Adherent-invasive Escherichia Coli Strains Research Articles

Norepinephrine affects the interaction of adherent-invasive Escherichia coli with intestinal epithelial cells

ABSTRACT Norepinephrine (NE), the stress hormone, stimulates many bacterial species’ growth and virulence, including Escherichia coli. However, the hormone’s impact on the adherent-invasive E. coli (AIEC) implicated in Crohn’s disease is poorly understood. In the study, we have investigated the effect of NE on the interaction of six AIEC strains isolated from an intestinal biopsy from 6 children with Crohn’s disease with Caco-2 cells. Our study focused on type 1 fimbria and CEACAM6 molecules serving as docking sites for these adhesins. The study results demonstrated that the hormone significantly increased the adherence and invasion of AIEC to Caco-2 cells in vitro. However, the effect was not associated with the impact of NE on the increased proliferation rate of AIEC or the fimA gene expression vital for their interaction with intestinal epithelial cells. Instead, the carcinoembryonic antigen-related cell-adhesion-molecule-6 (CEACAM6) level was increased significantly in NE-treated Caco-2 cells infected with AIEC in contrast to control uninfected NE-treated cells. These results indicated that NE influenced the interaction of AIEC with intestinal epithelium by increasing the level of CEACAM6 in epithelial cells, strengthening their adherence and invasion.

Adherent invasive Escherichia coli (AIEC) strain LF82, but not Candida albicans, plays a profibrogenic role in the intestine

BackgroundIntestinal fibrosis is a frequent complication of Crohn’s disease. However, the factors that cause chronicity and promote fibrogenesis are not yet understood.AimsIn the present study, we evaluated the profibrotic effects of adherent-invasive Escherichia coli (AIEC) LF82 strain and Candida albicans in the gut.MethodsColonic fibrosis was induced in C57BL/6 mice by administration of three cycles of 2.5% (w/v) dextran sulfate sodium (DSS) for 5 weeks. LF82 and C. albicans were administered orally once at the start of each week or each cycle, respectively. Expression of markers of myofibroblast activation was determined in TGF-β1-stimulated human intestinal epithelial cells (IECs).ResultsLF82 administration exacerbated fibrosis in DSS-treated mice, revealed by increased colonic collagen deposition and expression of the profibrotic genes Col1a1, Col3a1, Fn1 and Vim. This was accompanied by enhanced gene expression of proinflammatory cytokines and chemokines, as well as more recruited inflammatory cells into the intestine. LF82 also potentiated TGF-β1-stimulated epithelial–mesenchymal transition and myofibroblast activation in IECs, by further inducing gene expression of the main mesenchymal cell markers FN1 and VIM and downregulating the IEC marker OCLN. Proinflammatory cytokines were overexpressed with LF82 in TGF-β1-stimulated IECs. Conversely, C. albicans did not affect intestinal fibrosis progression in DSS-treated mice or myofibroblast activation in TGF-β1-stimulated IECs.ConclusionsThese results demonstrate that AIEC strain LF82, but not C. albicans, may play a major profibrogenic role in the gut.

Commonality of adherent-invasive Escherichia coli isolated from patients with extraintestinal infections, healthy individuals and the environment.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in inflammatory bowel disease (IBD) pathogenesis. These strains are defined by their ability to adhere to and invade intestinal epithelial cells, and to survive and replicate in macrophages. We postulated that AIEC strains may commonly inhabit the gut of healthy individuals (HI), cause extraintestinal infections, and be found in sewage treatment plants (STP) and surface waters (SW). A total of 808 E. coli strains isolated from HI; patients with community-acquired urinary tract infection (CA-UTI), septicaemia and urosepsis; STP; and SW, showing a diffuse adhesion pattern to Caco-2 cells were included in this study. Typing of the strains using a combination of RAPD-PCR and PhPlate fingerprinting grouped them into 48 common clones (CCs). Representatives of each CC were tested for the ability to invade Caco-2 cells, survive and replicate in macrophages, and for the presence of six virulence genes commonly found among AIEC strains. Twenty CCs were deemed AIEC based on their ability to survive and replicate in macrophages, while encoding htrA, dsbA and clbA genes. These CCs primarily originated from HI and CA-UTI patients but were also detected in secondary locations including STP and SW. Strains lacking intramacrophagic survival and replication abilities were regarded as diffusely adhering E. coli (DAEC). Certain clones of AIEC are common in the gut of HI whilst promoting CA-UTI. The survival and persistence of AIEC in STP and SW may have serious public health ramifications for individuals predisposed to IBD.

Antimicrobial Resistance Profiles of Adherent Invasive Escherichia coli Show Increased Resistance to β-Lactams.

The adherent invasive Escherichia coli (AIEC) pathotype has been associated with the aetiology of Crohn’s disease (CD). Scarce reports have shown the antimicrobial resistance (AMR) profiles of AIEC. Despite antibiotics not being recommended to treat CD, antimicrobial therapy could be useful in stratified patients, such as AIEC carriers. We examined the antimicrobial resistance profiles of AIEC strains to identify which therapies could be effective or confer a risk for such patients. Phenotypic resistance to 30 antimicrobials was tested according to CLSI standards. AIEC (n = 22) and non-pathogenic E. coli (non-AIEC) strains (n = 37) isolated from the gut mucosa of 31 CD patients and 18 controls were studied. De novo genome sequencing was carried out for 39 of the 59 strains, and AMR genes were searched using the DeepARG database in these genomes and 33 additional AIEC publicly available genomes. The strains isolated from CD and controls showed similar phenotypic AMR profiles. The genomic analysis did not reveal an increased prevalence of AMR genes. However, AIEC strains were more frequently resistant to β-lactams than non-AIEC strains (11 AIEC (50%) and 5 non-AIEC (22%) strains were resistant to at least one β-lactam; p < 0.042). Two AIEC strains were resistant to expanded-spectrum cephalosporins. One strain carried a plasmid-mediated AmpC β-lactamase (CMY-69), and the other presented mutations in the promotor of the intrinsic chromosomal AmpC related to the hyperproduction of this enzyme. The rest of the strains were resistant to β-lactams not including expanded-spectrum cephalosporins. The majority carried TEM-related β-lactamases. Genomic analysis including external AIEC revealed that the gene sul1 encoding for sulphonamide resistance was more frequent in AIEC strains than non-AIEC strains (34.6% vs. 9.5%, p = 0.030). AMR in AIEC is a matter of concern regarding the putative implication of the pathotype in CD. The high proportion of AIEC resistant to β-lactams warrants caution about the risk there may be in the use of these antimicrobials in AIEC-colonized CD patients.

Virulence determinants and genetic diversity of adherent-invasive Escherichia coli (AIEC) strains isolated from patients with Crohn's disease

BackgroundAdherent invasive Escherichia coli (AIEC) are implicated in the pathogenesis of inflammatory bowel diseases (IBD) particularly Crohn's disease (CD). The aim of this study is to isolate, identify, genotype, and characterize the virulence factors and the clinical significance of AIEC strains. MethodsIleal and colonic biopsies from 24 active CD patients and 15 healthy controls (HC) were collected. E. coli strains were identified by standard biochemical tests and confirmed by MALDI-TOF (bioMerieux, France) system. The AIEC phenotypes were determined by the adhesion, invasion, and survival within macrophages assays. The genetic virulence factors and genotyping characteristics were determined by PCR and PFGE respectively. The abundance and the antibiogram profile of E. coli strains was determined by qPCR and VITEK®2 (bioMerieux, France) automated system respectively. ResultsE. coli strains from 17 CD patients and 14 HC were isolated, 10 (59%) and 7 (50%) of them were identified as AIEC strains, respectively. We found that chuA and ratA genes were the most significant genetic markers associated with AIEC compared to non-AIEC strains isolated from CD patients and HC p = 0.0119, 0.0094 respectively. The majority of E. coli strains obtained from CD patients showed antibiotic resistance (71%) compared to HC (29%) against at least one antibiotic. The AIEC-like strains were more resistant to antibiotics compared to non-AIEC-like strains (53%) and (21%) respectively. ConclusionsWe have determined significant differences between AIEC strains and non-AIEC strains in terms of the prevalence of chuA and ratA virulence genes and the antibiotic resistance profiles. In addition, AIEC strains isolated from CD patients were found to be more resistant to penicillin/beta lactam and aminoglycoside antibiotics than AIEC strains isolated from HC 80%, 14% respectively.

Human Intestinal AIEC Strains Alter the Mucosal Microbiome and Establish a Niche for AIEC and Non‐AIEC Strains in Il10−/− Mice

Inflammatory bowel disease (IBD) affects over one million people in the United States and costs billions of dollars in direct medical expenses annually. This chronic inflammatory condition, which includes Crohn’s disease, currently has no medical cure. Additionally, patients with IBD are at a greater risk of developing inflammation‐associated colorectal cancer (I‐CRC). IBD and I‐CRC are associated with an increase of mucosally‐adhered bacteria, particularly Adherent‐Invasive Escherichia coli (AIEC). This proximity to the host can promote host‐directed pro‐inflammatory and pro‐carcinogenic activities by AIEC and co‐colonizing microbes. The overall aim of the project is to assess how colonization with Crohn’s‐associated clinicalE. coli strains alters the mucosal microbial community in the inflamed and non‐inflamed intestine.To this end, we have developed a polymicrobial colonization strategy that uses a novel barcoding technology to easily distinguish genetically similar, but functionally distinct sub‐strains of E. coli in a complex community. We have developed this strategy using a collection of well‐characterized clinical AIEC and non‐AIEC strains isolated from the intestinal mucosa of IBD and healthy patients. These strains are administered to formerly germ‐free mice (either inflammation susceptible Il10−/− or resistant WT) also with a murine fecal microbial transplant (FMT) to simulate physiologically‐relevant conditions. Our in vivo modeling has revealed that colonization with a pool of clinical isolates in the presence of the FMT enhances inflammation and promotes higher levels of E. coli colonizing the mucosa in inflammation‐susceptible mice. Importantly, colonization with these strains very significantly alters the composition of the mucosal microbiome, assessed by 16S rRNA sequencing, in inflamed but not uninflamed mice, including expansion of a co‐occuring pro‐inflammatory taxon and contraction of multiple protective species. These data suggest that colonization of the chronically inflamed with high levels of E. coli influence host health through both host‐microbe and microbe‐microbe interactions.Support or Funding InformationSupported by K01 award to JCA and the SPIRE NIH IRACDA program.

Comparative genomics of a subset of Adherent/Invasive Escherichia coli strains isolated from individuals without inflammatory bowel disease

There is increased evidence demonstrating the association between Crohn's Disease (CD), a type of Inflammatory Bowel Disease (IBD), and non-diarrheagenic Adherent/Invasive Escherichia coli (AIEC) isolates. AIEC strains are phenotypically characterized by their adhesion, invasion and intra-macrophage survival capabilities. In the present study, the genomes of five AIEC strains isolated from individuals without IBD (four from healthy donors and one from peritoneal liquid) were sequenced and compared with AIEC prototype strains (LF82 and NRG857c), and with extra-intestinal uropathogenic strain (UPEC CFT073). Non-IBD-AIEC strains showed an Average Nucleotide Identity up to 98% compared with control strains. Blast identities of the five non-IBD-AIEC strains were higher when compared to AIEC and UPEC reference strains than with another E. coli pathotypes, suggesting a relationship between them. The SNPs phylogeny grouped the five non-IBD-AIEC strains in one separated cluster, which indicates the emergence of these strains apart from the AIEC group. Additionally, four genomic islands not previously reported in AIEC strains were identified. An incomplete Type VI secretion system was found in non-IBD-AIEC strains; however, the Type II secretion system was complete. Several groups of genes reported in AIEC strains were searched in the five non-IBD-AIEC strains, and the presence of fimA, fliC, fuhD, chuA, irp2 and cvaC were confirmed. Other virulence factors were detected in non-IBD-AIEC strains, which were absent in AIEC reference strains, including EhaG, non-fimbrial adhesin 1, PapG, F17D-G, YehA/D, FeuC, IucD, CbtA, VgrG-1, Cnf1 and HlyE. Based on the differences in virulence determinants and SNPs, it is plausible to suggest that non-IBD AIEC strains belong to a different pathotype.

Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p < 0.028), whereas in AIEC strains ompA expression was the only increased when growing in association with cells (p = 0.032), but they did not significantly alter ompC and ompF expression under this condition (p > 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains.

Characteristics and pathogenic role of adherent-invasive Escherichia coli in inflammatory bowel disease: Potential impact on clinical outcomes

Adherent-invasive Escherichia coli (AIEC) has been reported as associated with the pathogenesis of inflammatory bowel disease (IBD). We aimed to investigate the characteristics of mucosa-associated E. coli and the clinical significance of AIEC in Korean IBD patients. E. coli strains were isolated from the mucosal tissues of 18 Crohn’s disease (CD) patients, 24 ulcerative colitis (UC) patients, and 9 healthy controls (HC). Adhesion, invasion, and survival assays were performed to evaluate phenotypic features of E. coli isolates and to identify AIEC. The presence of virulence genes and cytokine expression were examined using PCR. In addition, data on IBD-related hospitalization were collected. A total of 59 E. coli strains were isolated (25 from CD, 27 from UC, and 7 from HC). The average levels of adhesion, invasion, and survival were higher in E. coli strains from IBD patients than those from HC (adhesion: 1.65 vs. 0.71, p = 0.046; invasion: 1.68 vs. 0.52, p = 0.039; survival: 519.55 vs. 47.55, p = 0.363). Prevalence of AIEC in HC, CD and UC patients was 22.2%, 38.9% and 37.5%, respectively. E. coli isolates from IBD patients had various virulence genes and were associated with increased expression of TNF-α and IL-17. IBD-related hospitalization within 3 years was 18.8% in patients with AIEC and 11.5% in patients without AIEC. E. coli strains from IBD patients showed high levels of adhesion, invasion, and survival. AIEC strains were identified in both CD and UC patients at a similar rate. AIEC may be associated with sustaining inflammation in the pre-existing inflammatory mucosa.

Genetic and Phenotypic Features to Screen for Putative Adherent-Invasive Escherichia coli

To date no molecular tools are available to identify the adherent-invasive Escherichia coli (AIEC) pathotype, which has been associated with Crohn’s disease and colonizes the intestine of different hosts. Current techniques based on phenotypic screening of isolates are extremely time-consuming. The aim of this work was to search for signature traits to assist in rapid AIEC identification. The occurrence of at least 54 virulence genes (VGs), the resistance to 30 antibiotics and the distribution of FimH and ChiA amino acid substitutions was studied in a collection of 48 AIEC and 56 non-AIEC isolated from the intestine of humans and animals. χ2 test was used to find frequency differences according to origin of isolation, AIEC phenotype and phylogroup. Mann–Whitney test was applied to test association with adhesion and invasion indices. Binary logistic regression was performed to search for variables of predictive value. Animal strains (N = 45) were enriched in 12 VGs while 7 VGs were more predominant in human strains (N = 59). The prevalence of 15 VGs was higher in AIEC (N = 49) than in non-AIEC (N = 56) strains, but only pic gene was still differentially distributed when analyzing human and animal strains separately. Among human strains, three additional VGs presented higher frequency in AIEC strains (papGII/III, iss and vat; N = 22) than in non-AIEC strains (N = 37). No differences between AIEC/non-AIEC were found in FimH variants. In contrast, the ChiA sequence of LF82 was shared with the 35.5% of AIEC studied (N = 31) and only with the 7.4% of non-AIEC strains (N = 27; p = 0.027). Binary logistic regression analysis, using as input variables all the VGs and antibiotic resistances tested, revealed that typifying E. coli isolates using pic gene and ampicillin resistance was useful to correctly classify strains according to the phenotype with a 75.5% of accuracy. Although there is not a molecular signature fully specific and sensitive to identify the AIEC pathotype, we propose two features easy to be tested that could assist in AIEC screening. Future work using additional strain collections would be required to assess the applicability of this method.

Metabolic adaptation of adherent-invasive Escherichia coli to exposure to bile salts

The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of Crohn’s disease patients, adhere to intestinal epithelial cells, invade them and exacerbate intestinal inflammation. The high nutrient competition between the commensal microbiota and AIEC pathobiont requires the latter to occupy their own metabolic niches to survive and proliferate within the gut. In this study, a global RNA sequencing of AIEC strain LF82 has been used to observe the impact of bile salts on the expression of metabolic genes. The results showed a global up-regulation of genes involved in degradation and a down-regulation of those implicated in biosynthesis. The main up-regulated degradation pathways were ethanolamine, 1,2-propanediol and citrate utilization, as well as the methyl-citrate pathway. Our study reveals that ethanolamine utilization bestows a competitive advantage of AIEC strains that are metabolically capable of its degradation in the presence of bile salts. We observed that bile salts activated secondary metabolism pathways that communicate to provide an energy benefit to AIEC. Bile salts may be used by AIEC as an environmental signal to promote their colonization.

Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis.

Intestinal fibrosis is a severe complication in patients with Crohn’s disease (CD). Unfortunately, the trigger leading to the development of intestinal fibrosis in the context of CD remains elusive. Here, we show that colonization by a CD-associated pathobiont adherent-invasive Escherichia coli (AIEC) promotes the development of intestinal fibrosis. Exogenously inoculated AIEC strain LF82 and commensal E. coli HS were gradually eradicated from the intestine in healthy mice. In Salmonella- or dextran sodium sulfate-induced colitis models, AIEC exploited inflammation and stably colonize the gut. Consequently, persistent colonization by AIEC LF82 led to substantial fibrosis. In contrast, commensal E. coli HS was unable to derive a growth advantage from inflammation, thereby failing to colonize the inflamed intestine or promote intestinal fibrosis. AIEC colonization potentiated the expression of the IL-33 receptor ST2 in the intestinal epithelium, which is crucial for the development of intestinal fibrosis. The induction of ST2 by AIEC LF82 was mediated by flagellin, as the FliC mutant failed to induce ST2. These observations provide novel insights into pathobiont-driven intestinal fibrosis and can lead to the development of novel therapeutic approaches for the treatment of intestinal fibrosis in the context of CD that target AIEC and/or its downstream IL-33-ST2 signaling.

Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.

Mice harboring pathobiont-free microbiota do not develop intestinal inflammation that normally results from an innate immune deficiency.

BackgroundInability to maintain a stable and beneficial microbiota is associated with chronic gut inflammation, which classically manifests as colitis but may more commonly exist as low-grade inflammation that promotes metabolic syndrome. Alterations in microbiota, and associated inflammation, can originate from dysfunction in host proteins that manage the microbiota, such as the flagellin receptor TLR5. That the complete absence of a microbiota (i.e. germfree conditions) eliminates all evidence of inflammation in TLR5-deficient mice demonstrates that this model of gut inflammation is microbiota-dependent. We hypothesize that such microbiota dependency reflects an inability to manage pathobionts, such as Adherent-Invasive E. coli (AIEC). Herein, we examined the extent to which microbiota mismanagement and associated inflammation in TLR5-deficient mice would manifest in a limited and pathobiont-free microbiota. For this purpose, WT and TLR5-deficient mice were generated and maintained with the 8-member consortium of bacteria referred to as “Altered Schaedler Flora” (ASF). Such ASF animals were subsequently inoculated with AIEC reference strain LF82. Feces were assayed for bacterial loads, fecal lipopolysaccharide and flagellin loads, fecal inflammatory marker lipocalin-2 and microbiota composition.ResultsRelative to similarly maintained WT mice, mice lacking TLR5 (T5KO) did not display low-grade intestinal inflammation nor metabolic syndrome under ASF conditions. Concomitantly, the ASF microbial community was similar between WT and T5KO mice, while inoculation with AIEC strain LF82 resulted in alteration of the ASF community in T5KO mice compared to WT control animals. AIEC LF82 inoculation in ASF T5KO mice resulted in microbiota components having elevated levels of bioactive lipopolysaccharide and flagellin, a modest level of low-grade inflammation and increased adiposity.ConclusionsIn a limited-complexity pathobiont-free microbiota, loss of the flagellin receptor TLR5 does not impact microbiota composition nor its ability to promote inflammation. Addition of AIEC to this ecosystem perturbs microbiota composition, increases levels of lipopolysaccharide and flagellin, but only modestly promotes gut inflammation and adiposity, suggesting that the phenotypes previously associated with loss of this innate immune receptor require disruption of complex microbiota.

Adaptation of adherent-invasive E. coli to gut environment: Impact on flagellum expression and bacterial colonization ability

ABSTRACT The pathogenesis of Crohn's disease (CD) is multifactorial and involves genetic susceptibility, environmental triggers and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are flagellated bacteria more prevalent in CD patients than in healthy subjects and promote chronic intestinal inflammation. We aim at deciphering the role of flagella and flagellin modulation by intestinal conditions. AIEC flagellum expression is required for optimal adhesion to and invasion of intestinal epithelial cells. Interestingly, differential flagellin regulation was observed between commensal E. coli (HS) and AIEC (LF82) strains: flagellum expression by AIEC bacteria, in contrast to that of commensal E. coli, is enhanced under intestinal conditions (the presence of bile acids and mucins). Flagella are involved in the ability of the AIEC LF82 strain to cross a mucus layer in vitro and in vivo, conferring a selective advantage in penetrating the mucus layer and reaching the epithelial surface. In a CEABAC10 mouse model, a non-motile mutant (LF82-ΔfliC) exhibits reduced colonization that is restored by a dextran sodium sulfate treatment that alters mucus layer integrity. Moreover, a mutant that continuously secretes flagellin (LF82-ΔflgM) triggers a stronger inflammatory response than the wild-type strain, and the mutant's ability to colonize the CEABAC10 mouse model is decreased. Overexpression of flagellin in bacteria in contact with epithelial cells can be detrimental to their virulence by inducing acute inflammation that enhances AIEC clearance. AIEC pathobionts must finely modulate flagellum expression during the infection process, taking advantage of their specific virulence gene regulation to improve their adaptability and flexibility within the gut environment.

Comparative genomics reveals new single-nucleotide polymorphisms that can assist in identification of adherent-invasive Escherichia coli

Adherent-invasive Escherichia coli (AIEC) have been involved in Crohn’s disease (CD). Currently, AIEC are identified by time-consuming techniques based on in vitro infection of cell lines to determine their ability to adhere to and invade intestinal epithelial cells as well as to survive and replicate within macrophages. Our aim was to find signature sequences that can be used to identify the AIEC pathotype. Comparative genomics was performed between three E. coli strain pairs, each pair comprised one AIEC and one non-AIEC with identical pulsotype, sequence type and virulence gene carriage. Genetic differences were further analysed in 22 AIEC and 28 non-AIEC isolated from CD patients and controls. The strain pairs showed similar genome structures, and no gene was specific to AIEC. Three single nucleotide polymorphisms displayed different nucleotide distributions between AIEC and non-AIEC, and four correlated with increased adhesion and/or invasion indices. Here, we present a classification algorithm based on the identification of three allelic variants that can predict the AIEC phenotype with 84% accuracy. Our study corroborates the absence of an AIEC-specific genetic marker distributed across all AIEC strains. Nonetheless, point mutations putatively involved in the AIEC phenotype can be used for the molecular identification of the AIEC pathotype.

P011 ADHERENT-INVASIVE ESCHERICHIA COLI UTILIZES SERINE CATABOLISM TO OUTCOMPETE ITS COMMENSAL COMPETITORS

Crohn’s disease (CD) patients are often colonized by adherent-invasive Escherichia coli (AIEC). Although the colonization by AIEC is known to contribute to the pathogenesis of CD, the mechanisms by which AIEC thrives within the inflamed intestine in CD patients remain elusive. To elucidate the mechanisms by which AIEC outcompetes commensal non-pathogenic E. coli (cEC) in the intestine, in vivo competition of AIEC (strain LF82) and cEC (strain MG1655) was performed using co-colonization of the two strains in germ-free mice. Dextran sodium sulfate (DSS) was given to the co-colonized mice to mimic inflammatory environments. To identify the unique metabolic pathways that are utilized by AIEC during inflammation, transcriptomic changes in E. coli strains in the inflamed gut were analyzed by RNAseq. Amino acid (AA) defined diets were applied to bacterial in vivo competition to assess the impact of dietary serine on AIEC bloom. AIEC gained the growth advantage over cEC during DSS colitis. Intestinal inflammation reprogramed bacterial metabolic pathways from carbohydrate metabolism toward amino acid metabolism, particularly serine utilization, in AIEC. An isogenic mutant AIEC strain that lacks serine utilization genes failed to outcompete cEC in the inflamed gut. The deprivation of dietary serine also prevented outgrowth of AIEC in the inflamed but not in the steady-state gut in vivo. AIEC utilizes serine catabolism pathways to gain a growth advantage over cEC in the inflamed gut. Targeting unique metabolic pathways exploited by AIEC may selectively eliminate the pathogenic strains of gut bacteria without influencing beneficial commensal microbiota.

IL-17 is a protection effector against the adherent-invasive Escherichia coli in murine colitis

Inflammatory bowel disease (IBD) is caused by aberrant immune responses to the gut microbiota. Among the gut microbiota, adherent-invasive Escherichia Coli (AIEC) is thought to be the pathogen through invading the intestinal epithelial cells and causing inflammation. IL-17 secretion increase, induced by enhanced bacterial adhesion to the intestine epithelium, could on one hand protect the mucosa, but on the other hand, over amount of IL-17 initializes inflammation reactions that in turn damages the mucosa. The relationship between IL-17 and AIEC is still unclear. In this study, we tried to elucidate the function of IL-17 in AIEC-mediated colitis. Wild type (WT) and IL-17 knockout (IL-17 KO) mice were inoculated with AIEC strain E. coli LF82 and treated with dextran sodium sulphate (DSS). Histological examination of the colon was performed. Mucosa damage was assessed and scored. IL-22 and IL-17 in colon tissues were detected by ELISA, qPCR and immunohistochemistry methods. Transient AIEC colonization in IL-17 KO mice resulted in increased intestinal epithelial damage, systemic bacterial burden and mortality compared with WT controls. Moreover, IL-17 is required for the induction of IL-22 in the experimental animal models during AIEC strain E. coli LF82 colonization. These results indicate IL-17 plays a protective role in AIEC strain E. coli LF82 induced colitis by promoting IL-22 secretion.

Pathogenic Role of Associated Adherent-Invasive Escherichia coli in Crohn's Disease.

Several lines of evidence suggest that adherent-invasive Escherichia coli (AIEC) strains play an important role in Crohn's disease (CD). The objective of this study was to investigate the pathogenic role of two AIEC strains, LF82 and O83:H1, in CD patients. Organ cultures of colonic biopsies from patients were set up to assess the effects of LF82 and O83:H1 on the expression of CEACAM6, LAMP1, HLA-DR, ICAM1 by immunohistochemistry and of IL-8, IFNʏ, and TNF-α genes by RT-PCR. Moreover, on Caco2 cells, we analyzed the cell cycle, the expression of MGMT and DNMT1 genes, and DNA damage induced by LF82 and O83:H1, by FACS, RT-PCR, and DAPI staining, respectively. Epithelial and lamina propria mononuclear cells (LPMNC) expression of CEACAM6 and LAMP1 were higher in biopsies cultured in the presence of both O83:H1 and LF82 than in biopsies cultured with non-pathogenic E. coli. Both AIEC strains induced increased expression of ICAM-1 on blood vessels and HLA-DR on LPMNC. We observed higher levels of TNF-α, IFN-γ, and IL-8 transcripts in biopsies cultured with both AIEC strains than in those cultured with NP. Both LF82 and O83:H1, block the cell cycle into S phase, inducing DNA damage, and modulate the expression of DNMT1 and MGMT genes. Our data suggest that LF82 and 083:H1 strains of E. coli are able to increase in CD colonic biopsies the expression of all the pro-inflammatory cytokines and all the mucosal immune markers investigated. J. Cell. Physiol. 232: 2860-2868, 2017. © 2016 Wiley Periodicals, Inc.

QseC inhibition as an antivirulence approach for colitis-associated bacteria

Hosts and their microbes have established a sophisticated communication system over many millennia. Within mammalian hosts, this dynamic cross-talk is essential for maintaining intestinal homeostasis. In a genetically susceptible host, dysbiosis of the gut microbiome and dysregulated immune responses are central to the development of inflammatory bowel disease (IBD). Previous surveys of stool from the T-bet-/-Rag2-/- IBD mouse model revealed microbial features that discriminate between health and disease states. Enterobacteriaceae expansion and increased gene abundances for benzoate degradation, two-component systems, and bacterial motility proteins pointed to the potential involvement of a catecholamine-mediated bacterial signaling axis in colitis pathogenesis. Enterobacteriaceae sense and respond to microbiota-generated signals and host-derived catecholamines through the two-component quorum-sensing Escherichia coli regulators B and C (QseBC) system. On signal detection, QseC activates a cascade to induce virulence gene expression. Although a single pathogen has not been identified as a causative agent in IBD, adherent-invasive Escherichia coli (AIEC) have been implicated. Flagellar expression is necessary for the IBD-associated AIEC strain LF82 to establish colonization. Thus, we hypothesized that qseC inactivation could reduce LF82's virulence, and found that an absence of qseC leads to down-regulated flagellar expression and motility in vitro and reduced colonization in vivo. We extend these findings on the potential of QseC-based IBD therapeutics to three preclinical IBD models, wherein we observe that QseC blockade can effectively modulate colitogenic microbiotas to reduce intestinal inflammation. Collectively, our data support a role for QseC-mediated bacterial signaling in IBD pathogenesis and indicate that QseC inhibition may be a useful microbiota-targeted approach for disease management.