Increasing age, several co-morbidities, environmental contamination, antibiotic exposure and other intestinal perturbations appear to be the greatest risk factors for C. difficile infection (CDI). Therefore, elderly care home residents are considered particularly vulnerable to CDI. The main objective of this study was to evaluate and follow the prevalence of C. difficile in a Belgian nursing home. During a 4-month period, stool samples from a group of 23 elderly care home residents were collected weekly. A C. difficile microbiological detection scheme was performed along with an overall microbial biodiversity study of the faeces content by Targeted Metagenomic analysis. Culture of samples was performed in a selective medium cycloserine cefoxitin fructose cholate. An identification of the isolated colonies was done by PCR detection of tpi, tcdA, tcdB and cdtA genes. Toxic activity was confirmed by a cytotoxic immunoassay. Further characterization was performed by PCR ribotyping. The Metagenomic analysis was targeted on the v1-v3 hyper-variable region of 16S rDNA. The taxonomical assignment of the populations was performed with MOTHUR and Blast algorithms. Seven out of 23 (30.4%) residents were (at least one week) positive for C. difficile. The most common PCR-ribotype identified was 027. Targeted Metagenomic analyses reveals that each resident has his own bacterial imprint, which is stable during the entire study. Residents’ positives for C. difficile by classical microbiology showed an important proportion of C. difficile sequences. However, Metagenomics analysis can’t substitute targeted protocols. It was not used as a diagnostic tool to detect C. difficile but rather to determine the identification and correlations of the major bacterial populations that are present in the gut microbiota. In conclusion, this unique association of classical microbiology protocol with pyrosequencing allowed to follow C. difficile in patients and to identify several other bacterial populations whose abundance is correlated with ...