Investigation of the presence of panton valentine leucocidin (PVL) with polimerase chain reaction in staphylococcus aureus isolates

Staphylococcus aureus, is a pathogen causing many infections varying from skin infections depending on virulence factors to septicaemia. Panton-Valentine Leukocidin (PVL), one of the virulence factors of S. aureus, is a cytotoxin which causes tissue necrosis. Also, PVL is one of the important virulence factors of community acquired MRSA (methicillin resistant S. aureus) strains which appeared recently in several countries. These strains have the feature of forming soft tissue and lung infections accompanying with necrosis in healthy children and young adults. The tissue necrosis in these types of infections is reported as a consequence of PVL release. Our consumption/aim in this study is to investigate the presence of the related gene by using 433 bp long, pvl belonging primers which were obtained in clinical isolates in different countries (Ankara, İstanbul, İzmir) and to evaluate the clonal correlation/relation between the obtained PVL-positive strains with ?pulsed-field gel electrophoresis? (PFGE) technique.In our study, a multiplex PCR method was applied, which simultaneously determines nuc and plv genes by using luk-PV-1 and luk-PV-2 primers and S. aureus specific DNAase originated nuc-1 and nuc-2 primers. In total, 299 clinical samples which were collected from Dokuz Eylül University Medical Faculty Hospital, Hacettepe University Medical Faculty Child Hospital and İstanbul University Çapa Medical University Medical Faculty in 2005-2007 was examined with this method. Also to reveal the short term clonal correlation of PVL producing isolates, the clonal correlation were investigated with PFGE which is accepted as ``the gold standard??.The 11 of the 299 isolates (1 MRSA, 10 MSSA) (3.67 %) included in the study were determined as PVL-positive. As there was a difficulty in detecting of the routine sensibility of methisiline heteroresistant isolates, 16S/mec/nuc multipleks PCR was applied totally to the PVL-positive isolates at the same time with methisiline resistance investigation.Consequently, it was detected that the isolate suppossed to be MSSA from Hacettepe University Medical Faculty Child Hospital was in fact a MRSA isolate which is carrying mec gene.The rate between the MRSA isolates were detected as 1 % (1/99) and the rate between MSSA isolates were determined as 5 % (10/200). The only PVL-positive MRSA isolate was isolated from a blood sample obtained from Hacettepe University Medical Faculty Child Hospital. According to the centers, the distribution of PVL-positive MSSA isolates were as follows; 6 isolates from Dokuz Eylül University Medical Faculty Hospital, 3 isolates from Hacettepe University Medical Faculty Child Hospital and 1 isolate from Istanbul University Capa Medical University Medical Faculty. The rates according to the number of obtained MSSA strains were determined as 10 % (6/59) in Dokuz Eylül University Medical Faculty Hospital, 4.83 % (3/62) for MSSA and 2.6 % (1/38) for MRSA in Hacettepe University Medical Faculty Child Hospital and 1.25 % (1/79) in İstanbul University Çapa Medical University Medical Faculty.18 % (2/11) of PVL-positive MSSA isolates were isolated from blood (1 MSSA, 1MRSA) and 82 % (9/11) were isolated from wound cultures. The distribution of the rates of PVL-positive isolates/clinics for Dokuz Eylül University were; 50 % (3/6) in Gynaecologics and Obstetrics, 16.6 % (1/6) in Otorhinolaryngology, 16.6 % (1/6) in Brain Surgery and 16.6 % (1/6) Plastic Surgery services.The clonal relation between PVL-positive isolates was investigated with PFGE. It was reported that there was corralation between the four isolates obtained from Dokuz Eylül University. The macrorestriction patterns of these isolates were identical with each other in double groups. Between these two groups a single band pattern difference was found. The two of identic isolates were isolated from different services in two months period. One of the isolates obtained from Hacettepe University have similar macrorestriction pattern with these two groups. Other isolates were evaluated as completely different strains.As a result; the Panton-Valentine Leukocidin?s (PVL) was an indicator of especially community acquired methisiline resistant Staphylococcus aureus (CA-MRSA) strains and MSSA and MRSA isolate rates collected from different centers were found respectively 1.25-10 % and 1.25 % Besides these rates were not high; the detection of clonal correlation between isolates may indicate the potential of PVL-positive strains extension. As detecting the the methisiline resistance with routine methods might be difficult, applying molecular methods specific to mecA gene in PVL-positive isolates would be helpful for the characterization of the community acquired MRSA strains.Key words: Panton-Valentin Leucocidin (PVL), methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive S. aureus (MSSA), multiplex PCR, pulsed field gel electrophoresis (PFGE)