Cloning, Recombinant Production and Functional Analysis of Staphylococcal Phage Endolysins

Year 2023, , 14 - 22, 30.06.2023

Serap Pektaş , Osman Birol Özgümüş , İnci Durukan , Ümit Uzun , Ersin Karataş , Ali Osman Kılıç

https://doi.org/10.59518/farabimedj.1255123

Abstract

Staphylococci are pathogens that cause serious infections in humans and animals. Nosocomial infections caused by staphylococci, particularly methicillin-resistant Staphylococcus aureus (MRSA) strains, are mostly transmitted through healthcare workers, patients, or contaminated materials and food. In recent years, studies have been carried out to develop alternative antimicrobial strategies due to the inadequacy of existing antibiotics in the prevention of systemic, skin and implant-related biofilm infections caused by these multi-antibiotic resistant strains. One of these new approaches is the development of products containing the bacteriophage endolysin, which is particularly effective against multi-antibiotic-resistant bacteria. In this study, endolysin genes of bacteriophages (prophages) integrated into the chromosomes of Staphylococcus strains were amplified by polymerase chain reaction (PCR) and cloned into pET SUMO and pET-30b(+) vectors and produced recombinantly in E. coli. Anti-staphylococcal and antibiofilm activity of recombinant endolysins against S. aureus, S. epidermidis, and S. haemolyticus strains isolated from clinical specimens, were demonstrated using turbidity reduction, biofilm removal in microwell plates by crystal violet method, and capacity of endolysins to kill biofilm-forming bacteria by confocal microscopy imaging by live-dead staining. The combination of endolysin was shown to reduce bacterial culture turbidity by at least 50% at 60 minutes and biofilms by approximately 70% at 12 hours. These results show that endolysins have the potential to be used in the prevention of staphylococcal infections.

Keywords

Bacteriophage, Anti-biofilm, Endolysin, Confocal Microscopy, Staphylococcus

Supporting Institution

Karadeniz Teknik Üniversitesi

Project Number

TSA-2019-8344

Thanks

This work was supported by Office of Scientific Research Projects of Karadeniz Technical University.

Stafilokkal Faj Endolizinlerinin Klonlanması, Rekombinant Olarak Üretilmesi ve Fonksiyonel Analizleri

Abstract

Stafilokoklar insanlarda ve hayvanlarda ciddi enfeksiyonlara neden olan patojenlerdir. Stafilokokların, özellikle metisiline dirençli Staphylococcus aureus (MRSA) suşlarının neden olduğu nozokomiyal enfeksiyonlar çoğunlukla sağlık çalışanları, hastalar veya kontamine olmuş maddeler ve yiyecekler yoluyla bulaşır. Son yıllarda bu çoklu antibiyotik dirençli suşların neden olduğu sistemik, deri ve implant ilişkili biyofilm enfeksiyonlarının önlenmesinde mevcut antibiyotiklerin yetersizliği nedeniyle alternatif antimikrobiyal stratejiler geliştirmeye yönelik çalışmalar yapılmaktadır. Bu yeni yaklaşımlardan biri, çoklu antibiyotiğe dirençli bakterilere karşı özellikle etkili olan bakteriyofaj endolizin enzimini içeren ürünlerin geliştirilmesidir. Bu çalışmada, Staphylococcus suşlarının kromozomlarına entegre olmuş, bakteriyofajların (profaj) endolizin genleri polimeraz zincir reaksiyonu (PCR) ile çoğalaltılarak pET SUMO ve pET-30b(+) vektörlerine klonlandı ve E. coli’de rekombinant olarak üretildi. Rekombinant endolizinlerin, klinik örneklerden izole edilen S. aureus, S. epidermidis ve S. haemolyticus suşlarına karşı anti-stafilokokal ve antibiyofilm etkileri bulanıklılık azaltma, mikrokuyucuklu plaklarda kristal viyole yöntemi ile biyofilm uzaklaştırma ve konfokal mikroskopi yöntemi ile endolizinlerin biyofilmi oluşturan bakterileri öldürme kapasiteleri canlı ve ölü bakteri görüntüleme yöntemi ile saptandı. Endolizin kombinasyonunun bakteri kültür bulanıklılığını 60 dakikada en az %50 ve biyofilmleri 12 saatte yaklaşık %70 oranında azalttığı gösterildi. Bu sonuçlar endolizin enzimlerinin stafilokokal enfeksiyonların önlenmesinde kullanılma potansiyeline sahip olduğunu göstermektedir.

Keywords

Konfokal Mikroskopi, Staphylococcus, Bakteriyofaj, Biyofilm, Endolizin, Bacteriophage, Anti-biofilm, Endolysin, Confocal Microscopy

Project Number

TSA-2019-8344

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