Sex-Dependent Efficacy of Dietary Fiber in Pediatric Functional Abdominal Pain.

BACKGROUND & AIMS: Functional abdominal pain disorders (FAPDs) are more prevalent in female patients. Dietary fiber may alleviate FAPD symptoms; however, whether this effect is sex dependent remains unclear. We investigated the sex dependency of dietary fiber benefit on abdominal pain in children with FAPDs and explored the potential involvement of the gut microbiome. METHODS: In 2 cross-sectional cohorts of children with FAPDs (n = 209) and healthy control individuals (n = 105), we correlated dietary fiber intake with abdominal pain symptoms after stratifying by sex. We also performed sex-stratified and sex-interaction analyses on data from a double-blind trial in children with irritable bowel syndrome randomized to psyllium fiber (n = 39) or placebo (n = 49) for 6 weeks. Shotgun metagenomics was used to investigate gut microbiome community changes potentially linking dietary fiber intake with abdominal pain. RESULTS: In the cross-sectional cohorts, fiber intake inversely correlated with pain symptoms in boys (pain episodes: r = -0.24, P = .005; pain days: r = -0.24, P = 0.004) but not in girls. Similarly, in the randomized trial, psyllium fiber reduced the number of pain episodes in boys (P = .012) but not in girls. Generalized linear regression models confirmed that boys treated with psyllium fiber had greater reduction in pain episodes than girls (P = .007 for fiber × sex × time interaction). Age, sexual development, irritable bowel syndrome subtype, stool form, and microbiome composition were not significant determinants in the dietary fiber effects on pain reduction. CONCLUSIONS: Dietary fiber preferentially reduces abdominal pain frequency in boys, highlighting the importance of considering sex in future dietary intervention studies for FAPDs. (ClincialTrials.gov, Number NCT00526903).

Systems biology approach to functionally assess the Clostridioides difficile pangenome reveals genetic diversity with discriminatory power.

Combatting Clostridioides difficile infections, a dominant cause of hospital-associated infections with incidence and resulting deaths increasing worldwide, is complicated by the frequent emergence of new virulent strains. Here, we employ whole-genome sequencing, high-throughput phenotypic screenings, and genome-scale models of metabolism to evaluate the genetic diversity of 451 strains of C. difficile. Constructing the C. difficile pangenome based on this set revealed 9,924 distinct gene clusters, of which 2,899 (29%) are defined as core, 2,968 (30%) are defined as unique, and the remaining 4,057 (41%) are defined as accessory. We develop a strain typing method, sequence typing by accessory genome (STAG), that identifies 176 genetically distinct groups of strains and allows for explicit interrogation of accessory gene content. Thirty-five strains representative of the overall set were experimentally profiled on 95 different nutrient sources, revealing 26 distinct growth profiles and unique nutrient preferences; 451 strain-specific genome scale models of metabolism were constructed, allowing us to computationally probe phenotypic diversity in 28,864 unique conditions. The models create a mechanistic link between the observed phenotypes and strain-specific genetic differences and exhibit an ability to correctly predict growth in 76% of measured cases. The typing and model predictions are used to identify and contextualize discriminating genetic features and phenotypes that may contribute to the emergence of new problematic strains.

Neonatal antibiotics have long term sex-dependent effects on the enteric nervous system.

Infants and young children receive the highest exposures to antibiotics globally. Although there is building evidence that early life exposure to antibiotics increases susceptibility to various diseases including gut disorders later in life, the lasting impact of early life antibiotics on the physiology of the gut and its enteric nervous system (ENS) remains unclear. We treated neonatal mice with the antibiotic vancomycin during their first 10 postnatal days, then examined potential lasting effects of the antibiotic treatment on their colons during young adulthood (6 weeks old). We found that neonatal vancomycin treatment disrupted the gut functions of young adult female and male mice differently. Antibiotic-exposed females had significantly longer whole gut transit while antibiotic-treated males had significantly lower faecal weights compared to controls. Both male and female antibiotic-treated mice had greater percentages of faecal water content. Neonatal vancomycin treatment also had sexually dimorphic impacts on the neurochemistry and Ca2+ activity of young adult myenteric and submucosal neurons. Myenteric neurons of male mice were more disrupted than those of females, while opposing changes in submucosal neurons were seen in each sex. Neonatal vancomycin also induced sustained changes in colonic microbiota and lasting depletion of mucosal serotonin (5-HT) levels. Antibiotic impacts on microbiota and mucosal 5-HT were not sex-dependent, but we propose that the responses of the host to these changes are sex-specific. This first demonstration of long-term impacts of neonatal antibiotics on the ENS, gut microbiota and mucosal 5-HT has important implications for gut function and other physiological systems of the host. KEY POINTS: Early life exposure to antibiotics can increase susceptibility to diseases including functional gastrointestinal (GI) disorders later in life. Yet, the lasting impact of this common therapy on the gut and its enteric nervous system (ENS) remains unclear. We investigated the long-term impact of neonatal antibiotic treatment by treating mice with the antibiotic vancomycin during their neonatal period, then examining their colons during young adulthood. Adolescent female mice given neonatal vancomycin treatment had significantly longer whole gut transit times, while adolescent male and female mice treated with neonatal antibiotics had significantly wetter stools. Effects of neonatal vancomycin treatment on the neurochemistry and Ca2+ activity of myenteric and submucosal neurons were sexually dimorphic. Neonatal vancomycin also had lasting effects on the colonic microbiome and mucosal serotonin biosynthesis that were not sex-dependent. Different male and female responses to antibiotic-induced disruptions of the ENS, microbiota and mucosal serotonin biosynthesis can lead to sex-specific impacts on gut function.

Gut feelings: the microbiota-gut-brain axis on steroids.

The intricate connection between central and enteric nervous systems is well established with emerging evidence linking gut microbiota function as a significant new contributor to gut-brain axis signaling. Several microbial signals contribute to altered gut-brain communications, with steroids representing an important biological class that impacts central and enteric nervous system function. Neuroactive steroids contribute pathologically to neurological disorders, including dementia and depression, by modulating the activity of neuroreceptors. However, limited information is available on the influence of neuroactive steroids on the enteric nervous system and gastrointestinal function. In this review, we outline how steroids can modulate enteric nervous system function by focusing on their influence on different receptors that are present in the intestine in health and disease. We also highlight the potential role of the gut microbiota in modulating neuroactive steroid signaling along the gut-brain axis.

Neonatal Injury Increases Gut Permeability by Epigenetically Suppressing E-Cadherin in Adulthood.

Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a "double-hit" neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms.

Fecal Microbiota Transplantation Commonly Failed in Children With Co-Morbidities.

OBJECTIVES: Fecal microbiota transplantation (FMT) is arguably the most effective treatment for recurrent Clostridioides difficile infection (rCDI). Clinical reports on pediatric FMT have not systematically evaluated microbiome restoration in patients with co-morbidities. Here, we determined whether FMT recipient age and underlying co-morbidity influenced clinical outcomes and microbiome restoration when treated from shared fecal donor sources. METHODS: Eighteen rCDI patients participating in a single-center, open-label prospective cohort study received fecal preparation from a self-designated (single case) or two universal donors. Twelve age-matched healthy children and four pediatric ulcerative colitis (UC) cases from an independent serial FMT trial, but with a shared fecal donor were examined as controls for microbiome restoration using 16S rRNA gene sequencing of longitudinal fecal specimens. RESULTS: FMT was significantly more effective in rCDI recipients without underlying chronic co-morbidities where fecal microbiome composition in post-transplant responders was restored to levels of healthy children. Microbiome reconstitution was not associated with symptomatic resolution in some rCDI patients who had co-morbidities. Significant elevation in Bacteroidaceae, Bifidobacteriaceae, Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae was consistently observed in pediatric rCDI responders, while Enterobacteriaceae decreased, correlating with augmented complex carbohydrate degradation capacity. CONCLUSION: Recipient background disease was a significant risk factor influencing FMT outcomes. Special attention should be taken when considering FMT for pediatric rCDI patients with underlying co-morbidities.

Alterations of the Oral Microbiome and Cumulative Carbapenem Exposure Are Associated With Stenotrophomonas maltophilia Infection in Patients With Acute Myeloid Leukemia Receiving Chemotherapy.

BACKGROUND: Stenotrophomonas maltophilia is increasingly common in patients with acute myeloid leukemia (AML). Little is known about factors that drive S. maltophilia infection. We evaluated the microbiome and cumulative antibiotic use as predictors of S. maltophilia infection in AML patients receiving remission induction chemotherapy (RIC). METHODS: Subanalysis of a prospective, observational cohort of patients with AML receiving RIC between September 2013 and August 2015 was performed. Fecal and oral microbiome samples collected from initiation of RIC until neutrophil recovery were assessed for the relative abundance of Stenotrophomonas via 16S rRNA gene quantitation. The primary outcome, microbiologically proven S. maltophilia infection, was analyzed using a time-varying Cox proportional hazards model. RESULTS: Of 90 included patients, 8 (9%) developed S. maltophilia infection (pneumonia, n = 6; skin-soft tissue, n = 2); 4/8 (50%) patients were bacteremic; and 7/8 (88%) patients with S. maltophilia infection had detectable levels of Stenotrophomonas vs 22/82 (27%) without infection (P < .01). An oral Stenotrophomonas relative abundance of 36% predicted infection (sensitivity, 96%; specificity, 93%). No association of S. maltophilia infection with fecal relative abundance was found. Cumulative meropenem exposure was associated with increased infection risk (hazard ratio, 1.17; 95% confidence interval, 1.01-1.35; P = .03). CONCLUSIONS: Here, we identify the oral microbiome as a potential source for S. maltophilia infection and highlight cumulative carbapenem use as a risk factor for S. maltophilia in leukemia patients. These data suggest that real-time monitoring of the oral cavity might identify patients at risk for S. maltophilia infection.

Yeast ß-glucan reduces obesity-associated Bilophila abundance and modulates bile acid metabolism in healthy and high-fat diet mouse models.

Emerging evidence links dietary fiber with altered gut microbiota composition and bile acid signaling in maintaining metabolic health. Yeast ß-glucan (Y-BG) is a dietary supplement known for its immunomodulatory effect, yet its impact on the gut microbiota and bile acid composition remains unclear. This study investigated whether dietary forms of Y-BG modulate these gut-derived signals. We performed 4-wk dietary supplementation in healthy mice to evaluate the effects of different fiber composition (soluble vs. particulate Y-BG) and dose (0.1% vs. 2%). We found that 2% particulate Y-BG induced robust gut microbiota community shifts with elevated liver Cyp7a1 mRNA abundance and bile acid synthesis. These diet-induced responses were notably different when compared with the prebiotic inulin, and included a marked reduction in fecal Bilophila abundance which we demonstrated as translatable to obesity in population-scale American Gut and TwinsUK clinical cohorts. This prompted us to test whether 2% Y-BG maintained metabolic health in mice fed 60% HFD over 13 wk. Y-BG consistently altered the gut microbiota composition and reduced Bilophila abundance, with trends observed in improvement of metabolic phenotype. Notably, Y-BG improved insulin sensitization and this was associated with enhanced ileal Glpr1r mRNA accumulation and reduced Bilophila abundance. Collectively, our results demonstrate that Y-BG modulates gut microbiota community composition and bile acid signaling, but the dietary regime needs to be optimized to facilitate clinical improvement in metabolic phenotype in an aggressive high-fat diet animal model.NEW & NOTEWORTHY The study shows that dietary Y-BG supplementation modulated gut microbiota, bile acid metabolism and associated signaling pathways. Y-BG significantly reduced Bilophila abundance which is associated with obesity in human cohorts. Correlation analysis confirmed functional interactions between bile acid composition, gut microbiota, and metabolic phenotype, although clinical benefit did not reach significance in an aggressive obesity model. Gut microbiota and bile acids correlated with metabolic parameters, indicating future potential of dietary Y-BG modulation of metabolic pathways.

Systems biology evaluation of refractory Clostridioides difficile infection including multiple failures of fecal microbiota transplantation.

BACKGROUND: Fecal microbiota transplantation (FMT) aims to cure Clostridioides difficile infection (CDI) through reestablishing a healthy microbiome and restoring colonization resistance. Although often effective after one infusion, patients with continued microbiome disruptions may require multiple FMTs. In this N-of-1 study, we use a systems biology approach to evaluate CDI in a patient receiving chronic suppressive antibiotics with four failed FMTs over two years. METHODS: Seven stool samples were obtained between 2016-18 while the patient underwent five FMTs. Stool samples were cultured for C. difficile and underwent microbial characterization and functional gene analysis using shotgun metagenomics. C. difficile isolates were characterized through ribotyping, whole genome sequencing, metabolic pathway analysis, and minimum inhibitory concentration (MIC) determinations. RESULTS: Growing ten strains from each sample, the index and first four recurrent cultures were single strain ribotype F078-126, the fifth was a mixed culture of ribotypes F002 and F054, and the final culture was ribotype F002. One single nucleotide polymorphism (SNP) variant was identified in the RNA polymerase (RNAP) ß-subunit RpoB in the final isolated F078-126 strain when compared to previous F078-126 isolates. This SNV was associated with metabolic shifts but phenotypic differences in fidaxomicin MIC were not observed. Microbiome differences were observed over time during vancomycin therapy and after failed FMTs. CONCLUSION: This study highlights the importance of antimicrobial stewardship in patients receiving FMT. Continued antibiotics play a destructive role on a transplanted microbiome and applies selection pressure for resistance to the few antibiotics available to treat CDI.

Early life interaction between the microbiota and the enteric nervous system.

Recent studies on humans and their key experimental model, the mouse, have begun to uncover the importance of gastrointestinal (GI) microbiota and enteric nervous system (ENS) interactions during developmental windows spanning from conception to adolescence. Disruptions in GI microbiota and ENS during these windows by environmental factors, particularly antibiotic exposure, have been linked to increased susceptibility of the host to several diseases. Mouse models have provided new insights to potential signaling factors between the microbiota and ENS. We review very recent work on maturation of GI microbiota and ENS during three key developmental windows: embryogenesis, early postnatal, and postweaning periods. We discuss advances in understanding of interactions between the two systems and highlight research avenues for future studies.

Antibiotic exposure postweaning disrupts the neurochemistry and function of enteric neurons mediating colonic motor activity.

The period during and immediately after weaning is an important developmental window when marked shifts in gut microbiota can regulate the maturation of the enteric nervous system (ENS). Because microbiota-derived signals that modulate ENS development are poorly understood, we examined the physiological impact of the broad spectrum of antibiotic, vancomycin-administered postweaning on colonic motility, neurochemistry of enteric neurons, and neuronal excitability. The functional impact of vancomycin on enteric neurons was investigated by Ca2+ imaging in Wnt1-Cre;R26R-GCaMP3 reporter mice to characterize alterations in the submucosal and the myenteric plexus, which contains the neuronal circuitry controlling gut motility. 16S rDNA sequencing of fecal specimens after oral vancomycin demonstrated significant deviations in microbiota abundance, diversity, and community composition. Vancomycin significantly increased the relative family rank abundance of Akkermansiaceae, Lactobacillaceae, and Enterobacteriaceae at the expense of Lachnospiraceae and Bacteroidaceae. In sharp contrast to neonatal vancomycin exposure, microbiota compositional shifts in weaned animals were associated with slower colonic migrating motor complexes (CMMCs) without mucosal serotonin biosynthesis being altered. The slowing of CMMCs is linked to disruptions in the neurochemistry of the underlying enteric circuitry. This included significant reductions in cholinergic and calbindin+ myenteric neurons, neuronal nitric oxide synthase+ submucosal neurons, neurofilament M+ enteric neurons, and increased proportions of cholinergic submucosal neurons. The antibiotic treatment also increased transmission and responsiveness in myenteric and submucosal neurons that may enhance inhibitory motor pathways, leading to slower CMMCs. Differential vancomycin responses during neonatal and weaning periods in mice highlight the developmental-specific impact of antibiotics on colonic enteric circuitry and motility.

Microbiota stratification identifies disease-specific alterations in neuro-Behçet's disease and multiple sclerosis.

OBJECTIVES: Altered gut microbiota community dynamics are implicated in diverse human diseases including inflammatory disorders such as neuro-Behçet's disease (NBD) and multiple sclerosis (MS). Traditionally, microbiota communities are analysed uniformly across control and disease groups, but recent reports of subsample clustering indicate a potential need for analytical stratification. The objectives of this study are to analyse and compare faecal microbiota community signatures of ethno-geographical, age and gender matched adult healthy controls (HC), MS and NBD individuals. METHODS: Faecal microbiota community compositions in adult HC (n=14), NBD patients (n=13) and MS (n=13) were analysed by 16S rRNA gene sequencing and standard bioinformatics pipelines. Bipartite networks were then used to identify and re-analyse dominant compositional clusters in respective groups. RESULTS: We identified Prevotella and Bacteroides dominated subsample clusters in HC, MS, and NBD cohorts. Our study confirmed previous reports that Prevotella is a major dysbiotic target in these diseases. We demonstrate that subsample stratification is required to identify significant disease-associated microbiota community shifts with increased Clostridiales evident in Prevotella-stratified NBD and Bacteroides-stratified MS patients. CONCLUSIONS: Patient cohort stratification may be needed to facilitate identification of common microbiota community shifts for causation testing in disease.

Aging impairs protective host defenses against Clostridioides (Clostridium) difficile infection in mice by suppressing neutrophil and IL-22 mediated immunity.

BACKGROUND: Morbidity and mortality associated with Clostridioides (formerly Clostridium) difficile infection (CDI) rises progressively with advanced age (≥65 years) due in part to perturbations of the gut microbiota and immune dysfunction. Epidemiological data of community-acquired CDI suggests increased susceptibility may begin earlier during middle-age (45-64 years) but the causation remains unknown. METHODS: Middle-aged (12-14 months) and young (2-4 months) adult mice were infected with C. difficile, and disease severity, gut microbiome and innate immune response were compared. Cytokine reconstitution studies were performed in infected middle-aged mice. RESULTS: Infection of middle-aged mice with C. difficile led to greater disease compared to young controls, which was associated with increases in C. difficile burden and toxin titers, and elevated bacterial translocation. With the exception of an expansion of C. difficile in middle-aged mice, microbiome analysis revealed no age-related differences. In contrast, middle-aged mice displayed a significant defect in neutrophil recruitment to the colon, with diminished levels of innate immune cytokines IL-6, IL-23 and IL-22. Importantly, recombinant IL-22 administration during CDI reduced morbidity and prevented death in middle-aged mice. CONCLUSION: Increased susceptibility to C. difficile occurs in middle-aged mice modeling the community-acquired CDI demographics and is driven by an impaired innate immune response.

Next-Generation Probiotics Targeting Clostridium difficile through Precursor-Directed Antimicrobial Biosynthesis.

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.

Inhibiting Inducible Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice With Colitis.

BACKGROUND & AIMS: Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid- or dextran sodium sulfate-induced colitis and in Il10(-/-) mice. METHODS: Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10(-/-) mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice. RESULTS: Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid-induced colitis and associated bacterial translocation. CONCLUSIONS: Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.

Structural and functional changes within the gut microbiota and susceptibility to Clostridium difficile infection.

Alteration of the gut microbial community structure and function through antibiotic use increases susceptibility to colonization by Clostridium difficile and other enteric pathogens. However, the mechanisms that mediate colonization resistance remain elusive. As the leading definable cause of infectious diarrhea, toxigenic C. difficile represents a burden for patients and health care systems, underscoring the need for better diagnostics and treatment strategies. Next-generation sequence data has increased our understanding of how the gut microbiota is influenced by many factors including diet, disease, aging and drugs. However, a microbial-based biomarker differentiating C. difficile infection from antibiotic-associated diarrhea has not been identified. Metabolomics profiling, which is highly responsive to changes in physiological conditions, have shown promise in differentiating subtle disease phenotypes that exhibit a nearly identical microbiome community structure, suggesting metabolite-based biomarkers may be an ideal diagnostic for identifying patients with CDI. This review focuses on the current understanding of structural and functional changes to the gut microbiota during C. difficile infection obtained from studies assessing the microbiome and metabolome of samples from patients and murine models.

Probiotics as adjunctive therapy for preventing Clostridium difficile infection - What are we waiting for?

With the end of the golden era of antibiotic discovery, the emergence of a new post-antibiotic age threatens to thrust global health and modern medicine back to the pre-antibiotic era. Antibiotic overuse has resulted in the natural evolution and selection of multi-drug resistant bacteria. One major public health threat, Clostridium difficile, is now the single leading cause of hospital-acquired bacterial infections and is by far the most deadly enteric pathogen for the U.S. POPULATION: Due to the high morbidity and mortality and increasing incidence that coincides with antibiotic use, non-traditional therapeutics are ideal alternatives to current treatment methods and also provide an avenue towards prevention. Despite the need for alternative therapies to antibiotics and the safety of most probiotics on the market, researchers are inundated with regulatory issues that hinder the translational science required to push these therapies forward. This review discusses the regulatory challenges of probiotic research, expert opinion regarding the application of probiotics to C. difficile infection and the efficacy of probiotics in preventing this disease.

Administration of probiotic kefir to mice with Clostridium difficile infection exacerbates disease.

Lifeway(®) kefir, a fermented milk product containing 12 probiotic organisms, is reported to show promise as an alternative to fecal microbiota transplantation for recurrent Clostridium difficile infection (CDI). We employed a murine CDI model to study the probiotic protective mechanisms and unexpectedly determined that kefir drastically increased disease severity. Our results emphasize the need for further independent clinical testing of kefir as alternative therapy in recurrent CDI.

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.