Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile

Abstract

<i>Clostridium difficile</i> is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant <i>C. difficile</i> has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against <i>C. difficile</i> is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to <i>C. difficile</i>, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of <i>C. difficile</i>. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 <i>C. difficile</i> isolates to screen for susceptible bacterial hosts. The results revealed that all the <i>C. difficile</i> phages obtained in this work displayed a relatively narrow host range of 0-6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the <i>Myoviridae</i> family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for <i>C. difficile</i>.