Saccharomyces cerevisiae prevents postoperative recurrence of Crohn's disease modeled by ileocecal resection in HLA-B27 transgenic rats

Abstract

Background: Postoperative recurrence (POR) after ileocecal resection (ICR) affects most Crohn's disease patients within 3-5 years after surgery. Adherent-invasive Escherichia coli (AIEC) typified by the LF82 strain are pathobionts that are frequently detected in POR of Crohn's disease and have a potential role in the early stages of the disease pathogenesis. Saccharomyces cerevisiae CNCM I-3856 is a probiotic yeast reported to inhibit AIEC adhesion to intestinal epithelial cells and to favor their elimination from the gut.

Aim: To evaluate the efficacy of CNCM I-3856 in preventing POR induced by LF82 in an HLA-B27 transgenic (TgB27) rat model.

Methods: Sixty-four rats [strain F344, 38 TgB27, 26 control non-Tg (nTg)] underwent an ICR at the 12^th wk (W12) of life and were sacrificed at the 18^th wk (W18) of life. TgB27 rats were challenged daily with oral administration of LF82 (10⁹ colony forming units (CFUs)/day (d), n = 8), PBS (n = 5), CNCM I-3856 (10⁹ CFUs/d, n = 7) or a combination of LF82 and CNCM I-3856 (n = 18). nTg rats receiving LF82 (n = 5), PBS (n = 5), CNCM I-3856 (n = 7) or CNCM I-3856 and LF82 (n = 9) under the same conditions were used as controls. POR was analyzed using macroscopic (from 0 to 4) and histologic (from 0 to 6) scores. Luminal LF82 quantifications were performed weekly for each animal. Adherent LF82 and inflammatory/regulatory cytokines were quantified in biopsies at W12 and W18. Data are expressed as the median with the interquartile range.

Results: nTg animals did not develop POR. A total of 7/8 (87%) of the TgB27 rats receiving LF82 alone had POR (macroscopic score ≥ 2), which was significantly prevented by CNCM I-3856 administration [6/18 (33%) TgB27 rats, P = 0.01]. Macroscopic lesions were located 2 cm above the anastomosis in the TgB27 rats receiving LF82 alone and consisted of ulcerations with a score of 3.5 (2 - 4). Seven out of 18 TgB27 rats (39%) receiving CNCM I-3856 and LF82 had no macroscopic lesions. Compared to untreated TgB27 animals receiving LF82 alone, coadministration of CNCM I-3856 and LF82 significantly reduced the macroscopic [3.5 (2 - 4) vs 1 (0 - 3), P = 0.002] and histological lesions by more than 50% [4.5 (3.3 - 5.8) vs 2 (1.3 - 3), P = 0.003]. The levels of adherent LF82 were correlated with anastomotic macroscopic scores in TgB27 rats (r = 0.49, P = 0.006), with a higher risk of POR in animals having high levels of luminal LF82 (71.4% vs 25%, P = 0.02). Administration of CNCM I-3856 significantly reduced the levels of luminal and adherent LF82, increased the production of interleukin (IL)-10 and decreased the production of IL-23 and IL-17 in TgB27 rats.

Conclusion: In a reliable model of POR induced by LF82 in TgB27 rats, CNCM I-3856 prevents macroscopic POR by decreasing LF82 infection and gut inflammation.

Keywords: Colorectal surgery; Crohn's disease; Escherichia coli; Probiotic; Recurrence; Saccharomyces cerevisiae.

Figure 1

Study design. HLA-B27 transgenic rats (Tg) and wild-type rats (nTg) were randomized to receive phosphate buffered saline (n = 10), Saccharomyces cerevisiae (S. cerevisiae) CNCM I-3856 (n = 14), adherent-invasive Escherichia coli strain LF82 (n = 13), or S. cerevisiae CNCM I-3856 and LF82 (n = 27) by oral gavage from week (W) 10 or 11 to W18. Ileocecal resection was performed at W12, and animals were sacrificed at W18. Streptomycin (dotted line) was given on the last 3 d of W10 in all rats. Luminal (arrows) and/or adherent (dotted arrows) LF82 was quantified weekly during the 8-wk study. CFU: Colony-forming unit; PBS: Phosphate buffered saline; ICR: Ileocecal resection; d: Day.

Figure 2

Anastomotic macroscopic score (0-4).

Figure 3

Body weight evolution. A: Evolution of weight changes compared to body weight at W11 in nontransgenic (nTg) rats; B: Evolution of weight changes compared to body weight at W11 in transgenic (Tg) rats. ^bP < 0.01. LF82: Adherent-invasive Escherichia coli strain LF82; CNCM I-3856: Saccharomyces cerevisiae CNCM I-3856; PBS: Phosphate buffered saline.

Figure 4

Anastomotic macroscopic lesions and postoperative recurrence at sacrifice. A: Anastomotic macroscopic scores in the different groups of HLA-B27 transgenic (Tg) rats and wild-type (nTg) rats at sacrifice; B: % postoperative recurrence (anastomotic macroscopic score ≥ 2) at sacrifice in HLA-B27 Tg rats. ^aP < 0.05, ^bP < 0.01. LF82: Adherent-invasive Escherichia coli strain LF82; CNCM I-3856: Saccharomyces cerevisiae CNCM I-3856; PBS: Phosphate buffered saline.

Figure 5

Anastomotic histologic lesions at sacrifice. Anastomotic histologic scores in the different groups of HLA-B27 transgenic (Tg) rats and wild-type (nTg) rats at sacrifice. ^aP < 0.05, ^bP < 0.01. LF82: Adherent-invasive Escherichia coli strain LF82; CNCM I-3856: Saccharomyces cerevisiae CNCM I-3856; PBS: Phosphate buffered saline.

Figure 6

Levels of luminal adherent-invasive Escherichia coli LF82 at surgery and sacrifice. A: Luminal levels of adherent-invasive Escherichia coli strain LF82 at surgery [week (W) 12] and sacrifice (W18) in the different groups of HLA-B27 transgenic (Tg) rats and wild-type (nTg) rats; B: Luminal levels of LF82 at W12 and W18 in paired Tg rats receiving LF82 alone; C: Luminal levels of LF82 at W12 and W18 in paired Tg rats receiving Saccharomyces cerevisiae CNCM I-3856 and LF82. ^bP < 0.01, ^cP < 0.001. CFU: Colony-forming unit; log10: Decimal logarithm.

Figure 7

Levels of anastomotic adherent adherent-invasive Escherichia coli LF82 at surgery and sacrifice. A: Adherent levels of adherent-invasive Escherichia coli strain LF82 at surgery [week (W) 12] and sacrifice (W18) in the different groups of HLA-B27 transgenic (Tg) rats and wild-type (nTg) rats; B: Adherent levels of LF82 at W12 and W18 in paired Tg rats receiving LF82 alone; C: Adherent levels of LF82 at W12 and W18 in paired Tg rats receiving Saccharomyces cerevisiae CNCM I-3856 and LF82. ^cP < 0.001. CFU: Colony-forming unit; log10: Decimal logarithm.

Figure 8

Evolution of the levels of luminal adherent-invasive Escherichia coli LF82 after surgery. A: Weekly evaluation of the luminal LF82 Levels after surgery in HLA-B27 transgenic (Tg) rats and wild-type (nTg) rats receiving adherent-invasive Escherichia coli strain LF82 alone or Saccharomyces cerevisiae CNCM I-3856 and LF82; B: Global persistence of viable luminal LF82 after surgery and during the last 5 wk of the study in Tg rats receiving LF82 alone or CNCM I-3856 and LF82. ^cP < 0.001. CFU: Colony-forming unit; log10: Decimal logarithm.

Figure 9

Correlation between anastomotic macroscopic scores and adherent and luminal adherent-invasive Escherichia coli LF82 levels. A: Adherent levels of adherent-invasive Escherichia coli strain LF82 at sacrifice [week (W) 18] were correlated with anastomotic macroscopic scores at sacrifice in paired transgenic (Tg) animals receiving LF82 alone or in combination with Saccharomyces cerevisiae CNCM I-3856; B: At surgery (W12), the levels of adherent LF82 were correlated with luminal LF82 Levels in paired Tg animals receiving LF82 alone; C: At W18, the levels of adherent LF82 were correlated with luminal LF82 Levels in paired Tg animals receiving LF82 alone. ^aP < 0.05, ^bP < 0.01. CFU: Colony-forming unit; log10: Decimal logarithm.

Figure 10

Prognostic value of luminal adherent-invasive Escherichia coli LF82 levels in postoperative recurrence. A: Correlation between luminal adherent-invasive Escherichia coli strain LF82 Levels at week 14 and the risk of postoperative (POR) recurrence at W18 in transgenic (Tg) animals receiving LF82 alone or Saccharomyces cerevisiae (S. cerevisiae) CNCM I-3856 and LF82; B: Higher frequency of POR in highly infected (HI) Tg animals receiving LF82 alone or S. cerevisiae CNCM I-3856 and LF82 as defined by a cutoff value of 2.262 Log₁₀ CFUs (colony-forming units) of luminal LF82 per gram of stool at W14 in comparison with mildly infected (MI) Tg rats (71.4% vs 25%, P = 0.02). ^aP < 0.05. CFU: Colony-forming unit; log10: Decimal logarithm.

Figure 11

Interleukin-10 mRNA expression in the anastomotic mucosa. A: Interleukin (IL)-10 mRNA expression between surgery (week (W) 12) and sacrifice (W18) in paired Tg rats receiving adherent-invasive Escherichia coli strain LF82 alone; B: IL-10 mRNA expression between W12 and W18 in paired Tg rats receiving Saccharomyces cerevisiae (S. cerevisiae) CNCM I-3856 and LF82; C: IL-10 mRNA expression between W12 and W18 in paired Tg rats receiving S. cerevisiae CNCM I-3856 alone. ^aP < 0.05. β-act: β-actin.

Figure 12

Interleukin-23 mRNA expression in the anastomotic mucosa. A: Expression of interleukin (IL)-23 mRNA in the perianastomotic mucosa in all transgenic (Tg) and nontransgenic (nTg) groups at sacrifice; B: IL-23 mRNA expression between surgery [week (W) 12] and sacrifice (W18) in paired Tg rats receiving adherent-invasive Escherichia coli strain LF82 alone; C: IL-23 mRNA expression between W12 and W18 in paired Tg rats receiving coadministration of Saccharomyces cerevisiae (S. cerevisiae) CNCM I-3856 and LF82; D: IL-23 mRNA expression between W12 and W18 in paired Tg rats receiving S. cerevisiae CNCM I-3856 alone. ^aP < 0.05, ^bP < 0.01. β-act: β-actin; PBS: Phosphate buffered saline.

Figure 13

Interleukin-17 mRNA expression in the anastomotic mucosa. A: Expression of interleukin (IL)-17 mRNA in the perianastomotic mucosa in all transgenic (Tg) and nontransgenic (nTg) groups at sacrifice; B: IL-17 mRNA expression between surgery [week (W) 12] and sacrifice (W18) in paired Tg rats receiving adherent-invasive Escherichia coli strain LF82 alone; C: IL-17 mRNA expression between W12 and W18 in paired Tg rats receiving coadministration of Saccharomyces cerevisiae (S. cerevisiae) CNCM I-3856 and LF82; D: IL-17 mRNA expression between W12 and W18 in paired Tg rats receiving S. cerevisiae CNCM I-3856 alone. ^aP < 0.05. β-act: β-actin; PBS: Phosphate buffered saline.