| Summary: | Gut microbiota composition in patients with <i>Clostridioides difficile</i> colonization is not well investigated. We aimed to identify bacterial signatures associated with resistance and susceptibility to <i>C. difficile</i> colonization (CDC) and infection (CDI). Therefore, gut microbiota composition from patients with CDC (<i>n</i> = 41), with CDI (<i>n</i> = 41), and without CDC (controls, <i>n</i> = 43) was determined through 16S rRNA gene amplicon sequencing. Bacterial diversity was decreased in CDC and CDI patients (<i>p</i> < 0.01). Overall microbiota composition was significantly different between control, CDC, and CDI patients (<i>p</i> = 0.001). Relative abundance of <i>Clostridioides</i> (most likely <i>C. difficile</i>) increased stepwise from controls to CDC and CDI patients. In addition, differential abundance analysis revealed that CDI patients’ gut microbiota was characterized by significantly higher relative abundance of <i>Bacteroides</i> and <i>Veillonella</i> than CDC patients and controls. Control patients had significantly higher <i>Eubacterium hallii</i> and <i>Fusicatenibacter</i> abundance than colonized patients. Network analysis indicated that <i>Fusicatenibacter</i> was negatively associated with <i>Clostridioides</i> in CDI patients, while <i>Veillonella</i> was positively associated with <i>Clostridioides</i> in CDC patients. Bacterial microbiota diversity decreased in both CDC and CDI patients, but harbored a distinct microbiota. <i>Eubacterium hallii</i> and <i>Fusicatenibacter</i> may indicate resistance against <i>C. difficile</i> colonization and subsequent infection, while <i>Veillonella</i> may indicate susceptibility to colonization and infection by <i>C. difficile</i>.
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