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

Kafkas Universitesi Veteriner Fakultesi Dergisi ISSN: 1300-6045 e-ISSN: 1309-2251 Journal Home-Page: http://vetdergikafkas.org Kafkas Univ Vet Fak Derg 27 (1): 29-36, 2021 DOI: 10.9775/kvfd.2020.24680 RESEARCH ARTICLE Four Temperate Bacteriophages from Methicillin-resistant Staphylococcus aureus Show Broad Bactericidal and Biofilm Removal Activities [1] Mujib A. ABDURAHMAN 1,2,a İlknur TOSUN 1,b İnci DURUKAN 1,c Mona KHORSHIDTALAB 1,d Ali O. KILIÇ 1,e (*) This work was supported by the Scientific Research Projects Coordination Unit of Karadeniz Technical University (Grant Number: TDK-2015-5340) 1 Department of Medical Microbiology, Faculty of Medicine, Karadeniz Technical University, TR-61080 Trabzon - TURKEY 2 School of Natural and Computational Science, Dire Dawa University, P. O. Box 1362, Dire Dawa, ETHIOPIA [1] ORCIDs: a 0000-0001-6273-1536; b 0000-0002-6772-1043; c 0000-0002-9789-4738; d 0000-0001-8680-295X, e 0000-0002-5506-0866 Article ID: KVFD-2020-24680 Received: 05.07.2020 Accepted: 06.11.2020 Published Online: 09.11.2020 Abstract 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. Keywords: Bacteriophage, Siphoviridae, Staphylococcus, Biofilm, Host range Geniş Bakterisidal ve Biyofilm Giderme Etkisi Gösteren Metisiline Dirençli Staphylococcus aureus Kaynaklı Dört Yeni Ilıman Bakteriyofajın Tanımlanması Öz 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. Anahtar sözcükler: Bakteriyofaj, Siphoviridae, Staphylococcus, Biyofilm, Konak spektrumu How to cite this article? Abdurahman MA, Tosun İ, Durukan İ, Khorshidtalab M, Kılıç AO: Four temperate bacteriophages from methicillin-resistant Staphylococcus aureus show broad bactericidal and biofilm removal activities. Kafkas Univ Vet Fak Derg, 27 (1): 29-36, 2021. DOI: 10.9775/kvfd.2020.24680 * Corresponding Author ( ) Tel: +90 462 377 7760 E-mail: (AO Kılıç) This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) 30 Four Temperate Bacteriophages from ... Research Article Introduction Material and Methods Antimicrobial resistance (AR) in bacteria is a growing problem worldwide today and will remain a major threat to humans and animals as well as sustainable economic growth. In a recent report by the World Health Organization (WHO), 12 bacterial species were listed as global priority pathogens (GPP) including Staphylococcus aureus, emphasizing the immediate need for new antimicrobial alternatives [1]. This report discusses the current antibacterial therapy options, such as phage and phage endolysin therapy against infections caused by methicillin-resistant S. aureus (MRSA). Staphylococcus species are one of the most common bacteria of healthcare-associated infections and mainly responsible for infections related to catheters, implants, and medical devices [2]. Many of the healthcare-associated infections, particularly those that are caused by S. aureus including MRSA, are mainly transmitted from person to person through contaminated medical devices or via direct contact with colonized healthcare workers or patients. S. aureus as a member of ESKAPE (Enterococcus, S. aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli) organisms result in prolonged hospital stay, hospital-associated mortality, and substantial economic burden [3]. Contamination and infection of S. aureus is also a major problem in the agrifood sector, particu (...truncated)