Dietary fat promotes antibiotic-induced Clostridioides difficile mortality in mice

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea, and emerging evidence has linked dietary components with CDI pathogenesis, suggesting that dietary modulation may be an effective strategy for prevention. Here, we show that mice fed a high-fat/low-fiber “Western-type” diet (WD) had dramatically increased mortality in a murine model of antibiotic-induced CDI compared to a low-fat/low-fiber (LF/LF) diet and standard mouse chow controls. We found that the WD had a pro- C. difficile bile acid composition that was driven in part by higher levels of primary bile acids that are produced to digest fat, and a lower level of secondary bile acids that are produced by the gut microbiome. This lack of secondary bile acids was associated with a greater disturbance to the gut microbiome with antibiotics in both the WD and LF/LF diet compared to mouse chow. Mice fed the WD also had the highest level of toxin TcdA just prior to the onset of mortality, but not of TcdB or increased inflammation. These findings indicate that dietary intervention to decrease fat may complement previously proposed dietary intervention strategies to prevent CDI in high-risk individuals.

2 Introduction 5 The WD-fed mice showed a marked increase in mortality as compared to both the LF/LF 116 (HR 7.403 p = 0.0041) and chow-fed mice (HR 4.95 p = 0.00208) upon C. difficile exposure. 117 Mortality onset began at Day 4 in the WD and chow-fed mice and then continued to Day 8 in the 118 WD and to Day 6 in the chow-fed mice before stabilizing with the remaining mice appearing to 119 recover. The LF/LF diet-fed mice showed survival levels comparable to the chow-fed mice with 120 a slightly delayed onset of mortality (Fig. 1B). WD-fed diet mice did not show increased weight 121 loss compared to the other diets ( Figure S1). Qualitatively, WD-fed mice had more purulent and 122 liquid stools, and poorer grooming than LF/LF and chow-fed mice starting 2 days after infection. 123 Because our WD and LF/LF diet differed in sucrose content, we also tested a fourth diet that was 124 low in fat and fiber, but with sucrose equivalent to the WD (Table S1). Sucrose did not appear to 125 play a role in the increased mortality observed in the WD, as 100% survival was observed in 126 mice fed the low-fat/low-fiber/low-sucrose diet. (n = 10, one cage with 5 mice in two separate 127 experiments). 128

WD associated with increased C. difficile toxin TcdA but not TcdB or intestinal inflammation 129
To further explore the mechanisms of increased mortality in the WD-fed mice by 130 assessing factors that required the collection of host tissues, we conducted a second set of 131 experiments in which mice were sacrificed at day 3 post C. difficile gavage (cohort 2; Figure 1). 132 We chose 3 days post C. difficile gavage because this was just prior to the observed onset of 133 mortality in the first cohort across all 3 diets (Fig. 1B), and because we felt it was important to 134 compare all mice at a standard time point. 135 We measured cecal levels of C. difficile Toxins A (TcdA) and B (TcdB) by ELISA (see 136   Table S2 for cohort size and batch information). Interestingly, TcdA and not TcdB showed 137 differences with diet consistent with mortality patterns, with TcdA being much higher in the WD 138 6 compared to the LF/LF diet. The LF/LF diet also had slightly lower levels of TcdA than the 139 chow diet (Figure 2A), which is consistent with a delayed onset of mortality in the LF/LF diet 140 compared to chow (Figure 1). To understand whether intestinal inflammation was related to 141 toxin levels and differed between diets, we evaluated the transverse colon and cecum by 142 histology (Fig. 2B, D). Cecal and transverse colon tissues from mice sacrificed three days post-143 infection with C. difficile were fixed and stained with hematoxylin and eosin and were scored by 144 the Barthel and Dieleman scoring systems respectively by a trained histologist blinded to the 145 treatments and grouping of individuals 23,24 . The cecum and distal colon samples showed mild to 146 moderate inflammation, but the histologic damage did not differ across diet groups ( Fig. 2B; 147 representative histology Fig. 2D). To control for batch effects, we also assessed differences 148 between diet groups for both toxins and cecal/colon inflammation with linear regression that 149 included batch in the model (Table S2; Figure S2). These results were similar but showed 150 significantly increased inflammation in chow-fed versus WD-fed mice ( Figure S2). Cecal levels 151 of TcdB and not TcdA strongly correlated with cecal inflammation (Figure 2; Table S3). Taken 152 together, our results suggest that although TcdB levels are associated with higher intestinal 153 inflammation in these mice just prior to onset of mortality, the differences cannot explain the 154 increased mortality observed in the WD compared to low fat diets. Our data support a potential 155 role for TcdA in the increased mortality with the WD, but via a mechanism independent of 156 intestinal inflammation.

7
To further explore mechanism, we used targeted LC/MS to measure the levels of a pool 161 of 13 different bile acids in the aspirated cecal contents of a separate cohort of mice that were 162 sacrificed at 3 days post infection (Table S2). Bile acids have a complex relationship with C. 163 difficile germination and growth 13,26-29 . The primary bile acids TCA and CA can promote the 164 germination of C. difficile spores in vitro 13 and primary bile acids including CA are elevated in 165 individuals with first time or rCDI compared to controls 30,31 . The primary bile acid 166 chenodeoxycholic acid (CDCA) can block TCA-induced spore germination 27,28 and another 167 primary bile acid, Ursodeoxycholic acid (UDCA), can inhibit both C. difficile spore germination 168 and C. difficile growth 29 . Furthermore, the murine primary bile acids alpha muricholic acid 169 (a_MCA) and beta muricholic acid (b_MCA) can inhibit C. difficile spore germination and 170 growth 32 . Of particular interest in this study are also the secondary bile acids DCA and 171 lithocholate (LCA); these molecules are produced by the metabolic transformation of primary 172 bile acids by intestinal microbes 14 , can arrest the growth of vegetative C. difficile 13 , and are 173 lower in individuals with CDI 30,31 . We measured the levels of these bile acids with known 174 effects on C. difficile as well as 5 other taurine-conjugated bile acids ( Figure S3). To consider 175 known effects of bile acids on C. difficile growth and germination in our analyses, we binned the 176 bile acids that were inhibitors of C. difficile germination and/or growth (CDCA, UDCA, a_MCA, 177 b_MCA, LCA, DCA), and C. difficile germination promoters (TCA and CA). We also evaluated 178 the ratio of C. difficile promoters to inhibitors, as has been done previously 19 . 179 When comparing across diets, we were most interested in bile acid measures that showed 180 differential levels between the WD and both the LF/LF and chow diets, since the WD had high 181 CDI mortality compared to both the LF/LF and chow diets. C. difficile inhibitors were 182 significantly lower in the WD compared to the chow diet, but there was not a difference between 183 8 the WD and the LF/LF diet ( Figure 3A). C. difficile promoters had significantly higher levels in 184 the WD compared to chow but not compared to the LF/LF diet ( Figure 3A). Interestingly, the 185 ratio of promoters:inhibitors was significantly higher in the WD compared to both the LF/LF diet 186 and chow, consistent with mortality differences. We also analyzed whether each of the 13 bile 187 acids individually differed across diets, and diet significantly affected the levels of most ( Figure  188 S3). However, none were individually significantly different in the WD compared to both the 189 LF/LF and chow diet. While regressing cecal and colon inflammation scores ( Figure 3B) and 190 toxins TcdA and TcdB (data not shown) against bile acid summary measures, the only 191 significant relationship observed was a negative correlation between C. difficile inhibitors and 192 colon inflammation. Evaluating differences across diets using linear regression models that 193 included batch did not affect the interpretation of these results (Fig S2). Taken together, these 194 data support that bile acid pools were strongly influenced by diet, with the WD having the most 195 pro-C. difficile bile acid composition, but more work needs to be done to determine the degree to 196 which these differences were driving the higher mortality observed with the WD. 197 198

Cecal levels of SCFAs and their relationship to diet, secondary bile acids, cecal levels of C. 199 difficile toxins, and inflammation 200
To further explore potential mechanisms of increased mortality in the WD-fed mice we 201 also used targeted GC/MS to measure the levels of the SCFAs butyrate, propionate, and acetate 202 in the aspirated cecal contents in mice that were sacrificed at 3 days post infection (Table S2). butyrate also enhances C. difficile toxin production in vitro 15 33 . We also directly evaluated the 207 secondary bile acid DCA, since it can arrest the growth of vegetative C. difficile 13 and prior 208 studies have shown that secondary bile acid producers such as Clostridium scindens can protect 209 against CDI in mice 14 . 210 Butyrate, acetate, and DCA were all significantly higher in the chow diet compared to 211 both the LF/LF diet and WD ( Figures 3A and 4A). There was also a significant correlation 212 between levels of DCA and butyrate in a multivariate regression that accounted for differences 213 across diets ( Figure 4B). This is consistent with both DCA and butyrate having been linked with 214 the presence of a healthy protective gut microbiome composition and low levels of both have 215 been observed in individuals with rCDI 30,34 . Surprisingly, butyrate positively correlated with 216 TcdB ( Figure 4C) and cecal and colonic inflammation ( Figure 4D), but linear regression 217 indicated that this relationship was dependent on diet, being driven by a positive association in 218 the WD and LF/LF diet contexts only ( Figure 4C, Table S3). DCA also correlated with TcdB 219 levels and cecal and colonic inflammation in a diet dependent manner, with a positive 220 relationship in LF/LF and WD and the expected negative (protective) relationship only in chow 221 ( Figure 4D, Table S3). 222

A conventional chow diet increases homogeneity of response, resilience and alpha-diversity of 224 the gut microbiome after challenge with antibiotics and CDI compared to both purified diets 225
We next sought to understand how the composition of the fecal microbiome was affected 226 by diet during the course of antibiotic treatment and infection with C. difficile (Fig. 1A). Fecal 227 pellets were collected during experiment 1 upon arrival prior to diet change (Day -7), just prior 228 to the start of oral antibiotic delivery (Day 0), after 5 days of oral antibiotics (Day 5), and daily 229 through Day 10, which captured before and after the clindamycin injection given on day 7 and C.  9, chow-fed mice again displayed higher microbiome resilience than both the WD and LF/LF 245 diet groups. We also assessed the homogeneity of response to a disturbance among mice in the 246 same diet group. As an example, low homogeneity would occur if the mice within a diet group 247 showed high variability in the degree to which their gut microbiome changed upon antibiotic 248 exposure. We quantified this as the median pairwise weighted UniFrac distance for comparisons 249 within samples collected at the same time point from mice fed the same diet (Fig. 5C). Both the 250 WD and LF/LF diet showed much lower homogeneity of gut microbiome compositional 251 11 response to antibiotic challenge, particularly to the 5-day treatment with oral antibiotics (Day 5), 252 compared to chow-fed mice (Fig. 5C). 253 Similar patterns were seen when evaluating changes in alpha-diversity across the 254 experiment between each diet cohort. Figure 6 shows changes in phylogenetic entropy, which is 255 a measure of alpha diversity that considers species richness, evenness, and distinctness 36 . The 256 phylogenetic entropy of the WD-fed mice was lower than chow-fed mice after diet change and 257 this difference became more pronounced upon oral antibiotics and remained so through the rest 258 of the experimental timeline ( Fig. 6). Interestingly, the phylogenetic entropy of the LF/LF diet-259 fed mice remained equivalent to the chow-fed cohort with diet change but decreased to the same 260 level as the WD with antibiotic treatment (Fig. 6). 261 262

The WD and LF/LF diets had increased facultative anaerobe colonization and decreased 263 secondary bile acid and SCFA-producing bacteria compared to conventional chow diet 264
Low-diversity dysbiosis is a state of disturbance that is often characterized not only by 265 low alpha-diversity, but also by an increased ratio of facultative to strict anaerobes 37 . Low-266 diversity dysbiosis is associated with a number of diseases including rCDI 37 . We sought to 267 investigate whether the different diets tested influenced if the microbiome developed a We also used PICRUSt 38 to predict metagenomes using our 16S rRNA data to 1) 284 investigate trends in the prevalence of key genes in secondary bile and butyrate production over 285 the course of our experimental timeline and 2) predict which bacterial taxa were contributing 286 these genes. Because baiA, baiB, and baiCD are not available in PICRUSt2"s set of predicted 287 genes, we only used the genes for baiH (KEGG ID: K15873) and baiI (KEGG ID; K15874), 288 which are both genes in the bai operon 39 , to assess genomic potential for secondary bile acid 289 metabolism. Acetoacetate co-A transferase (but; K01034) and Butyrate Kinase (buk; KEGG ID: 290 K00929), which are the main pathways for fermentative production of butyrate in the gut 291 microbiome 40 , were used to assess butyrate production potential. Plotting these genes/pathways 292 over time reveals a significant effect of diet on their abundance and response to antibiotics (Fig.  293   7C). Although all diet groups showed a marked decrease in bile acid genes with oral antibiotics, 294 only the chow-fed mice displayed a recovery of secondary bile acid genes, though the source of 295 these genes switched from Lachnospiraceae UCG-006 to Blautia. This result is consistent with 296 13 our observation of higher cecal levels of secondary bile acids in chow-fed mice compared to 297 mice fed either the WD or LF/LF diets at 3 days post C. difficile gavage (Fig. 4A). 298 Butyrate coding capacity also differed between diet groups. Chow-fed mice showed 299 minimal change in the abundance of both the but and buk genes for fermentative butyrate 300 production during the time course while the WD mice had a decrease of 5 orders of magnitude 301 (Fig. 7D). The LF/LF diet-fed mice showed an intermediate phenotype with the resilience of the 302 butyrate pathway being mostly attributed to a butyrate kinase dependent pathway. The results for 303 but and not buk however are consistent with our measurements of cecal butyrate levels in these 304 mice 3 days post C. difficile gavage (Fig. 4A). This is consistent with but being regarded to be a 305 more important source of butyrate in the intestine 41 . 306 Since we had observed a strong positive correlation between cecal levels of butyrate and 307 the secondary bile acid DCA in our mass spectrometry data (Fig. 4B), we also determined 308 whether there was a relationship between butyrate and secondary bile acid coding capacity. We 309 found a highly significant association (p = 3.6x10 -5 ), with secondary bile acid producing genes 310 only predicted to be present in samples that also had high predicted levels of butyrate producing 311 genes (Fig. 7B). Influence of dietary protein has also been noted in a few studies. Specifically, one study 338 found a low-protein diet to be protective in an antibiotic-induced CDI murine model, with mice 339 fed a 2% protein diet having increased survival, decreased weight loss, and decreased overall 340 disease severity compared to mice fed a 20% protein defined diet 20 . Another study showed that a 341 15 diet poor in proline (an essential amino acid for C. difficile growth) prevented C. difficile 342 carriage 4 . Furthermore, in a recent study that evaluated both a high-fat/high-protein Atkins-type 343 diet and a high-fat/low-protein diet in a mouse model of antibiotic-induced CDI, the high-344 fat/high-protein diet promoted severe CDI and 100% mortality, while the high-fat/low-protein 345 diet had variable disease severity and survival, showing a strong effect of dietary protein but 346 indicating that the effects of fats were uncertain 18 . 347 Another had found that a diet that was high in refined carbohydrates and low in fiber had 348 improved CDI severity compared to mice fed a standard chow diet 18 . New data has suggested 349 that novel speciation of C. difficile may be selecting for strains that show increased sporulation 350 and host colonization capacity with sugar availability (glucose or fructose) 47. This work, 351 conducted with C. difficile strain (VPI 10463), did not show differences in mortality from CDI in 352 low-fat/low-fiber diets with different amounts of sucrose 18,19 . 353 Our results show that high dietary fat in the context of low dietary fiber had a strong 354 effect on CDI-induced mortality, with mechanisms distinct from a loss of beneficial microbial 355 metabolites. Evidence to suggest that a high-fat/low-fiber western-type diet could have a 356 profound effect on CDI was first presented over 20 years ago in experiments designed to study 357 the atherogenic properties of a Western diet in Syrian hamsters 44,45 . Significant mortality from 358 CDI was observed in hamsters fed a high-fat/low-fiber pro-atherogenic diet and not a typical 359 high-fiber/low-fat hamster diet, even in the absence of an antibiotic disturbance 44,45 . Another 360 recent study that conducted a study of antibiotic-induced CDI in a high-fat-diet (HFD) induced 361 obesity model found protracted disease in the HFD compared to a chow diet 19 , but not the severe 362 mortality that we observed with a high-fat/low-fiber diet. We posit that high dietary fat may have 363 a more profound influence on CDI than low dietary fiber since a prior study of MAC deficient 364 16 diets found that low fiber was associated with higher C. difficile carriage but did not describe the 365 severe disease/mortality that was observed here while using a similar mouse model 15 . However, 366 since we did not test a high-fat/high-fiber diet, it is unclear whether the high mortality that we 367 observed was due to a combination of high-fat and low-fiber in the diet, or just dietary fat. 368 Although these studies taken together support a potential synergy of high-fat and low-369 fiber leading to severe disease, it is important to note that these papers differ in many 370 experimental parameters including the source of the mice (which has been shown to influence 371 response to antibiotic perturbation and C. difficile clearance in mice 46 ), types of antibiotics used, 372 strain of C. difficile, and whether C. difficile was used as active growing bacteria (as done in our 373 study) or as spores. 374 375

The Role of Toxin Production and Inflammation 376
In order to further explore potential causes of death, we looked at both inflammation by 377 histology and levels of the toxins TcdA and TcdB by ELISA in cecal contents collected 3 days 378 post C. difficile infection, which was just prior to the onset of mortality in our longitudinal 379 cohort. Both TcdA and TcdB can disrupt cytoskeletal structure and tight junctions of target cells 380 48 and induce inflammation 49, 3 . We did not observe any differences in TcdB or cecal or colon 381 inflammation scores across diets. However, cecal levels of TcdB did correlate with cecal 382 inflammation, consistent with known effects of TcdB 48 50 . This supports that levels of TcdB 383 produced by C. difficile may indeed be causing pathology in these mice, but higher levels of 384 TcdB at Day 3 post CDI cannot alone explain the higher mortality that we began to observe at 385 fiber diet. Our results support a potential importance of TcdA and not TcdB in diet-associated 398 differences in CDI pathogenesis, but further studies that sample the toxin levels at more time 399 points over disease progression might prove illuminating. Indeed, another prior study that 400 showed higher CDI pathology in HFD-induced obesity model versus a regular chow diet did not 401 observe higher toxin levels (while binning TcdA and TcdB ELISA data) at day 3 post infection 402 (acute phase), but did find higher toxin levels and intestinal inflammation between diets at day 403 10 post infection, due to recovery occurring in the chow fed but not HFD-obese mice 19 . 404 Although it is possible that differences in TcdB and inflammation across diets in our study may 405 have emerged over time, it was not possible to evaluate this since we had much higher mortality 406 in our model, and most of our WD fed mice would have died by day 10. Further studies that use 407 complementary methods to measure toxin besides just ELISA, which can lack specificity for 408 TcdB in particular 52,53 , or with strains of C. difficile that produce TcdA or TcdB only would be 409 required for further validation 52 . Also, in these studies we measured toxin levels but were unable 410 18 to produce quality data regarding levels of C. difficile bacteria in the cecal materials. We thus 411 cannot evaluate whether these differences in toxin levels are driven by more bacteria or increased 412 toxin production by similar loads of bacteria. growth of vegetative C. difficile 13,29 . In line with these effects, reduced prevalence of the 426 secondary bile acid producer, Clostridium scindens in the fecal microbiome has been associated 427 with high incidence of CDI in both humans and in experimental mouse models, and gavaging 428 mice with C. scindens protected against CDI and restored intestinal secondary bile acid levels 14 . 429 Despite this strong evidence of a role of a protective effect of microbially produced secondary 430 bile metabolites in protection from CDI, this mechanism did not appear to be a sole driving 431 factor of the mortality that we observed in mice fed a WD, since the levels of these metabolites 432 were lowest in the mice fed the LF/LF diet even though the LF/LF mice did not experience 433 increased mortality. Levels of C. difficile inhibitors, which included the secondary bile acids 434 DCA and LCA, did negatively correlate with colonic inflammation, suggesting some degree of 435 protection in these mice. Functional interrogation of the microbiome using PICRUSt suggests 436 that the lack of secondary bile acids in the WD and LF/LF diet fed mice might be due to a lack of 437 recovery of secondary bile acid producing bacteria following antibiotic disturbance in both the 438 WD and LF/LF diet contexts. 439 We did find that the ratio of C. difficile promoters:inhibitors was significantly higher in 440 the WD compared to both the LF/LF and chow diets, consistent with mortality differences. Our 441 results support that a high-fat diet coupled with low-fiber and antibiotic treatment may provide a 442 "double hit" for shifting towards a pro-C. difficile bile acid poolwith dietary fat increasing 443 excretion of pro-C. difficile primary bile acids into the gut and antibiotic-induced gut 444 microbiome disturbance decreasing their conversion into protective secondary bile acids. In vitro 445 assays have demonstrated that variable mixtures of primary and secondary bile assays have 446 different impacts on C. difficile germination and growth 54 . However, more work needs to be 447 done to determine the degree to which these differences were driving the higher mortality 448 observed with the WD. A more convincing result would be if the C. difficile promoter:inhibitor 449 ratio also predicted C. difficile toxin production while controlling for diet, but this was not the 450 case (Fig. 3B). 451 We also found that diet had a significant effect on 4 of the 5 taurine conjugated bile acids 452 that we assayed, with TCA, T_b_MCA, TDCA, and TCDCA all showing a pattern of increased 453 levels in the WD compared to both the chow and LF/LF diets, but only comparisons of chow 454 versus WD reaching statistical significance (Fig. S3). It is probable that the further decrease in 455 levels of these taurine conjugated bile acids in the chow compared to the LF/LF diet is because 456 20 the primary bile acids that are produced by the host are converted by microbes to secondary bile 457 acids in only the chow diet. Our finding of increased TCA in the WD compared to chow is 458 consistent with a prior study that found that IL10-deficient mice fed a diet high in saturated fat, 459 had an increased proportion of taurine-conjugated bile acids compared to standard chow, and a 460 diet high in poly-unsaturated fats 21 . One prior study demonstrated that both TDCA and TCDCA 461 have pro-germinative effects on C. difficile, though in our study, their cecal concentrations were 462 orders of magnitude lower than TCA which is also a much stronger germinant 55 . 463 One weakness of our study is that we cannot differentiate between the complex changes 464

Effects of the microbiome and their metabolites 477
Our data suggests that a complex diet is critical for the resilience and homogeneity of 478 response of the gut microbiome after perturbation. In both cohorts of mice fed a purified diet that 479 was deficient in fiber, the gut microbiome was significantly more variable and slower to recover 480 to baseline after perturbation. We hypothesize that by supplying the gut with a preferred fuel 481 (fiber) for species associated with health (e.g. strict anaerobes), the community is able to resist 482 antibiotic induced changes and reconstitute more quickly once the pressure of antibiotic 483 treatment has been removed. Since the chow diet differed from the purified diets in many 484 components besides the levels of fiber, we cannot conclude from our study alone that increased 485 resilience to microbiome disturbance with antibiotics in chow is driven by differences in fiber. 486 However, our results are consistent with previous murine studies that have shown that low fiber 487 diets can increase antibiotic-induced microbiome disturbance and delay recovery from treatment 488 with ciprofloxacin 58 and that fiber supplementation can lead to a reduced disruption of the gut 489 microbiome to disturbance from amoxicillin 59 . 490 The increased resilience of gut microbiome composition to antibiotic disturbance was 491 also reflected through levels of the bacterially produced metabolites that we measured. Neither 492 the WD or LF/LF diets were able to maintain butyrate or secondary bile acid production 493 following antibiotic perturbation. Based on the correlation between butyrate and DCA 494 concentrations, we speculate that the lack of butyrate leads to increased luminal oxygen 495 concentrations that are unsuitable for Clostridium scindens and other secondary bile acid 496 producers. Prior work has shown that aerobic metabolism of butyrate by intestinal epithelial cells 497 is a key driver of intestinal hypoxia 60 . That there may be increased luminal oxygen 498 concentrations in the LF/LF and WD is consistent with our observation of a bloom in 499 Lactobacillales order, which is entirely composed of facultative anaerobes, after oral antibiotic 500 challenge in the WD and LF/LF diets but not chow. 501 22 While our data do not suggest a role for fiber in protection against mortality from CDI in 502 this mouse model since the LF/LF diet fed mice were protected without fiber in the diet, it would 503 be short-sighted to dismiss the beneficial role of fiber in maintaining a healthy gut microbiome 504 and resistance to CDI. Our model utilized a rather short-term diet change and an intense 505 antibiotic regimen. We also did not explore diets high in fat and high in fiber, where it is possible 506 that increased microbiome resilience to antibiotics due to fiber may protect from the detrimental 507 effects of fat. As discussed above, a fiber-deficient diet has been shown to hinder clearance of C. 508 showed that soluble fiber-supplementation drove hepatocellular carcinoma in mice in a manner 524 23 dependent on microbial fermentation to SCFAs, but this effect occurred when soluble fiber was 525 added to a compositionally defined diet and not to a conventional chow diet 65 . Our results 526 suggest that supplementation with soluble fibers such as inulin to prevent C. difficile may not 527 produce the desired result in individuals who are otherwise consuming highly refined diets. 528 529

Limitations of our study: 530
We have demonstrated a striking difference in diet-mediated mortality in an antibiotic-531 induced murine CDI model, but our study does have limitations. We did not explore how the 532 composition of fat influences these factors. Our WD composition represents a typical diet in the 533 United States based on population survey data. Further studies to determine if total fat intake or 534 specific types of fat drive our observed phenotype are needed. Furthermore, we only evaluated 535 the effects of fat in a low-fiber context. Evaluating a high-fat/high-fiber diet would elucidate 536 whether the expected beneficial effects of fiber on the microbiome would temper the negative 537 effects of high-fat. Comparisons between chow-fed mice and those receiving a purified diet are 538 limited due to the marked differences in the composition of macronutrients 66 . Since this is an 539 antibiotic-induced CDI model, our results only reflect effects of diet in the context of antibiotic 540 disturbance. Finally, we note that we induced CDI infection using a standardized amount of live 541 C. difficile, which is commonly used in murine studies of C. difficile 22  and toxin analysis, 2-7 independent experiments were conducted with separate starting dates 571 (Table S2). Within 24 hours, mouse feed was changed to one of three diets: standard chow, high-572 fat/low-fiber (WD), or LF/LF diet (all groups n=20 over 4 batches; Table S2). After seven days 573 of the new diet, we placed mice on a five-antibiotic cocktail (kanamycin (0.4 mg/ml), gentamicin 574 (0.035 mg/ml), colistin (850 U/ml), metronidazole (0.215 mg/ml), and vancomycin (0.045 575 mg/ml)) in their drinking water. Antibiotics were removed for 48 hours, after which we 576 administered an intraperitoneal injection of clindamycin in normal saline (10 mg/kg body 577 weight). Twenty-four hours after injection, we gavaged mice with 1.75x10 5 cfu of C. difficile 578 VPI 10463 in the vegetative stage. We weighed mice daily after removal of oral antibiotics and 579 they were euthanized if they lost >15% of body weight or were moribund. Fecal pellets were 580 collected at arrival (Day -7), after diet change and prior to oral antibiotics (Day 0) and then daily 581 after removal of oral antibiotics (Day 5-10). In a separate set of experiments, we performed the 582 same experimental protocol on 66 mice (chow = 20, low-fat/low-fiber = 20, WD = 26) over 7 583 different batches (see Table S2), but we sacrificed the mice 72 hours after infection and collected 584 cecal contents for SCFA, bile acid and toxin quantification and cecum and intestines for 585 histopathology. Mice for the second experiments were also obtained from Taconic Bioscience. 586 All mouse experiments were approved by the Institutional Animal Care and Use Committee and 587 complied with their guidelines and NIH Guide for the Care and Use of Laboratory Animals 588 (IACUC protocol #00249). 589 C. difficile growth: C. difficile strain VPI 10463 (ATCC, Manassas Virginia) was used 590 for all experiments. Frozen stocks were plated on to TCCFA agar plates (TekNova) and 591 incubated overnight in an anaerobic chamber (Coy, Grass Lake, Michigan). Single colonies were 592 picked and inoculated into BHI Media (Difco) and grown over night in anaerobic conditions. 593 samples sizes for 2 cohorts; more information on batching and n"s per assay is given in Table  945 S2). Cohort 1 was followed for 13 days post C. difficile gavage to monitor survival and gut 946 microbiome composition over time. Cohort 2 was sacrificed at 3 days post C. difficile gavage to 947 collect cecal contents for measurement of metabolites and toxin and colon and cecal mucosa for 948 histopathology (some assays were only conducted on a subset of Cohort 2; but all in at least 2 949 independent experiments; see Table S2 for details). Grey and orange boxes indicate the 950 timepoints at which samples were collected for the respective cohorts. (B) Survival curves on the 951