Mujib A. ABDURAHMAN, İlknur TOSUN, İnci DURUKAN, Mona KHORSHIDTALAB, Ali Osman KILIÇ

Four Temperate Bacteriophages from Methicillin-resistant Staphylococcus aureus Show Broad Bactericidal and Biofilm Removal Activities

The emergence of multi-drug resistance among many bacteria including zoonotic pathogens in the food chain poses a growing public health threat to humans, animals, and the environment worldwide. The inefficiency of current antibiotics to control these pathogens necessitated the development of alternative approaches, such as phage therapy, for the prevention and treatment of human and animal infections, food safety, and wastewater treatment. In this study, four temperate bacteriophages, designated as Trsa205, Trsa207, Trsa220, and Trsa222 were isolated by mitomycin C induction from methicillin-resistant Staphylococcus aureus (MRSA) strains. The phages were characterized based on their electron microscope morphology, burst size, host range, and biofilm removal potential. Based on their morphology, all four phages with isometric heads and long non-contractile tails belong to Siphoviridae family. The one-step growth curves of phages revealed that Trsa205 and Trsa207 have latent periods of about 20 min that results in a burst size of 30 and 45 virions/host cell, respectively, while Trsa220 and Trsa222 showed 25 min of latent period and produced 20 virus particles/cell. The agar-spot assay was used for phage host range determination, and biofilm removal activities were measured spectrophotometrically after crystal violet staining. It was found that at least two-thirds of 56 S. aureus strains (66%) could be lysed by phages when used in combination, and 20-38% by one of the phages. The four phages in combination were able to remove the S. aureus biofilms by 65%. Our results indicated that the newly identified bacteriophages have the potential to be used in phage therapy against multi-drug resistant S. aureus including MRSA and removal of biofilms.

Geniş Bakterisidal ve Biyofilm Giderme Etkisi Gösteren Metisiline Dirençli Staphylococcus aureus Kaynaklı Dört Yeni Ilıman Bakteriyofajın Tanımlanması

Çoklu antibiyotik direncinin besin zincirindeki zoonotik patojenler dahil tüm dünyada birçok bakteride ortaya çıkması insanlar, hayvanlar ve çevre için artan bir halk sağlığı tehdidi oluşturmaktadır. Bu patojenleri kontrol etmek için mevcut antibiyotiklerin yetersizliği, insan ve hayvan enfeksiyonlarının tedavisi, gıda güvenliği ve atık su arıtımı için faj terapisi gibi alternatif yaklaşımların geliştirilmesini gerektirmiştir. Bu çalışmada, metisiline dirençli Staphylococcus aureus (MRSA) suşlarından mitomisin C indüksiyonu ile Trsa205, Trsa207, Trsa220 ve Trsa222 olarak adlandırılan dört ılıman bakteriyofaj izole edilerek tanımlanmıştır. Fajlar, elektron mikroskop morfolojisi, konak hücre başına oluşan faj sayısı, konak genişliği ve biyofilm giderme potansiyellerine göre karakterize edildi. Morfolojilerine göre, izometrik başlı ve uzun kasılmayan kuyruklu dört fajın Siphoviridae ailesine ait oldukları belirlendi. Fajların tek aşamalı büyüme eğrilerine göre, Trsa205 ve Trsa207"nin 20 dakikalık latent periyotlara sahip olduğu ve sırasıyla hücre başına 30 ve 45 faj partikülü oluşturduğu, Trsa220 ve Trsa222"nin ise 25 dakikalık latent periyotu takiben hücre başına 20 virüs partikülü oluşturduğu saptandı. Faj konak genişliği tayini için agar-damlatma yöntemi kullanıldı ve kristal viyole boyamadan sonra biyofilm giderme aktiviteleri spektrofotometrik olarak ölçüldü. Fajların tek başlarına kullanıldıklarında 56 S. aureus suşunun %20-38"unu, dört faj birlikte kullanıldığında ise tüm suşların üçte ikisini (%66) enfekte ederek lize ettiği saptandı. Dört fajın kombine kullanıldığında S. aureus biyofilmini %65 oranında giderebildiği gösterildi. Sonuçlarımız, yeni tanımlanan bakteriyofajların, MRSA dahil çoklu ilaca dirençli S. aureus suşlarına karşı ve biyofilm giderme amacıyla faj tedavisinde kullanılma potansiyeline sahip olduğunu göstermiştir.

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