Clostridioides difficile (formerly Clostridium difficile) infection (CDI) can result from the disruption of the resident gut microbiota. Western diets and popular weight-loss diets drive large changes in the gut microbiome; however, the literature is conflicted with regard to the effect of diet on CDI. Using the hypervirulent strain C. difficile R20291 (RT027) in a mouse model of antibiotic-induced CDI, we assessed disease outcome and microbial community dynamics in mice fed two high-fat diets in comparison with a high-carbohydrate diet and a standard rodent diet. The two high-fat diets exacerbated CDI, with a high-fat/high-protein, Atkins-like diet leading to severe CDI and 100% mortality and a high-fat/low-protein, medium-chain-triglyceride (MCT)-like diet inducing highly variable CDI outcomes. In contrast, mice fed a high-carbohydrate diet were protected from CDI, despite the high levels of refined carbohydrate and low levels of fiber in the diet. A total of 28 members of the Lachnospiraceae and Ruminococcaceae decreased in abundance due to diet and/or antibiotic treatment; these organisms may compete with C. difficile for amino acids and protect healthy animals from CDI in the absence of antibiotics. Together, these data suggest that antibiotic treatment might lead to loss of C. difficile competitors and create a favorable environment for C. difficile proliferation and virulence with effects that are intensified by high-fat/high-protein diets; in contrast, high-carbohydrate diets might be protective regardless of the source of carbohydrate or of antibiotic-driven loss of C. difficile competitors. IMPORTANCE The role of Western and weight-loss diets with extreme macronutrient composition in the risk and progression of CDI is poorly understood. In a longitudinal study, we showed that a high-fat/high-protein, Atkins-type diet greatly exacerbated antibiotic-induced CDI, whereas a high-carbohydrate diet protected, despite the high monosaccharide and starch content. Our study results, therefore, suggest that popular high-fat/high-protein weight-loss diets may enhance CDI risk during antibiotic treatment, possibly due to the synergistic effects of a loss of the microorganisms that normally inhibit C. difficile overgrowth and an abundance of amino acids that promote C. difficile overgrowth. In contrast, a high-carbohydrate diet might be protective, despite reports on the recent evolution of enhanced carbohydrate metabolism in C. difficile.

14Clostridioides difficile (formerly Clostridium difficile) infection (CDI) can result from the 15 disruption of the resident gut microbiota. Western diets and popular weight-loss diets drive large 16 changes in the gut microbiome; however, the literature is conflicted with regard to the effect of 17 diet on CDI. Using the hypervirulent strain C. difficile R20291 (RT027) in a mouse model of 18 antibiotic-induced CDI, we assessed disease outcome and microbial community dynamics in 19 mice fed two high-fat diets in comparison with a high-carbohydrate diet and a standard rodent 20 diet. The two high-fat diets exacerbated CDI, with a high-fat/high-protein, Atkins-like diet 21 leading to severe CDI and 100% mortality, and a high-fat/low-protein, medium-chain 22 triglyceride (MCT)-like diet inducing highly variable CDI outcomes. In contrast, mice fed a 23 high-carbohydrate diet were protected from CDI, despite high refined carbohydrate and low fiber 24 content. 28 members of the Lachnospiraceae and Ruminococcaceae decreased in abundance due 25 to diet and/or antibiotic treatment; these organisms may compete with C. difficile for amino acids 26 and protect healthy animals from CDI in the absence of antibiotics. Together, these data suggest 27 that antibiotic treatment might lead to loss of C. difficile competitors and create a favorable 28 environment for C. difficile proliferation and virulence that is intensified by high-fat/high-protein 29 diets; in contrast, high-carbohydrate diets might be protective regardless of the source of 30 carbohydrate. 31Key words: C. difficile, microbiome, Atkins diet, high-fat diet, high-carbohydrate diet 32 33Clostridioides difficile (formerly Clostridium difficile) is an endospore-forming member of the 34 phylum Firmicutes that is the leading cause of antibiotic-associated and hospital-acquired 35 diarrhea. C. difficile infections (CDIs) make up > 70% of healthcare-associated gastrointestinal 36 infections, with symptoms ranging from mild diarrhea to severe infections resulting in ulcerative 37 colitis and toxic megacolon (1). Moreover, CDI is financially taxing on U.S. hospital 38 management (2) and is the cause of over 500,000 infections and 29,000 deaths annually, 39 according to a 2015 report (3). 40Stable and complex microbial communities in the gut act as a natural barrier against C. difficile 41 (4), but broad-spectrum antibiotics can disrupt the native microflora, allowing C. difficile to 42 multiply and cause CDI (5). Importantly, C. difficile has innate resistance to multiple antibiotics 43 and CDI is closely linked to administration of ampicillin, amoxicillin, cephalosporins, 44 clindamycin, and fluoroquinolones (6). In order to cause successful infection, C. difficile spores 45 must germinate, grow within the intestinal lumen, and produce toxins that mediate tissue damage 46 and inflammation (7). Specific chemical signals are needed for each of these steps. For example, 47 spore germination is promoted by variety of amino acids and primary bile salts, but inhibited by 48 se...

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA’s efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.

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