Margarita Martinez-Medina

Abnormal microbiota composition in the ileocolonic mucosa of Crohn's disease patients as revealed by polymerase chain reaction-denaturing gradient gel electrophoresis

Background Bacteria might play a role in the pathogenesis of Crohns disease (CD), and patients harbor a different type and density of gut microbiota compared with normal healthy subjects. Thus, the aim of this study was to compare the microbiota adhered to the mucosa of CD patients with that of healthy subjects. Methods Polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S rRNA gene fragments was used to identify the dominant bacterial species present in fresh biopsy samples obtained from the mucosa of 15 healthy and 19 CD subjects. Two patients suffering from ulcerative colitis and 1 suffering from ischemic colitis also were included. Results Individuals were clustered in 2 groups according to their molecular fingerprint, which differentiated the majority of CD specimens (88.2%) from the majority of healthy/ulcerative colitis/ischemic colitis specimens (82.3%). In addition, the patient‐to‐patient variability in microbiota was greater within the CD cluster than in the healthy/ulcerative colitis cluster (P = 0.000). One hundred forty‐one sequences were obtained from the PCR‐DGGE bands that were grouped into 58 different phylotypes, 8 of which were novel. BLAST analysis revealed that 74.5% of the sequences were similar to those of bacteria that have never been cultivated. In CD samples, prevalence values for Clostridium spp Ruminococcus torques and Escherichia coli were significantly higher, whereas Faecalibacterium was more frequently found in healthy specimens. Opportunistic pathogenic &ggr;‐proteobacteria were found occasionally, only in CD mucosal microbiota. Conclusions Microbiota attached to the ileocolonic mucosa of CD patients is distinguishable from that of healthy subjects. We postulate that individuals who are predisposed to CD are less able to regulate the microbial makeup of their intestines, which leads to an unstable microbial population.

Western diet induces dysbiosis with increased E coli in CEABAC10 mice, alters host barrier function favouring AIEC colonisation

Objective Western diet is a risk factor for Crohns disease (CD). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is abnormally expressed in CD patients. This allows adherent-invasive Escherichia coli (AIEC) to colonise the gut mucosa and leads to inflammation. We assessed the effects of a high fat/high sugar (HF/HS) Western diet on gut microbiota composition, barrier integrity and susceptibility to infection in transgenic CEABAC10 mice expressing human CEACAMs. Design Colonic microbiota composition and susceptibility of CEABAC10 mice to AIEC LF82 bacteria infection were determined in mice fed a conventional or HF/HS diet. Barrier function and inflammatory response were assessed by studying intestinal permeability, tight junction protein and mucin expression and localisation, and by determining histological score and levels of cytokine release. Results HF/HS diet led to dysbiosis in WT and transgenic CEABAC10 mice, with a particular increase in E coli population in HF/HS-fed CEABAC10 mice. These mice showed decreased mucus layer thickness, increased intestinal permeability, induction of Nod2 and Tlr5 gene transcription, and increased TNFα secretion. These modifications led to a higher ability of AIEC bacteria to colonise the gut mucosa and to induce inflammation. Conclusions Western diet induces changes in gut microbiota composition, alters host homeostasis and promotes AIEC gut colonisation in genetically susceptible mice. These results support the multifactorial aetiology of CD and highlight the importance of diet in CD pathogenesis.

Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohns disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH LF82 (expressing FimH with an AIEC-associated mutation) with fimH K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.

Similarity and Divergence among Adherent-Invasive Escherichia coli and Extraintestinal Pathogenic E. coli Strains

ABSTRACT Adherent-invasive Escherichia coli (AIEC) pathovar strains, which are associated with Crohns disease, share many genetic and phenotypic features with extraintestinal pathogenic E. coli (ExPEC) strains, but little is known about the level of genetic similarity between the two pathovars. We aimed to determine the frequency of strains with the “AIEC phenotype” among a collection of ExPEC strains and to further search for a common phylogenetic origin for the intestinal and extraintestinal AIEC strains. The adhesion, invasion, and intramacrophage replication capabilities (AIEC phenotype) of 63 ExPEC strains were determined. Correlations between virulence genotype and AIEC phenotype and between intestinal/extraintestinal origin, serotype, and phylogroup were evaluated for the 63 ExPEC and 23 intestinal AIEC strains. Phylogenetic relationships between extraintestinal and intestinal AIEC strains were determined using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Only four (6.35%) ExPEC strains, belonging to the O6:H1, O83:H1, and O25:H4 serotypes, were classified as having an AIEC phenotype. These strains were found to be genetically related to some intestinal AIEC strains of the same serotypes as revealed by MLST. No particular virulence gene sets correlated with the intestinal/extraintestinal origin of the strains or with the AIEC phenotype, whereas the gene sets did correlate with the serogroup. We identified two intestinal AIEC strains and one extraintestinal AIEC strain belonging to the O25:H4 serotype that also belonged to the emerging and virulent clonal group ST131. In conclusion, the ExPEC and AIEC pathovars share similar virulence gene sets, and certain strains are phylogenetically related. However, the majority of ExPEC strains did not behave like AIEC strains, thus confirming that the AIEC pathovar possesses virulence-specific features that, to date, are detectable only phenotypically.

Western diet induces a shift in microbiota composition enhancing susceptibility to Adherent-Invasive E. coli infection and intestinal inflammation.

Recent advances have shown that the abnormal inflammatory response observed in CD involves an interplay among intestinal microbiota, host genetics and environmental factors. The escalating consumption of fat and sugar in Western countries parallels an increased incidence of CD during the latter 20th century. The impact of a HF/HS diet in mice was evaluated for the gut micro-inflammation, intestinal microbiota composition, function and selection of an E. coli population. The HF/HS diet created a specific inflammatory environment in the gut, correlated with intestinal mucosa dysbiosis characterized by an overgrowth of pro-inflammatory Proteobacteria such as E. coli, a decrease in protective bacteria, and a significantly decreased of SCFA concentrations. The expression of GPR43, a SCFA receptor was reduced in mice treated with a HF/HS diet and reduced in CD patients compared with controls. Interestingly, mice treated with an agonist of GPR43 were protected against DSS-induced colitis. Finally, the transplantation of feces from HF/HS treated mice to GF mice increased susceptibility to AIEC infection. Together, our results demonstrate that a Western diet could aggravate the inflammatory process and that the activation of the GPR43 receptor pathway could be used as a new strategy to treat CD patients.

Escherichia coli in chronic inflammatory bowel diseases: An update on adherent invasive Escherichia coli pathogenicity.

Escherichia coli (E. coli), and particularly the adherent invasive E. coli (AIEC) pathotype, has been increasingly implicated in the ethiopathogenesis of Crohns disease (CD). E. coli strains with similar pathogenic features to AIEC have been associated with other intestinal disorders such as ulcerative colitis, colorectal cancer, and coeliac disease, but AIEC prevalence in these diseases remains largely unexplored. Since AIEC was described one decade ago, substantial progress has been made in deciphering its mechanisms of pathogenicity. However, the molecular bases that characterize the phenotypic properties of this pathotype are still not well resolved. A review of studies focused on E. coli populations in inflammatory bowel disease (IBD) is presented here and we discuss about the putative role of this species on each IBD subtype. Given the relevance of AIEC in CD pathogenesis, we present the latest research findings concerning AIEC host-microbe interactions and pathogenicity. We also review the existing data regarding the prevalence and abundance of AIEC in CD and its association with other intestinal diseases from humans and animals, in order to discuss the AIEC disease- and host-specificity. Finally, we highlight the fact that dietary components frequently found in industrialized countries may enhance AIEC colonization in the gut, which merits further investigation and the implementation of preventative measures.

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

BackgroundCrohns disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.ResultsSpecific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains (P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas 74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strains were more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) and invasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility (100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22 (26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA (26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongst biofilm producers.ConclusionThe principal contribution of the present work is the finding that biofilm formation capacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis.

Mucosa-associated Faecalibacterium prausnitzii phylotype richness is reduced in patients with inflammatory bowel disease.

ABSTRACT Faecalibacterium prausnitzii depletion in intestinal diseases has been extensively reported, but little is known about intraspecies variability. This work aims to determine if subjects with gastrointestinal disease host mucosa-associated F. prausnitzii populations different from those hosted by healthy individuals. A new species-specific PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting the 16S rRNA gene was developed to fingerprint F. prausnitzii populations in biopsy specimens from 31 healthy control (H) subjects and 36 Crohns disease (CD), 23 ulcerative colitis (UC), 6 irritable bowel syndrome (IBS), and 22 colorectal cancer (CRC) patients. The richness of F. prausnitzii subtypes was lower in inflammatory bowel disease (IBD) patients than in H subjects. The most prevalent operational taxonomic units (OTUs) consisted of four phylotypes (OTUs with a 99% 16S rRNA gene sequence similarity [OTU99]), which were shared by all groups of patients. Their distribution and the presence of some disease-specific F. prausnitzii phylotypes allowed us to differentiate the populations in IBD and CRC patients from that in H subjects. At the level of a minimum similarity of 97% (OTU97), two phylogroups accounted for 98% of the sequences. Phylogroup I was found in 87% of H subjects but in under 50% of IBD patients (P = 0.003). In contrast, phylogroup II was detected in >75% of IBD patients and in only 52% of H subjects (P = 0.005). This study reveals that even though the main members of the F. prausnitzii population are present in both H subjects and individuals with gut diseases, richness is reduced in the latter and an altered phylotype distribution exists between diseases. This approach may serve as a basis for addressing the suitability of F. prausnitzii phylotypes to be quantified as a putative biomarker of disease and depicting the importance of the loss of these subtypes in disease pathogenesis.

Faecalibacterium prausnitzii : from microbiology to diagnostics and prognostics

There is an increasing interest in Faecalibacterium prausnitzii, one of the most abundant bacterial species found in the gut, given its potentially important role in promoting gut health. Although some studies have phenotypically characterized strains of this species, it remains a challenge to determine which factors have a key role in maintaining the abundance of this bacterium in the gut. Besides, phylogenetic analysis has shown that at least two different F. prausnitzii phylogroups can be found within this species and their distribution is different between healthy subjects and patients with gut disorders. It also remains unknown whether or not there are other phylogroups within this species, and also if other Faecalibacterium species exist. Finally, many studies have shown that F. prausnitzii abundance is reduced in different intestinal disorders. It has been proposed that F. prausnitzii monitoring may therefore serve as biomarker to assist in gut diseases diagnostics. In this mini-review, we aim to serve as an overview of F. prausnitzii phylogeny, ecophysiology and diversity. In addition, strategies to modulate the abundance of F. prausnitzii in the gut as well as its application as a biomarker for diagnostics and prognostics of gut diseases are discussed. This species may be a useful potential biomarker to assist in ulcerative colitis and Crohn’s disease discrimination.

Anti-tumour Necrosis Factor Treatment with Adalimumab Induces Changes in the Microbiota of Crohn's Disease

BACKGROUND The composition of the intestinal microbiota is altered in Crohns disease [CD] patients. The objective of this study was to evaluate the qualitative and quantitative changes in the microbiota of CD patients in 3 months of treatment with adalimumab [ADA], and determine whether or not these changes are produced towards the recovery of the normal, healthy-like microbiota. METHODS The microbiota composition, and the Faecalibacterium prausnitzii / Escherichia coli quantitative relationship as dysbiosis indicator, were studied at baseline [T0], one month [T1], and 3 months [T3] after starting treatment using a polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE] of 16S rRNA gene fragments and quantitative PCR, respectively, in rectal mucosal biopsies from 15 CD patients and four healthy subjects. RESULTS T0 and T3 fingerprints were different in all patients; whereas T1 and T3 presented similar patterns. Recovered phylogroups were Firmicutes [79.1%], Bacteroides [12.5%], and Actinobacteria [6.25%]. The prevalence of E. coli decreased during treatment. Relative E. coli loads in CD samples were significantly reduced at every analysed step [T1 and T3] [p < 0.005] whereas no significant changes were observed in relative F. prausnitzii counts. CONCLUSION Treatment with ADA induces short-term changes in the microbiota composition which seem to parallel the partial recovery of the gut bacterial ecology, with recovery parameters tending to eubiosis recovery. The quantitative determination of dysbiosis-representative bacteria, such as E. coli, may provide a fast and reliable indicator of the healing state of the intestinal mucosa.