Investigation of PVL and MecA Gene Locies in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Nosocomial Samples of Hospital Patients and Hospital Staff  

Yaren H.1 , Saygin O.1 , Turaclar N.2 , Vural H.C.3 , Turhan A.B.3 , Tuncer E.I.4 , Arslan U.4
1 Department of Biology, Faculty of Science, Selcuk University, Konya, Turkey
2 Vocational School of Health Services, Selcuk University, Konya, Turkey
3 Faculty of Medicine, Medical Biology Department, Necmettin Erbakan University, Konya, Turkey
4 Faculty of Medicine, Microbiology Department, Selcuk University, Konya, Turkey
Author    Correspondence author
Genomics and Applied Biology, 2015, Vol. 6, No. 10   doi: 10.5376/gab.2015.06.0010
Received: 12 Nov., 2015    Accepted: 26 Dec., 2015    Published: 31 Dec., 2015
© 2015 BioPublisher Publishing Platform
Preferred citation for this article:

Yaren H., Saygin O., Turaclar N., Vural H.C., Turhan A.B., Tuncer E.I., and Arslan U., 2015, Investigation of PVL and MecA Gene Loci in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Nosocomial Samples of Hospital Patients and Hospital Staff, Genomics and Applied Biology, 6(10): 1-6 (doi: 10.5376/gab.2015.06.0010)

Abstract

Objective: Methicillin-resistant (MRSA) is a bacterium responsible for some infections difficult to treat in humans. Antibiotic resistance is the ability of microorganisms to resist the effects of antibiotic. MRSA is a strain of Staphylococcus aureus that is resistant to a large group of antibiotics by its definition. We studied in the Archeometry and Biotechnology Laboratory of Science Faculty in Selcuk University. In this study, the samples were taken from cultured nosocomial infections of 233 hospital staff and 477 hospital patients (total 710) in the Medical School Hospital of Selcuk University. We collected 2 Methicillin-susceptible Staphylococcus aureus (MSSA) samples from hospital patients and 40 Methicillin-resistant Staphylococcus aureus (MRSA) samples from hospital patients and staff (36 hospital patients 7.5%, 4 hospital staff 1.7%) for this research. Methods: We looked into present of mecA-1, mecA-2, PVL-1, PVL-2, and PVL-3 genes in each samples by PCR methods. mecA-1 and mecA-2 gene loci or band patterns in 40 MRSA samples was amplified by PCR. Results: mecA-1 and mecA-2 were observed in 40 MRSA, but not observed in 2 MSSA. PVL-1 gene was observed in 22 MRSA, PVL-2 gene was observed in 25 MRSA, PVL-3 gene was observed in 33 MRSA. PVL gene region was not observed in 2 MSSA strains. Molecular findings were verified the microbiologic results in the research. The mutation was not determined in MRSA originated from hospital patients and staff. Conclusion: The result of this study showed that the prevalence of MRSA was high in the hospital. It is an important sign to meet the challenges of the fight against epidemics.

Keywords
MRSA; mecA; PVL; MSSA; Nosocomial samples

1 Introduction
Staphylococcus aureus is a facultative anaerobic, gram-positive coccus which appears like grape clusters. S. aureus is catalase positive and can convert hydrogen peroxide to water and oxygen. This bacterium is an important cause of human diseases. Although staphylococcal diseases are most often associated with skin and soft tissue infections, their manifestations are numberless and include syndromes with low morbidity and mortality (Lowy, 1998; Yu et al., 1986). The infections of S. aureus are often fatal in the preantibiotic era; now it typically responds to a variety of antimicrobial agents (Peacock et al., 2001; Gordon and Lowy, 2008). However, the spread of multidrug-resistant strains of S. aureus in the health-care setting, particularly methicillin-resistant S. aureus (MRSA), have made these infections more difficult to treat (Cosgrove et al., 2003). Some strains of MRSA appear to be spreading in the community setting (Herold et al., 1998). The carriage of S. aureus has long been known to be one of the most strongly risk factors associated with subsequent infections (Martinez et al., 2009; Skinner and Keefer, 1941). The presence of nasal colonization of S. aureus can provide an indication of a higher risk for a subsequent infection, including with MRSA (Von et al., 2001; Wertheim et al., 2005). However, no population-based prevalence study has been conducted to measure S. aureus carriage, and reliable national population estimates are lacking. S. aureus is an opportunistic pathogen that can persist in the anterior nares of healthy humans (Miles et al., 1944; Nouwen et al., 2004; Von et al., 2001). Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a cause of infections in persons. MRSA has carried frequently PVL (Panton-Valentine leukocidin) gene which creates lytic pores in the cell membrane and kill neutrophils. PVL was encoded from genes lucF-PV and lucS-PV. So PVL was a toxin composed of two proteins (Diep et al., 2004; Witte et al., 2007). The difference between MRSA and methicillin-susceptible S. aureus (MSSA) is resistance to β-lactamase-stable β-lactam antibiotics. This is often associated with resistance to multiple other antibiotics, which limits the therapeutic options. MRSA has become an important pathogen in many hospitals (Lowy, 2003; Melzer et al., 2003; Peacock et al., 2001).


In this study, 710 samples were taken from hospital staff (233 persons) and hospital patients (477 persons) in the Medical School Hospital of Selcuk University. We isolated MRSA nosocomial samples from 4 hospital staff among 233 hospital staff and 36 hospital patients among 477 hospital patients (with total 710 persons) and MSSA from 2 hospital patients. We investigated especially 2 variants of mecA gene and 3 variants of PVL by PCR as microbiological and molecular. Then these were studied by SSCP methods whether or not there was a mutation in gene regions for each bacterium. We aimed to indicate PVL gene region which has virulence effect of MRSA strains.


2 Materials and Methods
2.1 Samples

This study was conducted between September 2007-January 2009 in Meram Medicine Faculty Microbiology Laboratory and Science Faculty Archaeometry and Biotechnology Laboratory of Selcuk University in Konya/Turkey. MRSA and MSSA strains were obtained from nosocomial samples of patients and hospital staff in Meram Medicine Faculty by using sterile swap. S. aureus ATCC 25923, S. aureus ATCC 29213 and MRSA LY19990053 were used as positive controls and taken from Microbiology Laboratory of Meram Medicine Faculty in Selcuk University/Turkiye.


2.2 Specimen collection and identification
Nosocomial samples were planted on sheep blood agar and incubated at 35-370C for 18-24 h on MRSA medium (Bio-merieux, Marcy l’Etoile, France). Staphylococcus aureus was diagnosed by gram dyeing, catalase, and coagulase tests. In addition Staphylococcus aureus was identified by API Staph (Bio-merieux, Marcy l’Etoile, France) after 24 h incubation and evaluated. These studies have shown that API Staph system identified the clinically isolated staphylococci as rapidly and accurately as conventional methods. These were verified on MRSA medium by composing green colonies.

 
2.3 DNA isolation protocol
The first day, 5 mL LB (Luria-Bertani) mediums were planted from unique colony. This was incubated at 370C for overnight. Second day, DNA was isolated and embarked. Bacterial colonies were added in 1 mL Tris-EDTA buffer by loop. Three different protocols were used for DNA extraction for each sample. The alkali lysis method was performed in the first protocol. 1.5 µL was taken from the mixture. It was centrifuged at 5000 ×rpm 5 mins. Supernatant was removed by micropipette. 10 µL Tris-EDTA buffer (pH8.0) was added onto pellet. It was centrifuged at 5000 ×rpm 5 min and supernatant was removed. 567 µL TE (Tris-EDTA) buffer and lysozyme (1 μL) (Sigma-Aldrich) were added and mixed by micropipette. The mixture was incubated at 370C for 30 mins in water bath. The cells were lysed by addition of 3 μL of 10% sodium dodecyl sulfate and 3 μL of proteinase K solution (Sigma-Aldrich), then it has a 15-mins incubation at 370C. 1 µL of 5 M NaCl and CTAB 80 µL were added and the mixture was incubated at 650C for 10 mins. The water phase was extracted with 780 µL chloroform: isoamyl alcohol (24:1) and the mixture was centrifuged at 10000 ×rpm for 5 minutes. Supernatant was taken to a clear tube. Phenol-chloroform-isoamyl alcohol (25:24:1) was added onto supernatant and the mixture was centrifuged at 10000 ×rpm for 5 minutes. Supernatant was taken to a clear tube. 0.6 volume isopropanol was added and the mixture was centrifuged at 10000 ×rpm for 5 minutes, and supernatant was removed. The pellet was washed with 10 μL of 70% ethanol and centrifuged at 5000 ×rpm for 2 minutes. Pellets were air dried and finally DNA was suspended in 100 μL of TE buffer. Purified DNA samples were stored at -200C until they were used.


EZ1 biorobot (Qiagen, Valencia, CA, USA) was performed in the second and third protocols. In the second protocol, 1.5 µL was taken from the each tube. It was centrifuged at 5000 ×rpm for 5 mins. Supernatant was removed by micropipette. 10 µL Tris-EDTA buffer (pH8.0) was added onto pellet. It was centrifuged at 5000 ×rpm for 5 mins and supernatant was removed. Then lysozyme (500 μL) (Sigma-Aldrich) was added and the mixture was incubated at 370C for 30 minutes. Isolation was performed in EZ1 biorobot DNA isolation equipment.


In the third protocol, 1.5 µL was taken from the each tube and centrifuged at 5000 ×g for 5 mins and supernatant was removed. 190 µL G2 buffer (Qiagen, D-40724) was added onto pellet. Then lysozyme (10 μL) (Sigma-Aldrich) was added and the mixture was incubated at 370C for 30 mins. Isolation was performed in EZ1 biorobot DNA isolation equipment. 1% melted agarose with 1× TAE buffer was allowed to cool and poured into the horizontal gel tank which the comb was installed in. Solidified agarose gel was placed to electrophoretic tank and tank was filled with 1× TAE buffer until 1-3 mm upon gel level. For visualizing the DNA extracts, 5 μL of each extract and 3 μL of bromophenol blue was electrophoresed in 1% agarose gels in 1× TAE buffer, which were then stained with ethidium bromide and examined under UV transilluminator.


2.4 DNA amplification methods
The primers which were used for the detection of mecA and PVL genes of S. aureus strain were listed in Table 1

 

 
Table 1 Bacterial primers sequences used in the study

 
Each primer was amplified separately with PCR reaction. PCR amplifications for mecA was composed of 2.5 μL PCR Buffer 10× (Invitrogen), 2.0 μL of MgCl2 (Invitrogen), 2.5 μL dNTP (Invitrogen), 0.6 μL of Taq DNA Polymerase (Invitrogen), 10 picomoles of each primer, 3 μL of the DNA sample, and sterile ultrapure water in order to reach 25 μL. PCR amplifications for PVL was composed of 2.5 μL PCR Buffer 10× (Invitrogen), 2.5 μL of MgCl2 (Invitrogen), 2.5 μL dNTP (Invitrogen), 0.8 μL of Taq DNA Polymerase (Invitrogen), 10 picomoles of each primer, 3 μL of the DNA sample, and sterile ultrapure water in order to reach 25 μL. PCR protocol was performed in mecA-1 using the following amplification cycles: denaturation for 5 minutes at 940C and 10 cycles at 940C for 30 seconds, 640C for 30 seconds, and 720C for 45 seconds and 25 cycles at 940C for 30 seconds, 500C for 45 seconds, and 720C for 2 minutes for annealing. Extension was performed at 720C for 10 minutes. PCR protocol was performed in mecA-2 using the following amplification cycles: denaturation for 10 minutes at 950C and 35 cycles at 950C for 30 seconds, 590C for 30 seconds, and 720C for 30 seconds for annealing. Extension was performed at 720C for 5 minutes. PCR protocol was performed in PVL-1 and PVL-3 using the following amplification cycles: denaturation for 1 minute at 950C and 35 cycles at 940C for 30 seconds, 520C for 30 seconds, and 720C for 30 seconds for annealing. Extension was performed at 720C for 10 minutes. PCR protocol was performed in PVL-2 using the following amplification cycles: denaturation for 1 minute at 950C and 35 cycles at 940C for 30 seconds, 540C for 30 seconds, and 720C for 30 seconds for annealing. Extension was performed at 720C for 10 minutes. The PCR-amplified samples were analysed by electrophoresis for 30 minutes at 125V with using Staphylococcus spp. and 2% agarose gel (Sigma Aldrich) added to ethidium bromide (0.5 mg/mL-Invitrogen) in TAE. The results were visualized by employing an image analyser (Positive controls were S. aureus ATCC 25923, S. aureus ATCC 29213, MRSA LY19990053 and PVL control strain).


2.5 SSCP analysis
The modified methods of SSCP-heteroduplex pattern analysis were used for the separations of amplified products(Jannati et al., 2013). On terminating electrophoresis, 8% non-denatured polyacrylamide gel was stained with silver nitrate solution. There were 142.5 gr and 7.5 gr bisacrylamide in 30% acrylamide stock solution. Then 35 mL acrylamide stock solution, 4 mL 5× TBE buffer, 1 mL glycerol and 10.65 mL dH2O were homogenized on magnetic mixing for 5 minute. 125 μL 10% Ammonium persulfate (APS) buffer and 12.5 μL Tetra Methyl Ethylene Daimine (TEMED) were added respectively. Polyacrylamide gel was spilled to vertical gel electrophoresis system (Biorad 10x10 cm) and comb was placed. Gel froze after one hour. PCR amplified products were denatured by formamide loading dye. Loading dye was prepared with 10 mL formamide, 10 mg xylene cyanol, 10 mg bromophenol blue, and 200 μL 0.5 M EDTA (pH8) and used as a denaturation buffer. 3 μL PCR product and 9 μL denaturation buffer were mixed and denatured at 95°C for 10 minutes on heater. PCR products were taken upon ice to obstruct renaturation after denaturation. These were loaded to polyacrylamide gel for 10 μL and 200V in vertical gel electrophoresis system.

 
3 Results
According to API test results, Staphylococcus aureus was identified from nasal mucosa and isolated 42 bacteria samples (40 MRSA, 2 MSSA). MIC values of methicillin-susceptible strains were determined by the resistance of oxacillin and vancomycin using E-tests for CLSI measure. MRSA strains were observed as oxacillin resistant and vancomycin susceptible.


According to PCR results mecA-1 and mecA-2 were observed in 40 MRSA, but not observed in 2 MSSA (Figure 1 and Figure 2).

 

 
Figure 1 Screening of PCR amplification mecA-1 genes
Note: M: 100 bp Marker (Fermentase); C1: ATCC 29213 Staphylococcus aureus; C2: ATCC 25923 
Staphylococcus aureus

 

 
Figure 2 Screening of PCR amplification mecA-2 genes
Note: M: 100 bp Marker (Fermentase); C1: ATCC 29213 Staphylococcus aureus; C2: ATCC 25923
Staphylococcus aureus

 

Molecular findings were verified by the microbiologic results in the research. PVL-1 gen was observed in 22 MRSA (Figure 3), PVL-2 gen was observed in 25 MRSA, PVL-3 gen was observed in 33 MRSA.

 

 
Figure 3 Screening of PCR amplification PVL-1 gene
Note: M: 100 bp Marker (Fermentase); C: LY19990053 MRSA positive, PVL positive

 
Then these were defined by SSCP methods if there was any mutation in regions for each bacterium in the study. There was no mutation in these Staphylococcus aureus strains or there wasn’t heterodublex band profile. PCR-SSCP method, although very sensitive, can only be used to detect mutations in small DNA fragments (Figure 4). Microbiologists and clinicians are faced with increasing options for the use of genotypic or phenotypic methods.

 

 
Figure 4 Screening of mecA-2 genes in PAGE
Note: M: 50 bp DNA marker (Fermentase); C: ATCC 29213
Staphylococcus aureus

 
45 bacteria strains (40 MRSA, 2 MSSA and 3 controls) were studied in nosocomial samples for identification of S. aureus carrying PVL gene as microbiological and molecular genetics. These were evaluated with regard to infection formation of bacteria.


4 Discussion
The aim of the study was to investigate of PVL and mecA gene locies in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from nosocomial samples of hospital patients and hospital staff. MRSA is a major nosocomial pathogen and investigating the prevalence of MRSA is important for public health.


Martinez et al., collected nasal swab cultures from 294 healthy volunteers in order to investigate the colonization rates of S. aureus in the community in south-eastern USA. The results showed that 21.4% (63/294) of healthy individuals were carrying Staphylococcus aureus and 0.68% (2/294) of them were carrying MRSA which has mecA gene. However, Gorwitz et al., reported that the rate of MRSA colonization was 1.5% in the healthy population. In our study, the rate of MRSA was 5.6% (40/710).


In the study of Jannati et al., 173 nurses were scanned for S. aureus nasal colonization and 8 of them were MRSA carriers. All of the MRSA strains were mecA positive, while 6 were oxacilin resistant and 2 were oxacilin susceptile.


Afroz et al. isolated 9 PVL positive MSSA strains among 33 MSSA and 26 MRSA strains by PCR. While researchers were defining S. aureus, they found that MRSA strains were PVL negative. In our study, mecA-1 and mecA-2 were observed in 40 MRSA, but not observed in 2 MSSA, and also MRSA strains were observed as oxacillin resistant and vancomycin susceptible. In addition, PVL-1 gen was observed in 22 MRSA, PVL-2 gen was observed in 25 MRSA, PVL-3 gen was observed in 33 MRSA. PVL gene region was not observed in 2 MSSA strains.


In conclusion, the result of this study showed that the prevalence of MRSA was high in the hospital. It is an important sign to meet the challenges of the fight against epidemics.


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