I.INTRODUCTION
Staphylococcus aureus (S. aureus) is a major causal origin of human diseases and accounts for more than 80% of purulent diseases in the environment of hospitals etc1).
S. aureus produces various material associated with infections and diseases such as teichoic acids of the cell wall component, exoenzymes (including staphylokinase, hyaluronidases, phosphatases, coagulases, catalases, proteases, nucleases and lipases), leukocidin, hemolysins and enterotoxin which causes food poisoning2). The Enterotoxin of S. aureus is a heat-resistant toxin and is known as a cause for diarrhea and vomiting in human beings. New types of enterotoxin (Seh, Seh, Sei, Sej, Sek, Sel, Sem, Sen, Seo) have been reported recently3). The hemolysinofS. aureus forms β-barrel pores in the cell membrane and is responsible for cell hemolysis with lack of contents. There are 4 types of hemolysin (α-, β-, γ-, δ-)4). The Panton-ValentineLeukocidin(PVL) of S. aureus is a fatal exotoxin to polymorphonuclear leukocytes and is associated with severe skin infections and the necrotizing pneumonia also. ProteinA(Spa) is involved in the adhesion induction for a vascular endothelial damage and intravascular diseases. The exfoliative toxin responsible for the peeling skin syndrome takes off the epithelial layer of the skin and causes a bullous impetigo5).
The various expressions and the combination of virulence factors have effects to the pathogenicity, persistence and the spread of infection also6). There fore, the analysis of virulence factors will help in the treatment of diseases causedby S. aureus. In this study was researched the drug resistance intensity of the S.aureus virulence factors. Also it was researched when the spread of those will be serious if detected in the dental area and what kind of specific virulence genes is acquired in clinics and hospitals. In addition, the distribution and the diversity of virulence genes who have a relationship between growing specific diseases and occurring factors were identified with the aim to prevent and to treat infections.
II.MATERIALS AND METHODS
1.Identification and characterization of S. aureus isolates
Between June and December 2010, the saliva from the oral cavity was collected from 129 outpatients with periodontitis. The group of patients between 20 and 50 years consisted of 88 patients from the S dental hospital and 41 patients from the E dental clinic both located in Seoul, Korea. The saliva was collected and sealed and then stored below -20°C until the use in the study. The isolation of bacteria was carried out according to the method of Genome analysis A Laboratory Manual (1997) and Murray et al. (2007) using PCR (polymerase chain reaction)6-8).
2.Genomic DNA extraction and PCR analysis test of virulence factors
DNA was extracted with the AccuPrep Genomic DNA Extraction Kit (Bioneer, Korea) and the virulence factors were performed by a PCR conditioning of primer as shown in Table 1. PCR amplification product was electrophoresed in a 1% agarose gel at 50 V, 600 mA for 50 min, stained with ethidium bromide (EtBr, Sigma, USA) 0.5 μg/ml and visualized using the Gel Doc XR+ transilluminator (Bio-Rad, USA)(Table 1).
III.RESULTS
1.Isolation of S. aureus
Between June and December 2010 were isolated 54 strains (41.9%) from saliva in the oral cavities collected from a total of 129 outpatients in Seoul. The patient group consisted of 88 patients from the S dental hospital and 41 patients from the E dental clinic.
2.Analysis of virulence-related gene
The virulence gene of 54 strains being isolated from the oral cavities of patients with periodontitis were shown in table 2. Enterotoxin of Seb was shown as the most frequent in 88.9% (48 strains) and Seg in 51.9% (28 strains), Sei in 44.4% (24 strains), Sea in 11.1% (6 strains), Sec in 1.8% (1 strain) followed in the order. Hemolysins of Hla, Hlb, and Hld were held as most frequent with 98.1% (53 strains), gamma-hemolysin mutated gene of HlgAC with 3.7% (2 strains), leukocidins of LukM 90.7% (49 strains) and LukD 88.9% (48 strains) were shown mainly. As a gene test result, fibronectin binding proteins of FnbA were the most frequently present in 83.3% (45 strains), laminin binding proteins of Eno were present in 100% (54 strains), fibrinogen binding proteins of Fib were present in 94.4% (51 strains) and elastin binding proteins of Ebp were present in 53.7% (29 strains) respectively. Capsular polysaccharide gene of Cap5 was shown as 50.0% (27) but there was no exfoliative toxin gene of Eta, Etb and toxic shock syndrome toxin of Tsst present(Table 2).
The diversity of virulence gene of the 54 strains of S. aureus was analyzed and the result was shown in Table 3. Among the 54 strains were three kinds of virulence factors with a total of 40.7% (22 strains) the most frequently which were Seb-Seg-Sei 35.2% (19 strains), Sea-Seb-Seg 4.9% (2 strains) Sea-Seg-Sei 2.4% (1 strain) in the order. The diversity of hemolysin gene was shown as 98.1% (53 strains) expressed with Hla-Hlb-Hld. The diversity of fibronectin binding proteins gene was shown as 59.3% (32 strains) expressed with FnbA-FnbB. The diversity of leukocidins gene was held as 88.9% (48 strains) expressed with LukD-LukM (Table 3).
IV.DISCUSSION
In the dental care environment, pathogens are floating around in the form of aerosols. They may contaminate care providers, the bodies of patients, instruments and the complete equipment of hospital. So infections are possible in many ways. S. aureus is reported as a major cause of nosocomial infections after dental care and is involved in the development of endocarditis, bacteremia, osteomyelitis, peritonitis and in the infection of soft tissues etc15).
As result of the serological analysis, Seg, Seh, Sei, Sej, Sek, Sel, Sem, Sen and Seo of enterotoxin were reported with heat resistance and a low molecular weight of 26,000-34,000 dalton based on the antigenicity. The general enterotoxin detection rate of 92.6% was in this study very high. Bania et al. detected 76% enterotoxin in S. aureus isolated from nasal cavities of hospital patients. Nashev et al. isolated 70% in S. aureus from the nasal cavities of the general population. In detail, Peck et al. isolated 22.4% enterotoxin among 165 strains which were collected from a domestic tertiary care hospital and the isolation rate in the domestic dental patients was higher.
Looking in detail, Seg, Sei and Seb type gene were detected highly; Peck et al. reported Seg and Sei as major enterotoxins, Nashev et al. reported Seg, Sei, Seb and Bania et al. reported Sec and Seg as predominant16-18). As an analysis result, Seg and Sei were detected globally. Hereby Korea and European countries showed a similar distribution.
S. aureus produces 4 kinds of hemolysin (α-, β-, γ- and δ-) and 5 kinds of membrane damages causing toxins leukocidin inclusive. Leukocidine affects platelets and monocytes and leads to a release of inflammatory cytokines. It is a potential virulence gene causing inflammatory diseases in the periodontium and opportunistic infections in surgery patients also. In this study, Hla, Hlb and Hld gene were detected with each 98%, 98% and 96% respectively. Strains with multiple genes of Hla-Hlb-Hld showed a 98% distribution. Jung et al. reported that 98% of clinical isolated MSRA strains were holding more than 2 kinds of hemolysin gene and 51% were holding Hla-Hlb-Hld-Hlg2 gene predominantly. In addition, Kouidhi et al. emphasized that 55% of isolated bacteria from children with dental caries owned α-toxin Hla gene19). This is the important pathogen as a toxic gene which affects the biofilm formation related with dental caries and leads to a worsening of gingivitis symptoms. S. aureus produces pathogens which were related with extracellular and cell wall associated components and may cause various diseases and produces many kinds of Cap also. Among them, type 5 and type 8 gene were produced with 70~80%20). Inthisstudy,Cap8 gene was not detected but Cap5 gene was detected with around 50%. Cap5 shows an universal aspect in Korea. The exfoliative toxin is a major toxin which is the cause for the staphylococcal scalded skin syndrome (SSSS) in new borns and children. It is serologically classified in Eta and Etb21).Inthisstudy,Eta and Etb gene were not detected at all which is in agreement with the results of Kim et al. who were studying the S. aureus isolated from inpatient’s blood in a tertiary care hospital and Salasia et al. who were studying the S. aureus isolated from milk of the province of central Java in Indonesia and the county of Hessen in Germany22,23). However, Lehnet al. reported some differences as Eta (0.5%) and Etb (0.0%) in isolated bacteria from blood cultures sampled in Germany. Meanwhile, In European and African regions were detected 90% of Eta and Etb and in Japan was mainly detected Etb24,25). For the toxin genes distribution are genetic and epidemiological differences responsible. Thus, the expression of these toxin genes is greatly regional affected. The leukocidal (synergohymenotropic) toxin including the Panton-Valentine leukocidin (PVL) contributes to amicroorganism infection by a phagocytosis inhibition of polymorphonuclear leukocytes.
In this study, lukD and lukM were detected with 88.9% and 90.7% respectively which is similar with the result of Ote et al. showing lukD (96%) and lukM (100%)10). The FnbA & B (fibronectin-binding protein A and B), the laminin binding protein (Eno), Fib (Fibrinogen binding protein) and Ebp (Elastin binding protein) are involved in S. aureus infections and mediate the bacteria adhesion to host tissues in addition to direct the toxic syndrome. The toxic syndrome produces toxins which are concerned with the plasma coagulation and the conjugation material in the body and plays an important role to initiate infections26). Inthisstudy, FnbA, FnbB, Fib, and Eno were detected with 83%, 66%, 94% and 100% respectively which is similar to the result of Kumar et al6). Th eyreportedaboutFnbA (100%), FnbB (15%), Fib (60%), Eno (100%) and demonstrated the importance of the disease-causing adhesion.
The isolates from periodontitis revealed a high proportion of virulence factors. But the genetic determinants of virulence factors did not reveal any significant relationship. It has been stated the genetic determinants of virulence factors are not residing on the same loci. The expression of virulence factors depends on their growth phase and on environmental stimuli, as a continuous secretion of the virulence factors would not be economic for the pathogen. The observations of this present study stressed out their non-significant associations also. In conclusion, almost all of the genetic components of the isolates and namely Hla, Hlb, Hld, Eno, Fnb, Fib and Luk appear as responsible for the expression of periodontitis. The other genetic determinants of virulence factors seems independent as an association could not be established between the tested isolates. Observations of this study are that exfoliative toxin, TSST and the capsular polysaccharide Cap8 gene are playing no role in the expression of periodontitis as they were not detected.
In conclusion, it is very important to identify the distribution and the diversity of toxin gene associated with the S. aureus expression in dental care patients with periodontitis directly for an effective prevention and treatment of dental diseases. The studies for the virulence factors of S. aureus were mainly conducted with inpatients. But such studies may be very inadequate for patients with periodontitis. Patients with dental diseases showed a high toxin gene expression so that the S. aureus in the area of dental care is judged as highly pathogen with a high infection rate. Additional studies about these toxin genes will be required in the future.
