Bacterial and Fungal Microbiota Changes Distinguish C. difficile Infection from Other Forms of Diarrhea: Results of a Prospective Inpatient Study

This study sought to characterize the bacterial and fungal microbiota changes associated with Clostridium difficile infection (CDI) among inpatients with diarrhea, in order to further explain the pathogenesis of this infection as well as to potentially guide new CDI therapies. Twenty‐four inpatients with diarrhea were enrolled, 12 of whom had CDI. Each patient underwent stool testing for CDI prior to being treated with difficile‐directed antibiotics, when appropriate. Clinical data was obtained from the medical record, while each stool sample underwent 16S rRNA and ITS sequencing for bacterial and fungal elements. An analysis of microbial community structures distinct to the CDI population was also performed. The results demonstrated no difference between the CDI and non‐CDI cohorts with respect to any previously reported CDI risk factors. Butyrogenic bacteria were enriched in both CDI and non‐CDI patients. A previously unreported finding of increased numbers of Akkermansia muciniphila in CDI patients was observed, an organism which degrades mucin and which therefore may provide a selective advantage toward CDI. Fungal elements of the genus Penicillium were predominant in CDI; these organisms produce antibacterial chemicals which may resist recovery of healthy microbiota. The most frequent CDI microbial community networks involved Peptostreptococcaceae and Enterococcus, with decreased population density of Bacteroides. Also presented in this study is the interaction among the bacterial and fungal communities. These results suggest that the development of CDI is associated with microbiota changes which are consistently associated with CDI in human subjects. These gut taxa contribute to the intestinal dysbiosis associated with C. difficile infection. Support or Funding Information DS received funding for this study from a Barsumian Trust Grant and from a CTSI grant.Principal Coordinates Analysis plot from a weighted UniFrac distance matrices: Control samples are displayed in blue, while CDI samples are in red. Distinct clustering can be observed between bacterial communities of these two groups. Sample S1 (no antibiotics) and S1A (antibiotics) are fecal samples from the same control individual. Sample CD3 (no antibiotics) and CD3A (antibiotics) are fecal samples from the same individual with CDI.2 Relative abundance of bacterial families in CDI and non‐CDI IndividualsThe relative abundance of the top 10 most abundant bacterial families for CDI ( n = 13) and control ( n = 13) fecal samples. Relative abundance of taxa was determined based on the relative abundance of 16S rRNA gene sequences assigned to a given bacterial family using the Greengenes database.4Venn diagram demonstrating distinct and shared OTUs between non‐CDI and CDI states.5Co‐occurrence network plot of bacterial taxa within the fecal microbiome of CDI patients. The co‐occurrence network plot generated within the Cytoscape plugin Conet reveals strong positive (Spearman's rho > 0.8) and strong negative (Spearman's rho < −0.8) correlations between OTUs identified within 13 CDI stool samples. Eighteen unique OTUs (indicated by rectangular nodes) possessing strong correlations with one another are shown. The 16S rRNA sequence of the Peptostreptococcaceae node highlighted in red matched Clostridium difficile strain 630 (query cover = 100%) when BLAST searched. Additional potential pathogenic OTUs are shown in orange, including Enterococcus and an unclassified Clostridiales . Edges connecting nodes highlighted in green are indicative of a strong positive correlation, whereas edges highlighted in red are indicative of a strong negative correlation.A subset of the full bipartit network: all bacteria shown by blue nodes were found to be Proteobacteria and they negatively correlate with the fungal Penicillium and unidentified29 fungal taxas while simultaneously positively correlating with Trichoderma and nectria.OTU network created in Cystoscape showing bacteria and fungi associated with each sample and fungi of interest highlighted. Taxa clustering towards the center appear in most of the samples while taxa on the periphery are unique to one or two samples. Candida is highlighted in green, Penicillium and unidentified29 are in red, and Trichoderma and Nectria are highlighted in blue.Kruskal Wallis non‐parametric test for significance created from CSS normalized OTU table to identify enriched fungal taxa within infected cohort with an alpha level of 0.05. All of the fungal taxa enriched in the healthy cohort belong to Candida subcategory while the infected cohort demonstrated an enrich Penicillium .Taxonomy P Value Infected Mean Noninfected MeanCandida sp WT_2012 0.02 436.78 Candida albicans 0.02 26.22 Candida sp WT_2012 0.02 7.78 Candida sp WT_2012 0.04 36.11 Penicillium 0.00 1.00