This Article

Citations


Creative Commons License
Except where otherwise noted, this work is licensed under Creative Commons Attribution-NonCommercial 4.0 International License.
This article, by Hamadan University of Medical Sciences , is licensed under a Creative Commons Attribution License .

Prevalence of the Panton-Valentine Leukocidin Gene in Clinical Isolates of Staphylococcus aureus Isolated From Hospitals the Ilam Province of Iran


1 Department of Biology, Faculty of Sciences, Ilam University of Medical Sciences, Ilam, IR Iran
*Corresponding author: Arman Rostamzad, Department of Biology, Faculty of Sciences, Ilam University of Medical Sciences, Ilam, IR Iran. Tel/Fax: +98-8432227022, E-mail: .
Avicenna Journal of Clinical Microbiology and Infection. 3(1): e32691 , DOI: 10.17795/ajcmi-32691
Article Type: Research Article; Received: Sep 1, 2015; Revised: Nov 10, 2015; Accepted: Nov 16, 2015; epub: Jan 23, 2016; collection: Feb 2016

Abstract


Background: Panton-Valentine leukocidin (luk-pv) is a cytotoxin that causes leukocyte destruction and tissue necrosis.

Objectives: The aim of this study was to determine the prevalence of the pv1, femA, and mecA genes in staphylococcus aureus isolates from clinical specimens in hospitals in Ilam, Iran.

Materials and Methods: One hundred and sixty Staphylococcus aureus isolates were collected from hospitals in Ilam, Iran, and phenotypic and genotypic examinations for methicillin-resistant S. aureus (MRSA) isolates were carried out. The antibiotic profiles of these isolates, and the minimal inhibitory concentration (MIC) of MRSA isolates was determined using the agar dilution method with vancomycin, cefoxitin, and oxacillin. All isolates were examined using polymerase chain reaction (PCR) primers for the femA, mecA, and Panton Valentine leukocidin (luk-pv) genes.

Results: The results showed 91 isolates (56.88%) were coagulase-positive, and 69 isolates (43.12%) were coagulase-negative Staphylococcus aureus (CNSA). Out of 91 (56.88%) coagulase-positive staphylococci, 32 isolates (35.16%) were resistant to cefoxitin, and 30 isolates (32.96%) were resistant to oxacillin, using disc diffusion method. PCR revealed the presence of the femA gene (510 bp band) in all coagulase-positive isolates (100%), and the mecA gene (513 bp band) was detected in 32 isolates (35.16%); out of 32 MRSA isolates, 13 isolates (40.62%) were positive for presence of the luk-pv gene (433 bp band).

Conclusions: The cefoxitin disk diffusion method showed the best results when compared to oxacillin disk, similar to results from detecting the mecA gene in PCR as a golden test. Studies on MRSA that carry the luk-pv gene should continue to provide significant insight into the prevalence and epidemiology of these important resistant pathogens. Also, the rate of pvl gene-producing isolates showed to be relatively high, and it should be detected in all staphylococcal infections.

Keywords: Methicillin Resistance; Staphylococcus aureus; Paton-Valentine; Ilam

1. Background


Staphylococcus aureus, frequently a member of the normal skin flora and the nasal cavity, often causes abscesses; infections of wounds to the skin, and soft tissue; osteomyelitis, endocarditis; pneumonia; and so on. It may also cause staphylococcal scalded skin syndrome, a severe disease in infants, or toxic shock syndrome (1). The virulence of Staphylococcus aureus depends on a variety of components, such as capsule and protein A, the clumping factor and coagulase, hemolysins, enterotoxins, toxic-shock syndrome [TSS] toxins, exfoliatins, and Panton-Valentine leukocidin [PVL] (2, 3). Panton Valentine leukocidin (PVL) is a cytotoxin, one of the β-pore forming toxins; its presence is associated with the increased virulence of certain strains of S. aureus. It is present in the majority of community-associated methicillin0resistant S. aureus (CA-MRSA) and is the cause of necrotic flesh eating lesions, an aggressive condition that often kills patients within 72 hours (4). PVL creates pores in the membranes of infected cells. PVL is produced from the genetic material of a bacteriophage that infects S. aureus, making it more virulent (5, 6). Epidemiological and clinical data provide compelling evidence that the high virulence potential of community-acquired MRSA is associated with genes like lukF-PV and lukS-PV (PVL), but direct evidence that PVL plays a role in pathogenesis has been limited (7). The presence of PVL in S.aureus appears to be associated with increased disease severity, ranging from cutaneous infections requiring surgical drainage to severe chronic osteomyelitis and severe necrotizing pneumonia, which could be fatal (8). The production of PVL is related to furuncles, cutaneous abscesses, and severe necrotic skin infections in school children and in certain communities (9).

2. Objectives


This study was conducted to examine Panton-valentine toxin production and methicillin resistance in clinical isolates of Staphylococcus aureus in Ilam Hospitals using the disc diffusion method and molecular method by PCR.

3. Materials and Methods


3.1. Samples Collection

A total of 16o non-duplicated S. aureus isolates used in this study were randomly collected from in-patient and outpatient populations of two government hospitals from Ilam, Iran between September 2012 and October 2013. Then, all isolates were identified as S. aureus using conventional microbiological tests. Pure stock cultures of all isolates were stored frozen at -76°C in Skim Milk broth, containing 10% glycerol (10).


3.2. Laboratory Methods

Specimens were diagnosed by Gram’s stain and were cultured on 10% sheep blood agar and MacConkey’s agar. Staphylococcal isolates were identified by Gram’s staining, catalase production, and haemolysis on blood agar, the oxidative-fermentative test, and the production of bound and free coagulase, mannitol fermentation and 7.5 percent NaCl tolerance, and heat labile DNase. Tube coagulase production is considered the “gold standard” for identifying S. aureus (10).


3.3. Antimicrobial Susceptibility Test

A disk diffusion test of Penicillin (10 U), vancomycin (30 μg), Ampicillin (10 µg), Gentamicin (10 μg), Erythromycin (15 μg), Clindamycin (2 μg), Amikacin (30 µg), ciprofloxacin (5 μg), Tetracycline (30 µg), and Co-trimoxazole (25 µg) (Mast, Merseyside, United Kingdom) was carried out using the Kirby-Bauer method according to CLSI Guidelines 2011. Mueller-Hinton agar plates with 2% NaCl were overlaid with the inoculums (turbidity equivalent to that of a 0.5 McFarland Standard) of the S. aureus clinical isolates. Zone diameters were measured at 24 (48 hours for vancomycin) following CLSI criteria (11). The minimal inhibitory concentration (MIC) was determined only for MRSA isolates by the agar dilution method. Briefly, gradient plates of Mueller-Hinton agar (Hi-media, India) were prepared with vancomycin, cefoxitin, and oxacillin (0.5 - 256 mg/L, Sigma- Aldrich). The 0.5 McFarland equivalent inoculum was prepared using an 18 - 24 hours-old culture spotted onto gradient plates. Plates were incubated overnight at 35°C for 24 hours before assessing the visible growth, as recommended by the national committee for clinical laboratory standards (11). MSSA (ATCC 6538) and MRSA (ATCC 33591) were included as control strains for disk diffusion (12).


3.4. Detection of Methicillin-Resistant Isolates

Detection and confirmation of methicillin-resistant S. aureus isolates was carried out using the disk diffusion method, and the following disks were used: oxacillin (1 μg) and cefoxitin (30 µg). After incubating the plates for 24 hours at 37°C, the antimicrobial activity was evaluated by determining the diameter of the inhibition zone, as recommended by the CLSI (9, 10). Then, results were compared to each other.


3.5. DNA Extraction

Total genomic DNA was extracted using phenol-chloroform isoamyl alcohol (Merck, Darmstadt, Germany). The specimens were pelleted, followed by adding 250 μL buffer I and buffer II containing RNase (Cinna Gen, Tehran, Iran). A 550 μL volume of phenol was added to the aliquots before being centrifuged at 10,000 g for 5 minutes. The supernatant clear phase was then collected into a new eppendorf tube, and the latter stages were repeated twice, in order to wash cell debris. A 0.1 volume of sodium acetate 0.1 M was then added to the tubes and washed twice using ethanol 100 and 80%, respectively. The tubes were then centrifuged at 12,000 g for 15 minutes. The pellet was finally dried and re-suspended in a 30 μL Tris-EDTA buffer (13). DNA extracts were stored at -20°C until required for PCR screening (13).


3.6. Detection of femA, mecA, and luk-pv Genes by PCR

In all coagulase-positive isolates, the resistant genes were detected by PCR. They included genes for femA, mecA, and Panton-valentine (luk-pv) producing toxin (14). The PCR primers used to detect the resistant genes are listed in Table 1 (1, 13). The PCR mixture was prepared in a final volume of 25 mL. The amplification mixture consisted of a 2.5 mL template DNA, 2 mL primers, 2 mL of a 10-fold concentrate PCR buffer, 2 mL dNTP, 0.5 mM MgCl2, 15 mL D.W. and 1 U of Taq DNA polymerase (CinnaGen, Tehran, Iran). A thermocycler (Master Cycler Gradient; Eppendorf, Hamburg, Germany) was programmed for the detection of femA genes with the following program: initial denaturation at 94°C for 3 minutes was followed by 30 cycles of amplification with 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds, except for the final cycle, which had an extension step of 4 minutes (14). The PCR products were submitted to electrophoresis on 1.5% agarose gel (MBI Fermentas) containing 0.4 mL of ethidium bromide and visualized by using UV trans illumination and photographed (BioDoc- Analyse; Biometra, Goettingen, Germany). The PCR program for the detection of the mecA gene was as follows: initial denaturation at 94°C for 3 minutes was followed by 30 cycles of amplification with 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, except for the final cycle, which had an extension step of 4 minutes (13). The PCR program for the detection of the luk-pv gene was as follows: initial denaturation step of 2 minutes at 94°C; 30 cycles of 15 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at 72°C; and a final elongation at 72°C for 7 minutes (1).


Table 1
Table 1
The Primer Sequencing for the Detection of femA and luk-pv1 Genes (1)

3.7. Statistical Analysis

All data analyses were performed using SPSS software version 11.5 for Windows. By using the w2-test and Fisher’s exact test, P values < 0.05 were considered statistically significant.

4. Results


4.1. Samples Collection

The clinical specimens were collected from blood (31%), wounds (20%), urine (21%), catheters (7%), sputum (12%), other sources (9%).


4.2. Diagnosis of Isolates Using Microbiological Methods

Out of 160 isolates of Staphylococci collected. The results showed 91 isolates (56.88%) were coagulase-positive, and 69 isolates (43.12%) were coagulase-negative for staphylococcus aureus (CNSA).


4.3. Antibiogram Profile of Isolates

Table 2 show the results of antibiotic resistance testing of the MRSA and CNSA isolates with the ten antibiotics studied. The patterns of MICs of oxacillin, cefoxitin, and vancomycin on the MRSA isolates were determined with concentrations varying from 4 µg/ml for vancomycin to 64 - 128 μg/mL for oxacillin and 32 µg/mL for cefoxitin.


Table 2.
Table 2.
Antibiotic-Resistant Pattern of MRSA Isolatesa

4.4. Detection of Methicillin-Resistant Isolates

Out of 91 (56.88%) coagulase-positive staphylococci, 32 isolates (35.16%) were resistant to cefoxitin, and 30 isolates (32.96%) were resistant to oxacillin, using disc diffusion method.


4.5. Detection of femA, mecA, and luk-pv Genes by PCR

PCR revealed the presence of the femA gene (510 bp band) in 91 isolates (100%); those were coagulase-positive, and in all isolates, those were resistant to cefoxitin when using the disk diffusion method (Figures 1 - 3). The mecA gene (513bp band) was detected in 32 isolates (35.16%); those were resistant to cefoxitin. Out of 32 MRSA isolates, 13 isolates (40.62%) were positive for the presence of the luk-pv gene (433 bp band).


Figure 1.
Figure 1.
Agarose Gel Electrophoresis for the Detection of the femA Gene (510 bp) in all Coagulase-Positive Staphylococcus aureus Strains by PCR

Figure 2.
Figure 2.
Agarose Gel Electrophoresis for the Detection of the luk-pv Gene (433 bp) in Staphylococcus aureus Strains by PCR

Figure 3.
Figure 3.
Agarose Gel Electrophoresis for the Detection of the mecA Gene (413 bp) in Staphylococcus aureus Strains by PCR

5. Discussion


Methicillin-resistant S. aureus produces a low affinity penicillin-binding protein (PBP2/ or PBP2a), in addition to usual PBP (15). Available data show that the structural genes of this PBP (mecA) are present in resistant strains, but not in susceptible ones (15). The mecA genes and its associated elements are located in the chromosome (16). There are two resistant mechanisms: oxacillin inactivation, which occurs with the high-level production of β-lactamase, and modified resistance, called MOD-SA, which results from the production of modified PBPs with altered resistance to oxacillin (9). In the present study, 160 S. aureus strains were collected from in-patient and outpatients of two government hospitals in Ilam, Iran. They were tested for MRSA and Panton-Valentine leukocidin toxin-producing S. aureus species. In the present study, the percentage of MRSA isolated from all patients was only 35.9%. Similar results were reported by Mohajeri et al. (2013) in Kermanshah, in western Iran, with a percentage of MRSA of 36% (17). Our results are in agreement with data collected by Salimnia and Brown (2005) among staphylococcus isolates in outpatient and inpatients at the Detroit medical center (DMC) and from Outreach specimens (18), and Malathi et al. in 2009, who found a percentage of 36.4% MRSA isolated from patients (19). The occurrence of MRSA results in this study was lower than some results in Iran and other countries. In Iran, a 56% prevalence of MRSA infection was reported by Japoni et al. in 2004, reporting from the Nemazi hospital in Shiraz (16). The prevalence of a 75% MRSA infection was reported by Izadi et al. in Tehran in 2012 (20). In the United States, a Texas hospital reported a 47% prevalence of MRSA infection in 2003 (21). In this study, the rate of Panton-Valentine leukocidin toxin-producing staphylococci was evaluated at 40.62% among MRSA isolates. Our data is in agreement with a survey conducted by Mariem et al. in two Tunisian university hospitals (22), and in agreement with a study by Al-Talib et al. conducted in Malaysia (23). Furthermore, the prevalence of MRSA-producing Luk-pv toxins observed here was lower than that reported in Belgium by Kaur et al. In their survey, the prevalence of MRSA isolates producing Luk-pv was reported at 85.1% (1). In Canada, in 2000, Mehrotra et al. reported on the prevalence of MRSA isolates producing Luk-PV toxin at a rate of 100% (24).


In our study, the oxacillin disk diffusion and cefoxitin disc diffusion methods were compared with PCR. The cefoxitin disk diffusion method showed 100% sensitivity and was superior to the oxacillin disk diffusion method (93.18% sensitivity) regarding the detection of oxacillin-resistant S. aureus using the PCR method for detecting the mecA gene. A survey conducted by Velasco et al. (25) showed that the cefoxitin disk diffusion method, recently recommended by the NCCLS and CLSI as a screening test, was the best method, showing 100% sensitivity and 98% specificity. Recent studies evaluating cefoxitin disks for the detection of MRSA also obtained good results, with a sensitivity of about 100% and specificity of 99% (26). Cauwelier et al. 2004, reported resistance to methicillin in 155 clinical MRSA isolates using different methods, including oxacillin and cefoxitin disks, latex agglutination, and an agar screening test. The cefoxitin disk diffusion method presented 100% sensitivity and 99% specificity, whereas the sensitivity of the oxacillin disk diffusion method fell to 97.1% (27). According to our data, compared to the gold standard (mecA gene detection), the cefoxitin disk diffusion method is preferable to the oxacillin disk diffusion method for the detection of MRSA. Furthermore, our data showed that the rate of pvl production among coagulase-positive S. aureus was 40%. In a survey conducted by Lina et al. a total of 172 strains were tested for the pvl gene, and they reported that 55% of isolates had this gene (28). Kaur et al. in 2012, reported that, from 70 species, 85.1% of S. aureus isolates produced the pvl gene (1). Al-Talib et al. in 2009, collected a total of 230 staphilococci species and pvl genes in any isolates no detected (23).

Acknowledgments

This study was supported by the University of Ilam, Iran. The authors are thankful to the Head of the Department of the Biology Research Center, Ilam University, Ilam, Iran.

Footnotes

Authors’ Contribution: Study concept and design: Arman Rostamzad; drafting of manuscript: Arman Rostamzad; critical revision of the manuscript for important intellectual content: Arman Rostamzad and Nabi Rostamneia.
Funding/Support: This article was financially supported by the Vice-Chancellor for Research Affairs of Ilam University.

References


Table 1

The Primer Sequencing for the Detection of femA and luk-pv1 Genes (1)

Primer Primer Sequence Size of Product, bp
Luk-PV1 433
F 5′- ATCATTAGGTAAAATGTCTGGACATGATCCA-3´
R 5′- GCATCAAGTGTATTGGATAGCAAAAGC-3′
Fem A 510
F 5'- AAAAAAGCACATAACAAGCG-3'
R 5'- GATAAAGAAGAAACGAGCAG-3'
mec A 513
F 5´- AAAATCGATGGTAAAGGTTGGC-3´
R 5'- AGTTCTGCAGTACCGGATTTGC-3´

Table 2.

Antibiotic-Resistant Pattern of MRSA Isolatesa

Antibiotic Pen Van Amp Gn E Da Ak Cip Te Ts
MRSA 98 (85.96) 0 100 (87.71) 55 (48.25) 62 (54.38) 37 (32.45) 24 (21.05) 48 (42.10) 59 (51.75) 48 (2.10)
CNSA 86 (100) 0 86 (100) 64 (74.41) 68 (79.06) 46 (53.48) 31 (36.04) 40 (46.51) 66 (76.74) 54 (62.79)
a Data are presented as No. (%).

Figure 1.

Agarose Gel Electrophoresis for the Detection of the femA Gene (510 bp) in all Coagulase-Positive Staphylococcus aureus Strains by PCR
Lanes 2, 3, 5, 6, 7, 8, 9, 10, positive strains; lane 4, negative strain (MSSA S. aureus ATCC 6538); lane 1, positive control (MRSA S. aureus ATCC 33591); lane M, molecular weight marker (100 bp).

Figure 3.

Agarose Gel Electrophoresis for the Detection of the mecA Gene (413 bp) in Staphylococcus aureus Strains by PCR
Lanes 4, 5, 6, 9, 10, positive strains; lanes 3, 7 and 8, negative strains; lane 2, positive control (MRSA S. aureus ATCC 33591); lane 1, negative control (MSSA S. aureus ATCC 6538); lane 19, water; lane M: molecular weight marker (100 bp).

Figure 2.

Agarose Gel Electrophoresis for the Detection of the luk-pv Gene (433 bp) in Staphylococcus aureus Strains by PCR
Lane M, molecular weight marker (100 bp); Lane 1, negative control; lanes 2, 3, positive strains to luk-pv genes; lanes 4, 5, negative strains.