Publication: Candida parapsilosis klinik izolatlarının morfotiplendirilmesi, genotiplendirilmesi ve farklı morfotiplerde bazı virülans faktörlerinin araştırılması
Abstract
Son yıllarda tüm dünyada Candida parapsilosis’e bağlı sistemik kandidiyaz olgularının görülme sıklığında artış vardır. Ancak bu enfeksiyonların epidemiyolojik değerlendirilmesi için belirlenmiş standart bir genotiplendirme yöntemi bulunmamaktadır. Bu çalışmada, morfotiplendirmenin C.parapsilosis enfeksiyonlarının epidemiyolojisinde, genotiplendirmenin yerine kullanılabilirliği ve belirli morfotiplerde olası virülans faktörlerinin [fosfolipaz, esteraz, asit proteinaz, sıvışık örtü (slime) üretimi ve hidrofobisite] belirlenmesi amaçlanmıştır. Buna yönelik olarak Marmara Üniversitesi Hastanesinde yatan hastaların kan (n= 40) ve idrar (n= 13) kültürlerinden izole edilen 53 C.parapsilosis suşunun Sabouraud-trifeniltetrazolyum agarda (STTZ) yüzey ve renk özellikleri, malt özütlü agarda (MA) ise yüzey özellikleri dikkate alınarak morfotiplendirmeleri yapılmış ve 10 farklı alt grup elde edilmiştir. İzolatların genotiplendirmesi için RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) yöntemi kullanılmış ve %90.6’sı tip II olmak üzere üç farklı genotip elde edilmiştir. Yumurta sarılı (pH: 4.2) besiyerinde yapılan deneylerin sonucunda hiçbir izolatta fosfolipaz üretimi saptanmamıştır. Tween-80’li agarda incelenen esteraz aktivitesi de, bir izolat dışında olumsuz olarak bulunmuştur. Salgısal asit proteinaz aktivitesinin saptanması amacıyla sığır serum albumini içeren (pH: 5.0) besiyeri kullanılmış ve suşların 40 (%75.4)’ında orta düzey pozitiflik (+), 7 (%13.2)’sinde ise kuvvetli pozitiflik (++) saptanırken, 6 (%11.3)’sı negatif sonuç vermiştir. Glukoz (%8) içeren Sabouraud buyyon (SB) kullanılarak sıvışık örtü üretimi değerlendirildiğinde, suşların %67.9’u negatif bulunmuş; %20.7’si zayıf (+), %7.5’i orta düzeyde (++) ve %3.7’si kuvvetli (+++) pozitif aktivite göstermiştir. Hekzadekanlı hidrokarbon aderens yöntemi ile izolatların yüzey hidrofobisite özellikleri araştırıldığında ise bir izolat az, diğerleri orta düzeyde hidrofobik olarak bulunmuştur. Elde edilen veriler kıyaslandığında morfotiplendirmenin genotiplendirmeye göre üstünlüğü belirlenememiştir. Ek olarak virülans faktörleri açısından da belirli bir morfotiple bağlantı kurulabilecek bir özellik saptanmamıştır.
In recent years there is an increase in frequency of systemic candidiasis cases caused by Candida parapsilosis. However there isn’t any standardized genotyping method to be used in epidemiology of those infections. In this study we aimed to determine utility of morphotyping instead of genotyping to study the epidemiology of 53 C.parapsilosis strains isolated from blood (n= 40) and urine (n= 13) cultures of inpatients at Marmara University Hospital, Istanbul, Turkey. The isolates were morphotyped according to their surface and color properties on Sabouraud-triphenyltetrazolium agar (STTZ) and surface characteristics on malt extract agar (MA) media and 10 different subgroups were obtained. In order to genotype the strains, RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) method was used and three different genotypes were obtained comprising mostly type II (90.6%). One of the putative virulence factors investigated in the isolates was phospholipase activity. Phospholipase production was not detected in any of the strains on egg-yolk agar (pH: 4.2). Esterase activity of the strains on Tween-80 agar was negative except for one. In order to observe acid proteinase activity, bovine serum albumin containing (pH: 5.0) agar was used and in 11.3% of the strains no acid proteinase activity was seen while in 75.5% moderate (+), in 13.2% strong (++) activity was detected. Slime production was investigated in 8% glucose containing Sabouraud broth (SB) medium and 67.9 % of the strains were found to be negative; while 20.7% were weakly (+), 7.5% were moderately (++) and 3.7% were strongly (+++) positive. Hydrophobicity of the strains was evaluated by using hexadecane hydrocarbon adherence assay and one isolate was weakly hydrophobic while the remaining strains were found to be moderately hydrophobic. We could not observe superiority of morphotyping over genotyping. Additionally no property was found to associate with a certain morphotype, in terms of virulence factors.
In recent years there is an increase in frequency of systemic candidiasis cases caused by Candida parapsilosis. However there isn’t any standardized genotyping method to be used in epidemiology of those infections. In this study we aimed to determine utility of morphotyping instead of genotyping to study the epidemiology of 53 C.parapsilosis strains isolated from blood (n= 40) and urine (n= 13) cultures of inpatients at Marmara University Hospital, Istanbul, Turkey. The isolates were morphotyped according to their surface and color properties on Sabouraud-triphenyltetrazolium agar (STTZ) and surface characteristics on malt extract agar (MA) media and 10 different subgroups were obtained. In order to genotype the strains, RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) method was used and three different genotypes were obtained comprising mostly type II (90.6%). One of the putative virulence factors investigated in the isolates was phospholipase activity. Phospholipase production was not detected in any of the strains on egg-yolk agar (pH: 4.2). Esterase activity of the strains on Tween-80 agar was negative except for one. In order to observe acid proteinase activity, bovine serum albumin containing (pH: 5.0) agar was used and in 11.3% of the strains no acid proteinase activity was seen while in 75.5% moderate (+), in 13.2% strong (++) activity was detected. Slime production was investigated in 8% glucose containing Sabouraud broth (SB) medium and 67.9 % of the strains were found to be negative; while 20.7% were weakly (+), 7.5% were moderately (++) and 3.7% were strongly (+++) positive. Hydrophobicity of the strains was evaluated by using hexadecane hydrocarbon adherence assay and one isolate was weakly hydrophobic while the remaining strains were found to be moderately hydrophobic. We could not observe superiority of morphotyping over genotyping. Additionally no property was found to associate with a certain morphotype, in terms of virulence factors.