AbstractBackgroundClostridium difficile (C. difficile)is a major source of healthcare-associated infection with a high risk of recurrence, attributable to many factors such as usage of antibiotics, older age and immunocompromised status of the patients.C. difficilehas also a highly diverse genome, which may contribute to its high virulence. Herein we examined whether the genome conservation, measured as non-synonymous to synonymous mutations ratio (dN/dS) in core genes, presence of single genes, plasmids and prophages increased the risk of reinfection in a subset of 134C. difficileisolates from our previous study in a singly hemato-oncology ward.MethodsC. difficileisolates were subjected to whole-genome sequencing (WGS) on Ion Torrent PGM sequencer. Genomes were assembled with MIRA5 and annotated with prokka and VRprofile. Logistic regression was used to asses the relationship between single gene presence and the odds of infection recurrence. DN/dS ratios were computed with codeml. Functional annotation was conducted with eggNOG-Mapper.ResultsWe have found that the presence of certain genes, associated with carbon metabolism and oxidative phosphorylation, increased the odds of infection recurrence. More core genes were under positive selective pressure in recurrent disease isolates – they were mostly associated with the metabolism of aminoacids. Finally, prophage elements were more prevalent in single infection isolates and plasmids did not influence the odds of recurrence.ConclusionsOur findings suggest higher genetic plasticity in isolates causing recurrent infection, associated mainly with metabolism. On the other hand, the presence of prophages seems to reduce the isolates’ virulence.