Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice

Abstract

Resident bacteria are incriminated in the pathogenesis of experimental colitis and inflammatory bowel diseases. We investigated the relative roles of various enteric bacteria populations in the induction and perpetuation of experimental colitis. HLA-B27 transgenic rats received antibiotics (ciprofloxacin, metronidazole, or vancomycin-imipenem) in drinking water or water alone in either prevention or treatment protocols. Mice were treated similarly with metronidazole or vancomycin-imipenem before or after receiving 5% dextran sodium sulfate (DSS). Germfree transgenic rats were colonized with specific-pathogen-free enteric bacteria grown overnight either in anaerobic or aerobic atmospheres. Nontransgenic rats colonized with anaerobic bacteria served as negative controls. Although preventive metronidazole significantly attenuated colitis in transgenic rats and DSS-treated mice, it had no therapeutic benefit once colitis was established. Ciprofloxacin also partially prevented but did not treat colitis in B27 transgenic rats. In both animal models vancomycin-imipenem most effectively prevented and treated colitis. Germfree transgenic rats reconstituted with enteric bacteria grown under anaerobic conditions had more aggressive colitis than those associated with aerobic bacteria. These results suggest that a subset of resident luminal bacteria induces colitis, but that a complex interaction of commensal aerobic and anaerobic bacteria provides the constant antigenic drive for chronic immune-mediated colonic inflammation.

FIG. 1

Blinded histologic scores of the ceca from B27 TG treated with different antibiotics starting before (4 weeks of age, prevention protocol) (□) and after (3 months of age, treatment protocol) () the onset of colitis. All rats were killed at 4 months of age. n = 8 or 9 rats in each group. ∗, P < 0.05 versus TG water controls. Cipro, ciprofloxacin; Metro, metronidazole; Vanco, vancomycin. Error bars indicate SEMs.

FIG. 2

(A) Photomicrograph of cecal inflammation in a 4-month-old SPF B27/β₂-microglobulin TG rat treated with water alone. (B) A TG rat treated with metronidazole after the onset of colitis (3 months of age) showed only slight reduction of cecal inflammation. (C) The cecum of a TG rat treated with vancomycin-imipenem after the onset of colitis (3 months of age) revealed substantially less cecal inflammation. (D) Normal cecum of a 4-month-old NT control rat treated with water alone.

FIG. 3

Tissue IL-1β protein concentrations of the ceca from B27 TG rats treated with different antibiotics starting before (4 weeks of age) (□) and after (3 months of age) () the onset of colitis. ∗, P < 0.05 versus TG water. Cipro, ciprofloxacin; Metro, metronidazole; Vanco, vancomycin. Error bars indicate SEMs.

FIG. 4

Blinded histologic scores of the ceca from BALB/c mice with DSS-induced colitis treated with different antibiotics starting 3 days before or 2 days after the onset of DSS administration. The 5% DSS was continued for a total of 7 days. ∗, P < 0.05 versus water. □, Prevention; , treatment; ■, water control. Error bars indicate SEMs.

FIG. 5

Total bacterial concentrations in the cecal contents of NT rats treated with different antibiotics for 4 weeks, measured in a counting chamber. ∗, P < 0.05 versus control; §, P < 0.05 versus ciprofloxacin (Cipro) and metronidazole (Metro). Vanco, vancomycin. Error bars indicate SEMs.

FIG. 6

Alteration of the ratio of anaerobic bacteria to aerobic bacteria by antibiotics compared to the ratio of anaerobic bacteria to aerobic bacteria in the water control group ([anaerobes/aerobes with antibiotics]/[anaerobes/aerobes for water control]). Antibiotic treatment resulted in an altered bacterial composition after 6 weeks. Compared with the water controls, vancomycin-imipenem reduced more anaerobes than aerobes as demonstrated by the decreased ratio of anaerobes to aerobes to 0.5 of that of the water control group. This alteration of bacterial composition was even exceeded by metronidazole, where the ratio of anaerobes to aerobes dropped to 1/10 of that of the water control group. In contrast ciprofloxacin reduced predominantly aerobes as shown by the increase of the anaerobic/aerobic ratio to 1.5-fold that of the water control group.

FIG. 7

Blinded histologic scores of the ceca from germfree B27 TG rats colonized for 1 month with cultures of SPF cecal bacteria incubated overnight under anaerobic or aerobic conditions. n = 6 to 9 rats/group. ∗, P < 0.05 versus anaerobic; §, P < 0.05 versus aerobic. Error bars indicate SEMs.