BackgroundCrohn'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.MethodsTo 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.ResultsE 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).ConclusionsTranslocation 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.
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.
ObjectiveColonic 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.DesignA 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).Results454-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.ConclusionsIBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.
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 mucosaassociated 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.
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.
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