Abstract :
[en] Clostridioides difficile is a spore-forming, anaerobic, Gram-positive bacterium that is the most common cause of antibiotic-associated diarrhoea in hospitals. The intestinal carriage of C. difficile can be asymptomatic or clinical. This bacterium is associated with various clinical signs ranging from mild diarrhoea to pseudomembranous colitis. Most strains of C. difficile produce two main toxins (A and B), which are associated with the development of acute diarrhoea and/or colitis.
An initial study was conducted on human faeces of three categories of individuals at three different sampling times: patients with inflammatory bowel disease (IBD), patients with C. difficile infection (CDI) and healthcare workers (HCW). The C. difficile carriage and 16S rDNA amplicon sequencing were assessed in the three clinical groups. The relationship between the groups and the faecal microbiota composition was investigated on three successive samples (IBD and HCW: every 3 months; CDI: 2 samples during the disease and 1 sample during remission after 3 months). The relationship between C. difficile carriage (C. difficile positive group and C. difficile negative group) was also studied. In the HCW group (n=3), no samples tested positive for C. difficile at three time points. In the IBD group (n=15), only two patients tested positive for C. difficile. In the CDI group (n=15), all samples were positive at sample time 1. In the CDI group, seven patients were still positive for C. difficile at sample time 2 and four remained positive at sample time 3. In the positive C. difficile carriage group, several genera were significantly increased: Enterococcus, Enterobacteriaceae_ge , Enterobacteriales_ge and Clostridioides. In the negative C. difficile group, more genera were significantly increased: Lachnospiraceae_NK4A136, Colidextribacter, Monoglobus, UCG.003, Oscillibacter, Oscillospiraceae_ge, Oscillospira, Coriobacteriales_ge, Marvinbryantia, Romboutsia, Ruminococcaceae_ge, Fusicatenibacter, Butyricicoccus, Clostridia_ge, Sutterella, Lachnospiraceae_UCG.004, Agathobacter, Clostridiales_ge, Lachnospira, Anaerostipes, Rhodospirillales_fa_ge .
In the second study, several growth curve assays were performed to monitor the behaviour of the spores and vegetative forms of a C. difficile PCR-ribotype 078 under different conditions mimicking the intestinal environment and the descending colon (6.6-6.9). The effect of faeces from four donors on C. difficile growth was investigated. No spore germination or growth was observed when the faeces were added, but C. difficile spores germinated in vitro when the pH was maintained between 6.6 and 6.9. This model contributed to the understanding that germination and growth of C. difficile are strongly pH-dependent. The evolution of the microbiota studied by 16S rDNA amplicon sequencing showed high proportions of Enterobacteriaceae and E. coli/Shigella when C. difficile grew, regardless of the inoculated faeces.
In the last study, an in vitro coculture model was established to study the effect of gut microbiota on C. difficile gene expression under anaerobic conditions. Three conditions were studied. Two different faeces were used to mimic the gut microbiota (FD1 and FD2) and a plate without faeces was used as a control (CT). Several parameters were analysed: the growth of C. difficile under different conditions, the effect of the bacterial microbiota on C. difficile gene transcription, the evolution of C. difficile gene transcription over time. Gene transcription was studied using RNA-Seq and RT-qPCR. The C. difficile growth was similar in the three conditions tested. The effect of bacterial microbiota did not significantly affect the growth. In the presence of bacterial microbiota, the expression of genes associated with germination, sporulation, and growth at the end of the exponential phase was downregulated.
In future, to study the interaction between C. difficile and the microbiota in more detail, the coculture model will be used with a bacterial consortium consisting of a mix of characterised strains, replacing the use of faecal samples.
