We investigated gut flora characteristics in patients with functional constipation (FC) and influences of short-term treatment with VSL#3 probiotic on flora and symptom improvement.
Thirty patients fulfilling Rome III criteria for FC and 30 controls were enrolled. Fecal samples were obtained before and after VSL#3 intake (one sachet twice daily for 2 weeks) and flora were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Symptom changes were also investigated.
The fold differences in
The gut flora is a collection of microorganisms that live within the intestine, creating a harmonious ecosystem with concentrations of up to 1011–1012 cells/g luminal contents and which represent approximately 60% of the fecal mass.1 The gut flora performs many important health-promoting functions such as metabolic activities, fermenting unused energy substrates, producing vitamins or hormones to direct the host to store fats, immune system development, and preventing growth of pathogenic bacteria.1–3 Moreover, recent studies have shown that gut flora may play a role in gut sensory and motor functions.4,5
Although a change in gut flora has been suggested as a possible pathogenesis of functional bowel disease, few studies exist on the relationship between functional constipation (FC) and gut flora. In a pediatric study, constipated children presented with a significant increase in
The influence of gut flora on FC can be assessed by examining the symptomatic effects of probiotics administered to FC patients. Several studies have suggested probiotics as a possible treatment agent.12–14 The therapeutic outcome of a specific probiotic in the management of patients with FC depends on various factors, including bacterial strains, treatment duration, administration form and dose, and host factors. Currently, due to the paucity of data, whether any particular probiotic is more effective in the treatment of FC remains inconclusive. Additionally, whether the improvement of symptoms is directly due to a change in gut flora itself is unclear.
Therefore, in this study we investigated the characteristics of gut microbiome in patients with FC using molecular methods and evaluated the influence of a short-term treatment with VSL#3 probiotic, consisting of multiple viable lactic acid bacteria, on gut flora and the improvement of symptom profiles. Similar studies have not been reported previously, thus we performed this pilot study in preparation for larger and more advanced studies.
We recruited 30 FC patients and 30 healthy controls from 6 university hospitals between October 2011 and August 2012. The inclusion criteria were as follows: 20–59 years of age; fulfilling Rome III criteria for FC for at least 5 years; availability of at least one gastrointestinal (GI) imaging study during the last 5 years; and availability for the entire study period. Healthy controls were enrolled after excluding of any GI complaints and colonoscopies were performed in controls over 40 years of age to eliminate other organic bowel diseases.
Subjects with other GI symptoms, a history of GI surgery, organic intestinal diseases, lactose malabsorption, pregnant or lactating, or severe systemic diseases including diabetes, cardiovascular problems, or neurologic diseases were excluded. Subjects with a history of antibiotic treatment or intentional probiotic consumption 1 month prior to starting this study were also excluded.
All participants provided written informed consent according to institutional guidelines, and the protocol was approved by the Institutional Review Board of the participating hospitals.
This study was registered at the Clinical Research Information Service (CRiS), registration number KCT0000448.
This was a non-randomized controlled study evaluating the characteristics of fecal flora in FC and the comparative effects of probiotics between the FC and control groups. In the protocol, a 1-week run-in observation period was followed by a 2-week treatment period, with fecal samples obtained before and after the treatment period. All subjects received a VSL#3 sachet (VSL Pharmaceuticals, Danisco, USA) twice daily for 2 weeks. Each VSL#3 sachet contained 450 billion lyophilized bacteria:
During the study period, subjects were required to record a daily diary of bowel function including complete spontaneous bowel movements (CSBMs) and stool consistency using a validated Bristol stool scale,15 as well as respond weekly to the following questions on the symptomatic relief of FC: “During the past seven days, have you experienced satisfactory relief of bowel movement frequency, stool consistency, or bloating?” Diet and drugs taken during the trial were recorded. Patients with FC were not strongly encouraged to quit their usual laxatives.
Subjects who showed symptomatic improvement of functional constipation were followed up monthly by telephone interview for 6 months.
Stool samples collected from all subjects were stored at −80°C for DNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR).
Fecal DNA was extracted using the QIAamp stool DNA Extraction kits (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. Quantitative RT-PCR was performed using the ABI SYBR Green PCR Mastermix from Applied Biosystems (Foster City, CA, USA), with primers amplifying the genes encoding 16S rRNA from specific bacterial groups including
This study included 30 patients with FC and 30 controls. Continuous variables are reported as medians with range and/or means ± SD, and categorical variables as relative frequencies.
The CSBM scores and Bristol scales are summarized as the means ± SD before and after VSL#3 ingestion. The comparative analysis of symptomatic scores was performed by paired
Fold differences of 5 bacterial species before and after probiotic treatment were compared using the Wilcoxon signed-rank test. Fold differences in the bacterial species between FC patients and controls were compared using the Mann-Whitney test. All
A total of 60 subjects (30 FC and 30 controls) provided fecal samples. Females represented 65% of the subjects, with a mean age of 33 years (range 22–42 years). The mean age of patients with FC was significantly greater than that of controls (35 ± 5 vs. 32 ± 3 years, respectively;
Fecal qRT-PCR showed a significant low value in
In controls, fold differences of
When the fluctuation of gut flora per person before and after ingesting VSL#3 were analyzed, the mean delta values of fold differences of beneficial bacteria were not significantly different between FC patients and controls (
The mean CSBM score per week in constipated patients increased significantly after VSL#3 ingestion for 2 weeks (6.3 ± 3.1/week), compared to before ingestion (2.5 ± 1.3/week,
In healthy controls, no significant difference between the mean CSBM scores before (6.6 ± 2.4/week) and after (7.6 ± 2.3/week) VSL#3 ingestion was observed (
The mean Bristol scores in constipated patients were significantly higher after VSL#3 ingestion for 2 weeks (4.1 ± 0.9) than after ingestion for 1 week (3.7 ± 0.9,
In healthy controls, no significant difference in the mean Bristol scores before (4.0 ± 0.7) and after (4.3 ± 0.8,
Relief of subjective CSBM frequency, stool consistency, and abdominal bloating in the FC group after ingesting VSL#3 for 2 weeks were reported in 70%, 60%, and 47% of patients, respectively.
After VSL#3 ingestion, 18 constipated patients who showed symptom improvement were followed up once per month by telephone interview. Constipation-related symptoms including hard stool or reduced defecation frequency, recurred in 11 patients (61%). Specifically, 9 patients (50%) complained of having the original symptom severity: the original hard stool level in 5 patients (28%) and reduced defecation frequency in 9 (44%). Of 11 patients with recurrence, 8 (72.7%) reported recurrence of constipation-related symptoms within 1 month (Fig. 6).
The rationale behind research on gut flora in constipated patients is related to the possibility of altering the delayed transit. Our study are meaningful not only because they revealed the quantitative difference in the main gut flora of patients with FC, but also because they show the effect of probiotics on FC, which is likely mediated by the gut flora. Moreover, this is the first study of the effectiveness of VSL#3 for FC.
There have been limited studies suggesting the effect of a change in gut flora on FC. A previous study showed that
The role that gut flora play in the pathogenesis of FC remains unclear. A microbiological study using traditional culture methods showed that some obligate bacteria, including
Several investigators have suggested that changes in the gut flora could alter the motor and secretory functions of the bowel. Probiotic studies in both animals and humans showed similar results in terms of stimulating motility, although the direct effect of probiotics on the gut flora has not been investigated.4,5,12,19,20
Our results also revealed a change in the gut flora in patients with FC. The fold differences in the concentrations of
In our study, the VSL#3 probiotic was administered with the expectation that it would change the gut flora, and improve bowel movement frequency, stool consistency, and bloating in patients. However, no significant changes in the fold differences of levels of the five bacterial taxa after ingesting VSL#3 were identified in the feces of patients with FC, although gut colonization by lactic acid bacteria in VSL#3 was confirmed in controls. The mean delta values of fold differences of beneficial bacteria were still not significantly different between FC patients and controls. It could be associated with the data showing that the changes of fold differences before and after treatment were not significant in FC but significant in control, although the fold differences in the concentrations of the beneficial bacteria in FC patients were significantly lower than in controls before ingesting VSL#3.
The possible reasons for the discrepancy between symptom improvement and gut floral changes after ingesting VSL#3 include that other bacterial species whose concentrations may have changed in constipated patients were not measured in this study, although these 5 bacterial taxa account for a substantial portion of the fecal flora. The other possibility is that physiologically active substances produced by the gut flora may have affected colon transit, regardless of the fold differences in the concentration of bacterial species.12,24–26 Several studies have demonstrated that administering probiotics or prebiotics such as
Bacterial colonization by short-term ingestion of probiotics may not be effective in a diseased bowel. The constipated subjects enrolled in this study had a long symptom history, and the laxatives previously used were not effective in relieving symptoms. Our study revealed that a 2-week ingestion of VSL#3 resulted in colonization of the gut in controls. Although CSBMs and stool consistency tended to increase, no clinically significant difference in bowel parameters was observed in controls. Further studies are needed to confirm the difference in colonization between controls and patients with FC after probiotic treatment, and to identify the underlying mechanism.
Much clinical experience and some data suggest that probiotics may be beneficial in many types of intestinal disorders, especially diarrheal diseases.30 Orally administered probiotics have been considered to target the motor and neural apparatus in post-infective gut dysfunctions.25 Probiotics including lactic acid bacteria were shown to be effective for the relief of abdominal bloating in patients with diarrhea-predominant irritable bowel syndrome (D-IBS), but did not show a significant alteration in gut transit.31,32 Studies involving the administration of a probiotic,
The VSL#3 probiotic was chosen because studies have suggested that in constipated patients, probiotics may be more effective if multiple strains are administered together.12,29,35 VSL#3 contains eight strains of live lactic acid bacteria that were selected to produce an optimal synergistic effect. In several D-IBS studies, lactic acid bacteria were shown to reduce abdominal bloating compared to a placebo and influence gut transit and bowel dysfunction,31,32 However, no reports are available on the effectiveness of VSL#3 for FC.
The duration of probiotic administration generally varies from 2 to 6 weeks or more, at least in D-IBS patients. We administered the probiotic to all subjects for 2 weeks. Administration for longer periods may have influenced patient compliance, and thus a shorter treatment periods was used. Instead of using a 2-week study period, the daily dose was doubled. None of the patients complained of side effects, and all showed good compliance with treatment.
The fact that 70% of the patients were satisfied with their symptomatic relief of bowel movement frequency is considered to be a very good outcome of VSL#3 treatment. However, it should be noted that at least 30% of constipated patients were unsatisfied with the double-dose VSL#3 treatment. In addition, 61% of patients who showed improvement had their constipation symptoms recur after treatment cessation, mostly within 1 month. Although the gut flora composition of an individual can fluctuate under different circumstances, such as dietary interventions, acute infection, and antibiotic treatment, it usually returns to normal or remains constant.1 According to our results, some constipated patients may derive substantial benefit from a combination of multiple probiotic strains. However, the treatment may need to be administered on a cyclical schedule or repeatedly because the resultant favorable effect is temporary.36
Quantitative analysis of fecal bacteria shows important differences in yield that are not always detectable by conventional culture techniques.1 The molecular method used in our study, however, had limitations in terms of estimating the composition of the colonic flora and providing a generalized view of the diverse intestinal flora compared with pyrosequencing methods. We used a comparative CT method to normalize the bacterial gene expression to total 16S rRNA in each fecal sample. This compensatory analysis is considered to be resonable to compare gut flora between groups in this study.
We also acknowledge a limitation of not involving a metabolomics study such as short chain fatty acids and pH. Additionally, the effect of VSL#3 on constipation symptoms was evaluated without a placebo group in this pilot study. However, a symptom assessment was conducted based mainly on CSBMs and stool consistency, which are relatively objective, as well as numerical counts. It is difficult to conclude that only a placebo effect was responsible for the improved symptoms of constipated patients with a long symptom history, and who had not responded to various laxatives.
Nevertheless, this pilot study is useful because it shows that gut flora can impact disturbed bowel function, as evidenced by the improvement of FC after short-term administration of a probiotic, as well the difference in gut flora concentrations between constipated patients and controls.
In conclusion, quantitative alterations of specific bacterial groups in the gut flora were found in patients with FC. VSL#3 could be effective in terms of improving clinical symptoms in constipated patients, although its direct effect on the gut flora of constipated patients remain unclear. More studies should aim to elucidate the mechanism underlying the effect of short-term treatment with VSL#3 on FC, particularly in light of its metabolomic effect on the gut flora.
Quantitative Polymerase Chain Reaction Primers
|Target bacteria||Primer sequence 5′-3′||Annealing temperature (°C)||PCR product size (base pair)|
Clinical Characteristics of Subjects
|FC patients||Healthy controls|
|Number of subjects||30||30|
|Age (mean ± SD, yr)||35 ± 5a||32 ± 3|
|BMI (mean ± SD, kg/m2)||21.0 ± 2.5||21.5 ± 2.9|
FC, functional constipation.