VSL#3^® May Reduce Abdominal Pain and Bloating in Ulcerative Colitis Remission with IBS-like Symptoms: An Exploratory Randomized, Double-Blind Placebo-Controlled Trial

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

 

 

I was invited to review the article “Probiotic VSL#3® may reduce abdominal pain and bloating in patients with ulcerative colitis in remission and functional bowel symptoms: results from a randomized, double-blind, placebo-controlled, explorative clinical trial “ by  Natalia Borruel Sainz, Xavier Serra-Ruiz, Francisco Guarner Aguilar*, Fabiana Castiglione, Olga Maria Nar- done, Lucrezia Laterza, Fabio Cascella, Antonio Gasbarrini, Agnieszka Dobrowolska, Alina Kanikowska, Pal Miheller, Orsolya Menyhárt, Daniele Noviello, Flavio Caprioli.

 

The subject is very highly rated amongst specialists and probiotics are a choice that will be strongly used in the near future.

I have carefully went through the manuscript and I have the following suggestions:

 

 

Please reformulate the title it is too long, and the attention focus is lost.

Line 35-What does it means quality of life measures? Please explain

Line 36

• Patient details please
• Admission criteria quick view, even if you presented them later
• Initial and final parameters
• Template of the informed consent

 

 

Line 40- which are the exploratory analyses?

Line 55- please list the Rome criteria

Line 71- bacterial names in Italics, please

Line 93-96- how you choosed the participating hospitals?

Line 115-138- it would be better if you can present data in a graph

Line 143-150- bacterial names in Italics please

Line 219- 55 patients is a small number to generate pertinent conclusions

 

 

Author Response

Comment 1: "Please reformulate the title it is too long, and the attention focus is lost."

Response: We thank the Reviewer for this suggestion. We agree that the original title was too long and that the focus of the manuscript could be improved. We have therefore shortened: VSL#3® may reduce abdominal pain and bloating in ulcerative colitis remission with IBS-like symptoms: an exploratory randomized, double-blind placebo-controlled trial

Comment 2: "Line 35-What does it means quality of life measures? Please explain"

Response: We thank the Reviewer for this comment. We agree that the term “quality-of-life measures” was insufficiently specific in the Abstract. We have revised the sentence to explicitly indicate the instruments used to assess quality of life, namely the IBS-specific Quality of Life questionnaire (IBS-QoL) and the Inflammatory Bowel Disease Questionnaire (IBDQ).

Comment 3: "

Line 36

• Patient details please
• Admission criteria quick view, even if you presented them later
• Initial and final parameters
• Template of the informed consent"

Response: We thank the Reviewer for this helpful suggestion. We agree that the Abstract should provide a clearer and more self-contained summary of the study population, key eligibility criteria, and main assessments. However, because the Abstract is limited to 250 words, we have added only the essential information required to orient the reader, while keeping the full details in the Methods and Results sections.

Specifically, the Abstract has been revised to state that adults with UC in stable clinical and endoscopic remission for ≥6 months and Rome IV C1 or C4 IBS-like symptoms were randomized to receive VSL#3® or placebo for 8 weeks. We also clarified the primary endpoint and listed the main secondary assessment tools, including IBS-SSS, SCCAI, fecal calprotectin, IBS-QoL, and IBDQ. Full patient details and baseline characteristics are reported in Table 1, patient disposition is shown in Figure 1, and complete inclusion and exclusion criteria are provided in the Methods section.

Regarding the informed consent template, all participants provided written informed consent before any study-related procedure, and the informed consent documentation was reviewed and approved by the relevant Ethics Committees as part of the study protocol. Given the word limit and the journal format, the template itself was not included in the Abstract. Informed consent template was shared with the Editor as per her request.

Comment 4: "Line 40- which are the exploratory analyses?"

Response: We thank the Reviewer for this comment. We have revised the Abstract to clarify that these were exploratory subgroup analyses focused on patients with abdominal pain or clinically relevant bloating at baseline.

Comment 5: "Line 55- please list the Rome criteria"

Response: We thank the Reviewer for this suggestion. We have added a concise description of the Rome IV C1 and C4 diagnostic criteria in the Inclusion criteria section, including the required symptom pattern and timing for irritable bowel syndrome and functional abdominal bloating/distension.

Comment 6: "Line 71- bacterial names in Italics, please"

Response: Bacterial genus and species names have been italicized throughout the manuscript, while strain identifiers have been left in regular font.

Comment 7: "Line 93-96- how you choosed the participating hospitals?"

Response: We thank the Reviewer for this comment. We have clarified in the Methods section that the participating hospitals were selected as referral IBD centers with experience in the clinical management of ulcerative colitis and in the conduct of clinical studies in IBD.

Comment 8: "Line 115-138- it would be better if you can present data in a graph"

Response: We thank the Reviewer for this suggestion. Patient disposition, including screening failures, randomization, and analysis populations, is presented graphically in Figure 1. Baseline demographic and clinical characteristics are retained in Table 1, as these variables are more clearly reported in tabular format with absolute numbers and percentages.

Comment 9: "Line 143-150- bacterial names in Italics please"

Bacterial genus and species names have been italicized throughout the manuscript, while strain identifiers have been left in regular font

Comment 10: " Line 219- 55 patients is a small number to generate pertinent conclusions"

Response: We agree with the Reviewer. The limited sample size is a major limitation of the study and precludes definitive conclusions. We have revised the Discussion and Conclusions to further emphasize that the study was prematurely interrupted because of slow recruitment, that only 55 patients were randomized compared with the planned sample size of 124, and that all efficacy findings should be interpreted as exploratory and hypothesis-generating rather than confirmatory.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors aimed to test whether Probiotic VSL#3® can alleviate IBS-like symptoms in the UC remission phase. Although the early termination of the clinical trial and the limited number of participants limit the study, the authors provided appropriate statistical analysis and interpreted the results with a modest tone. Despite the limitations, this study shows promising results of VSL#3 in alleviating IBS-like symptoms. Therefore, I have no further comments.

Author Response

Comment 1: "I have no further comments."

Response: We thank the Reviewer for the positive assessment of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This study explores the efficacy of the multistrain probiotic VSL#3® in patients with ulcerative colitis in remission who experience IBS-like symptoms. The randomized, double-blind, placebo-controlled design across multiple European centers is commendable, and the focus on functional bowel symptoms in quiescent UC addresses a clinically relevant gap. However, some points merit further consideration:

Major concerns

1.The study was prematurely interrupted, with only 55 patients randomized compared to the planned 124. This limitation substantially reduces statistical power and makes the findings exploratory rather than confirmatory. Could the authors discuss in more depth how this under-recruitment may have influenced the observed trends?

2.The Subject’s Global Assessment of Relief did not show significant differences between groups. Yet exploratory subgroup analyses suggested benefit in patients with higher baseline abdominal pain and bloating. The reliance on post hoc subgroup findings should be more cautiously framed.

3.While the inclusion criteria are well defined, the handling of missing data (e.g., imputation of non-responders) could be elaborated further to reassure readers about robustness.

4.The introduction mentions gut microbiota and gut–brain axis alterations as potential mechanisms. However, no microbiome analyses were performed. This weakens the mechanistic link. Could the authors acknowledge this limitation more explicitly?

5. The improvements in quality of life and symptom severity were numerically greater with VSL#3®, but not statistically significant. The discussion should better contextualize whether these changes are clinically meaningful despite the lack of statistical significance.

Minor concerns

1.Table 2 and Table 3 present responder rates and IBS-SSS scores clearly, but some percentages are based on small denominators. Adding absolute numbers alongside percentages consistently would aid clarity.

2.The manuscript alternates between “functional symptoms” and “IBS-like symptoms.” Standardizing terminology would improve readability.

3.The background cites meta-analyses on probiotics in IBS. It would strengthen the paper to include more recent systematic reviews or network meta-analyses to situate the findings in the broader evidence base.

4.The manuscript reports numerically greater improvements in IBDQ and IBS-QoL with VSL#3®. A more detailed breakdown of which domains improved (e.g., emotional, social, physical) would enrich interpretation.

Comments on the Quality of English Language

Nil.

Author Response

Major concerns

Comment 1: "The study was prematurely interrupted, with only 55 patients randomized compared to the planned 124. This limitation substantially reduces statistical power and makes the findings exploratory rather than confirmatory. Could the authors discuss in more depth how this under-recruitment may have influenced the observed trends?"

Response: We agree with the Reviewer. We have expanded the Discussion to clarify that premature discontinuation and under-recruitment substantially reduced statistical power, particularly for the primary responder-based endpoint.

Comment 2: "The Subject’s Global Assessment of Relief did not show significant differences between groups. Yet exploratory subgroup analyses suggested benefit in patients with higher baseline abdominal pain and bloating. The reliance on post hoc subgroup findings should be more cautiously framed."

Response: We agree with the Reviewer. We have revised the manuscript to more cautiously frame the subgroup analyses. We now explicitly state that analyses restricted to patients with baseline abdominal pain or clinically relevant bloating were exploratory and should be considered hypothesis-generating. We also revised the wording to avoid implying confirmatory efficacy based on these subgroup findings

Comment 3: "While the inclusion criteria are well defined, the handling of missing data (e.g., imputation of non-responders) could be elaborated further to reassure readers about robustness."

Response: We thank the reviewer for this comment. To further clarify the handling of missing data, we have expanded the Statistical Analysis section. For the primary endpoint (SGA response), analyses were performed both on available Week 8 data and using a conservative non-responder imputation approach, whereby subjects without a Week 8 assessment were classified as non-responders. For data analyzed by mixed nmodels all available longitudinal data were included in the mixed-effects model analyses, with missing post-baseline observations handled within the model estimation procedure and without explicit imputation. These clarifications have been added to the revised manuscript.

Comment 4: "The introduction mentions gut microbiota and gut–brain axis alterations as potential mechanisms. However, no microbiome analyses were performed. This weakens the mechanistic link. Could the authors acknowledge this limitation more explicitly?"

Response: We agree with the Reviewer. We have revised the Discussion to clarify that, although microbiota and gut–brain axis alterations provide a plausible rationale, no microbiome or mechanistic biomarker analyses were performed. Therefore, no mechanistic conclusions can be drawn, and the findings are limited to clinical and patient-reported outcomes.

Comment 5: "The improvements in quality of life and symptom severity were numerically greater with VSL#3®, but not statistically significant. The discussion should better contextualize whether these changes are clinically meaningful despite the lack of statistical significance"

Response: We agree with the Reviewer that this is an important point for the reader. We added a section in the Discussion to underline the generalizability of this data: “In this context, the improvement in quality of life and symptom severity that resulted numerically greater for VSL#3®, should be interpreted as explorative and not conclusive, as they should be confirmed in a adequately powered study”.

 

Minor concerns

Comment 1: "Table 2 and Table 3 present responder rates and IBS-SSS scores clearly, but some percentages are based on small denominators. Adding absolute numbers alongside percentages consistently would aid clarity."

Response: We thank the Reviewer for this comment. We would like to clarify that in Table 2, data are presented as both absolute numbers and their respective percentages; in Table 3, however, data are reported as mean and standard deviation"

 

Comment 2: "The manuscript alternates between “functional symptoms” and “IBS-like symptoms.” Standardizing terminology would improve readability."

Response: We thank the Reviewer for this comment. We have revised the manuscript to standardize terminology and use “IBS-like symptoms” throughout the text. The term “functional abdominal symptoms” has been retained only where needed as a broader descriptive term

Comment 3: "The background cites meta-analyses on probiotics in IBS. It would strengthen the paper to include more recent systematic reviews or network meta-analyses to situate the findings in the broader evidence base."

Response: We thank the Reviewer for this suggestion. We have revised the Introduction and Discussion to more explicitly cite and contextualize recent systematic reviews and meta-analyses on probiotics in IBS. Specifically, the cited evidence now includes recent meta-analytic studies published in 2023, 2024, and 2026, including three-level, network, and trial sequential meta-analytic approaches. We also clarified that these data mainly derive from primary IBS populations and should therefore be extrapolated cautiously to patients with ulcerative colitis in remission and IBS-like symptoms.

Comment 4: "The manuscript reports numerically greater improvements in IBDQ and IBS-QoL with VSL#3®. A more detailed breakdown of which domains improved (e.g., emotional, social, physical) would enrich interpretation."

Response: We thank the Reviewer for this suggestion. We have expanded the Results and Discussion to provide a clearer description of the IBS-QoL domains showing numerically greater or more sustained improvements with VSL#3®, including Interference with Activity, Health Worry, Relationship, Food Avoidance, and Social Reaction. Detailed domain-level results are reported in Supplementary Table 2. For IBDQ, we retained the interpretation at total-score level and for the prespecified bloating item, without adding unsupported domain-level conclusions

Reviewer 4 Report

Comments and Suggestions for Authors

This is an interesting and well-written paper on probiotic use against ulcerative colitis. The background provided is adequate, with many recent and relevant references. The aims are clear and the methodology is sound. Data presentation is also clear and informative. The results seem to confirm the authors' claims in Discussion. A few minor corrections:

1. Abstract: Briefly provide fold change values and stats for the significant severity reduction findings

2. Line 84: Describe the strain composition of the mix and the beneficial functions of each strain

3. Line 90: Briefly mention the objectives (experimental milestones) of this study.

4. Line 172: provide more details and a reference about the questionnaire

5. Discussion: Line 384, expand on meta-analysis [9]. How significant were these improvements?

6. Conclusions: are probiotics better than standard drug treatment against UC symptoms? any benefits or limitations? side effects?

Author Response

Comment 1: "Abstract: Briefly provide fold change values and stats for the significant severity reduction findings"

Response: We thank the Reviewer for this suggestion. Fold-change values were not calculated in the statistical analysis plan and are therefore not reported. To avoid overinterpretation in the Abstract, particularly given the exploratory nature of the study and the 250-word limit, we have retained a concise summary of the significant exploratory findings and clarified that the relevant quantitative results and p-values are reported in the Results section and corresponding tables.

 

Comment 2: "Line 84: Describe the strain composition of the mix and the beneficial functions of each strain"

Response: We thank the Reviewer for this comment. The strain composition of the VSL#3® formulation is already reported in the Materials and Methods section, under Treatment plan. Regarding the beneficial functions of individual strains, VSL#3® has been investigated and used as a multistrain formulation rather than as separate individual strains. Therefore, strain-specific beneficial effects are not available from the present study and would not be appropriate. We have clarified that the potential beneficial effects should be interpreted as referring to the multistrain formulation as a whole, not to individual strains.

 

Comment 3: "Line 90: Briefly mention the objectives (experimental milestones) of this study."

Response: We thank the Reviewer for this suggestion. We have revised the final sentence of the Introduction to briefly state the main study objective and the key assessment domains, including symptom relief, IBS symptom severity, UC activity, fecal calprotectin, and quality of life.

 

Comment 4: "Line 172: provide more details and a reference about the questionnaire"

Response:  We thank the Reviewer for this suggestion. We added both details and references about the IBDQ and IBS QoL questionnaire.

 

Comment 5: "Discussion: Line 384, expand on meta-analysis [9]. How significant were these improvements?"

Response: We thank the Reviewer for this suggestion. We have expanded the Discussion to better contextualize the meta-analytic evidence on probiotics in IBS. We now clarify that recent meta-analyses suggest modest improvements in global IBS symptoms compared with placebo, but that results are heterogeneous across probiotic strains, formulations, treatment durations, and outcome definitions. We also emphasize that these findings mainly derive from primary IBS populations and should be extrapolated cautiously to patients with ulcerative colitis in remission and IBS-like symptoms.

 

Comment 6: "Conclusions: are probiotics better than standard drug treatment against UC symptoms? any benefits or limitations? side effects?"

Response: We thank the Reviewer for this comment. The present study was not designed to compare probiotics with standard drug treatment for ulcerative colitis, and therefore no conclusion can be drawn regarding superiority or replacement of standard UC therapies. We have revised the Conclusions to clarify that VSL#3® may have a potential complementary, symptom-oriented role in patients with UC in remission and IBS-like symptoms, within the limitations of the exploratory design and reduced sample size. We also clarified that no safety concerns emerged during the study.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors

Thank you for taking in consideartion my suggestions as reviewer for the above mentioned paper.