The gut microbiota composition of intensive care unit (ICU) patients suffering from Clostridium difficile-positive diarrhea (CDpD) is poorly understood. This prospective study aims to use 16S rDNA (and metagenome) sequencing to compare the microbiota composition of 58 (and 5) ICU patients with CDpD (CDpD group), 33 (and 4) ICU patients with C. difficile-negative diarrhea (CDnD group), and 21 (and 5) healthy control subjects (control group), as well as CDpD patients in the A⁺B⁺ (N = 34; A/B: C. difficile TcdA/B), A^-B⁺ (N = 7), and A^-B^- (N = 17) subgroups. For 16S rDNA data, OTU clustering (tool: UPARSE), taxonomic assignment (tool: RDP classifier), α-diversity, and β-diversity analyses (tool: QIIME) were conducted. For metagenome data, metagenome assembly (tool: SOAPdenovo), gene calling (tools: MetaGeneMark, CD-HIT, and SoapAligner), unigene alignment (tool: DIAMOND), taxon difference analysis (tool: Metastats), and gene annotation (tool: DIAMOND) were performed. The microbial diversity of the CDpD group was lower than that of the CDnD and control groups. The abundances of 10 taxa (e.g., Deferribacteres, Cryptomycota, Acetothermia) were significantly higher in the CDpD group than in the CDnD group. The abundances of Saccharomycetes and Clostridia were significantly lower in CDpD in comparison with control. Some taxa were significantly different between the A⁺B⁺ and A^-B^- subgroups. CDpD might relate to a decrease in beneficial taxa (i.e., Saccharomycetes and Clostridia) and an increase in harmful taxa (e.g., Deferribacteres, Cryptomycota, Acetothermia) in gut microbiota of ICU patients. C. difficile toxin type might be slightly associated with gut microbiota composition.

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