Reviewer ’s report  
 
Title: Small intestinal microbiota composition and the prog nosis of infants with ileostomy 
resulting from distinct primary diseases  
 
Version: 2 Date: 21 Feb 2020  
 
Reviewer's report:  
 
Qian et al. here provide a revised manuscript on the composition of small -intestinal microbiota in 
neonates, which highlights microbial diversity in this understudied population. While some of 
the reviewers' concerns were addressed, the manuscript still contains major deficiencies 
regarding the presentation of data and - more importantly - statistical testing, which critically 
impede interpretation of the dataset:  
Individual points  
 
1. It would helpful if measures of alpha diversity (i.e. Shannon index) would be displayed 
per sample (i.e. in a dot plot) instead of providing only arithmetic means as currently shown in 
Table 1. This would allow a much better estimation about sample -to-sample variation.  
 
2. Page 6, line 127: Significance was not tested and thus a "significantly higher" 
abundance cannot be stated.  
 
3. Page 6, line 130: The authors state that "The two largest classes were  Escherichia -
Shigella and Klebsiella (Figure 2)." What does this refer to? Abundance in each patient sample? 
Broad appearance across different patients?  
 
4. I am highly concerned about the adequacy of the statistical tests, which were applied to 
determine t he influence of breast -feeding on clinical and microbiota -related parameters, 
particularly as these results are among the main claims of the authors. Firstly, no information 
about the applied tested is provided in the table legends or method sections. In t heir rebuttal 
letter, the authors imply that t -tests were performed. For the clinical endpoints "colitis" and 
"sepsis", t -tests are inadequate as these data represent frequencies (i.e. not numerical data), 
would best be represented in a contingency table a nd require appropriate statistical methods (e.g. 
Fisher's test). Using the numbers and frequencies provided in table 2 (i.e. 22 patients breast -fed, 
8 patients not breast -fed, rates of colitis 50% vs. 12.5% and rates of sepsis 82% vs. 50%), a 
Fisher's test  reveals non -significance for both colitis and sepsis. Moreover, it is unclear if more 
clinical parameters were determined (and potentially tested for association) and thus if correction 
for multiple testing may be required. While t tests may be appropriat e for growth rates (although 
a dot plot would be helpful to determine whether samples follow a normal distribution), 
differences were not statistically significant according to table 2 (p=0.091) and hence the claim 
on page 7 line 145 is incorrect. Further,  it is impossible to assess the meaning of the reported p 
values for the bacterial read outs (i.e. frequency of Bifidobacterium) as the authors do not state 
whether these are adjusted to account for multiple testing. In summary, the statistical analyses 
presented in Table 2 are inadequate and do not warrant the conclusions stated by the authors.  
 
5. While the discussion adequately refers to and interprets previous studies on small 
intestinal microbiota, large parts are only loosely connected to the results obtained in the present 
study.  
 
6. The authors state that "results are consistent with findings of previous studies; that is, 
enterobacteria always include Streptococcus and Veillonella spp. in the ecosystem, and these are 
the most active members of the ileal microbiota [3]." In light of these studies, how do the authors 
interpret the low abundance o f Veillonella in their samples (Fig. 1 and 2)?  
 
7. Page 9 lines 204 -206: The claimed correlation between Klebsiella/Enterobacter or 
Staphylococcus and gastrointestinal disease is not backed by the data presented.  
 
8. Page 10 lines 212 -213: "The results revea l that patients with small intestinal ostomy 
subsequent to different etiologies have specific microbial community characteristics". This is not 
supported by the presented data. There seems to be almost random clustering of samples in the 
hierarchical clust ering presented in Fig 2.  
 
9. The title of the study is misleading as no data are presented on the "prognosis of infants 
with ileostomy".  
 
Are the methods appropriate and well desc ribed?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy 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 manuscr ipt: 
 
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, f ees, funding, or salary from an 
organisation that may  in any way gain or lose financial ly from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either n ow 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 t o 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 int erests' 
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 understan d that my name will be included 
on my report to the authors and, if the ma nuscript is accepted f or publication, my named report 
including any attachments I upload will 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 in cluded 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: Small intestinal microbiota composition and the prog nosis of infants with ileostomy 
resulting from distinct primary diseases  
 
Version: 2 Date: 21 Feb 2020  
 
Reviewer's report:  
 
Qian et al. here provide a revised manuscript on the composition of small -intestinal microbiota in 
neonates, which highlights microbial diversity in this understudied population. While some of 
the reviewers' concerns were addressed, the manuscript still contains major deficiencies 
regarding the presentation of data and - more importantly - statistical testing, which critically 
impede interpretation of the dataset:  
Individual points  
 
1. It would helpful if measures of alpha diversity (i.e. Shannon index) would be displayed 
per sample (i.e. in a dot plot) instead of providing only arithmetic means as currently shown in 
Table 1. This would allow a much better estimation about sample -to-sample variation.  
 
2. Page 6, line 127: Significance was not tested and thus a "significantly higher" 
abundance cannot be stated.  
 
3. Page 6, line 130: The authors state that "The two largest classes were  Escherichia -
Shigella and Klebsiella (Figure 2)." What does this refer to? Abundance in each patient sample? 
Broad appearance across different patients?  
 
4. I am highly concerned about the adequacy of the statistical tests, which were applied to 
determine t he influence of breast -feeding on clinical and microbiota -related parameters, 
particularly as these results are among the main claims of the authors. Firstly, no information 
about the applied tested is provided in the table legends or method sections. In t heir rebuttal 
letter, the authors imply that t -tests were performed. For the clinical endpoints "colitis" and 
"sepsis", t -tests are inadequate as these data represent frequencies (i.e. not numerical data), 
would best be represented in a contingency table a nd require appropriate statistical methods (e.g. 
Fisher's test). Using the numbers and frequencies provided in table 2 (i.e. 22 patients breast -fed, 
8 patients not breast -fed, rates of colitis 50% vs. 12.5% and rates of sepsis 82% vs. 50%), a 
Fisher's test  reveals non -significance for both colitis and sepsis. Moreover, it is unclear if more 
clinical parameters were determined (and potentially tested for association) and thus if correction 
for multiple testing may be required. While t tests may be appropriat e for growth rates (although 
a dot plot would be helpful to determine whether samples follow a normal distribution), 
differences were not statistically significant according to table 2 (p=0.091) and hence the claim 
on page 7 line 145 is incorrect. Further,  it is impossible to assess the meaning of the reported p 
values for the bacterial read outs (i.e. frequency of Bifidobacterium) as the authors do not state 
whether these are adjusted to account for multiple testing. In summary, the statistical analyses 
presented in Table 2 are inadequate and do not warrant the conclusions stated by the authors.  
 
5. While the discussion adequately refers to and interprets previous studies on small 
intestinal microbiota, large parts are only loosely connected to the results obtained in the present 
study.  
 
6. The authors state that "results are consistent with findings of previous studies; that is, 
enterobacteria always include Streptococcus and Veillonella spp. in the ecosystem, and these are 
the most active members of the ileal microbiota [3]." In light of these studies, how do the authors 
interpret the low abundance o f Veillonella in their samples (Fig. 1 and 2)?  
 
7. Page 9 lines 204 -206: The claimed correlation between Klebsiella/Enterobacter or 
Staphylococcus and gastrointestinal disease is not backed by the data presented.  
 
8. Page 10 lines 212 -213: "The results revea l that patients with small intestinal ostomy 
subsequent to different etiologies have specific microbial community characteristics". This is not 
supported by the presented data. There seems to be almost random clustering of samples in the 
hierarchical clust ering presented in Fig 2.  
 
9. The title of the study is misleading as no data are presented on the "prognosis of infants 
with ileostomy".  
 
Are the methods appropriate and well desc ribed?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy 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 manuscr ipt: 
 
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, f ees, funding, or salary from an 
organisation that may  in any way gain or lose financial ly from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either n ow 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 t o 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 int erests' 
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 understan d that my name will be included 
on my report to the authors and, if the ma nuscript is accepted f or publication, my named report 
including any attachments I upload will 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 in cluded 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: Small intestinal microbiota composition and the prog nosis of infants with ileostomy 
resulting from distinct primary diseases  
 
Version: 2 Date: 21 Feb 2020  
 
Reviewer's report:  
 
Qian et al. here provide a revised manuscript on the composition of small -intestinal microbiota in 
neonates, which highlights microbial diversity in this understudied population. While some of 
the reviewers' concerns were addressed, the manuscript still contains major deficiencies 
regarding the presentation of data and - more importantly - statistical testing, which critically 
impede interpretation of the dataset:  
Individual points  
 
1. It would helpful if measures of alpha diversity (i.e. Shannon index) would be displayed 
per sample (i.e. in a dot plot) instead of providing only arithmetic means as currently shown in 
Table 1. This would allow a much better estimation about sample -to-sample variation.  
 
2. Page 6, line 127: Significance was not tested and thus a "significantly higher" 
abundance cannot be stated.  
 
3. Page 6, line 130: The authors state that "The two largest classes were  Escherichia -
Shigella and Klebsiella (Figure 2)." What does this refer to? Abundance in each patient sample? 
Broad appearance across different patients?  
 
4. I am highly concerned about the adequacy of the statistical tests, which were applied to 
determine t he influence of breast -feeding on clinical and microbiota -related parameters, 
particularly as these results are among the main claims of the authors. Firstly, no information 
about the applied tested is provided in the table legends or method sections. In t heir rebuttal 
letter, the authors imply that t -tests were performed. For the clinical endpoints "colitis" and 
"sepsis", t -tests are inadequate as these data represent frequencies (i.e. not numerical data), 
would best be represented in a contingency table a nd require appropriate statistical methods (e.g. 
Fisher's test). Using the numbers and frequencies provided in table 2 (i.e. 22 patients breast -fed, 
8 patients not breast -fed, rates of colitis 50% vs. 12.5% and rates of sepsis 82% vs. 50%), a 
Fisher's test  reveals non -significance for both colitis and sepsis. Moreover, it is unclear if more 
clinical parameters were determined (and potentially tested for association) and thus if correction 
for multiple testing may be required. While t tests may be appropriat e for growth rates (although 
a dot plot would be helpful to determine whether samples follow a normal distribution), 
differences were not statistically significant according to table 2 (p=0.091) and hence the claim 
on page 7 line 145 is incorrect. Further,  it is impossible to assess the meaning of the reported p 
values for the bacterial read outs (i.e. frequency of Bifidobacterium) as the authors do not state 
whether these are adjusted to account for multiple testing. In summary, the statistical analyses 
presented in Table 2 are inadequate and do not warrant the conclusions stated by the authors.  
 
5. While the discussion adequately refers to and interprets previous studies on small 
intestinal microbiota, large parts are only loosely connected to the results obtained in the present 
study.  
 
6. The authors state that "results are consistent with findings of previous studies; that is, 
enterobacteria always include Streptococcus and Veillonella spp. in the ecosystem, and these are 
the most active members of the ileal microbiota [3]." In light of these studies, how do the authors 
interpret the low abundance o f Veillonella in their samples (Fig. 1 and 2)?  
 
7. Page 9 lines 204 -206: The claimed correlation between Klebsiella/Enterobacter or 
Staphylococcus and gastrointestinal disease is not backed by the data presented.  
 
8. Page 10 lines 212 -213: "The results revea l that patients with small intestinal ostomy 
subsequent to different etiologies have specific microbial community characteristics". This is not 
supported by the presented data. There seems to be almost random clustering of samples in the 
hierarchical clust ering presented in Fig 2.  
 
9. The title of the study is misleading as no data are presented on the "prognosis of infants 
with ileostomy".  
 
Are the methods appropriate and well desc ribed?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy 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 manuscr ipt: 
 
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, f ees, funding, or salary from an 
organisation that may  in any way gain or lose financial ly from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, either n ow 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 t o 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 int erests' 
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 understan d that my name will be included 
on my report to the authors and, if the ma nuscript is accepted f or publication, my named report 
including any attachments I upload will 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 in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant