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Frequency of Fibronectin Binding Protein A and Panton-Valentine Leukocidin in Methicillin-Resistant Staphylococcus aureus Collected From Educational Hospitals in Qazvin, Iran


1 Department of Microbiology, Zanjan Branch, Islamic Azad University, Zanjan, IR Iran
2 Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, IR Iran
*Corresponding author: Amir Peymani, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, IR Iran. Tel: +98-2813324971, Fax: +98-2813324971, E-mail: a.peymani@gmail.com.
Biotechnology and Health Sciences. 2016 February; 3(1): e55934 , DOI: 10.17795/bhs-35939
Article Type: Research Article; Received: Jan 2, 2016; Accepted: Jan 11, 2016; epub: Feb 22, 2016; ppub: Feb 2016

Abstract


Background: Staphylococcus aureus is one of the most important organisms involved in nosocomial infection acquired by patients. In recent years, the appearance of methicillin-resistant S. aureus (MRSA) has turned the treatment of these infections into a serious challenge. Surface proteins, such as fibronectin binding proteins (FnBP), and the ability to produce Panton-Valentine leukocidin (PVL) are important factors in pathogenesis of this organism.

Objectives: The purpose of this study was to determine the prevalence of disease-associated genes in the clinical isolates of S. aureus encoding FNB and PVL, collected from the educational hospitals of Qazvin, Iran.

Patients and Methods: This was a descriptive, cross-sectional study in which a total of 103 isolates of methicillin-resistant S. aureus were collected from hospitalized patients in teaching hospitals of Qazvin, during 2013 - 2014. Initially, the identification of isolates was performed according to the standard laboratory methods, followed by confirming the presence of the femA gene, a gene specific to S. aureus. Later, the prevalence of virulence genes (fnb and pvl) was investigated by the PCR method, using specific primers. PCR products were sequenced to confirm the presence of the target genes.

Results: The results of this study showed that among 103 isolates of S. aureus resistant to methicillin, 88 isolates were positive for the presence of the pvl and fnb genes, with the fnb gene present in 86 (83.5%) isolates and the pvl gene only in 2 (1.9%) isolates.

Conclusions: The results of the present study indicate the presence of the pvl and fnb genes in the strains of S. aureus isolated from clinical specimens collected from the patients admitted to teaching hospitals in Qazvin. Considering the clinical significance of these organisms, and their potential in threatening public health systems, the identification, treatment, and infection control management of patients infected with these organisms is of prime necessity.

Keywords: Panton-Valentine Leukocidin; Fibronectin Binding Protein A; Staphylococcus aureus

1. Background


Staphylococcus aureus (S. aureus) is a facultative anaerobe, gram-positive coccus and part of the normal flora of the nose, skin, and gastrointestinal tract (1). This organism is one of the most common causes of nosocomial infections worldwide. Recently, the number of staphylococcal infections has been on the rise due to several reasons, including the spread of resistant strains, an increasing number of patients with immune deficiency, and the excessive use of aggressive medical devices such as catheters (2, 3). These organisms are involved in clinically important diseases including scalded skin syndrome, osteomyelitis, urinary tract infections, bacteremia, and toxic shock syndrome (4, 5). Although these bacteria are part of the normal flora of the human body in healthy people, resistance to antibacterial agents can lead to serious infections and even death. Methicillin-resistant S. aureus (MRSA) causes a variety of suppurative infections, which are resistant to antibiotic treatment. These organisms are unaffected by a penicillinase-resistant group of beta-lactam antibiotics (methicillin, nafcillin, and oxacillin) and cephalosporins, leading to redundancy of these antibacterial agents in treating infections caused by organisms (2). The prevalence of MRSA is high in nosocomial infections acquired at patient care centers, particularly in patients with open wounds and patients with immune deficiencies (6). The virulence of S. aureus is due to various factors such as surface adhesion molecules. Several studies have shown that surface proteins, such as fibronectin, play a crucial role in invasion of this organism to eukaryotic cells (7). The role and importance of attachment factors in a wide range of diseases caused by S. aureus has been studied, and fibronectins have been proven to play a significant role in tissue establishment of these organisms and development of serious diseases, such as keratitis and toxic arthritis, as well as in colonization on the surface of medical devices used for patients (8, 9). These surface proteins act as adhesins and have the ability to bind to extracellular matrix proteins of epithelial and endothelial surfaces. Fibronectin is secreted in the early phase of infection and specifically binds to extracellular matrix proteins of host cells. According to several studies, these proteins are involved in adhesion to damaged heart valves and increase the intracellular entry of the organisms through epithelial cells (10-12). Another virulence factor in S. aureus is Panton-Valentine leukocidin, which is considered a lethal factor for human neutrophils (13). These toxins often attack macrophages and polymorphonuclears and consist of a two-component protein (33 kDa and 34 kDa) that can be separated by electrophoresis into two fractions; both inactive alone. These two components of PVL have antigenic properties and can be modified into a toxoid. This toxin resists phagocytosis, increasing the invasive potential of S. aureus (14, 15). PVL is the only staphylococcal toxin that exclusively affects the leukocytes. In fact, this toxin can be considered an important virulence factor of S. aureus, as it has the ability to destroy leukocytes and eventually reduces the number of polymorphonuclears in the host’s body (16, 17). Considering the importance of S. aureus isolates in developing clinically important diseases, in the present study, the frequency of two genes encoding FNB and PVL as two significant virulence factors was investigated among the bacterial isolates collected from patients admitted to the teaching hospital in Qazvin. Identification of these factors provides healthcare authorities with valuable information to effectively treat infections, control, and eradicate these organisms in hospital environments.

2. Objectives


The purpose of this study was to determine the prevalence of disease-associated genes in the clinical isolates of S. aureus encoding the FNB and PVL virulence factors, collected from the educational hospitals of Qazvin.

3. Patients and Methods


3.1. Bacterial Isolation and Species Identification

In this cross-sectional study, a total of 103 isolates of S. aureus were collected from different clinical specimens of inpatients admitted to several teaching hospitals in Qazvin. These isolates were obtained from patients admitted to intensive care units (ICUs), internal medicine, neonatal and general surgery wards. Written informed consent was obtained from all subjects enrolled in this study. Initially, all isolates were identified using standard microbiological and laboratory methods, including growth on blood agar and type of hemolysis, gram stain, catalase test, slide and tube coagulase test, mannitol salt agar, and DNase test (18). Later, all isolates were identified by detection of the femA gene, which is specific to S. aureus.


3.2. Evaluation of Phenotypic Resistance to Methicillin

The disk diffusion method and agar-screening test were applied to identify MRSA. According to the clinical and laboratory standards institute (CLSI) guidelines, in the first method, the colonies of a 24 hour bacterial culture were added to normal saline to prepare a concentration equal to the turbidity present in the 0.5 McFarland standard tubes. Using the direct method, bacteria were cultured on Muller-Hinton agar medium, antibiotic disks (oxacillin 1 µg/mL; cefoxitin 30 µg/mL) were applied, and subsequently the agar plate was incubated at 37°C for 24 hours. After 24 hours following incubation, the diameter of the inhibition zones was measured and analyzed. A diameter of 22 mm for cefoxitin was considered as susceptible, while a diameter of 21 mm was considered resistant. For the oxacillin salt agar screening test, a Mueller Hinton agar medium containing 4% salt and 6 µg/mL oxacillin was made, followed by preparation of fresh bacterial culture equal to the 0.5 McFarland standard tube prepared using the direct method. Plates were placed on papers divided into 12 equal areas, followed by point cultivation of bacterial suspensions onto each area using sterile swabs. Twelve different isolates were cultured on each plate. Plates were incubated at 37°C for 24 hours. Later, the presence or absence of bacterial growth in the medium was assessed (19).


3.3. Detection of fnb and pvl Genes in MRSA Isolates

Isolation of fnb (encoding Fibronectin) and pvl (encoding Panton-Valentine leukocidin) genes was performed by total DNA extraction using a commercial extraction kit for gram-positive bacteria (Genomic DNA Extraction Kit, Bioneer Inc., South Korea), according to the manufacturer’s instructions. The DNA concentrations of the extracted samples were measured with a Nano Drop™ spectrophotometer using an absorbance ratio of A260/280. Later, the PCR technique was performed using specific primers for femA, fnb, and pvl genes (Table 1) (20-22).


Table 1.
Primers Used in PCR to Detect fnb, pvl and femA Genes

Amplification reactions were prepared in a total volume of 25 μL (24 μL of PCR master mix plus 1 μL of template DNA), including 50ng of genomic DNA, 5.0U of Taq DNA polymerase, 10 mM dNTP mix at a final concentration of 0.2 mM, 50 mM MgCl2 at a final concentration of 1.5 mM, 10 pM of each primer, and 1X PCR buffer (final concentration). PCR amplifications were applied in a thermocycler (Applied Biosystems, USA) as follows: initial denaturation temperature (96°C for 5 minutes) and 35 thermal cycles of 1 minute at 94°C, 1 minute at specific annealing temperature for each primer (58°C for femA gene, 55°C for fnb gene, and 56°C for pvl gene), and 1 minute at 72°C. A final extension step of 7 minutes at 72°C was performed. PCR products were electrophoresed on 1% agarose gel at 100 volts and later stained with ethidium bromide solution, and finally visualized in a gel documentation system (UVtec, UK). The purified PCR products were sequenced by the Macrogen Company (Seoul, South Korea) and the sequence alignment and analysis were performed online, using the BLAST program of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi).


3.4. Statistical Analysis

Data were summarized using mean ± SD (standard deviation), proportional frequency and confidence interval for microbiological, clinical, and demographic characteristics. All analyses were carried out using a statistical software package, SPSS for Windows version 16.0 (Chicago, IL, USA).

4. Results


Bacterial isolates of the present study were collected from different clinical samples including sputum (35%), wound (24.3%), blood (20.4%), urine (10.7%), and catheter (9.7%). Among these 37 samples (35.9%) were taken from women and 66 samples (64.1%) from men. Isolates were obtained from patients admitted to internal medicine (56) (54.4%), intensive care units (19) (18.4%), neonatal (21) (20.4%), and general surgery wards (7) (6.8%). All 103 isolates were positive for the presence of the femA gene, confirming that all isolates were S. aureus. The frequency of the fnb gene was 83.5%, a value mainly due to the organisms isolated from sputum samples (35%). Also, two isolates (1.9%), isolated from wound infections and sputum samples, were positive for the presence of pvl genes (Table 2).


Table 2.
Prevalence of pvl and fnb Genes Among 103 Isolates of S. aureus, Regarding Clinical Source and Hospital Wards a

5. Discussion


Currently, S. aureus is one of the most important pathogens in humans and is considered a major bacterial agent that is involved in the development of nosocomial infections. According to many studies, a long hospital stay is considered a risk factor development of staphylococcal infections (7). Patients infected with MRSA strains stay longer in hospitals compared to those who are infected with methicillin-susceptible S. aureus (MSSA). A long stay in a hospital makes patients with staphylococcal infections more susceptible to the development of severe infections of internal tissues such as septicemia and osteomyelitis. In addition, the mortality rate in patients with MRSA is significantly higher compared with patients infected with methicillin-susceptible isolates (5). The presence of fnb gene in S. aureus provides the organisms with the initial attachment and entry into epithelial cells (11). Also, the presence of PVL in S. aureus increases the pathogenicity of this organism, leading to the establishment of severe diseases including pneumonia and soft tissue infections (13). Based on the results of the present study, among 103 methicillin-resistant S. aureus isolates, the frequency of fnb and pvl genes were calculated as 83.5% and 1.9%, respectively. These results showed the low prevalence rate of the PVL toxin among clinical isolates of MRSA. This result has also been reported in some studies in which MRSA strains were positive for the presence of the pvl gene (23-25). In a recent study from Iran, Motamedi et al. (18) reported that the pvl gene was positive in 6% of S. aureus isolates, which were all negative for the mecA gene. Another study in Iran by Khosravi et al. (26), which was reported, showed that of 95 isolates of S. aureus collected from hospitalized burn patients, the presence of the pvl gene was confirmed in 6 (7.2%) MRSA strains and 4 (33.3%) MSSA strains, indicating a total PVL frequency of 10.5% among the isolates tested in their study. Chi et al. (27), studied 16 isolates of S. aureus collected from children in Taiwan with staphylococcal toxic shock syndrome (6 isolates) and scalded skin syndrome (10 isolates) for the presence of the fnb gene. They reported that the fnb gene was present in all 16 isolates, and the presence of the pvl gene was only confirmed in 4 isolates (67%). In another study by Neela et al. (28), 450 MRSA isolates were studied, and a frequency of 100% was found for the fnb gene, whereas no pvl gene was detected. Also, Rasmussen et al. (29), reported that all 46 nasal MSSA isolates studied were positive for the presence of the fnb gene, while none were positive for the pvl gene. In addition, the isolates obtained from patients with invasive staphylococcal infections showed a frequency of 100% for the presence of the fnb gene and a low frequency (1%) for the presence of the pvl gene. Peacock et al (23), demonstrated that of 178 samples taken from S.aureus carriers, 152 samples (98%) were fnb positive and 6 samples (4%) carried the pvl gene. In conclusion, the findings of this study showed that S. aureus isolates collected from patients in teaching hospitals of Qazvin were considerably positive for the presence of fnb and pvl genes. The co-existence of these two virulence factors plays a significant role in the invasion, pathogenesis, and the spread of these strains. Therefore, the application of an effective strategy to control infections, especially in hospitals, and proper management of treatment is required to prevent further spread of such organisms.

Acknowledgments

The authors would like to thank the acting head, and also other staff, of the cellular and molecular research center, Qazvin University of Medical Sciences, for their assistance with completing this research project.

Footnotes

Authors’ Contribution: Amir Peymani: conception and design of the study; Amir Peymani and Maria Taromian: laboratory work; Masoumeh Aslanimehr: data analysis and interpretation.

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Table 1.

Primers Used in PCR to Detect fnb, pvl and femA Genes

Targets Primer Sequences (5' - 3') Reference
fnb (20)
F: CACAACCAGCAAATATAG
R: CTGTGTGGTAATCAATGTC
pvl (21)
F: AGTGAACTTATCTTTCTATTGAAAAACACTC
R: GCATCAASTGTATTGGATAGCAAAAGC
femA (22)
F: AAAAAAGCACATAACAAGCG
R: GATAAAGAAGAAACCAGCAG

Table 2.

Prevalence of pvl and fnb Genes Among 103 Isolates of S. aureus, Regarding Clinical Source and Hospital Wards a

Variables Genes
fnb pvl
Hospital wards
Internal medicine 47 (45.6) 1 (1)
ICU 17 (16.5) 1 (1)
Neonatal disease 17 (16.5) N
Surgery 5 (4.8) N
Clinical specimens
Sputum 31 (30.1) 1 (1)
Blood 20 (19.4) N
Wound 17 (16.5) 1 (1)
Urine 10 (9.7) N
Catheter 8 (7.8) N
Abbreviations: ICU, intensive care unit; N, negative.
a Data are expressed as No. (%) or mean ± SD.