Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders (GI) worldwide, affecting approximately 11% of the global population [1
]. IBS is characterized by the co-occurrence of abdominal pain or discomfort, changes in bowel habits, and defecation [2
]. Based on bowel habits and stool consistency, IBS patients are grouped into three subtypes, namely the constipation-predominant IBS (IBS-C), diarrhea-predominant IBS (IBS-D), and the mixed bowel habits or cyclic patterns IBS (IBS-M) characterized by the occurrence of both constipation and diarrhea episodes [2
]. The physical symptoms of IBS are variable in severity, ranging from unpleasant to debilitating, and as a result, patients’ psychological well-being and quality of life can be adversely affected in more severe cases. This variability in the severity of IBS symptoms, coupled with our incomplete understanding of its pathophysiology, often results in extended delays for patients before receiving a diagnosis. This also leads to inconsistencies in the efficacy of symptom management regimes. Currently, the first step in IBS symptom management involves lifestyle and dietary modifications, such as the adoption of low fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) and gluten-free diets [3
]. In cases where dietary changes are ineffective, adjunctive pharmacotherapies such as antidiarrheal agents, antibiotics, laxatives, antispasmodics, or antidepressants are introduced. Pharmacological interventions often target specific but not all symptoms of IBS and may result in serious adverse events [4
]. Based on accumulating research evidence, gut microbial dysbiosis is thought to play a role in the pathophysiology of IBS and symptom severity. Consequently, promising leads in IBS management now include the potential use of specific probiotic bacteria to improve both the GI symptoms and psychological comorbidities in IBS [6
The potential use of probiotics in IBS management stems from their documented beneficial effects on GI health as well as their strong history of safe use [7
]. In addition, a lower abundance of Bifidobacterium
species has been observed in IBS patients [8
], and a reduction in IBS symptom severity following supplementation with probiotic strains from these genera was reported by several randomized controlled trials (RCTs) [9
]. For instance, Bifidobacterium lactis
DN-173 010, Lactobacillus paracasei
NCC2461, Lactobacillus acidophilus
NCFM, Bifidobacterium infantis
35624, and Bifidobacterium longum
NCC3001 have shown some efficacy at reducing GI symptoms of IBS, such as frequency of bowel movements (BMs), pain, and visceral hypersensitivity [11
]. Supplementation for 2 months with B. longum
W11 in combination with rifaximine, a broad-spectrum antibiotic, significantly reduced symptoms of IBS compared to rifaximine alone, as measured by a visual analogue scale [12
]. A clinical study exploring the effects of Lactobacillus paracasei
F19 on IBS symptoms reported an improvement in the bowel habits of participants with constipation or diarrhea and showed significant improvements in the frequency and intensity of self-reported pain [13
Probiotics were found to exert both strain-specific and dose-dependent effects on gut health [10
], but these effects are poorly characterized in IBS patients. Hence, characterizing the specific and dose-related effects of single probiotic strains in clinical trials could foster the development of more efficient probiotic blends tailored to IBS patients’ needs. Supplementation with Lactobacillus helveticus
R0052 and Bifidobacterium longum
R0175 has shown beneficial psychological effects in healthy human volunteers [15
] and patients with major depressive disorder [16
]. However, B. longum
R0175 supplementation has not been studied in IBS patients. Similarly, there are no previous studies on Lactobacillus paracasei
HA-196 in a population with IBS.
Therefore, the aim of the current study was to evaluate the efficacy of two single strain probiotics, B. longum R0175 (B. longum) and L. paracasei HA-196 (L. paracasei), in the management of GI and psychological symptoms in IBS patients.
2. Materials and Methods
2.1. Study Design
This study was approved by the Natural and Non-Prescription Health Products Directorate, Health Canada, Ottawa, Ontario on 23 June 2014. Research ethics board approval was granted on 7 July 2014 from the Institutional Review Board (IRB) Services, Aurora, Ontario. All participants in the study provided written informed consent at the screening visit and the study was conducted in accordance with the Declaration of Helsinki guidelines and its subsequent amendments. The trial was registered at ClinicalTrials.gov
(NCT02213172) and followed the CONSORT guidelines for randomized controlled trials [17
] (Supplementary Table S1
). The study was planned to only include participants with IBS-C. Due to low enrollment, the protocol was modified on 6 January 2016 to include participants with all IBS subtypes.
This randomized, double-blind, placebo-controlled, 3-arm parallel group study was conducted at KGK Science Inc., London, ON, Canada between September 2014 and February 2018. The study included a 2-week run-in period, during which regular bowel habits were reported, and an 8-week intervention period (day 1 to day 57), consisting of a total of four clinic visits (Figure 1
Clinical and qualitative assessments were conducted at screening, day 0 (baseline), 4 weeks and 12 weeks (end-of-study). At the screening visit, participants were provided with a 3-day food record and a 2-week daily diary to complete before the baseline visit. The daily diary assessed the number of spontaneous bowel movements (SBM; defined as a stool not induced by rescue medication), complete spontaneous bowel movements (CSBM; defined as an SBM associated with a sensation of complete evacuation), and stool consistency based on the Bristol stool scale [18
]. Participants were also instructed to collect a fecal sample (5–10 g) using the provided stool collection kits 3 days before taking the investigational product (IP). The fecal sample was to be frozen immediately upon collection and brought to the clinic before or at the baseline visit.
At all study visits, the Irritable Bowel Syndrome Symptom Severity Scale (IBS-SSS), Short Form (36) Health Survey v2TM (SF-36), Irritable Bowel Syndrome Quality of life (IBS-QOL), and Hospital Anxiety Depression Scale (HADS) questionnaires were administered and assessed. Additionally, 3-day food records and diaries were reviewed for daily IBS-SSS, concomitant therapies, adverse events, and study product use. Vital signs and anthropometric measurements were undertaken at each visit while hematology and clinical chemistry parameters for all safety endpoints were assessed at the screening and end-of-study visits.
At week 8, participants provided a fecal sample (5–10 g) collected within the 3 days preceding their last visit and frozen immediately after collection and then brought to the clinic.
Individuals diagnosed with IBS were recruited from Southwestern Ontario, Canada using the KGK Science Inc.’s internal participant database along with local electronic and physical advertisement devoid of gender or racial bias. Participants met the following inclusion criteria: aged 18 years or older, IBS diagnosed according to the Rome III criteria [19
], and willingness to discontinue probiotic consumption for the duration of the study. After the broadening of eligibility criteria to all subtypes, the distribution of IBS subtypes in each intervention group was determined after randomization using the IBS Rome III Questionnaire completed at screening.
Participants were excluded if they used medications to manage IBS symptoms or narcotics in the past month, history of gastrointestinal surgery, gastrointestinal disease (except hemorrhoids and uncomplicated diverticula) or family history of colorectal cancer, inflammatory bowel disease, or celiac sprue.
Each probiotic capsule contained 10 × 109 colony forming units (CFU) of either freeze-dried B. longum (Lot Numbers: NH131210-1VB and NH151104-ICP) or L. paracasei (Lot Numbers: NH131217-1VB and NH151106-ICP), with potato starch and magnesium stearate as excipients. The placebo (Lot Numbers: NH131226-ISC and NH151028-ICP) contained only potato starch and magnesium stearate. At the baseline visit, participants were instructed to start consuming one capsule of the investigational products with breakfast on the following day (Day 1), and then daily for 8 weeks. The investigational products and placebo were manufactured by Lallemand Health Solutions (LHS) and kept refrigerated (2–8 °C) before use at the study site.
2.4. Randomization and Blinding
Eligible participants were assigned a randomization code from a list generated by www.randomization.com
, and allocated to each intervention group in a 1:1:1 ratio. Except for 13 participants enrolled while the study was focused on IBS-C, IBS subtypes were not considered before randomization after the eligibility criteria were extended to include all subtypes. The investigational products were labelled in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines, applicable local regulatory guidelines, and included the applicable randomization number. All capsules were identical in size, shape, colour, and taste. Investigators, clinic staff and participants remained blinded for the duration of the study.
2.5. Assessments of IBS Symptom Severity, General Health and Psychological Well-Being
The primary outcome measure was the difference in the mean change in symptom severity between each probiotic group and the placebo group (calculated at week 4 and 8, over baseline; i.e., week 4–baseline and week 8–baseline). Symptom severity was assessed using the IBS-SSS.
The secondary outcomes included the difference in the mean change between each probiotic group and the placebo group (calculated at week 4 and 8, over baseline) in several aspects of general health, quality of life, and psychological well-being. The change in general health was assessed using the SF-36 questionnaire; a commonly used tool with 36-items to measure health-related quality of life in 8 domains [20
]. Levels of depression and anxiety were measured using the HADS questionnaire; an easy-to-use and validated tool developed for the detection of anxiety disorders and depression in a non-psychiatric hospital clinic setting [21
Other secondary outcomes were the difference in mean change between each probiotic group and placebo group (calculated at week 4 and 8, over baseline) for each of the following parameters: abdominal pain intensity, abdominal pain frequency, abdominal distension/tightness, bowel habit satisfaction, and IBS-SSS score for each IBS subtypes. Changes in the microbiome composition from baseline to week 8 for each probiotic group and the placebo group were also investigated. Additional secondary outcomes examined were the severity of straining and stool consistency according to the IBS daily diary and amount of rescue medication (Bisacodyl 5 mg, a laxative) used throughout the study for the relief of severe constipation (72 h since last BM or when symptoms of constipation became intolerable).
2.6. Fecal Microbe Composition Analysis
Stool samples collected at baseline and week 8 were used to confirm participant compliance and microbe composition through quantitative real-time polymerase chain reaction (qPCR). Total DNA from 250–350 mg of homogenized fecal samples was extracted using the QIAamp Fast DNA Stool Mini kit (QIAGEN, ON, Canada) with the following modifications: (1) two washes with 0.05 M phosphate buffer prior to the InhibitEx step and (2) a 0.1 mm zirconia/silica bead beating step (3 × 4 m/s for 1 min) prior to the centrifugation of samples to pellet particles. Ratios of A260/A280 were assessed to determine sample purity. Absolute quantification of the two probiotic strains and Bifidobacterium
genus and relative quantification of Akkermansia mucinphilia
and Faecalibaterium prausnitzii
was conducted. Template DNA generated for the standard curve formation involved spiking 109
of the appropriate lyophilized powder into a fecal mix and proceeding with the DNA extraction. DNA was diluted 10-fold to generate a standard curve ranging from 109
bacteria for L. paracasei
and B. longum
Primers for B. longum
R0175 forward: 5’-GTCGCCACATTTCATCGCAA-3’, reverse: 5’-GAGAGCTTCGATTGGCGAAC-3’; L. paracasei
HA-196 forward: 5’-ACCGAAGTCTATCACCCGGA-3’, reverse: 5’-TCGCCAAATTTGCTGTCGTG-3’; Bifidobacterium
genus forward: 5’-TGGAAGGTCTCGATGGAGGT-3’, reverse: 5’-CTGGACAAGCCGTTCCTGAT-3’). DNA was diluted 1/5 prior to qPCR. Each qPCR reaction contained 300 nM of the appropriate forward and reverse primer, 1X SYBR Select Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µL of diluted DNA. The epMotion 5075 liquid handling robot (Eppendorf, Hamburg, Germany) was used to dilute DNA from the fecal samples 5-fold, and add 9 mL of mastermix and 1 µL of diluted DNA to the reaction plate. The cycling conditions for the B. longum
R0175, L. paracasei
species, and 16S rRNA universal bacterial primers included a 2-min hold at 50 °C, a 2-min hold at 95 °C and 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The relative quantifications of A. muciniphila
and F. prausnitzii
was performed via species-specific primers and DNA was normalized by bacterial primers targeting the 16S rRNA gene. The primers and cycling conditions used for A. muciniphila
and F. prausnitzii
quantification were obtained from literature [22
]. The CFX384™ Touch Real-Time polymerase chain reaction (PCR) Detection System was used to perform quantitative PCR (qPCR) analyses and results were viewed using the CFX Maestro Software 1.1 (Bio-Rad, Hercules, CA).
Compliance was assessed by counting the returned IP at the final study visit. Percent compliance was calculated by determining the number of capsules consumed divided by the number expected to have been taken multiplied by 100. Participants were also asked every day in the IBS daily diary if they consumed the IP. In the event of a discrepancy between the information in the IBS daily diary and the amount of IP returned, compliance was determined based on the product returned unless an explanation for the loss of product was provided.
2.8. Statistical Analyses
The proposed sample size for this study was 285 enrolled participants, with 95 participants randomized into each of the three study arms in a double-blinded manner. The sample size calculation was based on a standard deviation (SD) of 60, a significance level of 5% (two-sided α), 83% power (β = 0.17), 20% attrition rate and a 110-point detectable difference in IBS-SSS scores from baseline to week 8, compared between probiotics and placebo. The assumptions were based on two previous trials, by Dapoigny et al. (2012) [25
] and Williams et al. (2009) [26
], which examined the effect of probiotic interventions with Lactobacillus
strains on the symptom severity scores in adult IBS patients.
All statistical analyses were completed using SAS® software Version 9.3 (SAS Institute, Cary, NC, USA) for Microsoft Windows, where probabilities ≤0.05 were considered statistically significant. A blinded interim analysis was conducted on the primary outcome (IBS-SSS) after 50% recruitment. The blind was maintained for all study personnel including the statistician. The per protocol (PP) population consisted of all participants who consumed at least 80% of either product dose, did not have any major protocol violations, and completed all study visits and procedures connected with measurement of the primary variable. The safety population consisted of all participants who received either product and on whom any post-randomization safety information was available. An efficacy analysis based on the PP population and IBS subtypes was performed. Variables were tested for normality and log-normality, and variables showing a log-normal distribution were analyzed in the logarithmic domain.
Appropriate non-parametric tests were used to analyze non-normal variables. Continuous, normally distributed variables were analyzed by linear mixed-effects models with visit (baseline, week 4, and week 8), intervention (L. paracasei, B. longum, and placebo groups), and their interaction as fixed effects, and subject as a random effect to capture the repeated observations on each subject. Additionally, within-group changes from Day 0 were obtained from the linear mixed-effects models.
In this randomized, double-blind, placebo-controlled study, both L. paracasei HA-196 and B. longum R0175 were found to be safe, and significantly improved participants’ experience of IBS-related symptoms after 8 weeks as reflected by reduced scores on the IBS-SSS. A similar decrease in symptom severity was also observed in the placebo group. The significant reduction in symptom severity was marked by a 30% decrease in L. paracasei and a 22% decrease in the B. longum groups compared to a 31% decrease in the placebo group. IBS-SSS is a widely used self-administered questionnaire that measures severity and frequency of abdominal pain, abdominal distension/tightness, bowel habit satisfaction, and quality of life. The total IBS-SSS score ranges from 0 to 500, with higher scores indicating more severe symptoms. A decrease in the IBS-SSS score indicates a reduction in symptom severity. When participants were grouped into IBS subtypes (IBS-C, IBS-D, IBS-M), a reduction in IBS-SSS was also observed in all three groups. However, L. paracasei supplemented IBS-C and IBS-D participants reported improvements in their bowel habits and stool consistency that were not observed in the placebo group.
In addition to IBS-SSS score, BM frequency was examined based on IBS subtype. At the end of the 8-week study, IBS-C participants in the L. paracasei group had significant improvements in both SBM and CSBM. Although not statistically significant, IBS-C participants in the placebo group reported fewer numbers of SBM and CSBM at the end of the study. For IBS-D participants, B. longum significantly reduced SBM compared to placebo from baseline to week 4, but this significant between-group difference was not sustained to the end of study. L. paracasei significantly improved the weekly frequency of SBM at week 8 in IBS-D participants compared to the placebo. Additionally, L. paracasei had a greater effect on reducing the number of CSBM in IBS-D participants than B. longum. As with IBS-C participants, the placebo did not alter bowel movement frequency with IBS-D. Although, not significantly different, L. paracasei supplemented IBS-M participants were the only group to report increases in SBM and CSBM after 8 weeks. IBS-M participants in the B. longum group did not report increases in SBM in participants, and those in the placebo had decreases in CSBM by the end of the study.
All participants with IBS who received L. paracasei reported improvements in stool consistency after 8 weeks, unlike IBS-C and IBS-M participants in the B. longum group as well as all participants given placebo. This shift in stool consistency in the L. paracasei groups was marked by a significant within-group reduction in stool consistency reported by IBS-D participants after 8 weeks. The monitoring of stool consistency and BM frequencies provide evidence of efficacy of the two probiotic strains, with L. paracasei being more efficacious in improving symptoms of IBS, particularly IBS-C and IBS-D.
An important aspect of this study was that of all IBS subtypes, the highest population of participants enrolled was those with IBS-M. IBS as a condition can be very transient in presentation of its type and intensity [27
]. Participants in this study often changed their subtype between screening and baseline run-in. Furthermore, the recording of IBS intensity is highly subjective making clinical studies in this area challenging. Despite the challenges associated with studies in this area, this study suggests that both L. paracasei
HA-196 and B. longum
R0175 play a role in mitigating the effects of IBS in certain subtypes. These data would suggest that probiotic efficacy may be dependent on the IBS subtype. This warrants further investigation and studies focused on specific IBS symptoms within the IBS symptomatology continuum rather than considering all IBS patients as a homogenous population.
Psychological comorbidities are commonly observed in IBS patients, who are significantly more likely to develop mental health conditions such as depressive disorders, anxiety, and sleep disorders [28
]. Considering this, probiotics consumption is viewed as a means to alleviate the psychological comorbidities of IBS [29
]. However, similar to the efficacy of probiotics and the physical symptoms of IBS, there is heterogeneity among studies linking probiotics and modulation of behaviour in humans. A narrative review of seven systematic reviews of RCTs in individuals with depressive symptoms noted that five reviews reported a beneficial effect of probiotics on depressive symptoms. However, the authors stated that heterogeneity in the type of populations studied warranted further studies in clinically diagnosed depression before more definitive conclusions could be made [30
]. More recently, a study on IBS participants with mild to moderate anxiety and/or depression reported that B. longum
NCC3001 improved depression scores based on the HADS scale. A reduction of two or more points was reported in 64% of the participants in the probiotic group compared to 32% in the placebo group (p
= 0.04) [31
], although anxiety or IBS symptoms scores were not different compared to placebo. In the current study, we did not observe any significant improvements in the HADS score nor in general health assessment (based on the SF-36) among IBS participants compared to placebo. However, participants in the B. longum
group reported having increased energy levels after 8 weeks, which can directly impact their willingness and ability to perform everyday tasks. Furthermore, participants in both B. longum
and L. paracasei
groups reported significant within-group improvements in social functioning after 8 weeks. In contrast, none of these improvements pertaining to mental health were found in participants receiving the placebo.
Ensuring compliance with the probiotic for the duration of the study is an essential component of examining efficacy. The ability to detect the probiotic strains in participant’s fecal samples can confirm the participant compliance calculated from the returned IP. Fecal samples from baseline and 8 weeks were analyzed by qPCR and revealed a high degree of compliance within the probiotic supplemented groups. In the placebo group, probiotic strain detection was not expected. However, a handful of participants in the placebo group did have detection, which may potentially be due to the participants harbouring similar sequences from the bacteria in their gut, which may occur naturally due to bacterial diversity.
The abundance of selected relevant bacteria, including A. muciniphila
, F. prausnitzii
species was also investigated in fecal samples by qPCR. Compared to baseline, participants in all three treatment groups had no significant changes in the two species and one genus quantified, though more participants had detection of Bifidobacterium
species in their fecal matter following 8-weeks of B. longum
supplementation. The latter may have contributed to the BM improvement observed in the B. longum
supplemented groups, as Bifidobacterium
species have been shown to improve bowel habits in all IBS subtypes [32
]. An increase in A. muciniphila
has been associated with a decrease in abdominal pain in IBS patients, and F. prausnitzii
has been shown to reduce visceral hypersensitivity in a non-inflammatory animal model of IBS [33
]. Thus, it is possible that the unchanged relative abundance of A. muciniphila
and F. prausnitzii
following probiotic supplementation may have contributed to the absence of significant differences in the IBS-SSS score between the groups. It also suggests that the potential mechanism by which L. paracasei
, exerted its impact on bowel habits and stool consistency may be independent of the specific bacterial species investigated.
A placebo effect of 33% was observed in this study, consistent with reports from previous IBS clinical trials. A review of RCTs evaluating complementary health products showed an overall placebo response of 43%, with higher placebo responses associated with longer treatment duration and greater number of clinic visits [35
]. A meta-analysis reported that the placebo response ranges from 16% to 71% in participants with IBS, with a population-weighted average of 40% in studies using natural health products and pharmaceutical interventions [36
]. The subjective nature of the assessments and the intrinsic nature of IBS patients who display high levels of suggestibility and a strong placebo effect may have contributed to the placebo effect seen in this study. Additionally, this placebo effect may partially explain why some outcomes assessed were significant at either week 4 or 8. The placebo effect in studies within this area of research also exemplifies the importance of a run-in period, which was included in this study. However, examining bowel habits for a longer period prior to intervening may be an important aspect of understanding probiotic efficacy while mitigating the placebo effect.
Other limitations of this study include the relatively small sample size of the population, more specifically when considering the IBS subtypes, which may have introduced variability in the responses and reduced effect sizes. Moreover, the severity of IBS symptoms using IBS-SSS was only captured at baseline, week 4, and week 8 and not at any of the intermediate weeks. A characteristic feature of IBS is that some weeks were worse than the others, and due to the lack of data from intermediate weeks, temporary improvements experienced by participants associated with probiotic supplementation may have been missed. Furthermore, the duration of IBS in participants prior to enrollment into the study was not accounted for, which may have influenced the outcomes measured. Additionally, participants in this study were diagnosed and classified by IBS subtype according to Rome III, which has now been updated to Rome IV [37
]. A recent study has shown differences in the diagnosis and classification of participants with IBS based on Rome III or Rome IV [38
].Of note, at the time of classification based on the Rome III questionnaire, no participants in this study were classified as the IBS-unclassified (IBS-U) subtype.
Low grade inflammation has been suggested to play a role in the pathogenesis of IBS. Abnormal mast cell activation in the gut has been associated with the development of IBS symptoms [39
]. Although beyond the scope of this study, it would be important to evaluate the anti-inflammatory effect of L. paracasei
or B. longum
in IBS patients. Overall, the results of the current study suggest a clinically relevant role for L. paracasei
or B. longum
as probiotics to reduce symptoms in IBS. Furthermore, they may add to the armamentarium of IBS management tools without the safety concerns that may accompany pharmaceuticals. The effects of each probiotic strain may be dependent on the IBS subtype, and future studies should target IBS-D and IBS-M for probiotic therapy.