|Year : 2016 | Volume
| Issue : 4 | Page : 476-482
Molecular characterisation of Panton–Valentine leucocidin-producing methicillin-resistant Staphylococcus aureus clones isolated from the main hospitals in Taif, KSA
EM Eed1, MM Ghonaim1, YM Hussein2, SS Al-Shehri3, AS Khalifa4
1 Department of Clinical Laboratories, Faculty of Applied Medical Sciences, Taif University, Taif, Saudi Arabia;Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menofia Governorate, Zagazig, Egypt
2 Department of Clinical Laboratories, Faculty of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Medical Biochemistry, Faculty of Medicine, Zagazig University, Zagazig, Egypt
3 Department of Clinical Laboratories, Faculty of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menofia Governorate, Zagazig, Egypt
4 Department of Clinical Pathology, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia
|Date of Submission||10-Dec-2015|
|Date of Acceptance||01-Aug-2016|
|Date of Web Publication||8-Dec-2016|
E M Eed
Department of Clinical Laboratories, Faculty of Applied Medical Sciences, Taif University, Taif, Saudi Arabia;Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menofia Governorate
Source of Support: None, Conflict of Interest: None
Introduction: Panton–Valentine leucocidin (PVL) is a bicomponent pore-forming cytolytic toxin encoded by the lukF-PV and lukS-PV genes. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) may carry the pvl genes which may be related to increased disease severity. This study aimed to characterise the PVL-producing MRSA recovered from different Taif Hospitals, Saudi Arabia. Methods: The study included 45 hospital-acquired-MRSA (HA-MRSA) and 26 CA-MRSA strains which were identified from 445 S. aureus strains isolated from different clinical samples. MRSA strains were identified by standard oxacillin salt agar screening procedure and by the detection of the mecA gene by the polymerase chain reaction (PCR). Detection of the S. aureus-specific femA, mecA and pvl genes was performed by multiplex PCR. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis was done for coagulase (coa) gene. Results: The staphylococcal cassette chromosome mec types of the 45 HA-MRSA strains were Type I (n = 24), Type II (n = 7) and Type III (n = 14) whereas the 26 CA-MRSA strains were Type IV (n = 14), Type V (n = 11) and one isolate was non-typeable. All the HA-MRSA and six CA-MRSA strains were PVL-negative PCR-RFLP analysis of coa gene showed that PVL-positive MRSA (n = 20) isolates showed six different patterns, and five patterns were shared by PVL-positive methicillin-susceptible S. aureus (MSSA). The eighth pattern was the most frequent in both MRSA and MSSA. Conclusion: PVL is more frequent among CA-MRSA than MSSA. All the HA-MRSA and 25% of CA-MRSA strains were negative for PVL. The pvl gene was related to the severity of infection but not related to coa gene RFLP pattern.
Keywords: Community-acquired methicillin-resistant Staphylococcus aureus, hospital acquired methicillin-resistant Staphylococcus aureus, Panton–Valentine leucocidin
|How to cite this article:|
Eed E M, Ghonaim M M, Hussein Y M, Al-Shehri S S, Khalifa A S. Molecular characterisation of Panton–Valentine leucocidin-producing methicillin-resistant Staphylococcus aureus clones isolated from the main hospitals in Taif, KSA. Indian J Med Microbiol 2016;34:476-82
|How to cite this URL:|
Eed E M, Ghonaim M M, Hussein Y M, Al-Shehri S S, Khalifa A S. Molecular characterisation of Panton–Valentine leucocidin-producing methicillin-resistant Staphylococcus aureus clones isolated from the main hospitals in Taif, KSA. Indian J Med Microbiol [serial online] 2016 [cited 2017 Feb 26];34:476-82. Available from: http://www.ijmm.org/text.asp?2016/34/4/476/195364
| ~ Introduction|| |
Staphylococcus aureus is considered a major human pathogen. Its primary reservoir is the anterior nares,,, and to lesser extent the skin and mucosa of healthy adults. Methicillin-resistant S. aureus (MRSA) was first reported 50 years ago and has become a major nosocomial pathogen worldwide. Methicillin resistance results from the introduction of mobile genetic element called staphylococcal cassette chromosome mec (SCCmec) that contains the antibiotic resistance gene mec A. The mec A gene codes for a variant type of penicillin-binding protein (PBP2a), which has a lower affinity for the β-lactam antibiotics.
There are two major strains of MRSA: hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA). They have been proven to be genetically distinct with respect to the SCCmec type; most HA-MRSA strains carry one of three types of SCCmec (Types I, II or III). On the other hand, CA-MRSA carry smaller SCCmec elements, usually Type IV, and to a lesser extent Type V. CA-MRSA is often less resistant to antibiotics than HA-MRSA., The increasing prevalence of CA-MRSA has highlighted the changing epidemiology of MRSA. In the Gulf region, MRSA has been considered as one of the most prevalent pathogens by Gulf Corporation Council countries. CA-MRSA can colonise healthy individuals and can also cause skin and soft tissue infections and necrotising pneumonia., CA-MRSA produces a wide variety of virulence factors that contribute to its pathogenesis as Panton–Valentine leucocidin (PVL).
PVL is a bicomponent cytolytic toxin encoded by two genetic elements called lukF-PV and lukS-PV genes, which are carried by a group of specific bacteriophages. After secretion of the two components, they form a pore-forming heptamer on neutrophil membranes, leading to either neutrophil lysis or apoptosis and contribute to tissue necrosis. PVL has been linked to skin, soft tissue infections and necrotising pneumonia. The toxin has also been linked to CA-MRSA disease worldwide; however, some CA-MRSA strains do not carry the pvl genes. It has been suggested that circulating CA-MRSA strains might be directly emerged from a historical PVL-producing penicillin-resistant clone phage type 80/81 that circulated in the 1960s and was highly virulent. PVL-positive methicillin-susceptible S. aureus (MSSA) has been reported in the 1950s as MSSA ST80 strains that were reported to cause necrotising pneumonia.,
In spite of the diversity of their genomic and antibiogram profile, virtually most of these newly emerging CA-MRSA strains have been reported to carry the PVL virulence genes. In addition, they possess a novel small mobile SCCmec Type IV or V which can be more easily transferred to other strains compared to the larger SCCmec types (Types I–III) that are prevalent in HA-MRSA strains., PVL is commonly found in CA-MRSA strains , and appears to be associated with increased disease severity.,, It has been suggested that screening for the PVL virulence factor in S. aureus may become a routine laboratory procedure.
This study was aimed to characterise the PVL-producing MRSA recovered from different hospitals located in Taif, Saudi Arabia. Characterisation of these strains was done phenotypically by conventional methods and genotypically by a multiplex polymerase chain reaction (PCR) for direct simultaneous detection of the S. aureus-specific femA, the mecA and the pvl genes. Gentotyping of the PVL-positive isolates by coagulase (coa) gene and SCCmec elements was also performed.
| ~ Methods|| |
S. aureus strains (n = 445) were isolated from different clinical samples obtained from the main hospitals at Taif, Saudi Arabia, from January 2013 to February 2015. Among them, 45 strains were identified as HA-MRSA whereas 26 strains were identified as CA-MRSA. The study proposal was approved by the Faculty of Applied Medical Sciences Ethics Committee, Taif University.
Strain identification and antibiogram
The isolates were identified by the conventional methods and were confirmed to be S. aureus by coagulase test. Antibiotic susceptibility profile for these isolates was determined and interpreted by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 2015.
Identification of methicillin-resistant Staphylococcus aureus strains
Methicillin resistance was tested by standard oxacillin salt agar screening plate procedure as indicated by the CLSI. Both known methicillin-resistant and methicillin-sensitive strains were included as controls. Methicillin resistance was further confirmed by the detection of the mecA gene by the PCR method as described below.
Culture and DNA extraction
The isolated strains were grown on LB culture at 37°C overnight. Genomic DNA was extracted by using a PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, USA) according to the manufacturer's instructions.
Detection of the Staphylococcus aureus-specific femA, mecA and pvl genes
Multiplex PCR assay targets the femA gene as a marker of S. aureus, the lukS/F- PV genes and the mecA gene (a determinant of methicillin resistance) with primers shown in [Table 1]. The optimised reaction conditions were adjusted as previously described. Multiplex PCR produced separate bands, corresponding to their respective molecular sizes that were easily recognisable in 1.5% agarose gel electrophoresis stained with ethidium bromide [Figure 1].
|Table 1: The primers sequences and the PCR products of the targeted genes|
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|Figure 1: Gel electrophoresis of multiplex polymerase chain reaction products. Lane 1 and 8: 100 bp DNA ladder, Lane 2: Panton–Valentine leucocidin-positive methicillin-resistant Staphylococcus aureus (433, 310 and 132 bp), Lane 3: Panton–Valentine leuckocidin-negative methicillin-resistant Staphylococcus aureus (433 and 132 bp), Lane 6: Panton–Valentine leucocidin-positive methicillin-susceptible Staphylococcus aureus (310 and 132 bp), Lane 7: Panton–Valentine leucocidin-negative methicillin-susceptible Staphylococcus aureus (132 bp only)|
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Staphylococcal cassette chromosome mec typing
SCCmec elements were typed using the multiplex PCR method as described previously, using the GeneAmp PCR kit (Applied Biosystems, Foster City, USA). This assay was based on detection of eight loci A to H selected on the basis mec element sequences.
Coagulase gene typing
PCR-restriction fragment length polymorphism (RFLP) was used to detect coa gene polymorphism of the PVL-positive MRSA and MSSA strains. The 3' end region of the coa gene was amplified by PCR as previously described by Lawrence et al. PCR amplification of the coa gene was performed using primers shown in [Table 1]. After amplification, 15 µL of the PCR products were digested for 15 min with 6 IU of restriction endonuclease Alul (Fermentas, Sunderland UK) according to the manufacturer's protocol. Restriction digest fragments were separated by agarose gel electrophoresis (2%).
| ~ Results|| |
Antimicrobial susceptibility patterns
All the isolated S. aureus strains were susceptible to vancomycin and linezolid. All the MRSA isolates were resistant to penicillin, oxacillin and cefoxitin. Various resistance patterns were demonstrated for the other antibiotics [Table 2] and [Table 3]. HA-MRSA strains were more resistant than CA-MRSA to the most tested non-β-lactam antibiotics, including ciprofloxacin, gentamicin, chloramphenicol and tetracycline. Two strains of MSSA were susceptible to penicillin and cefoxitin. PVL-positive MRSA was grouped into six different antibiogram patterns whereas PVL-positive MSSA was grouped into eight different patterns [Table 4] and [Table 5].
|Table 2: The distribution of pvl gene in the isolated MRSA and MSSA strains|
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|Table 3: The distribution of pvl gene among strains isolated from different clinical samples|
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|Table 4: Characters of PVL-positive MRSA strains, SCCmec type, PCR-RFLP of coa and mecA genes|
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|Table 5: Characters of PVL-positive MSSA strains, SCCmec type, PCR-RFLP of coa and mecA genes|
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Staphylococcal cassette chromosome mec typing
According to SCCmec typing, the 45 HA-MRSA strains were classified as Type I (n = 24), Type II (n = 7) and Type III (n = 14). On the other hand, the 26 CA-MRSA were classified as Type IV (n = 14) and Type V (n = 11). However, one isolate (MRSA-20) was non-typeable despite repeated attempts. All of HA-MRSA strains and only six isolates of the CA-MRSA were PVL-negative.
Polymerase chain reaction-restriction fragment length polymorphism analysis of coagulase gene
PCR products of coa gene amplification ranged from 648 to 810 bp. The restriction DNA digests were estimated by comparison with a 100-bp DNA marker. The coa gene of the PVL-positive MRSA (n = 20) isolates showed six different patterns [Table 4], the eighth pattern (162, 230, 324 bp) was the most frequent type and five patterns were shared by the PVL-positive MSSA [Table 5]. The eighth pattern was the most frequent type among both MRSA and MSSA (seven and five isolates, respectively).
| ~ Discussion|| |
The ability of S. aureus to cause diseases is attributed to a combination of many virulence factors as toxins, secreted exoproteins and cellular adhesins. Over 60% of the clinical isolates of S. aureus produce the PBP2a, which is responsible for the methicillin resistance.
In this study, MRSA accounted for 15.9% (n = 71) of the isolatedstrains, a rate which is comparable to other investigators. In contrast, an earlier study in Saudi Arabia demonstrated that MRSA accounted for 7.5% of all S. aureus isolates. Moreover, another study reported a much lower rate. However, Alli et al., reported that the prevalence of mecA gene was 42.3%. The prevalence of CA-MRSA in this study (5.8%) was relatively higher than other studies. This finding may be attributed to the constant influx of patients, visitors and healthcare workers, who constitute a reservoir for CA-MRSA into hospitals. The spread of these CA-MRSA isolates in both community and nosocomial settings has necessitated revised infection prevention and control guidelines. PVL has gained much importance in the recent years due to its association with the CA-MRSA infection. PVL is a phage-encoded exotoxin that has been found to be cytotoxic to rabbit and human neutrophils. The S. aureus strains with PVLhave been found to be rapidly spreading and cause serious skin and soft tissue infections such as pyomyositis, abscesses, breast abscesses, necrotising fasciitis and pneumonia.
In this study, a rapid triplex PCR which targeted the femA, mecA and pvl genes simultaneously was used for the specific detection of PVL productionand methicillin-resistance. All MRSA isolates (n = 71) were found to be positive for both femA and mecA. The pvl gene was detected in 76.9% (20/26) of CA-MRSA isolates and in 4.5% (17/374) of MSSA isolates. These results are similar to the rates (77–100%) reported in previous studies., Furthermore, Motamedi et al. reported that mecA and pvl genes were positive in 30% MRSA isolates while none of mecA positive isolates was positive for pvl gene. On the other hand, Abimanyu et al. reported that only 40% of CA-MRSA isolates were found to harbour the pvl gene. In addition, Alli et al., reported that the prevalence of the pvl gene was higher in MSSA than MRSA isolates (53.3% and 9.1%, respectively). Another study in Saudi Arabia showed that lukS/F was positive in 37.6% of CA-MRSA isolates. Also Muttaiyah et al. reported a prevalence rate of 37% among the clinical MSSA isolates. This worldwide spread of PVL-positive CA-MRSA is likely related to international travel. Tristan et al. reported that ST80 isolates recovered in France were mainly detected in patients who were originally from Algeria, where higher rate CA-MRSA was reported. This regional difference in PVL-producing CA-MRSA may be attributed to its smaller fitness burden, higher transmissibility and virulence compared to HA-MRSA and is epidemic in many countries. Although PVL is not the sole virulence determinant of CA-MRSA, rather one of several important factors that may play a key role in the successful dissemination of CA-MRSA.
In this study, the majority of the PVL-positive CA-MRSA (60%) isolates carried SCCmec Type IV. A previous study  reported that 73% of CA-MRSA isolates carried SCCmec Type IV. PCR-RFLP of the coa gene of PVL-positive MRSA revealed six different patterns while PVL-positive MSSA showed seven different patterns. Five patterns were found in the two groups while the eighth pattern was the most frequent type in both MRSA and MSSA (seven and five isolates, respectively).
In the present study, the correlation between coa genotype and PVL production was not statistically significant. Similar results were previously reported by Kobayashi et al., who found that MRSA and MSSA were classified into 6 and 12 RFLP patterns, respectively. Walker et al.,, showed that AluI digestion of the coa gene PCR products of 356 MRSA strains yielded 13 different RFLP patterns. In contrast to our results, Lawrence et al. showed that the strains were closely related and about 91.8% of the isolated strains had a unique coa gene RFLP pattern. Our results revealed that PVL-positive MRSA were associated with increased disease severity as blood stream (25%), surgical site infections (25%) and necrotising pneumonia (25%). Similar findings were also reported in previous studies.,,
| ~ Conclusion|| |
PVL is more commonly found among CA-MRSA compared to MSSA strains and is related to severe soft tissue infection. About 60% of the PVL-positive strains were SCCmec Type IV; however, there was no statistical correlation with coa gene RFLP pattern. Rapid and informative molecular typing is essential for early identification of PVL-positive MRSA strains to prevent spreading of these strains in hospitals. In the future, screening for the PVL as a virulence factor in S. aureus may become a routine laboratory procedure.
This study was funded by the Academic Research Center at Taif University, project number 1-436-4080.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]