Association of PVL Gene with SCCmec Typing in MRSA (both community associated and hospital-acquired) from a Tertiary Care Hospital, Lahore

Introduction: Antibiotic resistance is a major problem in staphylococcal infections. Resistance against Methicillin in S. aureus is due to the transfer of mobile cassettes of specific genes in S. aureus called Staphylococcal Cassette Chromosome mec (SCCmec). 13 types of SCCmec have been identified till now. Various clinical syndromes are associated with the presence of the PVL gene. Therefore, the present study was designed to check the prevalence of PVL gene and SCCmec types in CA-MRSA and HA-MRSA.
Materials and methods:This cross-sectional descriptive study was done in LGH/PGMI, Lahore. MRSA isolates were phenotypically confirmed by Cefoxitin (30) disc on Mueller-Hinton agar. DNA extraction was done by using a Genomic DNA purification kit. Detection of PVL gene and SCCmec types was done with PCR using specific primers.
Results:Out of 89 isolates, the maximum isolates were from pus swabs (45%), followed by wound swabs (15.5%). It was found that 55 (61.8%) were HA-MRSA isolates, while 34 (38.2%) were CA-MRSA isolates. PVL gene was found in 37% of isolates. Among PVL-positive samples, 36.4% were HA-MRSA, while 63.6% were CA-MRSA. The most common SCCmec in HA-MRSA was type III (69%), while in CA-MRSA, SCCmec type IVa was predominant (35.4%).
Conclusion: The present study provides insight into the prevalence of the PVL gene and different SCCmec types in CA-MRSA and HA-MRSA. The most common SCCmec type is linked with HA-MRSA.

Staphylococcus aureus, a ubiquitous pathogen, is a Gram-positive coccus, forming grape-like clusters, non-sporing, non-motile, catalase-positive facultative anaerobe, having a diameter of 0.5-1.5 µm. Antibiotic resistance is a major problem in staphylococcal infections, which may lead to an increase in the length of hospital stay and treatment failure.1 Infections caused by community-associated and hospital-acquired Methicillin-resistant Staphylococcus aureus (MRSA) have turned out to be a serious problem across the world.² MRSA isolates are frequently resistant to several non-β-lactam antibiotics, including macrolides, lincosamides, aminoglycosides, and tetracyclines.3Resistance against Methicillin in S. aureus is due to the transfer of mobile cassettes of specific genes in S. aureus called Staphylococcal Cassette Chromosome mec (SCCmec), and coagulase-negative staphylococci were the origin which transfer SCCmec cassette to S. aureus ST250. SCCmec consists of 3 components: (i) mec gene complex, (ii) Ccr (cassette chromosome recombinase) gene complex and (iii) J regions.4 The mecA and mecC genes in the SCCmec cassette are responsible for methicillin resistance. There are 13 types of SCCmec, and all are integrated at the attB site of the S. aureus genome, possessing significant characteristics.5 For SCCmec typing, conventional PCR techniques are more widely used; however, advanced techniques include whole genome sequencing and the use of bioinformatics tools for subsequent data analysis.
Many other virulence factors, such as Panton-Valentine leucocidin (PVL), which is encoded by lukF-PV and lukS-PV, are responsible for S. aureus's widespread infection rate. PVL encoded a pore-forming toxin that attacks white blood cells such as monocytes and neutrophils. It is associated with various clinical diseases, mainly skin infections and necrotizing pneumonia.6 PVL was considered to be associated with five predominant CA-MRSA strains epidemiologically.7 Among these, the USA300 clone is the major epidemic CA-MRSA strain globally, which is extremely pathogenic and causes intractable infections of the skin. Hence, the current study was designed to check the prevalence of PVl and different SCCmec types in clinical and Community-acquired isolates of Methicillin-resistant Staphylococcus aureus.

The present study was done in the Microbiology laboratory of PGMI/LGH and the Immunology Laboratory of UHS, Lahore, over one year from 01-09-2021 to 01-09-2022. It was a cross-sectional descriptive study design, and a non-probability consecutive sampling technique was used to collect the samples. The sample size was calculated according to the formula.
Level of confidence = 1-= 95% Anticipated population proportion margin of error = (P) = 36.1% Margin of Error = 10% Sample size = 89 All types of clinical samples that yielded MRSA were included in the present study. Samples from both genders were included. While S.aureus strains sensitive to Methicillin were excluded. Repetitive samples from the same patient were also excluded. Samples were collected and subcultured on blood and MacConkey agar. Confirmation of S.aureus was done by gram staining and different biochemical tests. Methicillin resistance was confirmed by 30 µg cefoxitin discs on Mueller-Hinton agar plates according to CLSI guidelines 2021. A bacterial suspension of 0.5 McFarland standard of turbidity was used. DNA extraction of confirmed MRSA isolates was done by using a genomic DNA purification kit (ThermoFisher Scientific, Germany) according to the manufacturer's guidelines. Primers used for the detection of different SCCmec types and PVL genes were designed by using LaserGene bioinformatics software. Primers were reconstituted with TE buffer. The working primer was prepared in a separate vial by adding 10µl of the stock primer in 90µl of the TE buffer 8. The set of primers used for PCR amplification is given in Table I. (Supplementary Material)
PCR amplification of SCCmec types and PVL gene was performed in a thermal cycler. Reaction mix of 20 µl was prepared as 10x PCR buffer (2.0 µl), dNTPs (0.5 µl), MgCl2 (1.2 µl), Forward and Reverse primer (2.0 µl) each, Template DNA (7.0 µl) Taq DNA polymerase (0.4 µl), ddH2O (4.9 µl). The PCR cycle was run at 95℃ for 4 min, followed by 30 cycles of 94℃ for 45 sec, 52℃ for 1 min, and 72℃ for 1 min, and a final extension at 72℃ for 10 min. All amplified products were run on 1.2% stained agarose gel along with a DNA ladder. (Sigma-Aldrich, UK.). Visualization of bands at specific molecular target sizes was confirmed in the Gel documentation system by UV illuminator. (BionexusInc, USA).11 Results were analyzed by using Microsoft Excel 2010 through frequencies and percentages

Among 89 confirmed isolates of MRSA, 45% were collected from pus swabs, followed by wound swabs (15%) and blood (12%). In contrast, 28% of isolates were collected from different specimens, including Fluids/aspirates, CSF, sputum, CVP tip, and urine. Among positive samples, it was found that 55 (61.8%) were HA-MRSA isolates, while 34 (38.2%) were CA-MRSA isolates. Among total samples, 37% (n=33) isolates were found to be positive for the PVL gene, among which 12 (36.4%) were HA-MRSA and 21 (63.6%) were CA-MRSA.

Association of PVL with different SCCmec types showed that among 47 isolates of SCCmec types I, II and III, 9(19.1%) were PVL positive, while 38 (80.9%) isolates were PVL negative. Among 40 isolates of IV subtypes and V, 33 (82.5%) were PVL-positive, while 7 (17.5%) were PVL-negative.

Methicillin-resistant S. aureus is one of the most communal organisms linked with nosocomial infections and has been recognized as a major problem for infection control.12 Acquiring resistance against commonly used antibiotics is mainly because of the presence of the PVL gene and different SCCmec types, which can be found in both HA-MRSA and CA-MRSA. In the present study, we determined the prevalence of the PVL gene and different SCCmec types in Community-associated and hospital-acquired MRSA isolates. In our study, the maximum number of MRSA isolates were isolated from pus samples (45%), followed by wound swabs (28%) and blood (12%). In accordance with our results, Maham Akram, in 2022, also reported 36.2% isolation of MRSA from pus samples 12. Various other studies also reported the maximum number of MRSA isolations from pus and wound swabs.^13,14
In the present study, among 89 MRSA isolates, 55 (61.8%) were HA-MRSA, while 34 (38.2%) were CA-MRSA isolates. A study conducted in China in 2022 also reported similar results in which community-associated MRSA (CA-MRSA) infections accounted for 24% of the total isolates. Similar results were reported from China in 2013, in which maximum numbers of MRSA isolates were found to be Hospital-acquired. ¹⁵
Panton-Valentine leucocidin is generally used as an indicator for Community-associated MRSA, accountable for skin and soft tissue infections 16. In our study, the PVL gene was present in 37% (n=33) isolates, among which 12 (36.4%) were HA-MRSA and 21 (63.6%) were CA-MRSA. A study conducted in 2022 reported a 35% prevalence of the PVL gene in MRSA isolates, which was in accordance with our results.18 However, a study conducted in India reported a 63% prevalence of the PVl gene in MRSA isolates, which indicates a higher prevalence than our study.19 Various reports from different countries reported lower pervasiveness of the PVL gene among isolates of MRSA (5% in France, 4.9% in the UK, 14.3% in Bangladesh)18, demonstrating how the prevalence of PVL differs significantly between different geographic areas and populations. Investigation of SCCmec types improves understanding of the epidemiology of MRSA and detection of relatedness among different strains. 98% of our isolates were typeable for SCCmec by PCR. Various studies around the world work on SCCmec typing by PCR to identify the most prevalent SCCmec type in their region and reported variations in typeability that were wholly less than 100%. Various researchers reported similar results to our findings of type ability (98% in Denmark, 97.4% in Portugal, 96.4% in Palestine).¹⁹
The high frequency of SCCmec type III (69%), followed by SCCmec type IVa (10.9%) and type II and IVc (5.5%) each in our study, was in accordance with the results of the study conducted in Alexandria, which reported 57% prevalence of SCCmec type III.20 Various other studies across the world also reported similar results.21,22 Conversely, studies conducted in Saudi Arabia and Kuwait reported SCCmec type IV as the most prevalent type.23,24 The discrepancy in the SCCmec types distribution from different geographic regions can be attributed to limited proficiencies of the conventional PCR detection method and differences in the specificity and sensitivity of primers used. In CA-MRSA, the most prevalent type was found to be type IVa (35.3%), followed by type IVc and IVe (17.6%) each in our study, which was in accordance with the study conducted in Japan in 2022 in which SCCmec type IV accounted for 44% of total samples in community-associated MRSA.25In our study, the presence of SCCmec type I, II and III in CA-MRSA demonstrated the spread of HA-MRSA in the Community, which was already reported in the previous studies conducted in China, the Philippines, Korea and Veitnam.¹⁷

The present study provides insight into the prevalence of the PVL gene and different SCCmec types in community-associated and hospital-acquired MRSA isolates. The most common SCCmec type linked with HA-MRSA was type III, while type IV was more prevalent in CA-MRSA. Increased association of the PVL gene with HA-MRSA is of major concern. More knowledge about the characterization of SCCmec types and transfer of PVL gene from CA-MRSA to HA-MRSA will help in better understanding for monitoring and managing MRSA infections.

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