Elaine O. Petrof

Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut

BackgroundFecal bacteriotherapy (‘stool transplant’) can be effective in treating recurrent Clostridium difficile infection, but concerns of donor infection transmission and patient acceptance limit its use. Here we describe the use of a stool substitute preparation, made from purified intestinal bacterial cultures derived from a single healthy donor, to treat recurrent C. difficile infection that had failed repeated standard antibiotics. Thirty-three isolates were recovered from a healthy donor stool sample. Two patients who had failed at least three courses of metronidazole or vancomycin underwent colonoscopy and the mixture was infused throughout the right and mid colon. Pre-treatment and post-treatment stool samples were analyzed by 16 S rRNA gene sequencing using the Ion Torrent platform.ResultsBoth patients were infected with the hyper virulent C. difficile strain, ribotype 078. Following stool substitute treatment, each patient reverted to their normal bowel pattern within 2 to 3 days and remained symptom-free at 6 months. The analysis demonstrated that rRNA sequences found in the stool substitute were rare in the pre-treatment stool samples but constituted over 25% of the sequences up to 6 months after treatment.ConclusionThis proof-of-principle study demonstrates that a stool substitute mixture comprising a multi-species community of bacteria is capable of curing antibiotic-resistant C. difficile colitis. This benefit correlates with major changes in stool microbial profile and these changes reflect isolates from the synthetic mixture.Trial registrationClinical trial registration number: CinicalTrials.gov NCT01372943

16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis

Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.

From stool transplants to next-generation microbiota therapeutics.

The epidemic of Clostridium difficile infection fueled by new virulent strains of the organism has led to increased use of fecal microbiota transplantation (FMT). The procedure is effective for even the most desperate cases after failure of multiple courses of antibiotics. The approach recognizes microbiota to be integral to normal human physiology, and microbiota being used in FMT represents a new class of therapeutics. Imbalance in the composition and altered activity of the microbiota are associated with many diseases. Consequently, there is growing interest in applying FMT to non-C difficile indications. However, this may succeed only if microbiota therapeutics are developed systematically, based on mechanistic understanding, and applying up-to-date principles of microbial ecology. We discuss 2 pathways in the development of this new therapeutic class: whole microbial communities separated from donor stool and an assembly of specific fecal microorganisms grown in vitro.

Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis

Objective Vitamin D and the vitamin D receptor (VDR) appear to be important immunological regulators of inflammatory bowel diseases (IBD). Defective autophagy has also been implicated in IBD, where interestingly, polymorphisms of genes such as ATG16L1 have been associated with increased risk. Although vitamin D, the microbiome and autophagy are all involved in pathogenesis of IBD, it remains unclear whether these processes are related or function independently. Design We investigated the effects and mechanisms of intestinal epithelial VDR in healthy and inflamed states using cell culture models, a conditional VDR knockout mouse model (VDRΔIEC), colitis models and human samples. Results Absence of intestinal epithelial VDR affects microbial assemblage and increases susceptibility to dextran sulfate sodium-induced colitis. Intestinal epithelial VDR downregulates expressions of ATG16L1 and lysozyme, and impairs antimicrobial function of Paneth cells. Gain and loss-of-function assays showed that VDR levels regulate ATG16L1 and lysozyme at the transcriptional and translational levels. Moreover, low levels of intestinal epithelial VDR correlated with reduced ATG16L1 and representation by intestinal Bacteroides in patients with IBD. Administration of the butyrate (a fermentation product of gut microbes) increases intestinal VDR expression and suppresses inflammation in a colitis model. Conclusions Our study demonstrates fundamental relationship between VDR, autophagy and gut microbial assemblage that is essential for maintaining intestinal homeostasis, but also in contributing to the pathophysiology of IBD. These insights can be leveraged to define therapeutic targets for restoring VDR expression and function.

Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells.

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA−) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.

Bacteria-Free Solution Derived from Lactobacillus plantarum Inhibits Multiple NF-KappaB Pathways and Inhibits Proteasome Function

Background: Bacteria play a role in inflammatory bowel disease and other forms of intestinal inflammation. Although much attention has focused on the search for a pathogen or inciting inflammatory bacteria, another possibility is a lack of beneficial bacteria that normally confer anti‐inflammatory properties in the gut. The purpose of this study was to determine whether normal commensal bacteria could inhibit inflammatory pathways important in intestinal inflammation. Methods: Conditioned media from Lactobacillus plantarum (Lp‐CM) and other gut bacteria was used to treat intestinal epithelial cell (YAMC) and macrophage (RAW 264.7) or primary culture murine dendritic cells. NF‐&kgr;B was activated through TNF‐Receptor, MyD88‐dependent and ‐independent pathways and effects of Lp‐CM on the NF‐&kgr;B pathway were assessed. NF‐&kgr;B binding activity was measured using ELISA and EMSA. 1&kgr;B expression was assessed by Western blot analysis, and proteasome activity determined using fluorescence‐based proteasome assays. MCP‐1 release was determined by ELISA. Results: Lp‐CM inhibited NF‐&kgr;B binding activity, degradation of I&kgr;B&agr; and the chymotrypsin‐like activity of the proteasome. Moreover, Lp‐CM directly inhibited the activity of purified mouse proteasomes. This effect was specific, since conditioned media from other bacteria had no inhibitory effect. Unlike other proteasome inhibitors, Lp‐CM was not toxic in cell death assays. Lp‐CM inhibited MCP‐1 release in all cell types tested. Conclusions: These studies confirm, and provide a mechanism for, the anti‐inflammatory effects of the probiotic and commensal bacterium Lactobacillus plantarum. The use of bacteria‐free Lp‐CM provides a novel strategy for treatment of intestinal inflammation which would eliminate the risk of bacteremia reported with conventional probiotics. Inflamm Bowel Dis 2009

Erythropoietin Protects Intestinal Epithelial Barrier Function and Lowers the Incidence of Experimental Neonatal Necrotizing Enterocolitis

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.

Fecal microbiota transplantation: in perspective.

There has been increasing interest in understanding the role of the human gut microbiome to elucidate the therapeutic potential of its manipulation. Fecal microbiota transplantation (FMT) is the administration of a solution of fecal matter from a donor into the intestinal tract of a recipient in order to directly change the recipient’s gut microbial composition and confer a health benefit. FMT has been used to successfully treat recurrent Clostridium difficile infection. There are preliminary indications to suggest that it may also carry therapeutic potential for other conditions such as inflammatory bowel disease, obesity, metabolic syndrome, and functional gastrointestinal disorders.

Probiotics and Gastrointestinal Disease: Clinical Evidence and Basic Science

Our intestinal microbiota serve many roles vital to the normal daily function of the human gastrointestinal tract. Many probiotics are derived from our intestinal bacteria, and have been shown to provide clinical benefit in a variety of gastrointestinal conditions. Current evidence indicates that probiotic effects are strain-specific, they do not act through the same mechanisms, and nor are all probiotics indicated for the same health conditions. However, they do share several common features in that they exert anti-inflammatory effects, they employ different strategies to antagonize competing microorganisms, and they induce cytoprotective changes in the host either through enhancement of barrier function, or through the upregulation of cytoprotective host proteins. In this review we focus on a few selected probiotics - a bacterial mixture (VSL#3), a Gram-negative probiotic (E. coli Nissle 1917), two Gram-positive probiotic bacteria (LGG, L. reuteri), and a yeast probiotic (S. boulardii) - for which sound clinical and mechanistic data is available. Safety of probiotic formulations is also discussed.

Mother's Milk-Induced Hsp70 Expression Preserves Intestinal Epithelial Barrier Function in an Immature Rat Pup Model

Preterm infants face many challenges in transitioning from the in utero to extrauterine environment while still immature. Failure of the preterm gut to successfully mature to accommodate bacteria and food substrate leads to significant morbidity such as neonatal necrotizing enterocolitis. The intestinal epithelial barrier plays a critical role in gut protection. Heat shock protein 70 (Hsp70) is an inducible cytoprotective molecule shown to protect the intestinal epithelium in adult models. To investigate the hypothesis that Hsp70 may be important for early protection of the immature intestine, Hsp70 expression was evaluated in intestine of immature rat pups. Data demonstrate that Hsp70 is induced by exposure to mothers milk. Hsp70 is found in mothers milk, and increased Hsp70 transcription is induced by mothers milk. This Hsp70 colocalizes with the tight junction protein ZO-1. Mothers milk-induced Hsp70 may contribute to maintenance of barrier function in the face of oxidant stress. Further understanding of the means by which mothers milk increases Hsp70 in the ileum will allow potential means of strengthening the intestinal barrier in at-risk preterm infants.