Interaction of adherent-invasive Escherichia coli with mammalian gut epithelial cells and the regulatory function of probiotics

Georgia Bradford

doi:10.25907/00996

Adherent-Invasive Escherichia coli (AIEC) are increasingly being linked to inflammatory bowel diseases (IBD) such as Crohn’s disease (CD), ulcerative colitis (UC), and the development of colorectal cancer (CRC). However, its exact role and pathogenicity remains poorly understood. AIEC strains are complex to characterise due to their patho-adapting abilities, adding to the growing concern of treating or preventing their colonisation. Probiotics are known for their potential therapeutic effects for those individuals with gastrointestinal pathogenic colonisations. However, their efficacy and modes of action against AIEC require further investigation. Using a co-culture of Caco-2 and HT29-MTX cell lines as an in vitro epithelium model and cell-free testing models, this project aimed to investigate the efficacy and modes of action of four individual probiotic strains and their combination in reducing AIEC colonisation of the human intestinal epithelium. The second aim of this project was to determine the primary virulence genes involved in AIEC pathogenesis and identify how AIEC differ from other pathotypes of E. coli. The ability of the probiotic strains or their combination to reduce AIEC interaction with the epithelial model was determined by challenging the strains with a wild type AIEC strain (F44A-1), isolated from a fatal case of CRC, both in co-inoculation and pre-inoculation assays. Adhesion, invasion and translocation testing of AIEC F44A-1 confirmed that strain is highly pathogenic, however all probiotic strains, individually and in combination significantly reduced their interaction with the cell model. However, the results demonstrated that the combination of strains did not significantly improve their efficacy against AIEC. The combination of strains was of equal or less effectiveness than the probiotic strains individually. The probiotics demonstrated that pre-inoculation of the strains generated the greatest inhibition of the AIEC, rather than co-inoculation, indicating their use as potential prophylactic treatment options. The modes of action used by the probiotic strains and their combination to inhibit AIEC colonisation were further tested when assessing for antimicrobial secretions and anti-biofilm properties. These studies demonstrated that significant reductions to AIEC occur before the pathogen interacts with the cell model, and that the probiotic-mediated inhibitory effects observed are species and environment specific. In view of the potential role of AIEC in the development of IBD, this project also investigated the pathogenicity of AIEC strain F44A-1 and a collection of 40 E. coli strains isolated from patients with IBD by testing their virulence gene (VG) profiles, interactions with the cell model and the correlation of these abilities with their phylogenetic grouping. The results indicated that all seven tested VGs (ibeA, afaC, lpfA, ompC, htrA, dsbA, and clbA) were present in varying profiles amongst the strains, with ibeA, htrA, and ompC genes carried in more the 85% of the strains. Gene expression studies were then conducted using F44A-1 and the cell model. The results indicated that ibeA and htrA genes were significantly upregulated, in addition to their high prevalence, this suggested that these genes may play a primary role in pathogenesis of AIEC. Whole genome sequencing (WGS) and bioinformatic analysis was conducted on F44A-1 and three selected AIEC strains based on the previous study. One strain (90-I) was later classified as Enterobacter hormaechei based on their WGS, whereas F44A-1 and the remaining two strains (86-I and 89-I) were confirmed as AIEC. Whilst strains 86-I and 89-I were revealed to be quite similar in their serotyping, phylogrouping, virulence factors and antimicrobial resistance, F44A-1 stood out. Annotation and other analysis tools identified genomic islands that contribute to its pathogenesis, specifically its virulence and antimicrobial resistance, with F44A-1 carrying genes that correspond with its phenotypic properties and other published work looking at AIEC. Genomic analysis of these strains allowed for detailed comparison between each strain and other previously published AIEC WGS from databases, revealing a better understanding of the role VGs play in pathogenesis of AIEC and their genetic diversity.

Interaction of adherent-invasive Escherichia coli with mammalian gut epithelial cells and the regulatory function of probiotics

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