Mary Beth Yacyshyn

Human Clostridium difficile infection: inhibition of NHE3 and microbiota profile.

Clostridium difficile infection (CDI) is principally responsible for hospital acquired, antibiotic-induced diarrhea and colitis and represents a significant financial burden on our healthcare system. Little is known about C. difficile proliferation requirements, and a better understanding of these parameters is critical for development of new therapeutic targets. In cell lines, C. difficile toxin B has been shown to inhibit Na(+)/H(+) exchanger 3 (NHE3) and loss of NHE3 in mice results in an altered intestinal environment coupled with a transformed gut microbiota composition. However, this has yet to be established in vivo in humans. We hypothesize that C. difficile toxin inhibits NHE3, resulting in alteration of the intestinal environment and gut microbiota. Our results demonstrate that CDI patient biopsy specimens have decreased NHE3 expression and CDI stool has elevated Na(+) and is more alkaline compared with stool from healthy individuals. CDI stool microbiota have increased Bacteroidetes and Proteobacteria and decreased Firmicutes phyla compared with healthy subjects. In vitro, C. difficile grows optimally in the presence of elevated Na(+) and alkaline pH, conditions that correlate to changes observed in CDI patients. To confirm that inhibition of NHE3 was specific to C. difficile, human intestinal organoids (HIOs) were injected with C. difficile or healthy and CDI stool supernatant. Injection of C. difficile and CDI stool decreased NHE3 mRNA and protein expression compared with healthy stool and control HIOs. Together these data demonstrate that C. difficile inhibits NHE3 in vivo, which creates an altered environment favored by C. difficile.

Human Clostridium difficile infection: altered mucus production and composition.

The majority of antibiotic-induced diarrhea is caused by Clostridium difficile (C. difficile). Hospitalizations for C. difficile infection (CDI) have tripled in the last decade, emphasizing the need to better understand how the organism colonizes the intestine and maintain infection. The mucus provides an interface for bacterial-host interactions and changes in intestinal mucus have been linked host health. To assess mucus production and composition in healthy and CDI patients, the main mucins MUC1 and MUC2 and mucus oligosaccharides were examined. Compared with healthy subjects, CDI patients demonstrated decreased MUC2 with no changes in surface MUC1. Although MUC1 did not change at the level of the epithelia, MUC1 was the primary constituent of secreted mucus in CDI patients. CDI mucus also exhibited decreased N-acetylgalactosamine (GalNAc), increased N-acetylglucosamine (GlcNAc), and increased terminal galactose residues. Increased galactose in CDI specimens is of particular interest since terminal galactose sugars are known as C. difficile toxin A receptor in animals. In vitro, C. difficile is capable of metabolizing fucose, mannose, galactose, GlcNAc, and GalNAc for growth under healthy stool conditions (low Na(+) concentration, pH 6.0). Injection of C. difficile into human intestinal organoids (HIOs) demonstrated that C. difficile alone is sufficient to reduce MUC2 production but is not capable of altering host mucus oligosaccharide composition. We also demonstrate that C. difficile binds preferentially to mucus extracted from CDI patients compared with healthy subjects. Our results provide insight into a mechanism of C. difficile colonization and may provide novel target(s) for the development of alternative therapeutic agents.

Editorial Commentary: The Role of Gut Inflammation in Recurrent Clostridium difficile–Associated Disease

Clostridium difficile is a scourge of nursing-home and hospital-based medicine. The bacterium itself is a grampositive, spore-forming, toxin-producing anaerobic gastrointestinal pathogen.With the development of NAP1 variants, it has developed a clinical impact of some significance with estimated annual costs to the national healthcare system of upward of half a million hospital and nursinghome infections, at least

The Role of Gut Inflammation in Recurrent CDAD

1 billion in patient costs, and 14 000 deaths per year in the United States [1, 2]. The personal costs are not trivial. Recurrent disease is common and life threatening. Antibiotic therapy fails in up to 25% of patients even after 10–15 days of therapy. Despite these statistics, we are only now beginning to understand the reasons for recurrent infection. The article by El Feghaly et al in the current issue of Clinical Infectious Diseases is the first to study cellular inflammatory components involved in persistent diarrhea in Clostridium difficile infection (CDI) [3]. The concept that bacteria persist and benefit from inflammation in their host is not a new one. Another gastrointestinal bacteria that triggers inflammation, as part of a nutritional strategy, is Helicobacter pylori. Much like C. difficile, this bacteria is commonly found among humans and produces a range of response from asymptomatic colonization to complicated, severe disease. The crux of CDI lies with the toxins produced by these bacteria. Clostridium difficile toxins TcdA, Tcdb, and CDT/ binary toxin initiate the host’s humoral response. Kelly et al have shown CDI resolution as well as protective immunity is related to level of serum immunoglobulin G (IgG) antitoxin [4, 5]. However, the antitoxin A IgG level at day 12 of the CDI did not appear to increase the predictability of continued, recurrent CDI in their cohort of patients [5, 6]. Several current clinical trials are examining the protective nature of the humoral response. The host’s immediate cellular response following onset of CDI and TcdA and TcdB secretion is the cytokine release of interleukin 8 (IL-8) from colonic epithelial cells and mobilization of regional immune effector cells (similar to H. pylori in the stomach). Onderdonk et al’s elegant study, with gnotobiotic mice monoassociated with C. difficile, demonstrated that the primary cellular immune response to toxin was edema and polymorphonuclear cell infiltrate, while also demonstrating the effectiveness of vancomycin and the rapid generation of resistance to clindamycin [7]. The resulting intense immune activation (primarily neutrophilic) results in the clinically classic endoscopic findings of the volcano lesion and pseudomembranes. Similar disruption in gut homeostasis is found in acute, active inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, where mucosal pathology is characterized by an influx of innate immune effector cells (primarily neutrophils, macrophages, dendritic cells, and natural killer T cells) producing an adaptive immune response cells including T and B cells. The immune response in these conditions is characterized by an imbalance of T-cell subgroups, including Th1, Th2, Th17, and Treg lymphocytes. With the increased evidence of bacterial dysbiosis and antigen processing in the pathogenesis of IBD, it is not surprising that similar aspects of gut inflammation are shared by both inflammatory diseases of the bowel characterized by gut flora changes and infectious diseases of the intestine characterized by often-times persistent inflammation of the intestine. Received 21 February 2013; accepted 25 February 2013; electronically published 13 March 2013. Correspondence: Bruce Yacyshyn, MD, Department of Medicine, University of Cincinnati School of Medicine, 231 Albert B. Sabin Way, ML 0595, Cincinnati, OH 45267 (bruce. yacyshyn@uc.edu). Clinical Infectious Diseases 2013;56(12):1722–3

Clostridium difficile recurrence is characterized by pro-inflammatory peripheral blood mononuclear cell (PBMC) phenotype

Clostridium difficile is a scourge of nursing-home and hospital-based medicine. The bacterium itself is a grampositive, spore-forming, toxin-producing anaerobic gastrointestinal pathogen.With the development of NAP1 variants, it has developed a clinical impact of some significance with estimated annual costs to the national healthcare system of upward of half a million hospital and nursinghome infections, at least

Gut microbiota and obesity: An opportunity to alter obesity through Fecal Microbiota Transplant (FMT)

1 billion in patient costs, and 14 000 deaths per year in the United States [1, 2]. The personal costs are not trivial. Recurrent disease is common and life threatening. Antibiotic therapy fails in up to 25% of patients even after 10–15 days of therapy. Despite these statistics, we are only now beginning to understand the reasons for recurrent infection. The article by El Feghaly et al in the current issue of Clinical Infectious Diseases is the first to study cellular inflammatory components involved in persistent diarrhea in Clostridium difficile infection (CDI) [3]. The concept that bacteria persist and benefit from inflammation in their host is not a new one. Another gastrointestinal bacteria that triggers inflammation, as part of a nutritional strategy, is Helicobacter pylori. Much like C. difficile, this bacteria is commonly found among humans and produces a range of response from asymptomatic colonization to complicated, severe disease. The crux of CDI lies with the toxins produced by these bacteria. Clostridium difficile toxins TcdA, Tcdb, and CDT/ binary toxin initiate the host’s humoral response. Kelly et al have shown CDI resolution as well as protective immunity is related to level of serum immunoglobulin G (IgG) antitoxin [4, 5]. However, the antitoxin A IgG level at day 12 of the CDI did not appear to increase the predictability of continued, recurrent CDI in their cohort of patients [5, 6]. Several current clinical trials are examining the protective nature of the humoral response. The host’s immediate cellular response following onset of CDI and TcdA and TcdB secretion is the cytokine release of interleukin 8 (IL-8) from colonic epithelial cells and mobilization of regional immune effector cells (similar to H. pylori in the stomach). Onderdonk et al’s elegant study, with gnotobiotic mice monoassociated with C. difficile, demonstrated that the primary cellular immune response to toxin was edema and polymorphonuclear cell infiltrate, while also demonstrating the effectiveness of vancomycin and the rapid generation of resistance to clindamycin [7]. The resulting intense immune activation (primarily neutrophilic) results in the clinically classic endoscopic findings of the volcano lesion and pseudomembranes. Similar disruption in gut homeostasis is found in acute, active inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, where mucosal pathology is characterized by an influx of innate immune effector cells (primarily neutrophils, macrophages, dendritic cells, and natural killer T cells) producing an adaptive immune response cells including T and B cells. The immune response in these conditions is characterized by an imbalance of T-cell subgroups, including Th1, Th2, Th17, and Treg lymphocytes. With the increased evidence of bacterial dysbiosis and antigen processing in the pathogenesis of IBD, it is not surprising that similar aspects of gut inflammation are shared by both inflammatory diseases of the bowel characterized by gut flora changes and infectious diseases of the intestine characterized by often-times persistent inflammation of the intestine. Received 21 February 2013; accepted 25 February 2013; electronically published 13 March 2013. Correspondence: Bruce Yacyshyn, MD, Department of Medicine, University of Cincinnati School of Medicine, 231 Albert B. Sabin Way, ML 0595, Cincinnati, OH 45267 (bruce. yacyshyn@uc.edu). Clinical Infectious Diseases 2013;56(12):1722–3

Clostridium difficile toxins A and B decrease intestinal SLC26A3 protein expression

Clostridium difficile infection (CDI) is a prevalent nosocomial and increasingly community-acquired problem. Little is known about the productive cellular response in patients. We used flow cytometry to define inflammatory (Th1 and Th17) and regulatory [Foxp3(+) T-regulatory (Treg)] cells present in circulating peripheral blood mononuclear cells (PBMC) from CDI patients. We consented 67 inpatients that tested either positive or negative for CDI and 16 healthy controls and compared their PBMC phenotypes. PBMC were collected, isolated, and stained for CD3, CD8 and either IL17 (Th17), IFN-γ (Th1) or Foxp3 (Treg) and analysed using flow cytometry. Twenty thousand events were collected in the lymphocyte gate (gate 1) and T-cell phenotypes were defined. CDI patients who clear the primary initial infection have greater numbers of non-CD3 PBMC. CDI patients who develop recurrence of CDI have a greater percentage of CD3(+)CD8(+), CD3(+)CD4(+)Foxp3 and fewer low granular CD3(-)Foxp3(+) PBMC. These patients have greater numbers of IFN-γ-producing lymphocytes, as well as PBMC phenotypes represented by increased IFN-γ- and IL17-co-expressing CD4(+)CD3(+). This initial pro-inflammatory phenotype decreases with repeated recurrence, demonstrating importance of timing of sample collection and history of symptoms. Patients with a history of recurrence had increased Foxp3(+)CD3(+)CD4(+) and IL17(+)CD3(+)CD4(+) populations. Hence, CDI recurrence is hallmarked by greater numbers of circulating CD3(+) lymphocytes skewed towards a Th1/Th17 inflammatory population as well as possible immune plasticity (Th17/Treg).

Metabolic Profiling of Human In-patient Stools: The Influence of C. difficile Infection and Recurrence

Obesity is a global pandemic with immense health consequences for individuals and societies. Multiple factors, including environmental influences and genetic predispositions, are known to affect the development of obesity. Despite an increasing understanding of the factors driving the obesity epidemic, therapeutic interventions to prevent or reverse obesity are limited in their impact. Manipulation of the human gut microbiome provides a new potential therapeutic approach in the fight against obesity. Specific gut bacteria and their metabolites are known to affect host metabolism and feeding behaviour, and dysbiosis of this biosystem may lead to metabolic syndrome. Potential therapies to alter the gut microbiota to treat obesity include dietary changes, supplementation of the diet with probiotic organisms and prebiotic compounds that influence bacterial growth, and the use of faecal microbiota transplant, in which gut microbiota from healthy individuals are introduced into the gut. In this review, we examine the growing scientific evidence supporting the mechanisms by which the human gut microbiota may influence carbohydrate metabolism and obesity, and the various possible therapies that may utilize the gut microbiota to help correct metabolic dysfunction.

Sa1276 Altered Mucus Oligosaccharide Composition in Human C. difficile Infection

Clostridium difficile infection (CDI) is the primary cause of nosocomial diarrhea in the United States. Although C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile-associated diarrhea remains poorly understood. Studies have shown that a decrease in both NHE3 (Na+/H+ exchanger) and DRA (downregulated in adenoma, Cl-/[Formula: see text] exchanger), resulting in decreased electrolyte absorption, is implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and downregulated in patients with CDI, but the role of DRA in CDI remains unknown. In the current studies, we examined the effects of C. difficile toxins TcdA and TcdB on DRA protein and mRNA levels in intestinal epithelial cells (IECs). Our data demonstrated that DRA protein levels were significantly reduced in response to TcdA and TcdB in IECs in culture. This effect was also specific to DRA, as NHE3 and PAT-1 (putative anion transporter 1) protein levels were unaffected by TcdA and TcdB. Additionally, purified TcdA and TcdA + TcdB, but not TcdB, resulted in a decrease in colonic DRA protein levels in a toxigenic mouse model of CDI. Finally, patients with recurrent CDI also exhibited significantly reduced expression of colonic DRA protein. Together, these findings indicate that C. difficile toxins markedly downregulate intestinal expression of DRA which may contribute to the diarrheal phenotype of CDI. NEW & NOTEWORTHY Our studies demonstrate, for the first time, that C. difficile toxins reduce DRA protein, but not mRNA, levels in intestinal epithelial cells. These findings suggest that a downregulation of DRA may be a critical factor in C. difficile infection-associated diarrhea.

Tu1254 Glitazones in the Treatment of Angioectasias: Regulating the TGF-β Signaling Pathway, PPARγ, Angiogenesis and Wound Repair Mechanisms

Abstract Background Initial C. difficile infection (CDI) and recurrence is dependent on host susceptibility. Hospital patients have a diverse range of other comorbidities and concomitant medications that cannot be duplicated in animal models. We hypothesize that the intersection of the luminal biome and host is represented by the metabolites. Their contribution facilitates CDI clearance or recurrence. Methods Discarded stool samples from 4 groups of in-patients (UC IRB 2014–8646) were used. Case controls (CC,n = 20) have diarrhea but are CDI-. Initial CDI+ (I, n = 20) clear infection. Initial CDI+ who become recurrent (IR, n = 18) have samples from initial infection. Known CDI+ Recurrent (KR, n = 10) have samples from second to fourth CDI. Stools were kept at 4°C until aliquoted, weighed and frozen at -80°C. We lyophilized each aliquot and performed metabolome analysis on 50 mg of dry sample. The NMR-Based Metabolomics Core used 1D NOESY and 1D CPMG NMR experiments to generate raw metabolic concentrations. Metabolite IDs were validated with 2D 1H-1H TOCSY and 2D 1H-13C HSQC experiments. Results 46 metabolites could be definitively identified and compared. Analysis of raw metabolite mM concentrations in CDI+ stools demonstrated more acetate (median ± median absolute deviation) (CC: 1.63 ± 1.54, I: 5.20 ± 3.21, IR: 4.21 ± 1.46, KR: 2.4 ± 2.15) and butyrate (CC: .1195 ± 0.1185, I: 0.4992 ± 0.3721, IR: 0.4196 ± 0.3832, KR: 0.2279 ± 0.2109). This suggests inability of host’s colon to productively use these SCFA. Levels of stool propionate appear to be linked to the recurrent environment (CC: 0.7620 ± 0.6562, I: 1.5162 ± 0.9189, IR: 0.6928 ± 0.5369, KR: 0.4490 ± 0.3406). Tryptophan catabolism has been linked to the CDI neutrophil response in mice. We detected decreased tryptophan in KR stool as well as lower numbers of circulating neutrophils (KR: 0.007 ± 0.007, CC: 0.0229 ± 0.0199, I: 0.0163 ± 0.0163, IR: 0.0246 ± 0.0201). This suggests altered host luminal metabolism can lead to biosystem changes Conclusion CDI in the human biosystem is complex with contribution of host comorbidities and medications. The luminal interface between biome and host is the metabolome. NMR metabolomics of human in-patient stools can provide a more relevant understanding of what conditions are necessary for CDI, its clearance and recurrence. Defeat of this costly infection needs alternative approaches. Disclosures M. B. Yacyshyn, Merck: Grant Investigator, Research grant; 
 B. Yacyshyn, Merck: Grant Investigator and Speaker’s Bureau, Research grant and Speaker honorarium