Clostridium difficile colonizes alternative nutrient niches during infection across distinct murine gut environments

5 Clostridium difficile infection (CDI) has grown to be the most prevalent cause of hospital 6 acquired infection in the United States. Susceptibility to CDI is induced by recent 7 antibiotic exposure, which is known to alter the structure of the gut microbiome and to 8 affect the availability of growth nutrients in the gut. We hypothesized that C. difficile is a 9 generalist that adapts its physiology to the nutrients available within the gut. We orally 10 challenged C57BL/6 mice that previously received one of three antibiotics with C. 11 difficile and demonstrated that it was able to colonize the cecum within 18 hours of 12 infection. However, levels of both spore and toxin production, which are known to be 13 affected by nutrient availability, varied between each antibiotic treatment group. To 14 more closely investigate the specific responses of C. difficile as it colonized the cecum, 15 we performed in vivo transcriptional analysis of C. difficile from cecal content of infected 16 mice. This approach revealed variation in expression of genes that drive life-cycle 17 switches as well as metabolic pathways associated with catabolizing a variety of carbon 18 sources such as carbohydrates, amino acids, and amino sugars. To assess which 19 substrates C. difficile was most likely exploiting in each antibiotic-perturbed microbiome, 20