La maladie de Crohn (MC) est caractérisée par une inflammation intestinale chronique affectant potentiellement n'importe quel segment du tube digestif. L’étiologie de la maladie reste encore inconnue, cependant, la théorie la plus largement acceptée repose sur une réponse inflammatoire anormale dirigée contre le microbiote intestinal chez un hôte génétiquement prédisposé. Plusieurs études ont démontré que la muqueuse iléale de patients atteints de MC est anormalement colonisée par des souches de Escherichia coli adhérentes et invasives (AIEC). Toutefois, à ce jour, au Brésil, aucune étude ne démontre la présence de telles souches d’E. coli chez les patients atteints de MC. Le but de cette étude était d'isoler et de caractériser les souches de E. coli chez les patients atteints de MC au Brésil. Les biopsies ont été réalisées sur 35 sujets, 24 atteints de MC et 11 contrôles. La colonisation par des entérobactéries associées à la muqueuse iléale de patients atteints de MC a été montré élevée par rapport au groupe contrôle. Parmi les 270 souches isolées, 241 ont été identifiées comme étant des E. coli : 183 à partir de patients atteints de MC et 58 des contrôles. La recherche de différents groupes phylogénétiques de E. coli a été réalisée par PCR. Il n'y a pas de différence significative entre la répartition des groupes phylogénétiques des souches de E. coli isolées dans le groupe témoin et les patients MC. Les capacités d'adhésion et d’invasion des souches aux cellules épithéliales intestinales humaines I-407 ont été analysées, aussi bien que sa capacité à survivre et se multiplier en macrophages humains THP-1. L'analyse moléculaire par PCR a également été réalisée pour la détection des facteurs de virulence et la présence de polymorphismes génétiques associées à des souches AIEC. Dans cette étude, seuls quelques- uns des isolats de E. coli présentaient des propriétés invasives et la capacité de survivre dans les macrophages. En outre, l’analyse de la séquence fimH des souches de E. coli isolées dans cette étude n'a pas révélé la sélection de polymorphismes dans l’adhésine FimH, comme décrit pour la collection de souches AIEC isolées chez des patients européens. Ces résultats ont donc permis de montrer que les souches isolées chez les patients atteints de MC brésiliens n’ont probablement pas encore co-évolué avec leur hôte pour développer un phénotype adhérent-invasif fort, mais il sera essentiel de suivre à l'avenir l'évolution des ces souches dans la population brésilienne pour comprendre la sélection et l'évolution du phénotype AIEC. / Crohn's disease (CD) is characterized by chronic intestinal inflammation potentially affecting any segment of the digestive tract. The etiology of the disease is still unknown, however, the most widely accepted theory relies on an abnormal inflammatory response directed against the gut microbiota in a genetically predisposed host. Several studies have shown that the ileal mucosa of patients with CD is abnormally colonized by adherent and invasive Escherichia coli strains (AIEC). However, to date, in Brazil, no study has demonstrated the presence of such E. coli strains. coli in patients with CD. The purpose of this study was to isolate and characterize E. coli strains in patients with CD in Brazil. Biopsies were performed on 35 subjects, 24 with MC and 11 controls. Colonization with enterobacteria associated with the ileal mucosa of MC patients was shown to be elevated relative to the control group. Of the 270 strains isolated, 241 were identified as E. coli: 183 from CD patients and 58 controls. The search for different phylogenetic groups of E. coli was performed by PCR. There is no significant difference between the distribution of phylogenetic groups of E. coli strains isolated in the control and MC patients. The adhesion and invasion abilities of strains to human intestinal epithelial cells I-407 were analyzed, as well as its ability to survive and multiply into human macrophages THP-1. PCR molecular analysis was also performed for the detection of virulence factors and the presence of genetic polymorphisms associated with AIEC strains. In this study, only a few of the E. coli isolates had invasive properties and the ability to survive in macrophages. In addition, analysis of the fimH sequence of E. coli strains isolated in this study did not reveal the selection of polymorphisms in the FimH adhesin, as described for the collection of AIEC strains isolated from European patients. These results have thus shown that strains isolated from patients with Brazilian CD probably have not yet co-evolved with their host to develop a strong adherent-invasive phenotype, but it will be essential to monitor the future evolution of these strains in the Brazilian population to understand the selection and evolution of the AIEC phenotype.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs – Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.