Clostridium difficile bacteriophage finding under normal and iron-limited conditions for the isolation of potential siderophore receptor-specific phages

Abstract

Clostridium difficile infection (CDI) represents a critical healthcare challenge. Phage therapy, known for its specificity of action, has emerged as a promising candidate for combating CDI, offering the potential to circumvent gut microbiome dysbiosis associated with conventional antibiotic regimens. However, research on Clostridium difficile (C. difficile) phage hunting has been relatively limited. Of the studies conducted, none have reported the successful isolation of freely existing C. difficile phages from environmental samples. Henceforth, this thesis explored free environmental phages as untapped sources of novel therapeutic properties, focusing on potential C. difficile phages specific to the highly conserved siderophore receptors. To that end, this study aimed to isolate C. difficile phages under both normal and iron-limited conditions. We report, under normal iron condition, the unprecedented isolation of free C. difficile phages from environmental samples, via a threefold phage enrichment protocol. Spot assays conducted revealed the presence of turbid halos, marking a potential discovery of depolymerase-producing C. difficile phage(s). Concurrently, we present a novel method utilizing Oxyrase® and AnaeroPack™ for the portable and efficient establishment of anaerobic conditions for C. difficile phage hunting. However, phage isolation under iron-limited condition proved unsuccessful, leaving the search for potential siderophore receptor-specific C. difficile phages inconclusive.