Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

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.

The Gut Bacterial Community Potentiates Clostridioides difficile Infection Severity.

The severity of Clostridioides difficile infections (CDI) has increased over the last few decades. Patient age, white blood cell count, and creatinine levels as well as C. difficile ribotype and toxin genes have been associated with disease severity. However, it is unclear whether specific members of the gut microbiota are associated with variations in disease severity. The gut microbiota is known to interact with C. difficile during infection. Perturbations to the gut microbiota are necessary for C. difficile to colonize the gut. The gut microbiota can inhibit C. difficile colonization through bile acid metabolism, nutrient consumption, and bacteriocin production. Here, we sought to demonstrate that members of the gut bacterial communities can also contribute to disease severity. We derived diverse gut communities by colonizing germfree mice with different human fecal communities. The mice were then infected with a single C. difficile ribotype 027 clinical isolate, which resulted in moribundity and histopathologic differences. The variation in severity was associated with the human fecal community that the mice received. Generally, bacterial populations with pathogenic potential, such as Enterococcus, Helicobacter, and Klebsiella, were associated with more-severe outcomes. Bacterial groups associated with fiber degradation and bile acid metabolism, such as Anaerotignum, Blautia, Lactonifactor, and Monoglobus, were associated with less-severe outcomes. These data indicate that, in addition to the host and C. difficile subtype, populations of gut bacteria can influence CDI disease severity. IMPORTANCE Clostridioides difficile colonization can be asymptomatic or develop into an infection ranging in severity from mild diarrhea to toxic megacolon, sepsis, and death. Models that predict severity and guide treatment decisions are based on clinical factors and C. difficile characteristics. Although the gut microbiome plays a role in protecting against CDI, its effect on CDI disease severity is unclear and has not been incorporated into disease severity models. We demonstrated that variation in the microbiome of mice colonized with human feces yielded a range of disease outcomes. These results revealed groups of bacteria associated with both severe and mild C. difficile infection outcomes. Gut bacterial community data from patients with CDI could improve our ability to identify patients at risk of developing more severe disease and improve interventions that target C. difficile and the gut bacteria to reduce host damage.

Binary Toxin Expression by Clostridioides difficile Is Associated With Worse Disease.

BACKGROUND: The incidence of Clostridioides difficile infection (CDI) has increased over the past 2 decades and is considered an urgent threat by the Centers for Disease Control and Prevention. Hypervirulent strains such as ribotype 027, which possess genes for the additional toxin C. difficile binary toxin (CDT), are contributing to increased morbidity and mortality. METHODS: We retrospectively tested stool from 215 CDI patients for CDT by enzyme-linked immunosorbent assay (ELISA). Stratifying patients by CDT status, we assessed if disease severity and clinical outcomes correlated with CDT positivity. Additionally, we completed quantitative PCR (PCR) DNA extracted from patient stool to detect cdtB gene. Lastly, we performed 16 S rRNA gene sequencing to examine if CDT-positive samples had an altered fecal microbiota. RESULTS: We found that patients with CdtB, the pore-forming component of CDT, detected in their stool by ELISA, were more likely to have severe disease with higher 90-day mortality. CDT-positive patients also had higher C. difficile bacterial burden and white blood cell counts. There was no significant difference in gut microbiome diversity between CDT-positive and -negative patients. CONCLUSIONS: Patients with fecal samples that were positive for CDT had increased disease severity and worse clinical outcomes. Utilization of PCR and testing for C. difficile toxins A and B may not reveal the entire picture when diagnosing CDI; detection of CDT-expressing strains is valuable in identifying patients at risk of more severe disease.

The IL-33-ILC2 pathway protects from amebic colitis.

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2-/- mice but not RAG2-/-γc-/- mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2-/- mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2-/-γc-/- mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis.

The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the United States. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hypervirulent isolate (strain [str.] R20291) and a historic strain (str. 630), validating both with in vitro and in vivo data sets. Growth simulations revealed significant correlations with measured carbon source usage (positive predictive value [PPV] ≥ 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the pentose phosphate pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in higher-order phenotypes like bacterial pathogenesis. IMPORTANCE Clostridioides difficile has become the leading single cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors. In the past, genome-scale metabolic network reconstruction (GENRE) analysis of bacteria has enabled systematic investigation of the genetic and metabolic properties that contribute to downstream virulence phenotypes. With this in mind, we generated and extensively curated C. difficile GENREs for both a well-studied laboratory strain (str. 630) and a more recently characterized hypervirulent isolate (str. R20291). In silico validation of both GENREs revealed high degrees of agreement with experimental gene essentiality and carbon source utilization data sets. Subsequent exploration of context-specific metabolism during both in vitro growth and infection revealed consistent patterns of metabolism which corresponded with experimentally measured increases in virulence factor expression. Our results support that differential C. difficile virulence is associated with distinct metabolic programs related to use of carbon sources and provide a platform for identification of novel therapeutic targets.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Protection from Lethal Clostridioides difficile Infection via Intraspecies Competition for Cogerminant.

Clostridioides difficile, a Gram-positive, spore-forming bacterium, is the primary cause of infectious nosocomial diarrhea. Antibiotics are a major risk factor for C. difficile infection (CDI), as they disrupt the gut microbial community, enabling increased germination of spores and growth of vegetative C. difficile To date, the only single-species bacterial preparation that has demonstrated efficacy in reducing recurrent CDI in humans is nontoxigenic C. difficile Using multiple infection models, we determined that precolonization with a less virulent strain is sufficient to protect from challenge with a lethal strain of C. difficile, surprisingly even in the absence of adaptive immunity. Additionally, we showed that protection is dependent on high levels of colonization by the less virulent strain and that it is mediated by exclusion of the invading strain. Our results suggest that reduction of amino acids, specifically glycine following colonization by the first strain of C. difficile, is sufficient to decrease germination of the second strain, thereby limiting colonization by the lethal strain.IMPORTANCE Antibiotic-associated colitis is often caused by infection with the bacterium Clostridioides difficile In this study, we found that reduction of the amino acid glycine by precolonization with a less virulent strain of C. difficile is sufficient to decrease germination of a second strain. This finding demonstrates that the axis of competition for nutrients can include multiple life stages. This work is important, as it is the first to identify a possible mechanism through which precolonization with C. difficile, a current clinical therapy, provides protection from reinfection. Furthermore, our work suggests that targeting nutrients utilized by all life stages could be an improved strategy for bacterial therapeutics that aim to restore colonization resistance in the gut.

Host Protective Mechanisms to Intestinal Amebiasis.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically.

Lewis Blood-group Antigens Are Associated With Altered Susceptibility to Shigellosis.

In a cohort of infants, we found that lack of the Lewis histo-blood group antigen was associated with increased susceptibility to shigellosis. Broadly inhibiting fucosylation in epithelial cells in vitro decreased invasion by Shigella flexneri. These results support a role for fucosylated glycans in susceptibility to shigellosis.

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

IL-33 drives group 2 innate lymphoid cell-mediated protection during Clostridium difficile infection.

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.

Colitis-Induced Th17 Cells Increase the Risk for Severe Subsequent Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the number one hospital-acquired infection in the United States. CDI is more common and severe in inflammatory bowel disease patients. Here, we studied the mechanism by which prior colitis exacerbates CDI. Mice were given dextran sulfate sodium (DSS) colitis, recovered for 2 weeks, and then were infected with C. difficile. Mortality and CDI severity were increased in DSS-treated mice compared to controls. Severe CDI is dependent on CD4+ T cells, which persist after colitis-associated inflammation subsides. Adoptive transfer of Th17 cells to naive mice is sufficient to increase CDI-associated mortality through elevated IL-17 production. Finally, in humans, the Th17 cytokines IL-6 and IL-23 associate with severe CDI, and patients with high serum IL-6 are 7.6 times more likely to die post infection. These findings establish a central role for Th17 cells in CDI pathogenesis following colitis and identify them as a potential target for preventing severe disease.

The Gut Microbiota Is Associated with Clearance of Clostridium difficile Infection Independent of Adaptive Immunity.

Clostridium (Clostridioides) difficile, a Gram-positive, anaerobic bacterium, is the leading single cause of nosocomial infections in the United States. A major risk factor for Clostridium difficile infection (CDI) is prior exposure to antibiotics, as they increase susceptibility to CDI by altering the membership of the microbial community enabling colonization. The importance of the gut microbiota in providing protection from CDI is underscored by the reported 80 to 90% success rate of fecal microbial transplants in treating recurrent infections. Adaptive immunity, specifically humoral immunity, is also sufficient to protect from both acute and recurrent CDI. However, the role of the adaptive immune system in mediating clearance of C. difficile has yet to be resolved. Using murine models of CDI, we found that adaptive immunity is dispensable for clearance of C. difficile However, random forest analysis using only two members of the resident bacterial community correctly identified animals that would go on to clear the infection with 66.7% accuracy. These findings indicate that the indigenous gut microbiota independent of adaptive immunity facilitates clearance of C. difficile from the murine gastrointestinal tract.IMPORTANCEClostridium difficile infection is a major cause of morbidity and mortality in hospitalized patients in the United States. Currently, the role of the adaptive immune response in modulating levels of C. difficile colonization is unresolved. This work suggests that the indigenous gut microbiota is a main factor that promotes clearance of C. difficile from the GI tract. Our results show that clearance of C. difficile can occur without contributions from the adaptive immune response. This study also has implications for the design of preclinical studies testing the efficacy of vaccines on clearance of bacterial pathogens, as inherent differences in the baseline community structure of animals may bias findings.

Clostridium difficile Alters the Structure and Metabolism of Distinct Cecal Microbiomes during Initial Infection To Promote Sustained Colonization.

Susceptibility to Clostridium difficile infection (CDI) is primarily associated with previous exposure to antibiotics, which compromise the structure and function of the gut bacterial community. Specific antibiotic classes correlate more strongly with recurrent or persistent C. difficile infection. As such, we utilized a mouse model of infection to explore the effect of distinct antibiotic classes on the impact that infection has on community-level transcription and metabolic signatures shortly following pathogen colonization and how those changes may associate with persistence of C. difficile Untargeted metabolomic analysis revealed that C. difficile infection had significantly larger impacts on the metabolic environment across cefoperazone- and streptomycin-pretreated mice, which became persistently colonized compared to clindamycin-pretreated mice, where infection quickly became undetectable. Through metagenome-enabled metatranscriptomics, we observed that transcripts for genes associated with carbon and energy acquisition were greatly reduced in infected animals, suggesting that those niches were instead occupied by C. difficile Furthermore, the largest changes in transcription were seen in the least abundant species, indicating that C. difficile may "attack the loser" in gut environments where sustained infection occurs more readily. Overall, our results suggest that C. difficile is able to restructure the nutrient-niche landscape in the gut to promote persistent infection.IMPORTANCEClostridium difficile has become the most common single cause of hospital-acquired infection over the last decade in the United States. Colonization resistance to the nosocomial pathogen is primarily provided by the gut microbiota, which is also involved in clearing the infection as the community recovers from perturbation. As distinct antibiotics are associated with different risk levels for CDI, we utilized a mouse model of infection with 3 separate antibiotic pretreatment regimens to generate alternative gut microbiomes that each allowed for C. difficile colonization but varied in clearance rate. To assess community-level dynamics, we implemented an integrative multi-omics approach that revealed that infection significantly changed many aspects of the gut community. The degree to which the community changed was inversely correlated with clearance during the first 6 days of infection, suggesting that C. difficile differentially modifies the gut environment to promote persistence. This is the first time that metagenome-enabled metatranscriptomics have been employed to study the behavior of a host-associated microbiota in response to an infection. Our results allow for a previously unseen understanding of the ecology associated with C. difficile infection and provide the groundwork for identification of context-specific probiotic therapies.

Clostridium difficile Colonizes Alternative Nutrient Niches during Infection across Distinct Murine Gut Microbiomes.

Clostridium difficile is the largest single cause of hospital-acquired infection in the United States. A major risk factor for Clostridium difficile infection (CDI) is prior exposure to antibiotics, as they disrupt the gut bacterial community which protects from C. difficile colonization. Multiple antibiotic classes have been associated with CDI susceptibility, many leading to distinct community structures stemming from variation in bacterial targets of action. These community structures present separate metabolic challenges to C. difficile. Therefore, we hypothesized that the pathogen adapts its physiology to the nutrients within different gut environments. Utilizing an in vivo CDI model, we demonstrated that C. difficile highly colonized ceca of mice pretreated with any of three antibiotics from distinct classes. Levels of C. difficile spore formation and toxin activity varied between animals based on the antibiotic pretreatment. These physiologic processes in C. difficile are partially regulated by environmental nutrient concentrations. To investigate metabolic responses of the bacterium in vivo, we performed transcriptomic analysis of C. difficile from ceca of infected mice across pretreatments. This revealed heterogeneous expression in numerous catabolic pathways for diverse growth substrates. To assess which resources C. difficile exploited, we developed a genome-scale metabolic model with a transcriptome-enabled metabolite scoring algorithm integrating network architecture. This platform identified nutrients that C. difficile used preferentially between pretreatments, which were validated through untargeted mass spectrometry of each microbiome. Our results supported the hypothesis that C. difficile inhabits alternative nutrient niches across cecal microbiomes with increased preference for nitrogen-containing carbon sources, particularly Stickland fermentation substrates and host-derived glycans. IMPORTANCE Infection by the bacterium Clostridium difficile causes an inflammatory diarrheal disease which can become life threatening and has grown to be the most prevalent nosocomial infection. Susceptibility to C. difficile infection is strongly associated with previous antibiotic treatment, which disrupts the gut microbiota and reduces its ability to prevent colonization. In this study, we demonstrated that C. difficile altered pathogenesis between hosts pretreated with antibiotics from separate classes and exploited different nutrient sources across these environments. Our metabolite score calculation also provides a platform to study nutrient requirements of pathogens during an infection. Our results suggest that C. difficile colonization resistance is mediated by multiple groups of bacteria competing for several subsets of nutrients and could explain why total reintroduction of competitors through fecal microbial transplant currently is the most effective treatment for recurrent CDI. This work could ultimately contribute to the identification of targeted, context-dependent measures that prevent or reduce C. difficile colonization, including pre- and probiotic therapies.

A whole new ball game: Stem cell-derived epithelia in the study of host-microbe interactions.

Recent advances in developmental and stem cell biology have resulted in techniques that enable the generation and maintenance of complex epithelium in vitro. While these models have been utilized to study host development and disease, a renewed appreciation of host-microbe interactions has sparked interest in employing these new techniques to study microbes at the epithelial interface. Here we review the current advances in host-microbe interactions that have resulted from experiments using these complex epithelia. Furthermore we highlight aspects of these techniques that warrant further development to facilitate the study of host-microbe interactions.

Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era.

IMPORTANCE: Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision making and public health reporting. Molecular tests are increasingly used to diagnose C difficile infection (CDI), but many molecular test-positive patients lack toxins that historically defined disease, making it unclear if they need treatment. OBJECTIVE: To determine the natural history and need for treatment of patients who are toxin immunoassay negative and polymerase chain reaction (PCR) positive (Tox-/PCR+) for CDI. DESIGN, SETTING, AND PARTICIPANTS: Prospective observational cohort study at a single academic medical center among 1416 hospitalized adults tested for C difficile toxins 72 hours or longer after admission between December 1, 2010, and October 20, 2012. The analysis was conducted in stages with revisions from April 27, 2013, to January 13, 2015. MAIN OUTCOMES AND MEASURES: Patients undergoing C difficile testing were grouped by US Food and Drug Administration-approved toxin and PCR tests as Tox+/PCR+, Tox-/PCR+, or Tox-/PCR-. Toxin results were reported clinically. Polymerase chain reaction results were not reported. The main study outcomes were duration of diarrhea during up to 14 days of treatment, rate of CDI-related complications (ie, colectomy, megacolon, or intensive care unit care) and CDI-related death within 30 days. RESULTS: Twenty-one percent (293 of 1416) of hospitalized adults tested for C difficile were positive by PCR, but 44.7% (131 of 293) had toxins detected by the clinical toxin test. At baseline, Tox-/PCR+ patients had lower C difficile bacterial load and less antibiotic exposure, fecal inflammation, and diarrhea than Tox+/PCR+ patients (P < .001 for all). The median duration of diarrhea was shorter in Tox-/PCR+ patients (2 days; interquartile range, 1-4 days) than in Tox+/PCR+ patients (3 days; interquartile range, 1-6 days) (P = .003) and was similar to that in Tox-/PCR- patients (2 days; interquartile range, 1-3 days), despite minimal empirical treatment of Tox-/PCR+ patients. No CDI-related complications occurred in Tox-/PCR+ patients vs 10 complications in Tox+/PCR+ patients (0% vs 7.6%, P < .001). One Tox-/PCR+ patient had recurrent CDI as a contributing factor to death within 30 days vs 11 CDI-related deaths in Tox+/PCR+ patients (0.6% vs 8.4%, P = .001). CONCLUSIONS AND RELEVANCE: Among hospitalized adults with suspected CDI, virtually all CDI-related complications and deaths occurred in patients with positive toxin immunoassay test results. Patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without C difficile by either method. Exclusive reliance on molecular tests for CDI diagnosis without tests for toxins or host response is likely to result in overdiagnosis, overtreatment, and increased health care costs.