Microbiome signatures of Clostridioides difficile toxin production and toxin gene presence: a shotgun metagenomic approach

Clostridioides difficile is an opportunistic gastrointestinal pathogen capable of asymptomatic colonization and causes diseases ranging from diarrhea to pseudo membranous colitis. Accurate diagnosis of C. difficile infection (CDI) is challenging and critical for treatment and control. We hypothesized that gut microbiome profiles could help distinguish C. difficile colonized patients with diarrhea from those with true CDI. We analyzed 172 stool samples from individuals who tested glutamate dehydrogenase positive for C. difficile. 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 C. difficile 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. Enterococcus species were abundant in all groups. The relative abundance of E. faecalis 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 Akkermansia muciniphila, Flavonifractor plautii, and Bifidobacterium adolescentis distinguished individuals with toxin-positive C. difficile. C. difficile abundance did not differentiate participants with true CDI from those who were colonized. We identified associations between the gut microbiome and C. difficile toxin gene presence and toxin production. These results highlight the potential for microbiome-informed diagnostics to improve CDI accuracy and guide treatment decisions.