Differential Expression of Clostridium difficile and phage genes during a one-step growth curve

Clostridium difficile is one of the most dangerous pathogens that causes significant human morbidity and mortality in hospital settings. Bacteriophages are viruses of bacteria that are ubiquitous in nature. Most strains of C. difficile carry one or more prophages that can support the virulence of the bacteria. However, during the lytic cycle, temperate phages can affect virulence genes expression. In order to investigate the impact of phage on the infection potential of C. difficile, high-throughput RNA sequencing reactions (RNA-seq) were conducted on a genome-wide transcriptomic analysis of the epidemic C. difficile strain R20291. The finding from RNA-seq analysis shows that at different growth time points the expression of a majority of strain R20291 genes changed for R20291 infected with phage CDHS1 compared with uninfected R20291. Those R20291 genes involved in core DNA binding and transcription were among the genes most highly expressed. Genes encoding metabolic processes and permease were among the very low expression levels group (i.e. were down-regulated). Both RNA-seq and qPCR techniques showed significant down regulation in genes coding toxin A, B and the binary toxin in C. difficile infected with phage CDHS1. Galleria mellonella larvae were used as an infection model to analyse phage effects on C. difficile virulence. This showed that larvae infected with lysogenic C. difficile containing φCDHS1 had reduced survival whilst less virulent bacteria which were resistant to φCDHS1 and wild type C. difficile, were less infective. In addition, the more pathogenic strains (lysogenic) produced greater immune responses in G. mellonella than the relatively less-pathogenic forms (resistant to φCDHS1 and wild type). This study suggests that temperate phage can mimic the virulence enhancing aspect for lytic phage.