Isolation and Characterization of Clostridium perfringens Bacteriophages and Optimization of Electro-Transformation Parameters for Clostridium difficile

Clostridium perfringens (C. perfringens) is responsible for a variety of diseases in humans and animals. Clostridium difficile (C. difficile) is the leading cause of antibiotic-associated diarrhoea. The available treatments for infections caused by both pathogens are not effective. Bacteriophage (phage) therapy is a promising treatment strategy to tackle and treat any infections or diseases caused by C. perfringens or C. difficile. The aim of this study was to isolate bacteriophages that infect C. perfringens and characterize them, and to optimize electroporation parameters for C. difficile with the end goal being to be able to genetically modify C. difficile phages. Five phages that infect C. perfringens were isolated and characterized from environmental samples. Two phages were sequenced and annotated, one was found to be a Podovirus and the other a Siphovirus. The Podovirus is a strictly lytic phage that does not possess any undesired genes such as the transduction gene, antibiotic resistance gene, and toxin genes. The endolysin of the Podovirus was cloned, expressed, and purified. The muralytic activity of the enzyme was confirmed by a zymogram. This endolysin has the ability to completely lyse 85.7 % of the tested Clostridium perfringens strains. A series of electroporation experiments were carried out using many experimental settings and varying parameters in order to deliver engineered phage and plasmid DNA genome to C. difficile. The electroporation refractory C. difficile R076 was treated with a cysteine protease inhibitor E- 64 and lysostaphin to facilitate electroporation. E-64 was able to reduce the thickness of the C. difficile surface layer proteins. The treatment with lysostaphin resulted in cell lysis. Unfortunately, all the attempts made to optimize the electroporation protocol for C. difficile were unsuccessful as no cells were transformed. However, the experimental observations provide a strong foundation for further work to develop an effective electro-transformation protocol for C. difficile.