Identifying metabolic adaptations characteristic of cardiotoxicity using paired transcriptomics and metabolomics data integrated with a computational model of heart metabolism.

Improvements in the diagnosis and treatment of cancer have revealed long-term side effects of chemotherapeutics, particularly cardiotoxicity. Here, we present paired transcriptomics and metabolomics data characterizing in vitro cardiotoxicity to three compounds: 5-fluorouracil, acetaminophen, and doxorubicin. Standard gene enrichment and metabolomics approaches identify some commonly affected pathways and metabolites but are not able to readily identify metabolic adaptations in response to cardiotoxicity. The paired data was integrated with a genome-scale metabolic network reconstruction of the heart to identify shifted metabolic functions, unique metabolic reactions, and changes in flux in metabolic reactions in response to these compounds. Using this approach, we confirm previously seen changes in the p53 pathway by doxorubicin and RNA synthesis by 5-fluorouracil, we find evidence for an increase in phospholipid metabolism in response to acetaminophen, and we see a shift in central carbon metabolism suggesting an increase in metabolic demand after treatment with doxorubicin and 5-fluorouracil.

Network analysis of toxin production in Clostridioides difficile identifies key metabolic dependencies.

Clostridioides difficile pathogenesis is mediated through its two toxin proteins, TcdA and TcdB, which induce intestinal epithelial cell death and inflammation. It is possible to alter C. difficile toxin production by changing various metabolite concentrations within the extracellular environment. However, it is unknown which intracellular metabolic pathways are involved and how they regulate toxin production. To investigate the response of intracellular metabolic pathways to diverse nutritional environments and toxin production states, we use previously published genome-scale metabolic models of C. difficile strains CD630 and CDR20291 (iCdG709 and iCdR703). We integrated publicly available transcriptomic data with the models using the RIPTiDe algorithm to create 16 unique contextualized C. difficile models representing a range of nutritional environments and toxin states. We used Random Forest with flux sampling and shadow pricing analyses to identify metabolic patterns correlated with toxin states and environment. Specifically, we found that arginine and ornithine uptake is particularly active in low toxin states. Additionally, uptake of arginine and ornithine is highly dependent on intracellular fatty acid and large polymer metabolite pools. We also applied the metabolic transformation algorithm (MTA) to identify model perturbations that shift metabolism from a high toxin state to a low toxin state. This analysis expands our understanding of toxin production in C. difficile and identifies metabolic dependencies that could be leveraged to mitigate disease severity.

Predicting changes in renal metabolism after compound exposure with a genome-scale metabolic model.

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence. To demonstrate the utility of iRno for predicting kidney function, we exposed primary rat renal proximal tubule epithelial cells to four compounds with varying levels of nephrotoxicity (acetaminophen, gentamicin, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six and twenty-four hours, and collected transcriptomics and metabolomics data to measure the metabolic effects of compound exposure. For the transcriptomics data, we observed changes in fatty acid metabolism and amino acid metabolism, as well as changes in existing markers of kidney function such as Clu (clusterin). The iRno metabolic network reconstruction was used to predict alterations in these same pathways after integrating transcriptomics data and was able to distinguish between select compound-specific effects on the proximal tubule epithelial cells. Genome-scale metabolic network reconstructions with coupled omics data can be used to predict changes in metabolism as a step towards identifying novel metabolic biomarkers of kidney function and dysfunction.

A novel mouse model of Campylobacter jejuni enteropathy and diarrhea.

Campylobacter infections are among the leading bacterial causes of diarrhea and of 'environmental enteropathy' (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.

Minimum Bactericidal Concentration of Ciprofloxacin to Pseudomonas aeruginosa Determined Rapidly Based on Pyocyanin Secretion.

Infections due to Pseudomonas aeruginosa (P. aeruginosa) often exhibit broad-spectrum resistance and persistence to common antibiotics. Persistence is especially problematic with immune-compromised subjects who are unable to eliminate the inhibited bacteria. Hence, antibiotics must be used at the appropriate minimum bactericidal concentration (MBC) rather than at minimum inhibitory concentration (MIC) levels. However, MBC determination by conventional methods requires a 24 h culture step in the antibiotic media to confirm inhibition, followed by a 24 h sub-culture step in antibiotic-free media to confirm the lack of bacterial growth. We show that electrochemical detection of pyocyanin (PYO), which is a redox-active bacterial metabolite secreted by P. aeruginosa, can be used to rapidly assess the critical ciprofloxacin level required for bactericidal deactivation of P. aeruginosa within just 2 hours in antibiotic-treated growth media. The detection sensitivity for PYO can be enhanced by using nanoporous gold that is modified with a self-assembled monolayer to lower interference from oxygen reduction, while maintaining a low charge transfer resistance level and preventing electrode fouling within biological sample matrices. In this manner, bactericidal efficacy of ciprofloxacin towards P. aeruginosa at the MBC level and bacterial persistence at the MIC level can be determined rapidly, as validated at later timepoints using bacterial subculture in antibiotic-free media.

Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with Giardia lamblia and enteroaggregative Escherichia coli.

Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.

Innate Immune Response and Outcome of Clostridium difficile Infection Are Dependent on Fecal Bacterial Composition in the Aged Host.

Background: Clostridium difficile infection (CDI) is a serious threat for an aging population. Using an aged mouse model, we evaluated the effect of age and the roles of innate immunity and intestinal microbiota. Methods: Aged (18 months) and young (8 weeks) mice were infected with C difficile, and disease severity, immune response, and intestinal microbiome were compared. The same experiment was repeated with intestinal microbiota exchange between aged and young mice before infection. Results: Higher mortality was observed in aged mice with weaker neutrophilic mobilization in blood and intestinal tissue and depressed proinflammatory cytokines in early infection. Microbiota exchange improved survival and early immune response in aged mice. Microbiome analysis revealed that aged mice have significant deficiencies in Bacteroidetes phylum and, specifically, Bacteroides, Alistipes, and rc4-4 genera, which were replenished by cage switching. Conclusions: Microbiota-dependent alteration in innate immune response early on during infection may explain poor outcome in aged host with CDI.

Amixicile Reduces Severity of Cryptosporidiosis but Does Not Have In Vitro Activity against Cryptosporidium.

Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition. Here, we show that amixicile, a highly selective water-soluble derivative of NTZ diminishes Cryptosporidium infection severity in a malnourished mouse model despite a lack of direct anticryptosporidial activity. We suggest that amixicile, by tamping down anaerobes associated with intestinal inflammation, reverses weight loss and indirectly mitigates infection-associated pathology.

Increased Urinary Trimethylamine N-Oxide Following Cryptosporidium Infection and Protein Malnutrition Independent of Microbiome Effects.

Cryptosporidium infections have been associated with growth stunting, even in the absence of diarrhea. Having previously detailed the effects of protein deficiency on both microbiome and metabolome in this model, we now describe the specific gut microbial and biochemical effects of Cryptosporidium infection. Protein-deficient mice were infected with Cryptosporidium parvum oocysts for 6-13 days and compared with uninfected controls. Following infection, there was an increase in the urinary excretion of choline- and amino-acid-derived metabolites. Conversely, infection reduced the excretion of the microbial-host cometabolite (3-hydroxyphenyl)propionate-sulfate and disrupted metabolites involved in the tricarboxylic acid (TCA) cycle. Correlation analysis of microbial and biochemical profiles resulted in associations between various microbiota members and TCA cycle metabolites, as well as some microbial-specific degradation products. However, no correlation was observed between the majority of the infection-associated metabolites and the fecal bacteria, suggesting that these biochemical perturbations are independent of concurrent changes in the relative abundance of members of the microbiota. We conclude that cryptosporidial infection in protein-deficient mice can mimic some metabolic changes seen in malnourished children and may help elucidate our understanding of long-term metabolic consequences of early childhood enteric infections.

Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection.

BACKGROUND: The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota. METHODS: Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin. RESULTS: Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment. CONCLUSIONS: Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

Treatment of Clostridium difficile infection using SQ641, a capuramycin analogue, increases post-treatment survival and improves clinical measures of disease in a murine model.

OBJECTIVES: Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus. METHODS: In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1-300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection. RESULTS: Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment. CONCLUSIONS: SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.

Role of leptin-mediated colonic inflammation in defense against Clostridium difficile colitis.

The role of leptin in the mucosal immune response to Clostridium difficile colitis, a leading cause of nosocomial infection, was studied in humans and in a murine model. Previously, a mutation in the receptor for leptin (LEPR) was shown to be associated with susceptibility to infectious colitis and liver abscess due to Entamoeba histolytica as well as to bacterial peritonitis. Here we discovered that European Americans homozygous for the same LEPR Q223R mutation (rs1137101), known to result in decreased STAT3 signaling, were at increased risk of C. difficile infection (odds ratio, 3.03; P = 0.015). The mechanism of increased susceptibility was studied in a murine model. Mice lacking a functional leptin receptor (db/db) had decreased clearance of C. difficile from the gut lumen and diminished inflammation. Mutation of tyrosine 1138 in the intracellular domain of LepRb that mediates signaling through the STAT3/SOCS3 pathway also resulted in decreased mucosal chemokine and cell recruitment. Collectively, these data support a protective mucosal immune function for leptin in C. difficile colitis partially mediated by a leptin-STAT3 inflammatory pathway that is defective in the LEPR Q223R mutation. Identification of the role of leptin in protection from C. difficile offers the potential for host-directed therapy and demonstrates a connection between metabolism and immunity.

Intestinal epithelial restitution after TcdB challenge and recovery from Clostridium difficile infection in mice with alanyl-glutamine treatment.

BACKGROUND: Clostridium difficile is an anaerobic bacterium that causes antibiotic-associated diarrhea. It produces toxin A and toxin B (TcdB), which cause injury to the gut epithelium. Glutamine is a fundamental fuel for enterocytes, maintaining intestinal mucosal health. Alanyl-glutamine (AQ) is a highly soluble dipeptide derivative of glutamine. We studied whether administration of AQ ameliorates the effects of TcdB in the intestinal cells and improves the outcome of C. difficile infection in mice. METHODS: WST-1 proliferation and cell-wounding-migration assays were assessed in IEC-6 cells exposed to TcdB, with or without AQ. Apoptosis and necrosis were assessed using Annexin V and flow cytometry. C57BL/6 mice were infected with VPI 10463 and treated with either vancomycin, AQ, or vancomycin with AQ. Intestinal tissues were collected for histopathologic analysis, apoptosis staining, and determination of myeloperoxidase activity. RESULTS: AQ increased proliferation in intestinal cells exposed to TcdB, improved migration at 24 and 48 hours, and reduced apoptosis in intestinal cells challenged with TcdB. Infected mice treated with vancomycin and AQ had better survival and histopathologic findings than mice treated with vancomycin alone. CONCLUSIONS: AQ may reduce intestinal mucosal injury in C. difficile-infected mice by partially reversing the effects of TcdB on enterocyte proliferation, migration, and apoptosis, thereby improving survival from C. difficile infection.

Model-driven characterization of functional diversity of Pseudomonas aeruginosa clinical isolates with broadly representative phenotypes.

Pseudomonas aeruginosa is a leading cause of infections in immunocompromised individuals and in healthcare settings. This study aims to understand the relationships between phenotypic diversity and the functional metabolic landscape of P. aeruginosa clinical isolates. To better understand the metabolic repertoire of P. aeruginosa in infection, we deeply profiled a representative set from a library of 971 clinical P. aeruginosa isolates with corresponding patient metadata and bacterial phenotypes. The genotypic clustering based on whole-genome sequencing of the isolates, multilocus sequence types, and the phenotypic clustering generated from a multi-parametric analysis were compared to each other to assess the genotype-phenotype correlation. Genome-scale metabolic network reconstructions were developed for each isolate through amendments to an existing PA14 network reconstruction. These network reconstructions show diverse metabolic functionalities and enhance the collective P. aeruginosa pangenome metabolic repertoire. Characterizing this rich set of clinical P. aeruginosa isolates allows for a deeper understanding of the genotypic and metabolic diversity of the pathogen in a clinical setting and lays a foundation for further investigation of the metabolic landscape of this pathogen and host-associated metabolic differences during infection.

In vivo physiological and transcriptional profiling reveals host responses to Clostridium difficile toxin A and toxin B.

Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.

Vancomycin treatment's association with delayed intestinal tissue injury, clostridial overgrowth, and recurrence of Clostridium difficile infection in mice.

Antibiotic treatment, including vancomycin, for Clostridium difficile infection (CDI) has been associated with recurrence of disease in up to 25% of infected persons. This study investigated the effects of vancomycin on the clinical outcomes, intestinal histopathology, and anaerobic community during and after treatment in a murine model of CDI. C57BL/6 mice were challenged with C. difficile strain VPI 10463 after pretreatment with an antibiotic cocktail. Twenty-four hours after infection, mice were treated daily with vancomycin, nitazoxanide, fidaxomicin, or metronidazaole for 5 days. Mice were monitored for either 6 or 12 days postinfection. Clinical, diarrhea, and histopathology scores were measured. Cecal contents or stool samples were assayed for clostridial or Bacteroides DNA and C. difficile toxins A and B. Vancomycin treatment of infected mice was associated with improved clinical, diarrhea, and histopathology scores and survival during treatment. However, after discontinuation of the drug, clinical scores and histopathology were worse in treated mice than in untreated infected controls. At the end of the study, 62% of the vancomycin-treated mice succumbed to recurrence, with an overall mortality rate equivalent to that of the untreated infected control group. Fidaxomicin-treated mice had outcomes similar to those of vancomycin-treated mice. C. difficile predominated over Bacteroides in cecal contents of vancomycin-treated mice, similar to findings for untreated infected mice. Decreasing the duration of vancomycin treatment from 5 days to 1 day decreased recurrence and deaths. In conclusion, vancomycin improved clinical scores and histopathology acutely but was associated with poor outcome posttreatment in C. difficile-infected mice. Decreasing vancomycin exposure may decrease relapse and improve survival in CDI.

Proposal for effective treatment of Shiga toxin-producing Escherichia coli infection in mice.

Previously, we reported that minocycline, kanamycin and norfloxacin improved the survival rate in the E32511 model that we developed (FEMS Immunol Med Microbiol 26, 101-108, 1999), but fosfomycin did not. In this study, we investigated the effectiveness of azithromycin (AZM) against Stx2d-producing EHEC O91:H21 strain B2F1 or Stx2c-producing Escherichia coli strain E32511 treated with mitomycin C in vivo. Recently, we reported the effectiveness of AZM in our model and AZM strongly inhibited the release of Stx2c from E32511 in vitro (PLOS ONE e58959, 2013). However, it was very difficult to completely eliminate E32511 in the mouse feces by treatment with AZM alone. In this report, only AZM or Daio effectively promoted survival of mice infected with B2F1 compared to untreated mice. Furthermore, Daio inhibited the colonization of GFP-expressing B2F1 in the mouse intestine. Similarly, a combination of AZM and Daio in the E32511-infected mice reduced E32511 in the mouse feces and significantly improved survival.

Model-driven characterization of functional diversity of Pseudomonas aeruginosa clinical isolates with broadly representative phenotypes.

Pseudomonas aeruginosa is a leading cause of infections in immunocompromised individuals and in healthcare settings. This study aims to understand the relationships between phenotypic diversity and the functional metabolic landscape of P. aeruginosa clinical isolates. To better understand the metabolic repertoire of P. aeruginosa in infection, we deeply profiled a representative set from a library of 971 clinical P. aeruginosa isolates with corresponding patient metadata and bacterial phenotypes. The genotypic clustering based on whole-genome sequencing of the isolates, multi-locus sequence types, and the phenotypic clustering generated from a multi-parametric analysis were compared to each other to assess the genotype-phenotype correlation. Genome-scale metabolic network reconstructions were developed for each isolate through amendments to an existing PA14 network reconstruction. These network reconstructions show diverse metabolic functionalities and enhance the collective P. aeruginosa pangenome metabolic repertoire. Characterizing this rich set of clinical P. aeruginosa isolates allows for a deeper understanding of the genotypic and metabolic diversity of the pathogen in a clinical setting and lays a foundation for further investigation of the metabolic landscape of this pathogen and host-associated metabolic differences during infection.

Systems-ecology designed bacterial consortium protects from severe Clostridioides difficile infection.

Fecal Microbiota Transplant (FMT) is an emerging therapy that has had remarkable success in treatment and prevention of recurrent Clostridioides difficile infection (rCDI). FMT has recently been associated with adverse outcomes such as inadvertent transfer of antimicrobial resistance, necessitating development of more targeted bacteriotherapies. To address this challenge, we developed a novel systems biology pipeline to identify candidate probiotic strains that would be predicted to interrupt C. difficile pathogenesis. Utilizing metagenomic characterization of human FMT donor samples, we identified those metabolic pathways most associated with successful FMTs and reconstructed the metabolism of encoding species to simulate interactions with C. difficile . This analysis resulted in predictions of high levels of cross-feeding for amino acids in species most associated with FMT success. Guided by these in silico models, we assembled consortia of bacteria with increased amino acid cross-feeding which were then validated in vitro . We subsequently tested the consortia in a murine model of CDI, demonstrating total protection from severe CDI through decreased toxin levels, recovered gut microbiota, and increased intestinal eosinophils. These results support the novel framework that amino acid cross-feeding is likely a critical mechanism in the initial resolution of CDI by FMT. Importantly, we conclude that our predictive platform based on predicted and testable metabolic interactions between the microbiota and C. difficile led to a rationally designed biotherapeutic framework that may be extended to other enteric infections.

Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model.

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-µg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.