Effect of Oral Administration of Butyrivibrio fibrisolvens MDT-1 on Experimental Enterocolitis in Mice

Bacterial strain and culture conditions. B. fibrisolvens MDT-1 was obtained as described previously (30). Clostridium butyricum MIYAIRI 588, Campylobacter coli (15 strains; isolated from farm animals), and Campylobacter jejuni (5 strains; isolated from farm animals) were provided by Meiji Seika Kaisha, Ltd. (Tokyo, Japan).

B. fibrisolvens and C. butyricum were grown in B. fibrisolvens medium, which consisted of a basal medium (14) supplemented with short-chain fatty acids, vitamins, and trace minerals (13). Culture conditions and procedures were previously described (14). Cell growth was estimated by changes in optical density at 600 nm. Cells were harvested at an optical density at 600 nm of 1.3 to 1.5 (late log growth) and cooled immediately on ice. Viable cells were counted using Hungate's roll tube method in a medium containing 2% agar (29). Viability was scored as the mean number of colonies from counts of five separate roll tubes for each sample.

Mice.Four-week-old male Jcl:ICR mice (CLEA Japan, Tokyo, Japan) were used for all experiments. Mice were housed in plastic cages with wire tops and permitted free access to a commercial diet (CE-2; CLEA Japan, Tokyo, Japan) and water. The diet consisted of a mixture of 249 g crude protein, 46 g crude fat, 37 g crude fiber, 67 g ash, and 514 g nitrogen-free extracts, with an energetic content of 829 kJ/kg feed. All of the animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Meiji University (IACUC-05-0007).

Oral administration of B. fibrisolvens and C. butyricum to mice. B. fibrisolvens MDT-1 and C. butyricum grown to late log stage were collected by centrifugation (5,000 × g, 10 min, 4°C) and washed with 0.8% NaCl. Washed cells resuspended in 0.8% NaCl (10¹³ CFU/liter) were administered to mice by gavage at a level of 0.1 ml or 10⁹ CFU · mouse⁻¹ · dose⁻¹ (or day⁻¹).

Animal experiment 1 (DSS-induced colitis).The protocol of this experiment is outlined in Fig. 1. Ten mice were used for each group. Using a stomach tube, mice in group A (control) were given 0.1 ml of 0.8% NaCl solution once a day for 3 days, followed by 7 days of drinking water. Group B mice were given 0.1 ml of 0.8% NaCl solution once a day for 3 days, followed by 7 days of drinking water containing 3% DSS (molecular weight, 40,000; MP Biomedicals, Germany) (32). Live MDT-1 and C. butyricum (10⁹ CFU/dose) were administered once a day for 3 days to group C and D mice, respectively, followed by 7 days of drinking water containing 3% DSS.

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FIG. 1.

Protocols for animal experiments 1 (A) and 2 (B). Group A, addition of 0.8% NaCl solution (N) for 10 days (control); group B, addition of 0.8% NaCl for 3 days and then DSS (D); group C, addition of B. fibrisolvens MDT-1 (B) for 3 days and then DSS; group D, addition of C. butyricum (Cb) for 3 days and then DSS; group E, addition of 0.8% NaCl for 10 days (control); group F, addition of 0.8% NaCl for 10 days and C. coli (Cc) on day 0; group G, addition of B. fibrisolvens MDT-1 for 10 days and C. coli on day 0. A, autopsy.

Daily clinical assessment of DSS-treated animals included measurement of the volume of drinking water consumed and body weight, evaluation of stool consistency, and the absence or presence of blood in the feces by ocular inspection. After 10 days, mice were sacrificed by cervical dislocation under ether anesthesia for autopsy. For each mouse, the liver and spleen were weighed separately, and the colon was removed. After exclusion of the cecum, colon length and weight were measured and tissue samples were then prepared for histological (five mice in each group) and MPO activity (the other five mice in each group) evaluations.

Disease activity index.Disease activity was quantified using clinical scores previously established by Hartmann et al. (18) that assess body weight loss, stool consistency, and rectal bleeding. For body weight, no loss was counted as 0 points, 1 to 5% loss as 1 point, 5 to 10% loss as 2 points, 10 to 20% loss as 3 points, and >20% loss as 4 points. For stool consistency, no points were given for well-formed pellets, 2 points were given for pasty and semiformed stools that did not stick to the anus, and 4 points were given for liquid stools that did stick to the anus. Bleeding was scored as 0 points for no blood observed, 2 points for a positive finding of blood, and 4 points for gross bleeding. The mean of these three scores represented the clinical score, with a theoretical range of 0 (healthy) to 4 (maximal severity of disease).

Histology.Histological examination was performed on samples of distal colon coming from 5 mice in each experimental group of 10 animals. Samples were fixed in 10% formalin before being embedded in paraffin. After deparaffinization, samples were cut into 5-μm-thick sections and stained with hematoxylin and eosin. The mucosal damage was evaluated independently by two investigators blinded to the study groups and quantified using mucosal damage scores previously established by Oda (28). These scores derived from the two investigators were averaged. The following three parameters were used: surface epithelial loss, crypt destruction, and inflammatory cell infiltration into the mucosa. A score of 0 to 4 was assigned to each of the three parameters according to the extent and severity of the change: 0, no change; 1, localized and mild; 2, localized and moderate; 3, extensive and moderate; and 4, extensive and severe. The sum of the scores from the three parameters was defined as the mucosal damage score for each animal. In addition, toluidine blue staining (pH 2.5) was also performed to evaluate sulfated polysaccharide in the mucosa (21).

MPO activity of colonic tissue.MPO activity was assayed according to Tian et al. (38). Briefly, the most distal 6-cm segment of the colon was mashed with mortar and pestle in 1% (wt/wt) HTAB (hexadecyltrimethylammonium bromide; Wako, Osaka, Japan) dissolved in 20 mM potassium phosphate buffer (pH 6.0, 50 mg of tissue/ml). The mashed tissue was sonicated, and the extract was then cleared by centrifugation (20,000 × g, 15 min, 4°C). The supernatant (0.01 ml) was mixed in a microplate well with 0.2 ml of 20 mM potassium phosphate buffer (pH 6.0) containing 20 mM guaiacol and 0.05% (wt/wt) hydrogen peroxide. Microplates were left at room temperature for 20 min, and then each reaction was terminated with 0.05 ml of 0.8 N sulfuric acid. The absorbance at 460 nm was measured with a multilabel counter (Arvo 1420; Wallac, Turku, Finland), and the increase in absorbance relative to that of a blank with no extract was used to assess MPO activity.

Inhibitory effect of MDT-1 on the growth of Campylobacter.The supernatant of MDT-1 cultures grown to late log stage was collected by centrifugation (20,000 × g, 10 min, 4°C) and filtered aseptically through a membrane filter (pore size, 0.22 μm). Brain/heart infusion agar (Becton Dickinson, New Jersey) containing 5% lysed horse blood (Nippon Bio-Test Laboratories, Tokyo, Japan) was supplemented to a level of 10% with either this supernatant or the plain growth medium used for B. fibrisolvens (as a control). The agar plates were then inoculated with individual strains of Campylobacter coli (15 strains) or Campylobacter jejuni (5 strains) and incubated at 42°C under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂) generated with CampyPak Plus (Becton Dickinson, New Jersey) in a GasPak jar (Becton Dickinson, New Jersey). Growth inhibition was judged by the significant reduction in colonies formed after 48 h of incubation.

Animal experiment 2 (effect of MDT-1 on infection with Campylobacter). C. coli 11580-3 was grown in a microaerophilic environment for 24 h at 42°C on brain/heart infusion agar supplemented with 5% lysed horse blood. Bacterial cells collected by scraping were resuspended in 0.8% NaCl solution (10⁷ CFU/ml).

The protocol of this experiment is outlined in Fig. 1. As in experiment 1, 10 mice were used for each group, and 0.1 ml of 0.8% NaCl solution was given to group E mice (control) once a day throughout the experimental period (10 days). Group F mice were similarly given 0.8% NaCl solution, but C. coli 11580-3 (10⁷ CFU) was administered on the fourth day (one dose). For group G mice, MDT-1 (10⁹ CFU/dose) was administered once a day for 10 days, and C. coli 11580-3 (10⁷ CFU) was administered on the fourth day. Stool consistency was scored during the experimental period. All mice were sacrificed 7 days after C. coli inoculation for autopsy. The colon was removed, and cecal contents were harvested by extrusion. The colonic tissue samples were then prepared for histological (five mice in each group) and MPO activity (the other five mice in each group) evaluations. The cecal contents were diluted with 0.8% NaCl solution (1:1), and 100-μl aliquots were plated on the Campylobacter agar with 5% antimicrobics and 10% sheep blood (Becton Dickinson, New Jersey). C. coli colonies were counted after 48 h of incubation in a 42°C microaerophilic environment.

Statistical analysis.Data were analyzed by one-way analysis of variance, and Tukey's test was used when the F test was significant. The Kruskal-Wallis test and Dunn's procedure as a multiple-comparison procedure were also performed to compare the means of nonparametric or abnormally distributed data. Differences with P values of <0.05 were considered significant. Data are expressed as means ± standard errors (SE). All statistical analyses were performed with the SigmaStat statistical analysis system (Jandel Scientific, San Rafael, CA).

Effect of MDT-1 on DSS-induced colitis.None of the mice in group A (control) exhibited any of the clinical signs of DSS-induced colitis, i.e., body weight loss, diarrhea, and bloody stool (Fig. 2).

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FIG. 2.

Effects of the oral administration of DSS, B. fibrisolvens MDT-1, and C. butyricum on the body weight (A), stool consistency score (B), fecal blood score (C), and disease activity index (DAI) (D) of mice. (○) Group A, DSS (−)/bacteria (−). (□) Group B, DSS (+)/bacteria (−). (•) Group C, DSS (+)/MDT-1 (+). (▪) Group D, DSS (+)/C. butyricum (+). + or − in parentheses indicates the presence or absence, respectively, of DSS, B. fibrisolvens MDT-1, and C. butyricum (bacteria). Data are expressed as means ± SE (n = 10). Different letters (a to c) at each time point indicate a significant difference between groups (P < 0.05).

No significant differences in the volume of drinking water between the four groups were observed during the experimental period. Mice treated with DSS demonstrated a significant decrease in body weight (group B) (Fig. 2A), which was reduced by administration of MDT-1 (group C) but not C. butyricum (group D). Diarrhea and bloody stool induced by treatment with DSS (group B) (Fig. 2B and C) were also alleviated significantly in mice administered MDT-1 (group C), but mice administered C. butyricum did not show a similar effect (group D). The presence of MDT-1 promoted a reduction in overall disease activity, as quantified by the disease activity index score (Fig. 2D). Furthermore, apparent colonic inflammation that was macroscopically observable in group B mice was less severe in group C mice (Fig. 3).

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FIG. 3.

Effect of the oral administration of DSS, B. fibrisolvens MDT-1, and C. butyricum on the histological damage score in mice. Bars indicate SE (n = 5). Different letters (a to c) indicate a significant difference between groups (P < 0.05).

The ratio of spleen to body weight, which increased greatly with DSS treatment alone, diminished significantly in mice administered MDT-1 (P < 0.05) (Table 1). Despite the fact that there were no significant changes in cecum or colorectum weight with DSS in either the absence or the presence of MDT-1 (Table 1), colorectum length was markedly reduced in DSS-treated mice. As a result, there was a corresponding increase in the ratio of colorectum weight to unit length, i.e., the colorectum was abnormally shortened and thickened. However, these DSS effects were diminished in mice administered MDT-1.

MPO activity in colonic tissue.MPO activity (per wet tissue weight) in colonic tissue increased greatly in mice treated with DSS, suggesting that DSS promotes an increase in neutrophil density that signals an inflammatory response. Administration of MDT-1 reduced the increase in MPO activity (Fig. 4), suggesting that MDT-1 can help alleviate DSS-induced inflammation. Again, C. butyricum had no alleviating effect.

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FIG. 4.

Effect of the oral administration of DSS, B. fibrisolvens MDT-1, and C. butyricum on the MPO activity in colonic tissue. *, change in absorbance/g of intestinal tissue. Bars indicate SE (n = 5). Different letters (a to c) indicate a significant difference between groups (P < 0.05).

Prevention of infection with Campylobacter spp. by MDT-1.Among the 15 strains of C. coli and 5 strains of C. jejuni examined, the addition of MDT-1 culture supernatant inhibited growth in 6 strains of C. coli and 3 strains of C. jejuni (data not shown).

Mice infected with one of the strains inhibited by MDT-1, C. coli 11580-3, lost body weight, and their cecal contents contained viable C. coli cells (Table 2), although no C. coli cells were detected in control mice. Administration of MDT-1 to mice alleviated the body weight loss and decreased the number of C. coli cells in the cecal contents to approximately 5% of that in nontreated, infected mice. Diarrhea induced by infection of C. coli (group F) was also alleviated significantly in mice administered MDT-1 before and during C. coli infection (group G) (Fig. 5).

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FIG. 5.

Effect of infection with C. coli 11580-3 and the oral administration of B. fibrisolvens MDT-1 on the stool consistency score in mice. (○) Group E. (□) Group F. (•) Group G. Data are expressed as means ± SE (n = 10). Different letters (a to c) at each time point indicate a significant difference between groups (P < 0.05).

MPO activity in colonic tissue increased greatly in mice infected with C. coli (Fig. 6). Administration of MDT-1 reduced the increase in MPO activity. Colonic tissue obtained from C. coli-infected mice shows acute inflammatory changes. The acute lesions were characterized by mild, focal, and nonulcerative infiltrates of neutrophils localized predominantly to the intercriptal lamina propria. Mice given MDT-1 showed smaller acute inflammatory changes induced by Campylobacter infection (data not shown).

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FIG. 6.

Effect of infection with C. coli 11580-3 and the oral administration of B. fibrisolvens MDT-1 on the MPO activity in colonic tissue. *, change in absorbance/g of intestinal tissue. Bars indicate SE (n = 5). Different letters (a and b) indicate a significant difference between groups (P < 0.05).