Molecular identification of methicillin resistance and virulence marker in staphylococcus aureus

Methicillin-Resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens in Sri Lanka. Life threatening infections with MRSA require rapid identification in order to initiate appropriate antimicrobial therapy. The PantonValentine leukocidin (PVL) is a virulence factor associated with severe MRSA infections for which routine detection is time consuming and dependent on the culture environment. 1 The current study was therefore carried out to develop a rapid method for detection of MRSA and the presence of PVL gene using a multiplex polymerase chain reaction (mPCR). In the first phase of this study, results of conventional and molecular methods were compared for 43 clinical isolates of Staphylococcus aureus received from Teaching Hospital Peradeniya (THP). In the second phase, an additional 43 clinical samples obtained from patients were directly tested using mPCR and results including detection time of conventional and molecular methods compared. femB was negative in 7 of 43 isolates identified as S aureus by conventional methods. Of the remaining 36 isolates, 12 were mecA positive with an oxacillin MIC of >2mg/L. The mecA gene was detected in 10 isolates identified as MSSA by conventional testing. The PVL gene was detected in 11 strains including both MRSA and MSSA. Direct mPCR on clinical samples was negative for all 3 genes in 15 of 43 samples from which S aureus was isolated. Of the remaining 28 samples, 24 were femB positive, 16 were mecA positive and 14 were positive for the PVL gene. mPCR is helpful for the rapid identification of methicillin resistance and PVL production is staphylococci but less helpful in direct testing of samples.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a specific strain of Staphylococcus aureus that has developed antibiotic resistance to all β-lactams.MRSA was first discovered in UK in 1961 and is now widespread, particularly in the hospital setting where it is commonly termed a superbug. 2In addition to MRSA traditionally being seen as a hospital-associated infection, in recent years community-acquired strains of MRSA (CA-MRSA) are being reported outside healthcare facilities throughout the world.These strains are often seen in healthy carriers -mostly among children, and often associated with skin and soft-tissue infections such as boils, styes and furunculosis. 3ome strains of CA-MRSA are particularly virulent and can cause life-threatening diseases such as pneumonia, mastitis, meningitis, osteomyelitis and endocarditis. 4sistance to methicillin is mediated by the gene mecA which codes for a unique penicillin binding protein PBP2A with reduced affinity for β-lactam rings (the primary active-site of the β-lactam antibiotics) and is transmitted by a mobile genetic element termed staphylococcal cassette chromosome mec (SCCmec). 5In addition, some strains of S. aureus over-express β-lactamase (BORSA) and some strains of S. aureus, known as MODSA, which possess a modification of existing penicillin-binding proteins ( rather than acquisition of a PBP2A) appear to be resistant to oxacillin despite being mecAnegative.It is felt that reporting them as MRSA is probably an overcall of resistance.Therefore, PCR detection of mecA is considered as the 'gold standard' for staphylococcal methicillin resistance. 2 Another gene, femB may further induce the expression of PBP2A for methicillin resistance in S. aureus and also acts as a specific species marker for identification of S. aureus in molecular assays. 6-MRSA strains tend to be more susceptible to non-ß lactam agents when compared with the hospital-acquired MRSA isolates. 2Many CA-MRSA have acquired the Panton-Valentine leukocidin (PVL) gene that produces a series of chemicals contributing to the invasiveness of these MRSA strains. 5PVL is a multicomponent protein cytotoxin which belongs to the pore-forming toxin family that forms an octameric pore in the affected membranes of human polymorphonuclear neutrophils (PMNs), macrophages and monocytes. 7PVL is mainly associated with primary skin infections, especially furuncles, and with other deep-seated infections including necrotizing community-acquired pneumonia, pyomyositis, osteomyelitis and septic arthritis. 7 Sri Lanka, MRSA was first reported from the Teaching Hospital, Peradeniya (THP) in October 1989 8 and has emerged as an important nosocomial pathogen over the past 20 years with up to 86% isolation rates in some hospitals. 9Infections with MRSA can give rise to increased morbidity and mortality, prolonged stay in hospital and increased cost of patient management.Isolation using selective media such as mannitol salt agar and detection of methicillin resistance by conventional antibiotic sensitivity testing and minimum inhibitory concentration (MIC) determination using dilution methods or E tests have been employed to detect MRSA. 10 These methods require 2-3 days for completion.
In addition, hetero-resistant strains may not always be detected using these methods as such isolates may express resistance only in some cells. 11More modern microbiological methods, such as DNA hybridization and the polymerase chain reaction (PCR) have focused on the detection of a DNA segment, mecA gene unique to MRSA.These assays are usually completed within one or two working days, thereby allowing a more rapid and accurate identification of methicillin resistance. 11Rapid detection and identification leads to early diagnosis and appropriate treatment which can significantly limit the duration and outcome of infection.The detection of PVL in S. aureus may also be helpful in explaining the severity of the infection.Further, aggressive therapy is required to treat S. aureus secreting PVL due to its severity of neutrophils, macrophages and tissue destruction thus warranting early detection.As PVL cytotoxin is difficult to detect by conventional methods, it is necessary to develop a molecular method that detects the PVL gene. 1 The present study sought to develop a rapid in-house extraction method for simultaneous detection of femB, mecA and PVL genes in clinical isolates and directly from patient samples using PCR.

Specimens
Forty-three clinical isolates identified as S. aureus by conventional laboratory methods collected from Teaching Hospital, Peradeniya (THP) between June and August 2007 were included in Phase 1 of the study.
In Phase 2, 43 patient samples [18 pus aspirates, 4 tracheal aspirates, 3 blood cultures, 1 liver abscess, 9 pus swabs, 3 umbilical swabs, 4 eye swabs and 1 ear swab] received between September to December 2007 from which S. aureus had been isolated and ABST performed in the routine microbiology laboratory-THP were included in the study.
A standard positive control of methicillin sensitive S aureus (NCTC 6571) and a standard negative control of Escherichia coli (NCTC 10418) were included with each batch tested. 12Minimum inhibitory concentration (MIC) and mecA detection were used for susceptibility testing.
The MIC of oxacillin was determined using the agar dilution method. 13The antibiotic concentration causing completely inhibition of growth was taken as the end point.The results for S. aureus were reported as susceptible (methicillin sensitive S aureus -MSSA) when MIC was < 2 mg/L and resistant (MRSA) when > 2mg/L. 14

Multiplex PCR for the detection of femB, mecA and PVL genes
Sample homogenization and DNA extraction were performed by the methods described by Gunasekera,M.B. 15 PCR assay was performed on purified genomic DNA extracted from clinical isolates as well as clinical specimens.Two sets of oligonucleotide primers were used to amplify mecA, femB and PVL genes.Primer sequences published by Jonas et al 2002 16 and Lina et al 1999 1 were employed for the detection of susceptible genes.Multiplex PCR was carried out with 5 and 2.5 µl of the extracted DNA in 25 µl of PCR amplification mix consisting of 10x Buffer, 25mM of MgCl 2, 2.5U of Taq polymerase, 0.25mM of dNTP, 0.4µM of each primer; mecA1, mecA2, luk PV1 and luk PV2 and 0.5 µM femB1 and femB2.After amplification for 35 cycles (45s of denaturation at 94 °C, 45s of annealing at 50 °C and 60s of extension at 72 °C), the amplicons were resolved by 1.5% agarose gel electrophoresis.(Fig. 1 & 2).

Sensitivity of PCR assay
To determine the lower limits of detection of MRSA, colony counts were performed on serial dilutions of a confirmed specimen from 10 0 to 10 -6 CFU/µl and parallel PCR was carried out on each dilution.PCR detection sensitivity was compared with the colony counts in order to determine the sensitivity of the mPCR assay. 10

Results
In the first step of this study, all 43 clinical isolates were conventionally confirmed as S. aureus.Using PCR, the femB gene was detected in 36 of the 43 isolates.
Out of the 43 isolates, 31 had MIC of methicillin <2 mg/L (MSSA) and 12 isolates were MRSA with values of 4 and > 128 mg/L.Using the PCR, all 12 (28%) MRSA strains identified by conventional methods gave positive results for the mecA gene.However, 10 of the 31 isolates identified as MSSA were also mecA positive.All 7 femB gene negative isolates were mecA positive (Figure 1).Eleven out of the 36 (30%) S. aureus isolates demonstrated the PVL gene.Ten of them were MSSA and 1 was MRSA (Table 1).

Figure1 Resolved DNA products from 7of 43 S. aureus clinical isolates
Of the S. aureus isolated from the 43 clinical samples tested in the second phase of the study, 18 (42%) were identified as MRSA by conventional disc diffusion testing with turn-around time of 48-72hrs.These 43 samples were directly examined for femB, mecA and PVL genes using PCR (Figure 2).An appreciable number of samples (15/43) were negative when tested directly for all 3 genes (Table 2).Of the remaining 28 specimens, femB gene was detected in 24(86%).The mecA gene was detected in 14 (78%) of the 18 samples from which MRSA was isolated.In addition, 2 samples from which MSSA was isolated were found positive for the mecA gene.Four samples which expressed the mecA gene were negative for femB gene.Fourteen of the 43 samples were positive for the PVL gene.7 of them were MSSA and 7 were MRSA.

Discussion
Detection of MRSA is important for patient care and appropriate utilization of infection control resources.In this evaluation, the molecular assay for MRSA detection was The current study demonstrated that femB gene was not detectable in 7 of 43 isolates identified as S. aureus in the clinical laboratory (Table 1).Kobayashi et al 17 reported that though femB gene is detectable only in S. aureus, an absence of femB gene does not mean that the isolate is not S. aureus.They have shown that up to 3% of S. aureus strains can give negative results for the femB gene even though they were positive for free coagulase and mannitol fermentation.Likewise, Mohanasoundaram and Lalitha 11 found that 6 (4%) of 150 MSSA strains which gave positive free coagulase and mannitol fermentation tests were femB negative.In contrast however, Sasaki et al 18 reported that other coagulase positive staphylococci species share similar phenotypic characteristics of S. aureus and occasionally cause human infection.These 7 isolates were excluded from the analysis due to this uncertainty of identification.
Oxacillin MIC was determined for the 43 isolates of S. aureus using the agar dilution method.Using break point MIC, 31 strains with MIC <2mg/L were identified as MSSA and the remaining 12 isolates with MIC>2mg/L as MRSA.Methicillin resistance is identified in the routine laboratory by disc diffusion and agar dilution methods which are simple and relatively cheap.Accurate determination of methicillin resistance in staphylococci by conventional tests is subject to variations in inoculum size, incubation time, medium pH, and medium salt concentration.In addition, difficulties occur when organisms have their MICs near the break-point.It is in such instances that alternative methods are requested for identification of such strains.MecA gene detection by PCR is used in the identification of methicillin resistance in both S. aureus and coagulase negative staphylococci.The mecA gene was detected in all 12 isolates identified as MRSA by conventional methods.
In addition, 10 strains identified as MSSA using MIC were mecA positive.Presently, there is a considerable debate about appropriate break points, and reports of correlation between mecA status and methicillin or oxacillin MICs are conflicting. 19,20Nevertheless, isolates that possess the mecA gene show borderline resistance due to hyper-production of β-lactamases and are either heterogeneous or homogenous in their expression of resistance.With homogenous expression, virtually all cells express resistance when tested by standard in vitro tests.However, testing of hetero-resistant isolates may result in some cells appearing susceptible and others resistant.The clinical problem with such isolates is that during chemotherapy with β-lactam antibiotics, selection of strains with PBP-2A could result in MRSA infection.A precise identification is therefore essential for reliable treatment of S. aureus infection.
The PVL gene was expressed by 11 of 43 (26%) S. aureus isolates.Ten of them were MSSA and 1 was MRSA.The PVL gene is encoded by the lukS-PV and lukF-PV genes, which are transmitted on a bacteriophage.PVL production has been described in MSSA and MRSA although lukS-PV and lukF-PV are not part of SCCmec.PVL is associated with severe necrotizing pneumonia and other soft tissue infections by destroying white cells.Lina et al reported that PVL is produced by <5 % of S. aureus strains. 1 The presence of the PVL gene in S. aureus strains provides a guide to the clinician that such strains may cause primary skin infections with or without necrotizing pneumonia, especially in young immune competent individuals. 21By testing for the PVL gene in the same PCR reaction, it is possible to gain further insight into the added virulence of the MSSA or MRSA strain.This information is not available when using the conventional methods.It is assumed that PVL is secreted only by S. aureus.Interestingly, 7 isolates with mecA gene and identified as S aureus by conventional testing were not positive for femB and PVL genes.These isolates could be either coagulase negative staphylococci or another species of coagulase positive staphylococci.However, the femB negative strains need further study.Isolation of S. aureus from clinical samples takes 24-48 hours.Direct molecular testing of samples would provide more rapid results which in certain clinical situations would be useful, particularly in the management of an outbreak or early appropriate treatment of severe infections.
A further 43 clinical samples from which S. aureus was isolated were directly tested by PCR for femB, mecA and PVL genes.The FemB gene was detected in 24 of 43 samples of which 17(40%) were pus aspirates, 3 (7%) blood cultures, 3 (7%) tracheal aspirates and 1(2%) liver abscess.As shown in Table 2, samples received as swabs appear to be insensitive for direct molecular testing with detection in only 2 such samples (7%).Some possible reasons for the poor detection could be due to a low bacterial load which is below the required sensitivity of the PCR assay, cotton material in the swab acting as an inhibitor in the PCR assay or due to the storage condition of samples at room temperature for several days.
The mecA gene was detected in 14 of the 18 samples from which MRSA had been isolated by conventional methods.Four swabs which yielded MRSA using conventional isolation methods were negative for femB, mecA and PVL genes.
Two samples from which MSSA was isolated were positive for the mecA gene.This highlights the issue, that disc diffusion or agar dilution testing performed in routine laboratories might fail to identify some strains of S. aureus which are MRSA as discussed previously.
Fourteen of the 28 samples tested directly were found to be positive for PVL gene.PVL detected directly has diagnostic and therapeutic implications in severe PVL-related staphylococcal syndromes.It is noteworthy that none of the femB negative samples contained the PVL gene.
When comparing the assay times with conventional and PCR based methods, conventional testing of samples takes 3 to 4 days to confirm a MRSA.With an established PCR assay, confirmation of an isolate as MRSA is obtained in 4 hours which would be of clinical significance, particularly in patients with severe staphylococcal sepsis.
This PCR assay is a rapid method which simultaneously identifies the S. aureus specific femB gene, the mecA gene that confers methicillin resistance and the PVL gene which determines the presence of PVL cytotoxin virulence factor.However, the relatively high negative findings when testing samples rather than isolates needs further study.This study demonstrated the presence of the PVL gene for the first time in Sri Lankan isolates of S. aureus. 22A subsequent study of S aureus colonization of skin in patients with atopic dermatitis in a different locality of Sri Lanka also demonstrated the presence of the PVL gene in 5 of 8 MRSA strains tested, 23 suggesting that these strains could be widely dispersed in the country.National surveillance of both hospital and community acquired MRSA and the distribution of the PVL genes is needed to plan strategies for control as well as empiric management of patients with severe staphylococcal sepsis.

Conclusion
In conclusion, this study shows that the conventional methods for detection of methicillin resistance viz., disc diffusion and agar dilution are time and labour consuming.Multiplex PCR is a simple, rapid and a good confirmatory test, but its use is limited to molecular diagnostic laboratories due to the need for technical expertise and cost.

Figure 1 :
Figure 1: Agarose gel electrophoresis showing the PCR patterns of 651-bp, 433-bp and 310bp amplified genes of femB, mecA and PVL respectively from clinical isolates with

Figure 2
Figure 2 Resolved DNA products from 7 of 43 S. aureus clinical samples PVL 433 bp mecA 310 bp developed using both clinical isolates and patient samples and compared with conventional methods.