Klinik Candida izolatlarında karvakrolün antimikrobiyal etkilerinin araştırılması ve immunoelektron mikroskobik yöntem ile görüntülenmesi

Abstract

Candida türleri yüzeyel ve sistemik hastalığa sebep olurlar. Candida biyofilmleri de hastane kaynaklı ve vücut içi araçlarla ilişkili enfeksiyonlar için önemli bir sorundur. Birçok antifungal ajanın etkinliği, sitotoksisitesi ve ilaca karşı artan direnç oranı nedeniyle sınırlıdır. Son yıllarda uçucu yağlar gibi bazı doğal ürünlerin antimikrobiyal ve antibiofilm etkinliği dikkat çekticidir. Karvakrol [2-metil-5-(1-metiletil) fenol], kekikteki fenolik bileşenlerden biridir ve güçlü antimikrobiyal aktiviteye sahiptir. Şu ana kadar, klinik Candida suşlarında karvakrolün antimikrobiyal etkilerini tartışan sınırlı sayıda çalışma vardır. Çalışmamızda, karvakrolün klinik Candida izolatları üzerindeki etkilerini mikrobiyolojik ve geçirimli elektron mikroskobu yöntemleri ile araştırmak ve karvakrolün fungal hücre duvarı üzerindeki etkilerinin hifal duvar proteini olan Hwp1 proteini kullanılarak immünoelektron mikroskobik yöntemle araştırılması hedeflenmiştir. Bu çalışmada, 24 klinik izolat ve 1 referans (C. albicans ATCC 14053) kullanılmıştır. Karvakrolun minimum inhibisyon konsantrasyonu (MİK), broth mikrodilüsyon yöntemi kullanılarak belirlenmiştir. MİK sonuçları, test edilen tüm izolatlar için MİK ≤% 0.031 (hacim / hacim) göstermiştir. Geçirimli elektron mikroskobu çalışmaları için izolatlar ½ MİK konsantrasyonunda 48 saat boyunca karvakrole maruz bırakılmış ve sonuçlar kontrol ile karşılaştırılmıştır. Bulgularımıza göre, karvakrol, Candida izolatlarında çok düşük MİK değerleri ile yüksek antifungal potansiyel göstermiştir. Elektron mikroskobik olarak, MİK değerinde ve yüksek konsantrasyonlarda herhangi bir büyüme gözlenmemiştir; Hücresel hasar, alt MİK konsantrasyonlarında da belirlen miştir. Karvakrolün hücrelere geri dönüşümsüz bir hasar verdiği gösterilmiştir. Bulgularımıza göre, karvakrolün çalışılan tüm konsantrasyonlarda hücre büyümesini ya da hifal büyümeyi artırıcı etkisi olmadığını gözlemlenmiştir. İmmünogold etiketleme sonuçları, karvakrolün hücre duvarındaki etkisini gözlemlemek için kullanılmış; Karvakrol uygulanan hücrelerde immun etiketleme çok azalmış olmasına rağmen dağılmış stoplazma içerisinde de yer yer Hwp1 proteinin varlığı gözlemlenmiştir. Gelecekteki çalışmalar için Candida infeksiyonlarının topikal tedavisinde karvakrolün umut verici bir ilaç potansiyeli bulunmakta ve konu ile ilgili detaylı çalışmalara gereksinim duyulmaktadır.

Keywords

• Candida
• biyofilm
• karvakrol

Investigation of the antimicrobial effects of carvacrol in clinical Candida isolates and imaging by immunoelectron microscopic method

Abstract

Candida species can cause superficial and systemic disease and their biofilms have become an important problem for both hospital-acquired and device-associated infections. The efficacy of many antifungal agents are limited by their cytotoxicity and increasing rate of drug-resistant. In last years, the antimicrobial and antibiofilm activity of some natural products such as essential oils have drawn attention. Carvacrol [2-methyl-5-(1-methylethyl)phenol] is one of the phenolic components of thyme and have strong antimicrobial activity. Thus far, limited reports have discussed the antimicrobial effects of carvacrol on clinical Candida strains. In our study, it was aimed to investigate the effects of carvacrol on clinical Candida isolates by microbiological and transmission electronmicroscopic methods and to investigate the effects of carvacrol on the fungal cell wall by immunoelectron microscopic method using the hyphal wall protein Hwp1 protein. In this study, 24 clinical isolates and 1 reference strain (C. albicans ATCC 14053) were used. The minimum inhibitory concentration (MIC) of carvacrol was determined using the broth microdilution method. MIC results showed a MIC ⩽ 0.031% (vol/vol) for all isolates tested. For transmission electron microscopic studies, isolates were also exposured to the carvacrol at concentration of 1⁄2 MIC for 48 hours and results were compared with the control. According to our results, carvacrol showed high antifungal potential with very low MIC values on Candida isolates. Electron microscopically, no growth was observed at the MIC value and the higher concentrations; cellular damage was also determined at sub MIC concentrations. It has been shown that carvacrol causes irreversible damage to cells. We observed that carvacrol did not increase cell growth or hyphal growth at all studied concentrations. The immunogold labeling results were used to observe the effect of carvacrol on the cell wall. Although immune labeling was greatly reduced in carvacrol treated cells, the presence of Hwp1 protein was also observed in the scattered cytoplasm. Usage of carvacrol in the topical treatment of Candida infections with further study it was determined that a potentially promising drug and detailed studies on the subject are needed.

Keywords

• Candida
• biofilm
• carvacrol

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