Comprehensive probiogenomics analysis of the commensal Escherichia coli CEC15 as a potential probiotic strain

Abstract

Background: Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis.

Results: CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis.

Conclusions: These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

Keywords: Escherichia coli CEC15; Escherichia coli Nissle 1917; Gastrointestinal tract; Genomics; Immunomodulation; Mucositis; Probiogenomics; Probiotics.

Fig. 1

Phylogenomic tree of Escherichia coli strains. The phylogenomic analysis was based on 1,000 single-copies genes shared among all the strains. CEC15 and EcN strains are highlighted by a blue and a green box, respectively. Strains highlighted in red are pathogenic strains, while highlights in yellow and gray indicates commensal and environmental strains, respectively. The strains in purple are from the commercial probiotic Synbioflor2®

Fig. 2

Schematic circular representation of CEC15 (A) and EcN (B) genomic islands. Pathogenicity Island (PAI), Metabolic Island (MI), Resistance Island (RI), and Prophage regions were found on the genome. Figure generated by BRIG software. Circles, from the inside-out, indicate chromosome size (black circle), the GC skew positive (green) and negative (purple), the GC content (in black indicating higher content outwards and lower content inwards), and the chromosome (blue in figure A for CEC15 and red in figure B for EcN) with the location of PAI (blue), MI (green), RI (red), and prophage regions (orange)

Fig. 3

Hemolytic activity assay of E. coli strains. Strains Staphylococcus aureus IT2 (1), S. aureus Bk (2), CEC15 (3), and EcN (4) were spotted on sheep-blood agar and incubated overnight, the presence of a halo was observed for the two control strains (1 and 2) but not for the tested strains in this study (3 and 4)

Fig. 4

Bacterial survival in the simulated human digestive tract. Both strains were submitted to an artificial digestion process and, at each step, aliquots were collected to estimate the quantity of viable bacteria. CFU counting were made before the experiment begins (Initial), at the start of gastric phase (pH adjusted to 3 - T1), at the end of gastric phase and beginning of intestinal phase (120 min in pH 3 - T2), and at the end of intestinal phase (pH restored to 7 and 120 min incubation - T3). The lines represent the CFU count in each step of the digestion processes while the bars represent the viability in percentage relative to the initial CFU. Data are expressed as mean and standard deviation of three independent experiments

Fig. 5

Adhesive profile of CEC15 and EcN strains. The presence of fimbriae/pili and flagella in CEC15 and EcN strains could be confirmed by Transmission electron microscopy of fresh (A and D) and fixated (B and E), and by scanning electron microscopy (SEM) of fixated samples (C and F) of CEC15 and EcN, respectively. A significant quantity of proteins were found on sheared samples of both strains, about half of them being shared between strains (G). The heatmap (H) present the percentage of COG-classified proteins presented in each condition, according to the code: [C] Energy production and conversion; [D] Cell cycle control, cell division, chromosome partitioning; [E] Amino Acid metabolism and transport; [F] Nucleotide metabolism and transport; [G] Carbohydrate metabolism and transport; [H] Coenzyme metabolism; [I] Lipid metabolism; [J] Translation; [K] Transcription; [L] Replication and repair; [M] Cell wall/membrane/envelop biogenesis; [N] Cell motility; [O] Post-translational modification, protein turnover, chaperone functions; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport and catabolism; [S] Function Unknown; [T] Signal Transduction; [U] Intracellular trafficking and secretion. The effectiveness of these adhesins were tested by adhesion assay on Caco-2 cells (I) were CEC15 presented a better adhesive profile (23.31%) than EcN (1.46%). White arrows indicate the presence of fimbriae/pili. Black arrows indicate flagella. Scale in all pictures is equivalent to 1µm and the pictures were taken on amplification of 30,000 for TEM and 15,000 for SEM

Fig. 6

Modulation of immunoregulatory and barrier-related genes expression in Caco-2 cells. The relative gene expression of genes related to immunomodulation and intestinal barrier (Il1b (A and B), Il8 (C and D), Mcp1 (E and F), Nfkb1a (G and H), Ptgs2 (I and J), Tnf (K and L), Ocln (M and N), and Muc2 (O and P)) on CEC15- and EcN-treated cells, respectively, were evaluated with the Gapdh, B2m, and Hprt1 genes as reference (2^{- ΔΔct}). Statistical analyses were performed by One-way ANOVA with Tukey’s post-test on GraphPad Prism 7.0. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. NC: negative control; CEC15-SN: CEC15 supernatant; CEC15-M100: CEC15 treatment at MOI 100; CEC15-M10: CEC15 treatment at MOI 10; EcN-SN: EcN supernatant; EcN-M100: EcN treatment at MOI 100; EcN-M10: EcN15 treatment at MOI 10

Fig. 7

Clinical and histopathological aspects of E. coli strains’ administration. Mice (n = 8) were administered either sterile PBS, CEC15 (10¹⁰ CFU/mice/day) or EcN (10¹⁰ CFU/mice/day) for 12 days with administration of 5-FU (300 mg/kg) or PBS on the day 10. The results above show the weight variation (day 10-13) (A) and the morphological characteristics, such as intestinal permeability (B) and tissue neutrophilic infiltration (C). The structural damage caused by the 5-FU administration and the partial protection promoted by CEC15, as well as the unmodified morphology on the control groups and of the EcN treatment after mucositis induction can be observed on the slides (D), dyed with hematoxylin and eosin (Magnification of 20X). The histopathologic inflammatory scoring based on villous atrophy, rupture of the surface enterocyte borders, depletion of calyceal cells, loss of crypt architecture, destruction of crypt cells, abscess formation in the crypts, infiltration of lymphocytes and polymorphonuclear cells, dilation of capillaries and lymphatic vessels, and thickening with edema formation in the submucosa and external muscle layers. Histological features were scored on a scale of 0 (average) to 3 (max damage), and points were summed for each animal accordingly (E), villus height (F), and the depth of the crypts (G) were measured from these slides. Statistical analyses were performed by One-way ANOVA with Tukey’s post-test on GraphPad Prism 7.0. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. NC: negative control; CEC15: healthy CEC15-treated; EcN: healthy EcN-treated; MUC: mucositis control; CEC15/MUC: mucositis CEC15-treated; EcN/MUC: mucositis EcN-treated

Fig. 8

Alpha diversity and abundance of OTU of intestinal bacteria. A Alpha diversity shown by Shannon index estimated for each group: NC: negative control (n = 6); CEC15: healthy CEC15-treated (n = 7); EcN: healthy EcN-treated (n = 7); MUC: mucositis control (n = 4); CEC/MUC: mucositis CEC15-treated (n = 5); EcN/MUC: mucositis EcN-treated (n = 5). B Relative abundance of intestinal microbiota at the phylum level among the groups. C Heatmap analysis of the bacterial genus distribution among the 34 samples based on hierarchical clustering. One-way ANOVA and Bonferroni multiple comparisons test. Different letters indicate significative differences, p value < 0.05

Fig. 9

Relative abundance of the main genus of fecal bacteria. A–L abundances of genus in the feces of mice in different groups. Data are expressed in absolute OTU reads (± S.E.M). Different letters indicate statistical significance (P < 0.05; one-way ANOVA and Bonferroni multiple comparisons test). NC: negative control (n = 6); CEC15: healthy CEC15-treated (n = 7); EcN: healthy EcN-treated (n = 7); MUC: mucositis control (n = 4); CEC/MUC: mucositis CEC15-treated (n = 5); EcN/MUC: mucositis EcN-treated (n = 5)