Novel insights into phage biology of the pathogenClostridioides difficilebased on the active virome

Abstract

AbstractThe global pathogenClostridioides difficileis a well-studied organism, and researchers work on unravelling its fundamental virulence mechanisms and biology. Prophages have been demonstrated to influenceC. difficiletoxin expression and contribute to the distribution of advantageous genes. All this underlines the importance of prophages inC. difficilevirulence. Although severalC. difficileprophages were sequenced and characterized, investigations on the entire active virome of a strain are still missing. Phages were mainly isolated after mitomycin C-induction, which does not resemble natural stressor forC. difficile.We examined active prophages from differentC. difficilestrains after cultivation in the absence of mitomycin C by sequencing and characterization of particle-protected DNA. Phage particles were collected after standard cultivation, or after cultivation in the presence of the secondary bile salt deoxycholate (DCA). DCA is a natural stressor forC. difficileand a potential prophage-inducing agent. We also investigated differences in prophage activity between clinical and non-clinicalC. difficilestrains. Our experiments demonstrated that spontaneous prophage release is common inC. difficile, and that DCA presence induces prophages. Fourteen different, active phages were identified by this experimental procedure. We could not identify a definitive connection between clinical background and phage activity. However, one phage exhibited distinctively higher activity upon DCA-induction in the clinical strain than in the corresponding non-clinical strain, although the phage is identical in both strains. We recorded that enveloped DNA mapped to genome regions with characteristics of mobile genetic elements other than prophages. This pointed to mechanisms of DNA mobility that are not well-studied inC. difficileso far. We also detected phage-mediated lateral transduction of bacterial DNA, which is the first described case inC. difficile. This study significantly contributes to our knowledge on prophage activity inC. difficileand revealed novel aspects onC. difficile(phage) biology.