Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria play a key role; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns which may be critical to the development of this disease. Fecal samples from twenty preterm infants, ten with NEC and ten matched controls (including four twin pairs) were obtained from patients in a single site Level III neonatal intensive care unit. Bacterial DNA from individual fecal samples were 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 which may accentuate the impact of single dominant microorganisms favored by empiric and wide-spread use of antibiotics.

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

IMPORTANCE Clostridium difficile infection (CDI) is a major burden in health care and community settings. CDI recurrence is of particular concern because of limited treatment options and associated clinical and infection control issues. Fecal microbiota transplantation (FMT) is a promising, but not readily available, intervention. OBJECTIVE To determine whether frozen-and-thawed (frozen, experimental) FMT is noninferior to fresh (standard) FMT in terms of clinical efficacy among patients with recurrent or refractory CDI and to assess the safety of both types of FMT. DESIGN, SETTING, AND PARTICIPANTS Randomized, double-blind, noninferiority trial enrolling 232 adults with recurrent or refractory CDI, conducted between July 2012 and September 2014 at 6 academic medical centers in Canada. INTERVENTIONS Patients were randomly allocated to receive frozen (n = 114) or fresh (n = 118) FMT via enema. MAIN OUTCOMES AND MEASURES The primary outcome measures were clinical resolution of diarrhea without relapse at 13 weeks and adverse events. Noninferiority margin was set at 15%. RESULTS A total of 219 patients (n = 108 in the frozen FMT group and n = 111 in the fresh FMT group) were included in the modified intention-to-treat (mITT) population and 178 (frozen FMT: n = 91, fresh FMT: n = 87) in the per-protocol population. In the per-protocol population, the proportion of patients with clinical resolution was 83.5% for the frozen FMT group and 85.1% for the fresh FMT group (difference, −1.6% [95% CI,-10.5% to ϱ]; P = .01 for noninferiority). In the mITT population the clinical resolution was 75.0% for the frozen FMT group and 70.3% for the fresh FMT group (difference, 4.7% [95% CI,-5.2% to ϱ]; P < .001 for noninferiority). There were no differences in the proportion of adverse or serious adverse events between the treatment groups. CONCLUSIONS AND RELEVANCE Among adults with recurrent or refractory CDI, the use of frozen compared with fresh FMT did not result in worse proportion of clinical resolution of diarrhea. Given the potential advantages of providing frozen FMT, its use is a reasonable option in this setting.

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 down-regulates expressions of ATG16L1 and lysozyme, and impairs anti-microbial 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 IBD patients. 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.

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AvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-B (NF-B) pathway, IB␣ and ␤-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cellfree system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IB␣ and ␤-catenin in epithelial cells. AvrA deubiquitinates IB␣, which blocks its degradation and leads to the inhibition of NF-B activation. Target genes of the NF-B pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of ␤-catenin. Target genes of the ␤-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased ␤-catenin further negatively regulates the NF-B pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-B and ␤-catenin pathways. (Am J Pathol

Conditioned media from the probiotic Lactobacillus GG (LGG-CM) induce heat shock protein (Hsp) expression in intestinal epithelial cells. LGG-CM induces both Hsp25 and Hsp72 in a time- and concentration-dependent manner. These effects are mediated by a low-molecular-weight peptide that is acid and heat stable. DNA microarray experiments demonstrate that Hsp72 is one of the most highly upregulated genes in response to LGG-CM treatment. Real-time PCR and electrophoretic mobility shift assay confirm that regulation of Hsp induction is at least in part transcriptional in nature, involving heat shock factor-1. Although Hsps are not induced for hours after exposure, transient exposure to LGG-CM is sufficient to initiate the signal for Hsp induction, suggesting that signal transduction pathways may be involved. Experiments confirm that LGG-CM modulates the activity of certain signaling pathways in intestinal epithelial cells by activating MAP kinases. Inhibitors of p38 and JNK block the expression of Hsp72 normally induced by LGG-CM. Functional studies indicate that LGG-CM treatment of gut epithelial cells protects them from oxidant stress, perhaps by preserving cytoskeletal integrity. By inducing the expression of cytoprotective Hsps in gut epithelial cells, and by activating signal transduction pathways, the peptide product(s) secreted by LGG may contribute to the beneficial clinical effects attributed to this probiotic.

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.