Soluble Non-Starch Polysaccharides From Plantain (Musa x paradisiaca L.) Diminish Epithelial Impact of Clostridioides difficile.

Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea. Adhesion of this Gram-positive pathogen to the intestinal epithelium is a crucial step in CDI, with recurrence and relapse of disease dependent on epithelial interaction of its endospores. Close proximity, or adhesion of, hypervirulent strains to the intestinal mucosa are also likely to be necessary for the release of C. difficile toxins, which when internalized, result in intestinal epithelial cell rounding, damage, inflammation, loss of barrier function and diarrhoea. Interrupting these C. difficile-epithelium interactions could therefore represent a promising therapeutic strategy to prevent and treat CDI. Intake of dietary fibre is widely recognised as being beneficial for intestinal health, and we have previously shown that soluble non-starch polysaccharides (NSP) from plantain banana (Musa spp.), can block epithelial adhesion and invasion of a number of gut pathogens, such as E. coli and Salmonellae. Here, we assessed the action of plantain NSP, and a range of alternative soluble plant fibres, for inhibitory action on epithelial interactions of C. difficile clinical isolates, purified endospore preparations and toxins. We found that plantain NSP possessed ability to disrupt epithelial adhesion of C. difficile vegetative cells and spores, with inhibitory activity against C. difficile found within the acidic (pectin-rich) polysaccharide component, through interaction with the intestinal epithelium. Similar activity was found with NSP purified from broccoli and leek, although seen to be less potent than NSP from plantain. Whilst plantain NSP could not block the interaction and intracellular action of purified C. difficile toxins, it significantly diminished the epithelial impact of C. difficile, reducing both bacteria and toxin induced inflammation, activation of caspase 3/7 and cytotoxicity in human intestinal cell-line and murine intestinal organoid cultures. Dietary supplementation with soluble NSP from plantain may therefore confer a protective effect in CDI patients by preventing adhesion of C. difficile to the mucosa, i.e. a "contrabiotic" effect, and diminishing its epithelial impact. This suggests that plantain soluble dietary fibre may be a therapeutically effective nutritional product for use in the prevention or treatment of CDI and antibiotic-associated diarrhoea.

Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation.

BACKGROUND: Perturbations of the intestinal microbiome, termed dysbiosis, are linked to intestinal inflammation. Isolation of adherent-invasive Escherichia coli (AIEC) from intestines of patients with Crohn's disease (CD), dogs with granulomatous colitis, and mice with acute ileitis suggests these bacteria share pathoadaptive virulence factors that promote inflammation. METHODS: To identify genes associated with AIEC, we sequenced the genomes of phylogenetically diverse AIEC strains isolated from people with CD (4), dogs with granulomatous colitis (2), and mice with ileitis (2) and 1 non-AIEC strain from CD ileum and compared them with 38 genome sequences of E. coli and Shigella. We then determined the prevalence of AIEC-associated genes in 49 E. coli strains from patients with CD and controls and correlated genotype with invasion of intestinal epithelial cells, persistence within macrophages, AIEC pathotype, and growth in standardized conditions. RESULTS: Genes encoding propanediol utilization (pdu operon) and iron acquisition (yersiniabactin, chu operon) were overrepresented in AIEC relative to nonpathogenic E. coli. PduC (propanediol dehydratase) was enriched in CD-derived AIEC, correlated with increased cellular invasion, and persistence in vitro and was increasingly expressed in fucose-containing media. Growth of AIEC required iron, and the presence of chuA (heme acquisition) correlated with persistence in macrophages. CD-associated AIEC with lpfA 154 (long polar fimbriae) demonstrated increased invasion of epithelial cells and translocation across M cells. CONCLUSIONS: Our findings provide novel insights into the genetic basis of the AIEC pathotype, supporting the concept that AIEC are equipped to exploit and promote intestinal inflammation and reveal potential targets for intervention against AIEC and inflammation-associated dysbiosis.

Dietary supplementation with soluble plantain non-starch polysaccharides inhibits intestinal invasion of Salmonella Typhimurium in the chicken.

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1-99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5-10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64-81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75-90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis.

Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer.

OBJECTIVE: Colonic mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates. DESIGN: A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome). RESULTS: 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin. CONCLUSIONS: IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.

Hypothesis: Increased consumption of emulsifiers as an explanation for the rising incidence of Crohn's disease.

Crohn's disease (CD) incidence has increased over the past fifty years but the explanation is unclear. CD can be brought into remission by liquid enteral feeding, but the mechanism for this response is unknown. We suggest that consumption of emulsifiers in processed foods may promote CD by increasing bacterial translocation. This is supported by evidence that (i) geographical variation in CD correlates with emulsifier consumption as does the increasing incidence of CD in Japan; (ii) although CD incidence also correlates with fat consumption, the response to enteral feeding is not affected by the fat content of the feed and (iii) very small concentrations of the emulsifier polysorbate 80 enhance bacterial translocation across intestinal epithelia. Undigested emulsifiers may increase bacterial translocation, particularly in the small intestine where the mucus layer is discontinuous. The hypothesis should be testable by trials of enteral feeding with/without emulsifiers.

Soluble plantain fibre blocks adhesion and M-cell translocation of intestinal pathogens.

Dietary fibres may have prebiotic effects mediated by promotion of beneficial bacteria. This study explores the possibility that soluble plant fibre may also improve health by inhibiting epithelial adhesion and translocation by pathogenic bacteria. We have focussed on soluble non-starch polysaccharide (NSP) from plantain bananas (Musa spp.) which previous studies showed to be particularly effective at blocking Escherichia coli epithelial adherence. In vitro and ex vivo studies assessed the ability of plantain NSP to inhibit epithelial cell adhesion and invasion of various bacterial pathogens, and to inhibit their translocation through microfold (M)-cells and human Peyer's patches mounted in Ussing chambers. Plantain NSP showed dose-related inhibition of epithelial adhesion and M-cell translocation by a range of pathogens. At 5mg/ml, a concentration readily achievable in the gut lumen, plantain NSP inhibited adhesion to Caco2 cells by Salmonella Typhimurium (85.0 ± 8.2%, P<.01), Shigella sonnei (46.6 ± 29.3%, P<.01), enterotoxigenic E.coli (56.1 ± 23.7%, P<.05) and Clostridium difficile (67.6 ± 12.3%, P<.001), but did not inhibit adhesion by enteropathogenic E.coli. Plantain NSP also inhibited invasion of Caco2 cells by S. Typhimurium (80.2 ± 9.7%) and Sh. sonnei (46.7 ± 13.4%); P<.01. Plantain NSP, 5mg/ml, also inhibited translocation of S. Typhimurium and Sh. sonnei across M-cells by 73.3 ± 5.2% and 46.4 ± 7.7% respectively (P<.05). Similarly, S. Typhimurium translocation across Peyer's patches was reduced 65.9 ± 8.1% by plantain NSP (P<.01). Soluble plantain fibre can block epithelial adhesion and M-cell translocation of intestinal pathogens. This represents an important novel mechanism by which soluble dietary fibres can promote intestinal health and prevent infective diarrhoea.

Translocation of Crohn's disease Escherichia coli across M-cells: contrasting effects of soluble plant fibres and emulsifiers.

BACKGROUND: Crohn's disease is common in developed nations where the typical diet is low in fibre and high in processed food. Primary lesions overlie Peyer's patches and colonic lymphoid follicles where bacterial invasion through M-cells occurs. We have assessed the effect of soluble non-starch polysaccharide (NSP) and food emulsifiers on translocation of Escherichia coli across M-cells. METHODS: To assess effects of soluble plant fibres and food emulsifiers on translocation of mucosa-associated E coli isolates from Crohn's disease patients and from non-Crohn's controls, we used M-cell monolayers, generated by co-culture of Caco2-cl1 and Raji B cells, and human Peyer's patches mounted in Ussing chambers. RESULTS: E coli translocation increased across M-cells compared to parent Caco2-cl1 monocultures; 15.8-fold (IQR 6.2-32.0) for Crohn's disease E coli (N=8) and 6.7-fold (IQR 3.7-21.0) for control isolates (N=5). Electron microscopy confirmed E coli within M-cells. Plantain and broccoli NSP markedly reduced E coli translocation across M-cells at 5 mg/ml (range 45.3-82.6% inhibition, p<0.01); apple and leek NSP had no significant effect. Polysorbate-80, 0.01% vol/vol, increased E coli translocation through Caco2-cl1 monolayers 59-fold (p<0.05) and, at higher concentrations, increased translocation across M-cells. Similarly, E coli translocation across human Peyer's patches was reduced 45±7% by soluble plantain NSP (5 mg/ml) and increased 2-fold by polysorbate-80 (0.1% vol/vol). CONCLUSIONS: Translocation of E coli across M-cells is reduced by soluble plant fibres, particularly plantain and broccoli, but increased by the emulsifier Polysorbate-80. These effects occur at relevant concentrations and may contribute to the impact of dietary factors on Crohn's disease pathogenesis.

Replication of Colonic Crohn's Disease Mucosal Escherichia coli Isolates within Macrophages and Their Susceptibility to Antibiotics.

There is increasing evidence that Escherichia coli organisms are important in Crohn's disease (CD) pathogenesis. In CD tissue they are found within macrophages, and the adherent-invasive CD ileal E. coli isolate LF82 can replicate inside macrophage phagolysosomes. This study investigates replication and antibiotic susceptibility of CD colonic E. coli isolates inside macrophages. Replication of CD colonic E. coli within J774-A1 murine macrophages and human monocyte-derived macrophages (HMDM) was assessed by culture and lysis after gentamicin killing of noninternalized bacteria and verified by electron microscopy (EM). All seven CD colonic isolates tested replicated within J774-A1 macrophages by 3 h (6.36-fold +/- 0.7-fold increase; n = 7 isolates) to a similar extent to CD ileal E. coli LF82 (6.8-fold +/- 0.8-fold) but significantly more than control patient isolates (5.2-fold +/- 0.25-fold; n = 6; P = 0.006) and E. coli K-12 (1.0-fold +/- 0.1-fold; P < 0.0001). Replication of CD E. coli HM605 within HMDM (3.9-fold +/- 0.7-fold) exceeded that for K-12 (1.4-fold +/- 0.2-fold; P = 0.03). EM showed replicating E. coli within macrophage vacuoles. Killing of HM605 within J774-A1 macrophages following a 3-h incubation with antibiotics at published peak serum concentrations (C(max)) was as follows: for ciprofloxacin, 99.5% +/- 0.2%; rifampin, 85.1% +/- 6.6%; tetracycline, 62.8% +/- 6.1%; clarithromycin, 62.1% +/- 5.6% (all P < 0.0001); sulfamethoxazole, 61.3% +/- 7.0% (P = 0.0007); trimethoprim, 56.3% +/- 3.4% (P < 0.0001); and azithromycin, 41.0% +/- 10.5% (P = 0.03). Ampicillin was not effective against intracellular E. coli. Triple antibiotic combinations were assessed at 10% C(max), with ciprofloxacin, tetracycline, and trimethoprim causing 97% +/- 0.0% killing versus 86% +/- 2.0% for ciprofloxacin alone. Colonic mucosa-associated E. coli, particularly CD isolates, replicate within macrophages. Clinical trials are indicated to assess the efficacy of a combination antibiotic therapy targeting intramacrophage E. coli.

Characterization of epithelial IL-8 response to inflammatory bowel disease mucosal E. coli and its inhibition by mesalamine.

BACKGROUND: Mucosally adherent E. coli are found in inflammatory bowel disease (IBD) and colon cancer. They promote release of the proinflammatory cytokine interleukin-8 (IL-8). We explored mechanisms for this release and its inhibition by drugs. METHODS: IL-8 release from colon epithelial cells in response to mucosal E. coli isolates from IBD, colon cancer, and controls was characterized at the cellular and molecular level. RESULTS: IL-8 response of HT29 cells was greater with Crohn's disease (689 +/- 298 [mean +/- SD] pg IL-8/mL at 4 hours, n = 7) and colon cancer isolates (532 +/- 415 pg/mL, n = 14) than with ulcerative colitis (236 +/- 58 pg/mL, n = 6) or control isolates (236 +/- 100 pg/mL, n = 6, P < 0.0001). Bacterial supernatants contained shed flagellin that triggered IL-8 release. For whole bacteria the IL-8 response to E. coli that agglutinate red blood cells (548 +/- 428 pg IL-8/mL, n = 16), a function that correlates with epithelial invasion, was greater than for nonhemagglutinators (281 +/- 253 pg/mL, n = 17; P < 0.0001). This was particularly marked among E. coli that, although flagellate, could not release IL-8 from TLR5-transfected HEK293 cells. IL-8 release was mediated by extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and inhibited by mesalamine, but not hydrocortisone, at therapeutic concentrations. CONCLUSIONS: Mucosa-associated E. coli shed flagellin that elicits epithelial IL-8 release but this may only become relevant when the mucosal barrier is weakened to expose basolateral TLR5. Adherent and invasive IBD and colon cancer E. coli isolates also elicit a flagellin-independent IL-8 response that may be relevant when the mucosal barrier is intact. The IL-8 release is MAPK-dependent and inhibited by mesalamine.

Microbial mannan inhibits bacterial killing by macrophages: a possible pathogenic mechanism for Crohn's disease.

BACKGROUND & AIMS: Crohn's disease (CD) is mimicked by inherited phagocyte disorders and is associated with circulating antibodies against yeast mannan (anti-Saccharomyces cerevisiae antibody; ASCA). We speculated that mannans might impair phagocyte function. METHODS: S cerevisiae mannan was assessed for its effects on human peripheral blood neutrophils, adherent monocytes, and monocyte-derived macrophages (MDM). RESULTS: Mannan caused dose-related increased survival of CD Escherichia coli HM605 within adherent monocytes from 24% +/- 10.5% (control) to 114% +/- 22.7% with mannan 1 mg/mL at 2 hours (mean +/- SEM, n = 9; P = .0002). Electron microscopy showed E coli HM605 surviving and probably replicating within macrophage vesicles. Mannan (1 mg/mL) inhibited the respiratory burst in neutrophils and monocytes (both P = .002) and bacterial killing within MDM (P < .001). E coli survival was increased within macrophages from TLR4(-/-) (126% +/- 3.5% survival at 2 hours) and MyD88(-/-) (134.8% +/- 6.5%) mice compared with wild-type mice (both P < .0001). Mannan had no additional effect, showing that TLR4 and MyD88 are involved in bacterial killing by macrophages and its inhibition by mannan. Putative CD-associated micro-organisms were screened for the ASCA mannan epitope by Galanthus nivalis lectin (GNA) blotting. ASCA epitope was expressed by Candida albicans and Mycobacterium paratuberculosis but not by Mycobacterium tuberculosis or E coli. Supernatants from M paratuberculosis culture inhibited killing of E coli HM605 by adherent human monocytes and murine macrophages. The inhibitory activity was removed by GNA-affinity chromatography. CONCLUSIONS: Suppression of mucosal phagocyte function by microbial mannans, possibly of Mycobacterial origin, may contribute to CD pathogenesis.