Reviewer ’s report  
 
Title:  Effect of administration of a probiotic preparation on gut microbiota and immune response 
in healthy women in India  
 
Version: 1 Date: 03 Dec 2017 
 
Reviewer:  Maria L.  Marco  
 
Reviewer’s report:  
 
This paper describes a small human study including 14 healthy participants w ho consumed the 
probiotic supplement VSL#3 for 4 weeks. The fecal microbiota and serum immune cells and 
cytokine levels were assessed prior to and at the end of the 4 weeks as well as again another 4 
weeks later. There was no observable change in the fecal  microbiota composition and only 
modest variations immune response parameters were found. There is value to the study 
performed, particularly in that it targeted healthy individuals. However, the data analyses are 
inadequate and therefore this brings the s ubsequent conclusions into question.  
 
Major concern: A time series experiment was performed (0, 4, and 8 weeks), and therefore the 
data are not independent from each other. Intra -individual variation should be examined 
comparing the immune and gut microbio ta changes over time. For example, the "significant" 
changes in Th17 and TNF levels might have been due to small (non -significant) lateral changes 
among a fraction of the individuals. For the 16S rRNA assessments, intra -individual comparisons 
to baseline m ight help to resolve the reported lack of increase among Bifidobacterium, 
Lactobacillus and Streptococcus genera expected with VSL#3 consumption. Therefore, data 
analyses and resulting statistical tests were inappropriately applied throughout the manuscrip t. 
 
 
Other concerns:  
 
No significant differences between the gut microbiota were found. Therefore, lengthy figures 2 
and 3 are unnecessary. The figures should be condensed and curated.  
 
L10 The emergence of new techniques has NOT led to alterations in the gut  microbiota. The 
sentence should be revised for accuracy  
 
L40-45 The statement "probiotics are used for treatment of…" is inadequate because of the 
citations used. Animal studies are not equivalent to human studies. The use of probiotics for 
treatment of l upus is not wide -spread or even well supported.  
 
L45-50 This statement is not supported by the citation (a 2004 paper on synbiotics and patients 
with cirrhosis)  
 
L20-21 Spelling errors (enrollment, enroll)  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not,  please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either n ow or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating  to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload wi ll be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy  of the journal  
Reviewer ’s report  
 
Title:  Effect of administration of a probiotic preparation on gut microbiota and immune response 
in healthy women in India  
 
Version: 1 Date: 03 Dec 2017 
 
Reviewer:  Maria L.  Marco  
 
Reviewer’s report:  
 
This paper describes a small human study including 14 healthy participants w ho consumed the 
probiotic supplement VSL#3 for 4 weeks. The fecal microbiota and serum immune cells and 
cytokine levels were assessed prior to and at the end of the 4 weeks as well as again another 4 
weeks later. There was no observable change in the fecal  microbiota composition and only 
modest variations immune response parameters were found. There is value to the study 
performed, particularly in that it targeted healthy individuals. However, the data analyses are 
inadequate and therefore this brings the s ubsequent conclusions into question.  
 
Major concern: A time series experiment was performed (0, 4, and 8 weeks), and therefore the 
data are not independent from each other. Intra -individual variation should be examined 
comparing the immune and gut microbio ta changes over time. For example, the "significant" 
changes in Th17 and TNF levels might have been due to small (non -significant) lateral changes 
among a fraction of the individuals. For the 16S rRNA assessments, intra -individual comparisons 
to baseline m ight help to resolve the reported lack of increase among Bifidobacterium, 
Lactobacillus and Streptococcus genera expected with VSL#3 consumption. Therefore, data 
analyses and resulting statistical tests were inappropriately applied throughout the manuscrip t. 
 
 
Other concerns:  
 
No significant differences between the gut microbiota were found. Therefore, lengthy figures 2 
and 3 are unnecessary. The figures should be condensed and curated.  
 
L10 The emergence of new techniques has NOT led to alterations in the gut  microbiota. The 
sentence should be revised for accuracy  
 
L40-45 The statement "probiotics are used for treatment of…" is inadequate because of the 
citations used. Animal studies are not equivalent to human studies. The use of probiotics for 
treatment of l upus is not wide -spread or even well supported.  
 
L45-50 This statement is not supported by the citation (a 2004 paper on synbiotics and patients 
with cirrhosis)  
 
L20-21 Spelling errors (enrollment, enroll)  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not,  please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either n ow or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating  to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload wi ll be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy  of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title:  Effect of administration of a probiotic preparation on gut microbiota and immune response 
in healthy women in India  
 
Version: 1 Date: 03 Dec 2017 
 
Reviewer:  Maria L.  Marco  
 
Reviewer’s report:  
 
This paper describes a small human study including 14 healthy participants w ho consumed the 
probiotic supplement VSL#3 for 4 weeks. The fecal microbiota and serum immune cells and 
cytokine levels were assessed prior to and at the end of the 4 weeks as well as again another 4 
weeks later. There was no observable change in the fecal  microbiota composition and only 
modest variations immune response parameters were found. There is value to the study 
performed, particularly in that it targeted healthy individuals. However, the data analyses are 
inadequate and therefore this brings the s ubsequent conclusions into question.  
 
Major concern: A time series experiment was performed (0, 4, and 8 weeks), and therefore the 
data are not independent from each other. Intra -individual variation should be examined 
comparing the immune and gut microbio ta changes over time. For example, the "significant" 
changes in Th17 and TNF levels might have been due to small (non -significant) lateral changes 
among a fraction of the individuals. For the 16S rRNA assessments, intra -individual comparisons 
to baseline m ight help to resolve the reported lack of increase among Bifidobacterium, 
Lactobacillus and Streptococcus genera expected with VSL#3 consumption. Therefore, data 
analyses and resulting statistical tests were inappropriately applied throughout the manuscrip t. 
 
 
Other concerns:  
 
No significant differences between the gut microbiota were found. Therefore, lengthy figures 2 
and 3 are unnecessary. The figures should be condensed and curated.  
 
L10 The emergence of new techniques has NOT led to alterations in the gut  microbiota. The 
sentence should be revised for accuracy  
 
L40-45 The statement "probiotics are used for treatment of…" is inadequate because of the 
citations used. Animal studies are not equivalent to human studies. The use of probiotics for 
treatment of l upus is not wide -spread or even well supported.  
 
L45-50 This statement is not supported by the citation (a 2004 paper on synbiotics and patients 
with cirrhosis)  
 
L20-21 Spelling errors (enrollment, enroll)  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not,  please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either n ow or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating  to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload wi ll be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy  of the journal  
assistant