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The etiology of Crohn's disease (CD) is multifactorial. Bacterial and fungal microbiota are involved in the onset and/or progression of the disease. A bacterial dysbiosis in CD patients is accepted; however, less is known about the mycobiome and the relationships between the two communities. We investigated the interkingdom relationships, their metabolic consequences, and the changes in the fungal community during relapse and remission in CD.Two cohorts were evaluated: a British cohort (n = 63) comprising CD and ulcerative colitis patients, and controls. The fungal and bacterial communities of biopsy and fecal samples were analyzed, with the fecal volatiles; datasets were also integrated; and a Dutch cohort (n = 41) comprising CD patients and healthy controls was analyzed for stability of the gut mycobiome.A dysbiosis of the bacterial community was observed in biopsies and stool. Results suggest Bacteroides is likely key in CD and may modulate Candida colonization. A dysbiosis of the fungal community was observed only in the Dutch cohort; Malassezia and Candida were increased in patients taking immunosuppressants. Longitudinal analysis showed an increase in Cyberlindnera in relapse. Saccharomyces was dominant in all fecal samples, but not in biopsies, some of which did not yield fungal reads; amino acid degradation was the main metabolic change associated with CD and both bacteria and fungi might be implicated.We have shown that Bacteroides and yeasts may play a role in CD; understanding their role and relationship in the disease would shed new light on the development and treatment of CD.

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy., (Copyright © 2019 Papoutsopoulou, Burkitt, Bergey, England, Hough, Schmidt, Spiller, White, Paszek, Jackson, Martins Dos Santos, Sellge, Pritchard, Campbell, Müller and Probert.)

Objective: Infliximab is important in the therapeutic arsenal of Crohn's disease (CD). However, its effect on mucosal barrier function is not fully understood. Adherent-invasive Escherichia coli (AIEC) are important in CD pathophysiology, but the transmucosal uptake routes are partly unknown. We investigated effects of infliximab on uptake of colon-specific AIEC HM427 across CD colonic mucosa., Materials and Methods: Endoscopic biopsies from non-inflamed colon of seven patients with CD, before and after two infliximab infusions, and eight non-inflammation controls, were mounted in Ussing chambers. Paracellular permeability ( 51 Cr-EDTA) and transmucosal passage of GFP-expressing HM427 were studied. Mechanisms of HM427 transepithelial transport were investigated in Caco-2 monolayers treated with TNF, in the presence of infliximab and/or endocytosis inhibitors., Results: Before infliximab treatment, colonic passage of HM427 [CD: 2475 CFU (450-3000); controls 1163(225-1950)] and 51 Cr-EDTA permeability were increased in CD (p < .05), but were restored to control levels by infliximab (CD: 150 (18.8-1069)). In TNF-exposed Caco-2 monolayers HM427 transport and lipid rafts/HM427 co-localization was decreased by infliximab. The lipid raft inhibitor methyl-β-cyclodextrin decreased HM427 transport., Conclusion: Infliximab restored the colonic barrier to AIEC in CD; an effect partially mediated by blocking lipid rafts in epithelial cells. This ability likely contributes to infliximab's clinical efficacy in colonic CD.

Background: Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly adherent and invasive Escherichia coli that replicate inside macrophage phagolysosomes. We compared CD and healthy control (HC) macrophages for their abilities to kill E. coli and generate neutrophil chemoattractants and also assessed the effects of hydroxychloroquine (HCQ) and vitamin D on killing of phagocytosed E. coli., Methods: Peripheral blood monocyte-derived macrophages from CD and HC were compared for bacterial killing and generation of neutrophil chemoattractants in response to CD-derived E. coli. Escherichia coli replication was also assessed in the presence and absence of HCQ, alone and with antibiotics, and vitamin D., Results: Monocyte-derived macrophages from patients with CD were similar to HC in allowing replication of phagocytosed CD-derived E. coli: HM605 {CD: N = 10, mean fold replication in 3 hr = 1.08 (95% confidence interval [CI], 0.39-1.78); HC: N = 9, 1.50 (95% CI, 1.02-1.97); P = 0.15} and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD = 2.55 [95% CI, 2.31-2.80]; HC = 2.65 [95% CI, 2.46-2.85], P = 0.42). HCQ and 1,25 OH2-vitamin D3 both caused dose-dependent inhibition of intramacrophage E. coli replication 3-hour postinfection; HCQ: 73.9% inhibition (P < 0.001) at 1 μg/mL, accompanied by raised intraphagosomal pH, and 1,25 OH2-vitamin D3: 80.7% inhibition (P < 0.05) at 80 nM. HCQ had synergistic effects with doxycycline and ciprofloxacin., Conclusions: CD and HC macrophages perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics.

Multiple studies have demonstrated alterations in the intestinal microbial community (termed the microbiome) in Crohn's disease (CD) and several lines of evidence suggest these changes may have a significant role in disease pathogenesis. In active and quiescent disease, both the faecal and mucosa-associated microbiome are discordant with matched controls with reduced biodiversity, changes in dominant organisms and increased temporal variation described. Mucosa-associated adherent, invasive Escherichia coli (E. coli) (AIEC), pro-inflammatory and resistant to killing by mucosal macrophages, appear to be particularly important. AIEC possess several virulence factors which may confer pathogenic potential in CD. Type-1 pili (FimH) allow adherence to intestinal cells via cell-surface carcinoembryonic antigen-related cell adhesion molecules and possession of long polar fimbrae promotes translocation across the intestinal mucosa via microfold (M)-cells of the follicle-associated epithelium. Resistance to stress genes (htrA, dsbA and hfq) and tolerance of an acidic pH may contribute to survival within the phagolysosomal environment. Here we review the current understanding of the role of mucosa-associated E. coli in Crohn's pathogenesis, the role of the innate immune system, factors which may contribute to prolonged bacterial survival and therapeutic strategies to target intracellular E. coli.

Adherent-invasive Escherichia coli strains are increasingly being associated with intestinal pathologies. Here we present the genome sequence of E. coli HM605, a strain isolated from colonic biopsy specimens of a patient with Crohn's disease.

Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC)--a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyer's patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2(-/-) mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients.

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.

Nucleotide-binding oligomerization domain-containing-2 (NOD2) acts as a bacterial sensor in dendritic cells (DCs), but it is not clear how bacterial recognition links with antigen presentation after NOD2 stimulation. NOD2 variants are associated with Crohn's disease, where breakdown in self-recognition of commensal bacteria leads to gastrointestinal inflammation. Here we show NOD2 triggering by muramyldipeptide induces autophagy in DCs. This effect requires receptor-interacting serine-threonine kinase-2 (RIPK-2), autophagy-related protein-5 (ATG5), ATG7 and ATG16L1 but not NLR family, pyrin domain containing-3 (NALP3).We show that NOD2-mediated autophagy is required for both bacterial handling and generation of major histocompatibility complex (MHC) class II antigen-specific CD4(+) T cell responses in DCs. DCs from individuals with Crohn's disease expressing Crohn's disease-associated NOD2 or ATG16L1 risk variants are defective in autophagy induction, bacterial trafficking and antigen presentation. Our findings link two Crohn's disease-associated susceptibility genes in a single functional pathway and reveal defects in this pathway in Crohn's disease DCs that could lead to bacterial persistence via impaired lysosomal destruction and immune mediated clearance.

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.

Adherent and invasive mucosa-associated Escherichia coli have been implicated in the pathogenesis of colon cancer and inflammatory bowel diseases. It has been reported that such isolates share features of extraintestinal E. coli (ExPEC) and particularly uropathogenic E. coli (UPEC). We used suppression subtractive hybridization (SSH) to subtract the genome of E. coli K-12 from that of a colon cancer mucosal E. coli isolate. Of the subtracted sequences, 53 % were present in the genomes of one or more of three sequenced UPEC strains but absent from the genome of an enterohaemorrhagic E. coli (EHEC) strain. Of the subtracted sequences, 80 % matched at least one UPEC genome, whereas only 4 % were absent from the UPEC genomes but present in the genome of the EHEC strain. A further genomic subtraction against the UPEC strain 536 enriched for sequences matching mobile genetic elements, other ExPEC strains, and other UPEC strains or commensals, rather than strains associated with gastrointestinal disease. We analysed the distribution of selected subtracted sequences and UPEC-associated pathogenicity islands (PAIs) amongst a panel of mucosa-associated E. coli isolated from colonoscopic biopsies of patients with colon cancer, patients with Crohn's disease and controls. This enabled us to identify a group of isolates from colon cancer (30-40 %) carrying multiple genes previously categorized as UPEC-specific and implicated in virulence.

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.

Background & Aims: Altered mucosal glycosylation in inflammatory bowel disease and colon cancer could affect mucosal bacterial adherence. This study aimed to quantify and characterize mucosa-associated and intramucosal bacteria, particularly Escherichia coli, in these conditions., Methods: Mucosa-associated bacteria were isolated, after dithiothreitol mucolysis, from biopsy samples obtained at colonoscopy (Crohn's disease, n = 14 patients; ulcerative colitis, n = 21; noninflamed controls, n = 24) and at surgical resection (colon cancer, n = 21). Intramucosal bacteria were grown after gentamicin treatment followed by hypotonic lysis., Results: Mucosa-associated and intramucosal bacteria were cultured more commonly in Crohn's disease (79%, P = 0.03; and 71%, P < 0.01, respectively), but not ulcerative colitis (38% and 48%), than in noninflamed controls (42% and 29%) and were commonly cultured from colon cancers (71% and 57%). Mucosa-associated E. coli, which accounted for 53% of isolates, were more common in Crohn's disease (6/14; 43%) than in noninflamed controls (4/24, 17%), as also were intramucosal E. coli: Crohn's disease, 29%; controls, 9%. E. coli expressed hemagglutinins in 39% of Crohn's cases and 38% of cancers but only 4% of controls, and this correlated (P = 0.01) with adherence to the I407 and HT29 cell lines. Invasion was cell-line dependent. E. coli, including nonadherent isolates, induced interleukin-8 release from the cell lines. E. coli adhesins showed no blood group specificity, excepting 1 cancer isolate (HM44) with specificity for the Thomsen-Friedenreich antigen, but they could be blocked by soluble plantain fiber., Conclusions: These studies support a central role for mucosally adherent bacteria in the pathogenesis of Crohn's disease and colon cancer. Soluble plant fibers that inhibit their adherence have therapeutic potential.

Previous studies have shown the presence in faeces of sulphatases, sialidases, glycosidases, and proteases relevant to mucus degradation, but the relative role of these enzymes in the degradation of colonic mucus has been unclear. A total mucinase assay using 14C threonine biologically labelled human colonic mucin as substrate was therefore developed in this study. Faecal mucinase activity of a pooled normal faecal filtrate was capable of removing 80% of the 14C threonine label from mucin within eight hours incubation, but 20% remained intact despite prolonged incubation. The pH profile of mucinase activity is broad (pH 4.5-9.5) suggesting contribution from multiple enzymes. Mucinase activity was reduced by preincubation with 100 micrograms/ml chymostatin (82.8%), 0.5 mg/ml EDTA (91.6%), and 4 g/l bismuth subsalicylate (72.0%). All 55 faecal samples studied contained detectable mucinase activity, measured as dpm release/micrograms protein/hour, which was greater in samples from patients with ulcerative colitis (n = 17, median 52.7, interquartile range 32.9-66.9), than controls (n = 26, 34.4, 26.8-40.4, p < 0.02) or patients with Crohn's disease (n = 12, 35.5, 17.5-55.7, p < 0.05). There was, however, no significant difference in faecal mucinase activity between inactive and active ulcerative colitis. These results suggest that faecal mucinase activity is one factor contributing to the thin mucus layer in ulcerative colitis and represents a potential target for drug treatment.

Heat-extracted antigens from seven species of Bacteroides were used in passive hemagglutination and counterimmunoelectrophoretic tests. Sera from 87 normal persons (group I) and 15 patients with ulcerative colitis (group II) were of low and equal reactivity in passive hemagglutination tests; all positive tests were eliminated by 2-mercaptoethanol reduction of the sera. When these same sera were tested by counterimmunoelectrophoresis with six of the Bacteroides antigens, no significant difference in the percentage of positive reactions was noted. However, using the chi-square test, the seventh antigen, prepared from Bacteroides vulgatus, successfully distinguished the two populations at the 0.025 level. Counterimmunoelectrophoretic tests with the B. vulgatus antigen also provided a means to separate the patients in group II with active disease from those in remission at a P value of 0.01. All the sera from 12 patients with defined Crohn's disease activity indexes reacted with the B. vulgatus antigen in counterimmunoelectrophoretic tests. Reduction and alkylation of patient sera with 2-mercaptoethanol and iodoacetamide removed detectable antibody in 78% of the samples, which suggested a dominant role of immunoglobulin M in the response to Bacteroides antigens.