Role of Cytolethal Distending Toxin in Altered Stool Form and Bowel Phenotypes in a Rat Model of Post-infectious Irritable Bowel Syndrome.

BACKGROUND/AIMS: Campylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes. METHODS: Adult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejunicdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC). RESULTS: All 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni. CONCLUSIONS: In this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.

Methanobrevibacter smithii is the predominant methanogen in patients with constipation-predominant IBS and methane on breath.

PURPOSE: Among irritable bowel syndrome (IBS) patients, breath methane producers overwhelmingly have constipation predominance (C-IBS). Although the most common methanogen in humans is Methanobrevibacter smithii, incidence and type of methanogenic bacteria in C-IBS patients are unknown. METHODS: By use of a questionnaire and lactulose breath testing, subjects with Rome II C-IBS and methane (>3 ppm) were selected (n = 9). The control group included subjects with IBS who had no breath methane (n = 10). Presence of bacterial DNA was assessed in a stool sample of each subject by quantitative-PCR using universal 16S rDNA primer. M. smithii was quantified by use of a specific rpoB gene primer. RESULTS: M. smithii was detected in both methane and non-methane subjects. However, counts and relative proportion of M. smithii were significantly higher for methane-positive than for methane-negative subjects (1.8 × 10(7) ± 3.0 × 10(7) vs 3.2 × 10(5) ± 7.6 × 10(5) copies/g wet stool, P < 0.001; and 7.1 ± 6.3 % vs 0.24 ± 0.47 %, P = 0.02 respectively). The minimum threshold of M. smithii resulting in positive lactulose breath testing for methane was 4.2 × 10(5) copies/g wet stool or 1.2 % of total stool bacteria. Finally, area-under-curve for breath methane correlated significantly with both absolute quantity and percentage of M. smithii in stool (R = 0.76; P < 0.001 and R = 0.77; P < 0.001 respectively). CONCLUSIONS: M. smithii is the predominant methanogen in C-IBS patients with methane on breath testing. The number and proportion of M. smithii in stool correlate well with amount of breath methane.

Intestinal methane production in obese individuals is associated with a higher body mass index.

BACKGROUND: Obesity is an epidemic that affects 1 in 3 individuals in the United States, and recent evidence suggests that enteric microbiota may play a significant role in the development of obesity. This study evaluated the association between methanogenic archaea and obesity in human subjects. METHODS: Subjects with a body mass index (BMI) of 30 kg/m² or higher were prospectively recruited from the weight loss program of a tertiary care medical center. Subjects who met the study's inclusion criteria were asked to complete a questionnaire that included a series of visual analogue scores for bowel symptom severities. Subjects then provided a single end-expiratory breath sample to quantitate methane levels. Bivariate and multivariate analyses were used to determine associations with BMI. RESULTS: A total of 58 patients qualified for enrollment. Twenty percent of patients (n = 12) had breath test results that were positive for methane (>3 parts per million [ppm]), with a mean breath methane concentration of 12.2±3.1 ppm. BMI was significantly higher in methane-positive subjects (45.2±2.3 kg/m²) than in methane-negative subjects (38.5±0.8 kg/m²; P=.001). Methane-positive subjects also had a greater severity of constipation than methane-negative subjects (21.3±6.4 vs 9.5±2.4; P=.043). Multiple regression analysis illustrated a significant association between BMI and methane, constipation, and antidepressant use. However, methane remained an independent predictor of elevated BMI when controlling for antidepressant use (P<.001) and when controlling for both constipation and antidepressant use (6.55 kg/m² greater BMI; P=.003). CONCLUSION: This is the first human study to demonstrate that a higher concentration of methane detected by breath testing is a predictor of significantly greater obesity in overweight subjects.

Acute and chronic histological changes of the small bowel secondary to C. jejuni infection in a rat model for post-infectious IBS.

BACKGROUND: Campylobacter jejuni has been implicated in the pathogenesis of post-infectious irritable bowel syndrome (PI-IBS) in humans, effects which may be because of cytolethal distending toxin (CDT). In this study, we characterized both acute and chronic-phase histological changes of the small bowel in rats exposed to wild-type C. jejuni 81-176, or a strain that does not produce CDT, by using a validated rat model of PI-IBS. METHODS: Sprague-Dawley rats were given 1.0 × 10(8) CFU of either wild-type C. jejuni 81-176 (C+, PI/C+) or the CDT-negative strain (CDT-), or vehicle alone (Control). Acute-phase rats (C+, CDT-) were euthanized on days 2, 4, 8, 16, and 32. Chronic-phase rats (PI/C+, Control) were euthanized 3 months after clearing the initial infection. Segments of duodenum, jejunum, and ileum were resected and the contents plated for C. jejuni culture, and tissue sections were stained for histology. RESULTS: We observed preferential infection of the ileum and jejunum by Campylobacter jejuni. Compared with controls, epithelial cell basal membrane ballooning, villous tip disruption, and reduced villous-to-crypt ratios were observed for both C+ and CDT- rats. Villous widening, the only result significantly different in C+ vs. CDT- rats, was greatest at day 4 (134.1 ± 21.12 µm vs. 109.9 ± 10.6 µm for CDT-, P < 0.01). Little or no cellular inflammatory changes were seen during acute C. jejuni infection. Three months after clearing the initial infection, no histological changes remained. CONCLUSION: Significant histological changes, with the absence of inflammatory cells, are seen in the duodenum, jejunum, and ileum of rats during acute infection with C. jejuni. These changes occurred irrespective of the presence or absence of the CDT toxin.

Antibiotic prophylaxis prevents the development of a post-infectious phenotype in a new rat model of post-infectious IBS.

BACKGROUND: A recent post-infectious rat model with Campylobacter jejuni 81-176 has replicated the events noted in humans with post-infectious irritable bowel syndrome (IBS). In this study, we test whether prophylactic treatment with the antibiotic rifaximin will prevent the development of long-term altered bowel function in this model. METHODS: Sprague-Dawley rats were divided into two groups. Both groups were gavaged with a 1 mL solution of 10(8) cfu/mL of C. jejuni. However, one group was also prophylactically gavaged with a solution of rifaximin 200 mg per day for 3 days (the day before gavage, the day of gavage, and the day after gavage with C. jejuni). Fresh stool was collected from rats daily until two consecutive stool cultures were negative for C. jejuni. The rats were then housed for 3 months. At the end of 3 months, fresh stool was collected on three consecutive days to determine stool % wet weight and stool consistency on a stool score. RESULTS: Rats that received rifaximin antibiotic prophylaxis had a greater rate of stool shedding of C. jejuni. However, the mean duration of colonization was shorter in the rifaximin-treated group (10.3 ± 7.1 days) compared to rats receiving no prophylaxis (12.6 ± 5.9 days) (P < 0.01). After 3 months, rats that did not receive rifaximin had a greater variability in stool % wet weight (P < 0.01). Furthermore, the average stool consistency over 3 days of measurement was closer to normal in the rifaximin-treated rats, with a consistency of 1.1 ± 0.3, compared to 1.5 ± 0.4 in rats receiving no prophylaxis (P < 0.00001). CONCLUSIONS: Prophylactic treatment of rats with the antibiotic rifaximin in a new animal model of post-infectious IBS with C. jejuni mitigated the development of long-term altered stool form and function.

Polycystic ovary syndrome is associated with an increased prevalence of irritable bowel syndrome.

INTRODUCTION: Polycystic ovary syndrome (PCOS) affects 5-10% of reproductive-aged women. Irritable bowel syndrome (IBS) is a chronic intestinal disorder that affects up to 20% of adults, more often women. We evaluate if there is a relationship between these common conditions. METHODS: Polycystic ovary syndrome and control subjects were prospectively recruited. A questionnaire was given to determine their gastrointestinal symptoms. Body mass index (BMI) and percent body fat were also calculated. RESULTS: About 65 female subjects completed the study. Among the 36 PCOS patients, 15 subjects (42%) had IBS, compared to 3 subjects (10%) among controls (p < 0.01). Control subjects were leaner (BMI: 27.5 +/- 1.1 vs. 31.4 +/- 1.2 kg/m(2), p < 0.05) than PCOS patients. Among women with PCOS, those with IBS had a higher BMI (32.9 +/- 2.0 kg/m(2)) compared to those with PCOS but no IBS (30.3 +/- 1.6 kg/m(2)) and controls (27.5 +/- 1.1 kg/m(2); p < 0.05). This difference was true even after correcting for BMI and age (p < 0.01). CONCLUSIONS: Women with PCOS have a higher prevalence of IBS compared to healthy controls. When IBS is present with PCOS, a higher BMI and percent body fat is seen compared to PCOS alone.

A combination of rifaximin and neomycin is most effective in treating irritable bowel syndrome patients with methane on lactulose breath test.

AIM: There is a growing interest in methane and its association with constipation in functional bowel disease. Neomycin-based treatment of methane-positive subjects has resulted in improvement of constipation. Rifaximin, although superior for the treatment of irritable bowel syndrome compared with other antibiotics, seems less effective in methane-positive subjects. In this study, we evaluate 3 different antibiotic treatments in patients who have a methane-positive breath test: rifaximin only, neomycin only, and the combination of neomycin and rifaximin. METHODS: A retrospective chart review was conducted on patients with methane on their lactulose breath test (> or =3 ppm of methane) who received one of the following antibiotic treatments: 500 mg b.i.d. for 10 days of neomycin alone, 400 mg t.i.d. for 10 days of rifaximin alone, or a combination of both rifaximin and neomycin for 10 days. All patients must have received antibiotic treatment after their initial consultation at the medical center and, in addition, had at least 1 follow-up to evaluate the effects of the treatment. After inclusion/exclusion criteria were met, all charts were evaluated to determine if the subject was a responder to the antibiotic therapy. This included clinical symptom improvement and eradication of methane on their breath test. RESULTS: Of the subjects receiving the treatment of rifaximin and neomycin (n=27), 85% had a clinical response, compared with 63% of subjects in the neomycin only group (n=8) (P=0.15) and 56% of subjects in the rifaximin only group (n=39) (P=0.01). When comparing the neomycin group with the rifaximin group, the difference was nonsignificant. When evaluating methane eradication results, 87% of subjects taking the rifaximin and neomycin combination eradicated the methane on their breath test. This is compared with 33% of subjects in the neomycin group that eradicated the methane (P=0.001), and only 28% of subjects in the rifaximin group (P=0.001). Of the patients who did not eliminate the methane with only rifaximin treatment, 66% of those who subsequently used the rifaximin and neomycin treatment were able to normalize their breath test. CONCLUSIONS: The combination of rifaximin and neomycin is more effective in treating methane-producing subjects-in both clinical response and methane elimination.

Evaluating breath methane as a diagnostic test for constipation-predominant IBS.

Studies suggest that subjects with IBS have altered gut flora. Among these findings, methane production is more commonly associated with constipation-predominant symptoms. In this study, we prospectively evaluated the role of methane as a diagnostic test. Consecutive Rome I positive IBS patients referred for a lactulose breath test were eligible to participate. After exclusion criteria, subjects completed a symptom questionnaire grading bloating, diarrhea, and constipation on a VAS scale (0-100 mm). Once completed, a physician interviewed the subjects and rated the subject accordingly, and also determined whether the patient had C-IBS, D-IBS, or neither. Subjects and physicians were blinded to the results of the breath test. The presence of methane in the breath test was compared to the results of the scoring by subjects and physicians. A total of 56 Rome I positive IBS subjects were enrolled. During breath testing, 28 subjects produced methane. Good agreement between physician's evaluation and the patient's was seen (diarrhea = 0.69; constipation = 0.69; bloating = 0.62). The severity of constipation was noted to be greater in the methane group (49.3 +/- 28.7) than in the non-methane group (25.3 +/- 31.47) (P < 0.01). In contrast, diarrhea was less severe in the methane group (12.3 +/- 21.0) than the non-methane group (36.7 +/- 32.4) (P < 0.01). Out of the 56 patients, 23 C-IBS subjects were identified by the physician. When methane was used to predict the assignment of C-IBS compared to non-C-IBS, it had a sensitivity of 91.7% and a specificity of 81.3% (OR = 47.7, CI = 9.4-232, P < 0.00001). In conclusion, methane is a potential diagnostic test for the identification of C-IBS and may guide treatment.

New clinical method for distinguishing D-IBS from other gastrointestinal conditions causing diarrhea: the LA/IBS diagnostic strategy.

Modern methods of diagnosing diarrhea-predominant irritable bowel syndrome (D-IBS) require a "diagnosis of exclusion" approach. In this study we aim to test the diagnostic ability of using the fluctuation of frequency and consistency of bowel patterns in IBS to discriminate it from other causes of diarrhea. Eligible subjects were asked to complete a questionnaire on the changes in form and frequency of bowel habits by time. The primary endpoint was to evaluate the diagnostic effectiveness of having irregularly irregular bowel function and form as more characteristic of IBS versus non-IBS causes. Patients were prospectively recruited from a tertiary care GI clinic. Subjects had to have diarrhea as their primary complaint. In the case of IBS, D-IBS subjects were recruited. Subjects with celiac disease, Crohn's and ulcerative colitis were recruited for comparison and were categorically called "non-IBS." Non-IBS subjects could not have a recent history of blood in stool or a history of bowel surgery, fistulae or narcotic use. Sixty-two IBS and 37 non-IBS subjects were recruited. Among the 62 IBS subjects, 49 (79%) stated that their bowel habits varied in form and frequency on a daily basis compared to 35% in non-IBS subjects (OR = 8.9, CI = 3.5-22.5, P < 0.00001). When subjects were compared by the number of different stool forms they had witnessed in the prior week, IBS subjects noted 3.58 +/- 0.19 types and non-IBS reported 2.35 +/- 0.16 (P < 0.00001). Using > or = 3 stool forms per week as a method of discriminating IBS from non-IBS, 50 out of 62 subjects with IBS (81%) reported this greater number of forms compared to 15 out of 37 (41%) non-IBS subjects (sensitivity = 0.81; specificity = 0.60). The use of this simple tool that identifies an irregularly irregular bowel form and function is successful in separating D-IBS from non-IBS subjects.