Probiotic Properties of Escherichia coli Nissle in Human Intestinal Organoids

EHEC and UPEC destroy the epithelial barrier. HIOs were injected with 10³ CFU of Nissle, UPEC, or EHEC along with the fluorescent dye FITC-dextran. (A) The retention of fluorescence was monitored microscopically over time. Bars, 100 μm. Representative images of experiments performed in triplicate are shown. (B) The epithelial barrier function was assessed by quantifying the retention of fluorescence using ImageJ software, and the fluorescence is plotted as the mean ± SD (n = 3). The statistical significance (determined by an unpaired t test) of the results for Nissle versus those for EHEC was assessed at 18 h postinfection (**, P < 0.003), and the statistical significance of the results for Nissle versus those for UPEC was assessed at 72 h postinfection (*, P < 0.02).

Epithelial damage of infected HIOs. HIOs were microinjected with 10³ CFU of Nissle, EHEC, or UPEC and harvested at the indicated times postinjection. Cryosections of infected HIOs were stained for DNA (blue; DAPI), bacteria (green; anti-E. coli for Nissle and UPEC, anti-O157 for EHEC), and F-actin (red; phalloidin) and assessed by confocal microscopy. The lumen is labeled. Bars, 20 μm. Representative images of experiments performed in triplicate are shown. (A) Nissle at 18 hours; (B) EHEC at 18 hours; (C) Nissle at 72 hours; (D) UPEC at 72 hours.

Nissle and EHEC coinfection. (A) Experimental timeline. HIOs were injected with 10³ CFU of Nissle. After 12 h, half were challenged with 10³ CFU of EHEC. (B) Plot of the mean number of CFU ± SD (n = 3) determined at the indicated times. (C) Cryosection of coinfected HIOs at 42 h (30 h after challenge with EHEC) stained for DNA (blue; DAPI), bacteria (green; anti-O157), and F-actin (red; phalloidin). Bar, 20 μm. A representative image of an experiment performed in triplicate is shown.

Nissle and UPEC coinfection. (A) Experimental timeline. HIOs were injected with 10³ CFU of Nissle, and 24 h later, half were challenged with 10³ CFU of UPEC. (B) Plot of the mean number of CFU ± SD (n = 3) determined at the indicated times. (C) Cryosection of coinfected HIOs at 96 h (72 h after challenge with UPEC) stained for DNA (blue; DAPI), bacteria (green; anti-E. coli), and F-actin (red; phalloidin). Bar, 20 μm. A representative image of an experiment performed in triplicate is shown.

Nissle protects the epithelial barrier function from infection with pathogenic UPEC and EHEC. HIOs were microinjected with 10³ CFU of Nissle along with FITC-dextran and challenged with 10³ CFU of EHEC at 12 h or UPEC at 24 h. (A) The retention of fluorescence was monitored microscopically over time. Each column represents a single organoid imaged at the indicated time. Bars (displayed only on the final image), 100 μm. Representative images of experiments performed in triplicate are shown. (B) The epithelial barrier function was assessed by quantifying the retention of fluorescence using ImageJ software, and the results are plotted as the mean ± SD, with at least 3 repeats being performed. Differences were not statistically significant at 72 h by analysis of variance with Bonferroni’s multiple-comparison posttest.

E-cadherin expression. Cryosections from saline-injected controls, single-strain infections, and coinfection experiments (as described in Fig. 4 and 5) were stained for E-cadherin. A white box was sized to outline the epithelial cells stained for E-cadherin in the control HIOs. Keeping the boxed area constant, fluorescence was quantified for three random epithelial areas for each organoid, and the results were averaged. Statistical analysis was performed using the averages for the three independent repeats (n = 3). (A) Saline, 72 h postinjection; (B) Nissle, 72 h postinjection; (C) EHEC, 18 h postinjection; (D) Nissle and EHEC, coinfection at 18 h; (E) UPEC, 72 h postinjection; (F) Nissle and UPEC, coinfection at 72 h. White boxes indicate the regions used to quantify fluorescence. Bars, 20 μm. (G and H) Fluorescence was quantified using ImageJ software and plotted as the mean ± SD (n = 3). (G) E-cadherin fluorescence in the EHEC challenge; (H) E-cadherin fluorescence in the UPEC challenge. Statistical significance was assessed with GraphPad Prism (version 5) software, using one-way analysis of variance with Bonferroni’s posttest. *, P = 0.1 to 0.01; ***, P < 0.0001.

Nissle protects from apoptosis. HIOs were microinjected with 10³ CFU of Nissle, EHEC alone, or UPEC alone or coinfected, as described in Fig. 4 and 5. The outer border of the HIO, including the mesenchymal layer, was marked, and the caspase 3 total fluorescence within the area was determined. Values were normalized by dividing the total fluorescence intensity by the area of the organoid. Each infection was performed in triplicate. (A to F) Cryosections of infected HIOs were stained for DNA (blue) and the apoptotic marker activated caspase 3 (red). (A) Nissle, 18 h postinfection; (B) EHEC, 18 h postinfection; (C) Nissle and EHEC, coinfection at 18 h; (D) Nissle, 72 h postinfection; (E) UPEC, 72 h postinfection; (F) Nissle and UPEC, coinfection at 72 h. Bars, 50 μm. L, lumen; M, mesenchyme. (G) Fluorescence was quantified using ImageJ software and plotted as the mean ± SD (n = 3). Statistical significance was assessed with GraphPad Prism (version 5) software, using one-way analysis of variance with Bonferroni’s posttest. The results of comparisons between the pathogen with Nissle and the pathogen without Nissle are indicated. *, P = 0.1 to 0.01; **, P = 0.001 to 0.01.

Nissle influences ROS production. HIOs were microinjected with saline; 10³ CFU of Nissle, EHEC alone, or UPEC alone; or coinfected, as described in Fig. 4 and 5. (A to F) HIOs were injected with an ROS detection reagent, and ROS fluorescence was measured. (A) Saline, 72 h postinjection; (B) Nissle, 72 h postinjection; (C) EHEC, 18 h postinjection; (D) Nissle and EHEC, coinfection at 18 h; (E) UPEC, 72 h postinjection; (F) Nissle and UPEC, coinfection at 72 h. Bars, 50 μm. (G) Fluorescence intensity was quantified using ImageJ software and plotted as the mean ± SD (n = 3). Statistical significance was assessed with GraphPad Prism (version 5) software, using one-way analysis of variance with Bonferroni’s posttest. *, P = 0.1 to 0.01; **, P = 0.001 to 0.01; ***, P < 0.0001. (H) The number of CFU recovered was determined at the indicated time points (n = 3).

Strain competition in broth culture. (A) Phage life cycle. (Top) Lytic infection results in phage production and bacterial death; (bottom) the repressor produced by lysogenic phage can prevent lytic infection and bacterial death. (B to F) LB broth or filter-sterilized culture supernatant was inoculated with 10³ CFU of a single strain or strains mixed 1:1, as indicated, and the numbers of CFU were assessed over time. (B) LB broth was inoculated with Nissle, EHEC, or both. The growth of Nissle was suppressed by EHEC. (C) Nissle was inoculated into LB broth. Nissle and a colony of Nissle that survived coculture with EHEC (Nissle::Stx2PT) were inoculated into the culture supernatant from EHEC. The growth of Nissle was suppressed by the EHEC supernatant. (D) Nissle was inoculated into LB broth or the supernatant from Nissle::Stx2PT. The growth of Nissle was suppressed by the Nissle::Stx2PT supernatant. (E) LB broth was inoculated with Nissle, EDL933, or both. The growth of neither strain was suppressed in mixed culture. (F) LB broth was inoculated with Nissle, UPEC, or both. The growth of neither strain was suppressed in mixed culture.