The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis

We thank John D. Ward and Lindsey N. Kaydo for their technical support and Dr. Wei Xin for the histological scoring of ileitis severity. ARP is an Assistant Professor of Medicine at CWRU School of Medicine. Metagenomic sequencing was conducted in the laboratory of Dr. Skip Virgin at Washington University, School of Medicine, St. Louis, MO. Raw sequencing data files will be available upon request.Peer reviewedPostprin

Crohn's Disease-Associated Adherent-Invasive <em>Escherichia coli</em> Adhesion Is Enhanced by Exposure to the Ubiquitous Dietary Polysaccharide Maltodextrin

<div><p>Crohn's disease (CD) is associated with intestinal dysbiosis evidenced by an altered microbiome forming thick biofilms on the epithelium. Additionally, adherent-invasive <em>E. coli</em> (AIEC) strains are frequently isolated from ileal lesions of CD patients indicating a potential role for these strains in disease pathogenesis. The composition and characteristics of the host microbiome are influenced by environmental factors, particularly diet. Polysaccharides added to food as emulsifiers, stabilizers or bulking agents have been linked to bacteria-associated intestinal disorders. The escalating consumption of polysaccharides in Western diets parallels an increased incidence of CD during the latter 20^th century. In this study, the effect of a polysaccharide panel on adhesiveness of the CD-associated AIEC strain LF82 was analyzed to determine if these food additives promote disease-associated bacterial phenotypes. Maltodextrin (MDX), a polysaccharide derived from starch hydrolysis, markedly enhanced LF82 specific biofilm formation. Biofilm formation of multiple other <em>E. coli</em> strains was also promoted by MDX. MDX-induced <em>E. coli</em> biofilm formation was independent of polysaccharide chain length indicating a requirement for MDX metabolism. MDX exposure induced type I pili expression, which was required for MDX-enhanced biofilm formation. MDX also increased bacterial adhesion to human intestinal epithelial cell monolayers in a mechanism dependent on type 1 pili and independent of the cellular receptor CEACAM6, suggesting a novel mechanism of epithelial cell adhesion. Analysis of mucosa-associated bacteria from individuals with and without CD showed increased prevalence of <em>malX</em>, a gene essential for MDX metabolism, uniquely in the ileum of CD patients. These findings demonstrate that the ubiquitous dietary component MDX enhances <em>E. coli</em> adhesion and suggests a mechanism by which Western diets rich in specific polysaccharides may promote dysbiosis of gut microbes and contribute to disease susceptibility.</p> </div

Deregulation of intestinal anti-microbial defense by the dietary additive, maltodextrin

Inflammatory bowel disease (IBD) is a complex, multi-factorial disease thought to arise from an inappropriate immune response to commensal bacteria in a genetically susceptible person that results in chronic, cyclical, intestinal inflammation. Dietary and environmental factors are implicated in the initiation and perpetuation of IBD; however, a singular causative agent has not been identified. As of now, the role of environmental priming or triggers in IBD onset and pathogenesis are not well understood, but these factors appear to synergize with other disease susceptibility factors. In previous work, we determined that the polysaccharide dietary additive, maltodextrin (MDX), impairs cellular anti-bacterial responses and suppresses intestinal anti-microbial defense mechanisms. In this addendum, we review potential mechanisms for dietary deregulation of intestinal homeostasis, postulate how dietary and genetic risk factors may combine to result in disease pathogenesis, and discuss these ideas in the context of recent findings related to dietary interventions for IBD

Bacterial strains.

<p>Bacterial strains.</p

MDX promotes biofilm formation of multiple <i>E. coli</i> strains in a process dependent on MDX metabolism.

<p>(A) Specific biofilm formation of a panel of <i>E. coli</i> strains. (B) Micrographs of crystal violet stained biofilms from A. (C) Specific biofilm formation of LF82 in medium supplemented with MDX of different chain lengths. (D) Micrographs of crystal violet stained biofilms from C. Average ±SD shown. *p<0.05, **p<0.01, n.d. = none detected.</p

MDX increases biofilm formation via type 1 pili.

<p>(A) Scanning electron micrographs of LF82 biofilms. Arrowheads indicate bacteria-plastic (white) or inter-bacterial (black) adhesins. (B) Assessment of the LF82 <i>fim</i> operon by PCR to determine type 1 pili expression. (C) Specific biofilm formation of LF82 in the presence or absence of 2% mannose. (D) Micrographs of crystal violet stained biofilms from C. (E) Specific biofilm formation of LF82 and isogenic mutant strains. (F) Micrographs of crystal violet stained biofilms from E. Average ±SD shown. *p<0.05, **p<0.01.</p

MDX-enhanced LF82 adhesion to epithelial cells occurs via a mechanism independent of CEACAM6.

<p>(A) Immunoblots of CEACAM6 expression. (B) Adhesion of LF82 to Caco2 cell lines stably expressing shRNAs. Average ±SD shown. **p<0.01, ***p<0.001 relative to glucose. (C) Immunoblots of CEACAM6 expression. (D) Immunoflurescent confocal micrographs of LF82 adhered to Caco2 cells used in B. Green = LF82, blue = nuclei.</p

Bacteria with the <i>malX</i> gene are more prevalent in the mucosa of ileal CD.

<p>(A) Prevalence of the <i>malX</i> gene normalized to total <i>Eubacterial</i> DNA (<i>Eub</i>) amplified from mucosal samples by qPCR. (B) Prevalence of <i>E. coli</i> 16S DNA (<i>E. coli</i>) as measured in A. Mean indicated with bar. *p<0.0175.</p

MDX selectively enhances LF82 adhesion to Raw264.7 macrophages.

<p>(A) Total amount of Raw264.7 cell-associated LF82. (B) Intracellular LF82 recovered from Raw264.7 cells. (C) Immunofluorescent confocal micrographs of LF82 (red) infected Raw264.7 cells (green). Nuclei  =  blue. Average ±SD shown. **p<0.01, ***p<0.001 relative to glucose.</p

MDX enhances epithelial cell adhesion in a type 1 pili-dependent manner.

<p>(A) Adhesion of LF82 to HT29 monolayers pre-incubated with 2% mannose. (B) Adhesion of LF82 isogenic mutants to HT29 monolayers. Average ±SD shown. *p<0.05, **p<0.01 relative to glucose.</p