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Full http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210564/
Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features.
October, 2014
Department of Public Health and Infectious Diseases, University of Rome, Italy
Conte MP, Longhi C, Marazzato M, Conte AL, Aleandri M, Lepanto MS, Zagaglia C, Nicoletti M, Aloi M, Totino V, Palamara AT, Schippa S
BACKGROUND:
Adherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn's disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features.
RESULTS:
A total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ2 = 6.167 P = 0.013), kpsMT II (χ2 = 6.167 P = 0.013), fyuA (χ2 = 6.167 P = 0.013), and ibeA (χ2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls.
CONCLUSIONS:
In conclusion, the AIEC-like abundance in CD patients and the presence of virulence genes only in CD patients further support the hypothesis that this pathovar may play an etiologic role in the insurgeonce and/or in the persistence in CD. These results are further supported by the recent findings indicating that colonization of the intestinal mucosa by AIEC strains, may initiate chronic inflammation of susceptible hosts by altering the gut microbiota [23]. Moreover, it has been recently reported a higher incidence of IBD in patients feed with fat-rich Western diet. It has been suggested that a fat-rich diet may induce changes in the composition of intestinal microbiota that, in turn, may influence the colonization and persistence of AIEC strains in genetically susceptible hosts [39]. These findings open up new scenarios addressed at the development of new strategies for the prevention and control of IBD not only based at eliminating AIEC strains from the inflamed intestinal mucosa but also throughout calibrated nutritional interventions.
Adhesion phenotypes assay. Phase contrast fields showing Hep-2 and Caco-2 cell monolayers infected with two representative AIEC strains isolated in this work (PP45 and DF6) and with reference AIEC LF82 strain. All 27 AIEC strains showed an aggregative (AA) patterns of adherence. Magnification, 1000 X.
Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn's disease patients: phenotypic and genetic pathogenic features.
October, 2014
Department of Public Health and Infectious Diseases, University of Rome, Italy
Conte MP, Longhi C, Marazzato M, Conte AL, Aleandri M, Lepanto MS, Zagaglia C, Nicoletti M, Aloi M, Totino V, Palamara AT, Schippa S
BACKGROUND:
Adherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn's disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features.
RESULTS:
A total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ2 = 6.167 P = 0.013), kpsMT II (χ2 = 6.167 P = 0.013), fyuA (χ2 = 6.167 P = 0.013), and ibeA (χ2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls.
CONCLUSIONS:
In conclusion, the AIEC-like abundance in CD patients and the presence of virulence genes only in CD patients further support the hypothesis that this pathovar may play an etiologic role in the insurgeonce and/or in the persistence in CD. These results are further supported by the recent findings indicating that colonization of the intestinal mucosa by AIEC strains, may initiate chronic inflammation of susceptible hosts by altering the gut microbiota [23]. Moreover, it has been recently reported a higher incidence of IBD in patients feed with fat-rich Western diet. It has been suggested that a fat-rich diet may induce changes in the composition of intestinal microbiota that, in turn, may influence the colonization and persistence of AIEC strains in genetically susceptible hosts [39]. These findings open up new scenarios addressed at the development of new strategies for the prevention and control of IBD not only based at eliminating AIEC strains from the inflamed intestinal mucosa but also throughout calibrated nutritional interventions.
Adhesion phenotypes assay. Phase contrast fields showing Hep-2 and Caco-2 cell monolayers infected with two representative AIEC strains isolated in this work (PP45 and DF6) and with reference AIEC LF82 strain. All 27 AIEC strains showed an aggregative (AA) patterns of adherence. Magnification, 1000 X.
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