Microbiome signatures of <i>Clostridioides difficile</i> toxin production and toxin gene presence: a shotgun metagenomic approach

<i>Clostridioides difficile</i> is an opportunistic gastrointestinal pathogen capable of asymptomatic colonization and causes diseases ranging from diarrhea to pseudomembranous colitis. Accurate diagnosis of <i>C. difficile</i> infection (CDI) is challenging and critical for treatment and control. We hypothesized that gut microbiome profiles could help distinguish <i>C. difficile</i> colonized patients with diarrhea from those with true CDI. We analyzed 172 stool samples from individuals who tested glutamate dehydrogenase positive for <i>C. difficile</i>. Participants were categorized by toxin status (i.e., toxin positive or negative) and then further classified into three toxin groups based on the production of toxin, and if not produced, whether the <i>C. difficile</i> strain carried toxin-encoding genes. We examined associations between patient characteristics, prior antibiotics exposure, microbiome community structure and function, and toxin categories. Thirty-five percent of toxin-negative participants received antibiotics despite not meeting the criteria for true CDI. <i>Enterococcus</i> species were abundant in all groups. The relative abundance of <i>E. faecalis</i> was higher among individuals with prior antibiotics exposure. Alpha and beta diversity did not differ by toxin group. After controlling for prior antibiotics exposure and previous CDI episode, the abundance of <i>Akkermansia muciniphila, Flavonifractor plautii</i>, and <i>Bifidobacterium adolescentis</i> distinguished individuals with toxin-positive <i>C. difficile. C. difficile</i> abundance did not differentiate participants with true CDI from those who were colonized. We identified associations between the gut microbiome and <i>C. difficile</i> toxin gene presence and toxin production. These results highlight the potential for microbiome-informed diagnostics to improve CDI accuracy and guide treatment decisions.IMPORTANCE<i>Clostridioides difficile</i> colonizes humans and causes diarrhea in community and hospital settings. <i>C. difficile</i> infection (CDI) is a toxin-mediated disease, and its diagnosis is challenging. The goal of this study was to determine whether differences in the gut microbiome could help distinguish between colonized individuals and those with CDI. We examined stool samples and data from 172 individuals categorized into three groups based on the detection of toxin and, if not detected, whether toxin-encoding genes were present in the <i>C. difficile</i> strain. We identified bacteria, such as <i>Enterococcus faecalis</i>, that were more abundant in people who had used antibiotics. While the diversity of the gut microbiome did not differ by toxin group, specific gut bacteria, antibiotic resistance genes, and metabolic pathways were associated with toxin group. Our findings suggest that considering the full gut microbiome and factors like past antibiotic use could help improve the diagnosis and treatment of CDI.