Longitudinal Gut Microbiome Changes Associated with Transitions from C. difficile Negative to C. difficile Positive on Surveillance Tests.

Clostridioides difficile is an obligate anaerobe and is primarily transmitted via the fecal-oral route. Data characterizing the microbiome changes accompanying transitions from non-colonized to C. difficile colonized subjects are currently lacking. In this retrospective cohort study, we examined 16S rRNA gene sequencing data in a total of 481 fecal samples belonging to 107 patients. Based on C. difficile status over time, patients were categorized as Negative-to-Positive, Negative Control, and Positive Control. A linear mixed effects model was fitted to investigate the changes in the Shannon α-diversity index over time. Zero-inflated negative binomial/Poisson mixed effects models or generalized linear mixed models with negative binomial/Poisson distribution were used to investigate the changes in taxon counts over time among different groups. A total of 107 patients were eligible for the study. The median number of stool samples per patient was 3 (IQR 2-4). A total of 42 patients transitioned from C. difficile negative to positive (Negative-to-Positive), 47 patients remained negative throughout their tests (Negative Control) and 18 were always C. difficile positive (Positive Control). A significant difference in microbiome composition between the last negative samples and the first positive samples were shown in Negative-to-Positive patients, ANOSIM p = 0.022. In Negative-to-Positive patients, the phylum Pseudomonadota and family Enterobacteriaceae increased significantly in the first positive samples compared to the last negative samples, p = 0.0075 and p = 0.0094, respectively. Within the first 21 days, Actinomycetota decreased significantly over time in the Positive Control group compared to the other two groups (p < 0.001) while Bacillota decreased in both the Negative-to-Positive group and Positive Control. These results demonstrate that the transition from C. difficile negative to C. difficile positive is associated with alterations in gut microbial communities and their compositional patterns over time. Moreover, these changes play an important role in both the emergence and intensification of the gut microbiome dysbiosis in patients who transitioned from C. difficile negative to positive and those who always tested positive.