of S. aureus in comparison to PCR.[24-26] Though, the detection rate of fem-A gene varies slightly in different geographical regions, but expression of fem-A gene is a unique feature of S. aureus and it can be reliably used as a marker in the differentiation of S. aureus from CoNS. The good performance of conventional techniques in the detection of S. aureus in comparison to PCR (fem-A) suggest that the conventional methods still remain a method of choice in the accurate detection of S. aureus.

On further evaluating the involvement of fem-A gene in the expression of methicillin resistance, it was found that 43.58% isolates were fem-A, mec-A positive (PCR confirmed MRSA) and 56.41% isolates were fem-A positive, mec-A negative (PCR confirmed MSSA) isolates. This is in concordance to other studies where prevalence of fem-A gene in mec-A negative isolates were found to be higher[24] or almost equivalent[25] to mec-A positive isolates. This finding indicate that the influence of fem-A gene on the methicillin resistance in the S. aureus strains is slight as prevalence of fem-A gene was found higher in MSSA (mec-A negative) in comparison to MRSA (mec-A positive). From these results, it has become evident that methicillin resistance in S. aureus may be significantly regulated by other genes like mecR1 and mec I genes or some other unidentiifed factors, rather than fem-A gene.

The heterogeneous nature of methicillin resistance in S. aureus limits the accuracy and reliability of phenotypic methods such as DD and dilution methods.[10] In the present study, among the phenotypic methods, the performance of oxacillin agar dilution (sensitivity 92.15%, specificity 90.90%) was found to be better in comparison to cefoxitin DD (sensitivity 86.27%, specificity 83.33%) and oxacillin DD (sensitivity 70.58%, specificity 75.75%) in the detection of mec-A mediated resistance. In contrast, recently, many studies have reported 100% accuracy of cefoxitin DD test (100% ssensitivity and specificity),[49] in comparison to oxacillin DD (sensitivity 100%, specificity 56%),[4] (sensitivity 87.5%, specificity 100%),[9] and oxacillin agar dilution (sensitivity 100%, specificity 90%).[4] Similarly, Bosselemez-Tmaz et al., reported better performance of cefoxitin DD (sensitivity 99.19%, specificity 100%) over oxacillin DD (sensitivity 95.96%, specificity 100%) in comparison to PCR.[8] A few studies reported that the oxacillin DD (100% sensitivity and specificity) approaches the accuracy of PCR in the detection of MRSA.[7] However, in concordance to the present study, one study reported that none of the phenotypic methods was reliable for the detection of the methicillin resistance in S. aureus.[10]

In the present study, the higher false negativity of DD methods (oxacillin 29.41%, and cefoxitin 13.7%) was reported in comparison to 7.84% of oxacillin agar dilution. All four isolates were oxacillin sensitive S. aureus (OSSA) by all three phenotypic methods but mec-A positive, suggesting these isolates probably non-PBP 2’ producing strains have been detected earlier and referred as cryptically methicillin resistant strain.[2427] This was supported by the fact that MIC value of all four isolates was in the range of 1-2 μg/ml. Therefore it is recommended that the isolates with MIC value in this range should be further confirmed with PCR, as clinical problem with such strains like beta-lactam antibiotics induced production of PBP 2’ may be seen during chemotherapy, leading the conversion of the strain into oxacillin resistant S. aureus (ORSA) as demonstrated in vitro.[28]

The percentage of cryptic isolates reported in our study, is comparatively less than the study conducted by Kobayashi et al., (16.7%),[24] but higher than the study conducted by Nikbakht et al., (3.75%).[27]

The rest 11 (21.56%) isolates were OSSA by oxacillin DD and 3 (5.8%) by cefoxitin DD methods, but all were ORSA by oxacillin agar dilution; suggesting it as heteroresistant strains of MRSA instead of cryptic strains. Heteroresistant strains have usual mechanism for methicillin resistance, but few expresses in vitro, suggesting that the expression of resistance sub-population to be suppressed by over expression of susceptible sub-populations.[9] This too corroborated by the fact, that all isolates had MIC value in the range of 4-8 μg/ml. The specificity of the DD methods is often affected by high false positive isolates. The high false positivity of DD methods (oxacillin 24.24% and cefoxitin 16.66%) in the present study could be due to hyper-production of beta-lactamase, leading to phenotypic expression of oxacillin resistance. Such strains are called BORSA strains, because despite the absence of genetic mechanism for methicillin resistance (mec-A gene), these starins express resistance phenotypically.[4] However, wide variation was seen in the MIC value of BORSA strains. The MIC value all false positive isolates by all three phenotypic methods (9.09%) was in the range of 4-8 μg/ml. Similar oxacillin MIC range was reported earlier for very high percentage (45%) of BORSA strains as detected by oxacillin DD,[4] but no such isolates had been reported by cefoxitin DD.

The rest 5 (7.57%) isolates which were resistant to both DD methods but sensitive to oxacillin agar dilution (MIC-2 μg/ml) and another 5 (7.57%) isolates resistant to oxacillin DD but sensitive to cefoxitin DD and oxacillin agar dilution (MIC of 2 μg/ml). The results suggest that such isolates may have another mechanism of oxacillin resistant than PBP 2a for methicillin resistance. Such strains are labelled as modified S. aureus (MODSA) strains, which possess a modification of existing penicillin binding proteins rather than acquisition of new PBP as in the mechanism for classical MRSA. Probably, these MODSA strains under antibiotic pressure may evolve into fully resistant isolates in future, as MIC value of such isolates (2 μg/ml) were higher than the isolates (0.125-1 μg/ml) which were sensitive by all three phenotypic methods in concordance with PCR; and labelled as true MSSA. Occurrence of various types of strains among S. aureus population complicated the accurate detection of MRSA. Though the cefoxitin was proved to be better predictor of mec-A mediated resistance and the performance was equivalent to PCR,[489] it was also found to be unsatisfactory. However, not absolute but better co-relation was seen between oxacillin agar dilution and PCR in the detection of mec-mediated resistance. The determination of the MIC of those isolates showing discrepancy results with DD methods give additional information about the different strains of S. aureus that may complicate the treatment and patient management. Comparing the MIC value of mec-A positive and negative isolates it was found that majority of isolates showing discrepant results with DD methods was in the MIC range of 2-8 μg/ml, suggesting this MIC range to be critical (doubtful) breakpoint and the isolates with MIC within this range should always be confirmed further with PCR.

The screening of pvl gene among MRSA has gained importance in recent years due to high involvement of pvl toxin in CA-MRSA infections.[1629] The three major genotypic markers that distinguishes CA-from HA-MRSA isolates are: Their genetic lineage (ST), the architecture of mobile genetic element (SCCmec type) and presence of PVL toxin.[15] Almost 100% of CA-MRSA strains possess the pvl gene compared to <5% in HA-MRSA and MSSA.[30] Thus many researchers have highlighted the pvl gene as a reliable marker for CA-MRSA infections.[162931-32]

In the present study, pvl gene was included to validate the molecular definition of CA-MRSA and test the hypothesis that “PVL toxin is a reliable marker for CA-MRSA infections”. We found the prevalence of pvl gene as 46.15% (54/117), 94.44% (51/54), and 5.55% (3/54) among S. aureus, MRSA and MSSA isolates respectively. All MRSA were positive for pvl gene irrespective of their types (CA-36 and HA-15), This is in contrast to several studies, which reported presence of pvl toxin only in CA-MRSA and none from HA-MRSA isolates.[2931-33] But our study is in agreement with few studies reporting that the presence of PVL toxin cannot be used as a sole marker for CA-MRSA.[3435] Considering the high detection of pvl gene in MRSA (irrespective of their types) than MSSA, it can be rather hypothesize that MRSA is an important reservoir of pvl gene and is now being slowly acquired by MSSA strains.

It is concluded that phenotypic methods still remain the preferred choice for the species identification, but for the detection of MRSA none of the phenotypic methods showed 100% accuracy with PCR. However, considering the better performance of oxacillin agar dilution and also the additional information provided by this method for the isolates showing discrepancy results with DD methods, it is suggested that a combination of tests should be used and PCR method should be applied for confirmation of resistance, as assay has an advantage of rapid and simultaneous detection of mec-A mediated resistance along with the detection of any other gene (fem-A and pvl)) with multiplex PCR techniques.

Future work suggested

Further molecular typing of the MRSA isolates will be helpful to validate the molecular definition of CA- and HA-MRSA isolates, as pvl gene one of the important genotypic marker for CA-MRSA, was found to be an unreliable marker to differentiate it from HA-MRSA. It will be helpful in taking appropriate measures in control and prevention of further spread of MRSA.