Streptococcus pyogenes İzolatlarının Virülans Faktörlerinin Araştırılması ve “Multiple Locus Variable Number Tandem Repeat Fingerprinting (MLVF)” Yöntemi ile Tiplendirilmesi

Investigation of Streptococcus pyogenes Virulence Factors and Typing by Multiple Locus Variable Number Tandem Repeat Fingerprinting (MLVF) Method

Hatice TÜRK DAĞI,Şerife YÜKSEKKAYA,Tuba SEYHAN,Duygu FINDIK,İnci TUNCER,Uğur ARSLAN

Özet

Streptococcus pyogenes, konağın boğaz ve cildini kolonize eden, farenjit, tonsillit ve impetigo gibi hafif enfeksiyonların yanı sıra streptokoksik toksik şok sendromu, septisemi ve nekrotizan fasiit gibi şiddetli invaziv enfeksiyonlara neden olan ve çeşitli virülans faktörleri üreten önemli bir bakteriyel patojendir. Bu çalışmada tonsillo-farenjitli hastaların boğaz kültürlerinden elde edilen S.pyogenes izolatlarının antibiyotiklere duyarlılıklarının saptanması, virülans faktörlerinin [pirojenik ekzotoksin genleri (speA, C, G, H, I, J, K, L, M, smeZ ve ssa), deoksiribonükleaz genleri (sdaB, spd3, sdc ve sdaD), proteaz genleri (speB, spyCEP ve scpA) ve inhibitör genlerinin (mac ve sic)] belirlenmesi ve “multiple locus variable number tandem repeat fingerprinting (MLVF)” yöntemiyle tiplendirilmesi amaçlanmıştır. Bu araştırmada 150 adet S.pyogenes izolatı konvansiyonel yöntemler ve streptokok grup A lateks kiti (Biomerieux, Fransa) ile tanımlanmış, antibiyotik duyarlılıkları Kirby-Bauer disk difüzyon yöntemiyle “Clinical and Laboratory Standards Institute- CLSI” önerilerine göre çalışılmış ve değerlendirilmiştir. DNA izolasyonu, ticari bir DNA izolasyon kiti (Qiagen, Almanya) kullanılarak üretici firma önerileri doğrultusunda yapılmıştır. Virülans genleri multipleks polimeraz zincir reaksiyonu (PCR) yöntemiyle belirlenmiş, MLVF analizi bakteri genomu içinde yer alan tekrarlayan dizilere özgül primerler kullanılarak multipleks PCR yöntemiyle gerçekleştirilmiştir. Çalışma sonunda S.pyogenes izolatlarının tamamının penisilin G, sefotaksim, seftriakson, eritromisin, klindamisin,

Abstract

Streptococcus pyogenes is an important bacterial pathogen that colonizes the throat and skin of human beings and causes a wide variety of diseases ranging from mild infections like pharyngitis, tonsillitis and impetigo to severe invasive infections such streptococcal toxic shock syndrome, septicemia, and necrotizing fasciitis, and produces a wide variety of virulence factors. The aim of this study was to investigate the antibiotic resistance, virulence genes; [pyrogenic exotoxin genes (speA, C, G, H, I, J, K, L, M, smeZ and ssa), deoxyribonuclease genes (sdaB, spd3, sdc ve sdaD), protease genes (speB, spyCEP ve scpA) and inhibitor genes (mac and sic)] of S.pyogenes strains isolated from throat cultures of patients with symptomatic tonsillo-pharyngitis and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method. One hundred and fifty S.pyogenes isolates were identified by conventional methods and streptococcus group A latex kit (Biomerieux, France). Antibiotic susceptibility tests were performed by Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute. DNA isolation was performed by using a commercial DNA isolation kit (Qiagen, Germany) in accordance with manufacturer’s recommendations. The virulence genes were determined by multiplex PCR. MLVF method was performed with multiplex PCR using specific primers for repeated sequences within bacterial genome. All of the S.pyogenes isolates were susceptible to penicillin G, cefotaxime, ceftriaxone, chloramphenicol, clindamycin, erythromycin, levofloxacin, vancomycin and linezolid. Among streptococcal pyrogenic exotoxin genes the most frequent gene was smeZ (90.0%) followed by speG (88.0%), speC (58.7%), ssa (42.7%), speA (33.3%), speJ (24.0%), speK (18.7%), speH (14.0%), speI (13.3%), speL and speM (9.3%). Of the DNase genes, sdaB was detected in all strains (100%), spd3, sdc, sdaD genes were determined as 64.7%, 36.0%, 24.7% respectively. Protease genes (speB, spyCEP, scpA) and mac gene from the inhibitor genes were positive in all strains, and sic gene was positive in only 3 (2.0%) of the isolates. Thirty-two different patterns that contained two or more isolates were determined by MLVF analysis. Ninety one isolates were included in any of the 32 different patterns, while 59 isolates were defined as sporadic isolates. In conclusion, S.pyogenes isolates collected from throat cultures of patients with symptomatic tonsillo-pharyngitis in Konya/Turkey were susceptible to all antibiotics studied and have carried a very high rate of virulence factors. However the isolates were mostly clonally unrelated and sporadic. This study is the first report in Turkey, in which S.pyogenes isolates were typed by the MLVF method and a large number of virulence factors were investigated.