Induction of experimental ulcerative colitis by Fusobacterium varium isolated from colonic mucosa of patients with ulcerative colitis

Abstract

Background: Bacteria are implicated in certain forms of model chronic colitis but the identity and role of bacteria in human ulcerative colitis (UC) are uncertain.

Aims: To isolate pathogenic bacteria from inflamed mucosa of patients with UC, to examine whether the bacteria have a toxin to Vero cells, and to determine whether the toxin induces UC-like lesions in animals.

Methods: Bacteria were isolated from UC patients and supernatants from cultures were filtered and tested for cytotoxicity to Vero cells. Bacterial cells producing the cytotoxic supernatants were examined by polymerase chain reaction for verotoxin genes. Culture supernatants of cytotoxic strains were examined by high performance liquid chromatography for organic acid concentrations. Mice were given enemas containing organic acid at the mean concentration in the supernatants of cytotoxic strains to ascertain whether colonic lesions appear in UC.

Results: Only supernatants from cultures of Fusobacterium varium killed Vero cells. Bacterial cells lacked verotoxin genes. Bacterial culture supernatants contained high concentrations of n-butyric acid and the mean concentration (32 mmol/l) was cytotoxic to Vero cells. Twenty four hours after mice were given enemas containing either butyric acid or F varium culture supernatants, colonic ulcers with crypt abscesses, inflammatory cell infiltration, and apoptotic changes were observed.

Conclusions: Butyric acid in culture supernatants from cultures of F varium caused UC-like lesions in mice. This study indicates that F varium may be one of the elusive pathogenic factors in UC.

Figure 1

Cytotoxic effects of culture supernatant of one strain of F varium on Vero cells. Cells had long filamentous tendrils, were rounded, and many were detaching into the culture medium. Culture supernatant of E coli O157:H7 EDL-931, which produces verotoxin, had the same effects (not shown). Uninoculated culture broths gave no cytotoxicity.

Figure 2

In vivo effects of butyric acid, culture supernatant of F varium, and control substances on colorectal mucosa of mice. (A) Crypt abscess formation in a mouse treated with butyric acid. Inflammatory cell infiltration and early erosive changes are apparent in the upper portion of the colorectal mucosa. A crypt abscess (arrow) is noted. Pathological index of 3 (24 hours after butyrate enema, haematoxylin and eosin (H&E), original magnification ×400). (B) Early mucosal changes in a mouse treated with a culture supernatant of F varium. Degenerative changes are evident in the superficial layer of the colorectal mucosa. Several apoptotic bodies (arrows) are present in the lower portions of the intestinal crypts. Mild inflammatory cell infiltration can be seen in the lamina propria. Pathological index of 2 (24 hours after supernatant enema, H&E, original magnification ×400). (C) Early diffuse erosion in a mouse treated with a culture supernatant of F varium. Diffuse erosive change is apparent in the colorectum, which shows luminal inflammatory exudation. Severe inflammatory cell infiltration can be seen in the lamina propria, with extensive gland loss. Pathological index of 4 (supernatant enema, H&E, original magnification ×400). (D) In this mouse and other mice treated with culture supernatant or butyric acid, many apoptotic cells are evident with terminal uridine DNA nick end labelling (TUNEL) in the lower part of the intestinal crypts (butyric acid enema, original magnification ×300).

Figure 3

Pathological and apoptotic indices of ulcerative colitis-like lesions caused in the colorectum by F varium culture supernatant or butyric acid enemas. The mean pathological index of mice treated with F varium (3.3) or butyric acid (3.5) was higher than that of mice treated with HCl (p=0.021 and 0.019), ABCM broth (both p<0.001), or phosphate buffered saline (PBS) (both p<0.001). Similarly, the mean apoptotic indices for mice treated with F varium (24.0%) or butyric acid (20.8%) were higher than for HCl (p=0.0045 and 0.012), ABCM broth (both p<0.001), or PBS (both p<0.001). The mean apoptotic index of mice treated with HCl (10.1%) was higher than that of ABCM broth or PBS (both p<0.001). Values are mean (SD); statistical analyses were performed using the Mann-Whitney U test with Bonferroni’s correction.