Reviewer ’s report  
Title: Faecal bacterial microbiota in patients with cirrhosis a nd the effect of lactulose  
administration  
 
Version: 0 Date: 01 Aug 2017  
 
Reviewer: Zlatan Mujagic  
 
Reviewer's report:  
The study deals with the important topic of the role of microbiota in patients with liver cirrhosis 
and the effects of lactulose on faecal microbiota composition, assessed using 16S rRNA gene 
sequencing. The research question is very relevant, as hepatic e ncephalopathy is one of the major 
complications of liver cirrhosis and is treated using lactulose or rifaximin, both targeting gut 
microbiota. On the other hand, the mechanisms involved and the exact role of the gut microbiota 
is very poorly understood. Th erefore, better understanding of this role, which is the aim of the 
study, could lead to improvement of the treatment strategies.  
 
The article is well written and backgrounds, methods and results are well presented. 
Furthermore, the results are well discu ssed including a fair approach to available literature. 
Overall, I am positive about the study and support the publication of this manuscript. However, I 
do have several remarks and suggestions for revision.  
 
1) Research question.  
The last sentence of th e introduction does describe what is done. But could the authors state a 
clear research question and possibly include a hypothesis in the introduction section. As I see it, 
there are 2 research questions in this paper; regarding the difference in faecal mi crobiota 
composition between cirrhosis patients and healthy controls, and the possible change of gut 
microbiota composition in cirrhosis patients after treatment with lactulose.  
 
2) Methods.  
The inclusion and exclusion criteria are described by the author s. But, could the authors provide 
more information on the following points:  
- Via which route were the patients included? Out -patient clinics, hospital or GP patients, 
inpatients, one or more centres?  
- In which time period were the subjects included?  
- How were the controls included? Public advertisements? Via the included patients (family 
members)?   
- How were the healthy controls defined as healthy? Did they use any medication, or did they 
have any current or previous diseases or disorders? Was a physi cal examination conducted or 
did the researchers include oral medical history taking?  
 
3) Methods.  
Stool sample collection was just shortly mentioned. Could the authors indicate how the samples 
were collected, transported and stored until analyses? When w ere the samples frozen? And was 
this done in the same way for all subjects and both study groups?  
 
4) Terminology.  
The authors use both the terms gut microbiota and gut flora in the manuscript. While the term gut 
flora has been used in previous literature , there is widespread consensus that this term should not 
be used in scientific papers. I suggest to replace flora throughout the manuscript by microbiota.  
 
5) Results, Table 1  
Could the normal value for each parameter be added to the table? As these can differ between 
countries, and furthermore, in many countries different units are used.  
 
6) Results, Table 1  
The proportion of patients with hepatitis B and C as aetiology of cirrhosis, as well as 
cryptogenic, is quite high, compared to European or the US studies, where alcohol and fatty liver 
are much more frequent causes of liver cirrhosis. Is this due to demographic differences or due to 
the used inclusion criteria, in particular the criterion for 'no co -existing disease', which could 
have led to exclusi on of NAFLD patients with diabetes mellitus by example?  
This should be discussed by the authors in the discussion section.  
 
7) Identified grammatical/spelling errors and small remarks:  
- Line 217: At the end of the sentence the parentheses should be removed.  
- Line 293: "mixture of bacteria present in an individual's gut - collectively referred to as the 
'gut microbiota".  
Actually, bacteria are the largest part of gut microbiota, but t here are other micro -organisms as 
well. This should be stated.  
- Line 297: It should state 'among others', as gut microbiota is shown to be involved in more 
diseases and disorders than listed here.  
- Line 304: also here 'among others'.  
 
8) Discussion.  
The discussion section is well written and a thorough comparison to relevant literature is 
provided. Furthermore, the authors show insight in the limitations of the study and address this 
well. I do have one small note on this point. Although the authors do  acknowledge the small 
sample number as a possible limitation, they argue that the paired sample design reduces a 
possible selection bias due to the small study population. I do agree partly with the authors on 
this point.  
Liver cirrhosis, as the authors show in table 1, is based on very different etiological factors and is 
characterised by a range of complications and scales of severity. This indicates that the patient 
population is very heterogeneous. Therefore, to provide robust conclusions with regard to 
differences in microbiota composition and diversity between patients and controls, large sample 
numbers are required. This should be included in the discussion section. Also as advise for future 
studies.  
 
9) Discussion.  
The authors have used the PICRUS t analysis to investigate possible metabolic function of the 
microbiota. In the discussion, it should be indicated clearly that based on the 16s rRNA gene 
sequencing data only an estimate of function can be provided, as it is not measured by this 
method. F urthermore, it should be stated that more useful techniques, including metagenomics 
sequencing and metabolic profiling of faecal water and urine, are available, and should be used 
in future studies that focus on the current patient population to study the metabolic functionality 
and activity of gut microbiota.  
 
10) Discussion.  
The authors should discuss the point of hepatic encephalopathy. How many of the subjects 
included developed HE during the study period, if any. And should the main research question 
be (possible in future studies) to study gut microbiota of subjects pre and post HE who are 
treated with lactulose, possibly with a subgroup analysis that includes a group of patients well 
responsive to lactulose treatment compared to those who do not resp ond to therapy. Such an 
approach would lead to better understanding of the role of gut microbiota in development of HE 
in cirrhotic patients and could provide leads for patient selection with regard to therapy.  
This should be discussed in some degree in t he discussion section as the authors should include a 
section with their advice for future studies.  
 
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 adequa tely supported by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommende d, 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 interest s 
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 pub lication of this  
manuscript, either now 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 cu rrently 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 de tails below.  
 
No competing interests of any kind  
 
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 a ttachments 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 comment s 
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: Faecal bacterial microbiota in patients with cirrhosis a nd the effect of lactulose  
administration  
 
Version: 0 Date: 01 Aug 2017  
 
Reviewer: Zlatan Mujagic  
 
Reviewer's report:  
The study deals with the important topic of the role of microbiota in patients with liver cirrhosis 
and the effects of lactulose on faecal microbiota composition, assessed using 16S rRNA gene 
sequencing. The research question is very relevant, as hepatic e ncephalopathy is one of the major 
complications of liver cirrhosis and is treated using lactulose or rifaximin, both targeting gut 
microbiota. On the other hand, the mechanisms involved and the exact role of the gut microbiota 
is very poorly understood. Th erefore, better understanding of this role, which is the aim of the 
study, could lead to improvement of the treatment strategies.  
 
The article is well written and backgrounds, methods and results are well presented. 
Furthermore, the results are well discu ssed including a fair approach to available literature. 
Overall, I am positive about the study and support the publication of this manuscript. However, I 
do have several remarks and suggestions for revision.  
 
1) Research question.  
The last sentence of th e introduction does describe what is done. But could the authors state a 
clear research question and possibly include a hypothesis in the introduction section. As I see it, 
there are 2 research questions in this paper; regarding the difference in faecal mi crobiota 
composition between cirrhosis patients and healthy controls, and the possible change of gut 
microbiota composition in cirrhosis patients after treatment with lactulose.  
 
2) Methods.  
The inclusion and exclusion criteria are described by the author s. But, could the authors provide 
more information on the following points:  
- Via which route were the patients included? Out -patient clinics, hospital or GP patients, 
inpatients, one or more centres?  
- In which time period were the subjects included?  
- How were the controls included? Public advertisements? Via the included patients (family 
members)?   
- How were the healthy controls defined as healthy? Did they use any medication, or did they 
have any current or previous diseases or disorders? Was a physi cal examination conducted or 
did the researchers include oral medical history taking?  
 
3) Methods.  
Stool sample collection was just shortly mentioned. Could the authors indicate how the samples 
were collected, transported and stored until analyses? When w ere the samples frozen? And was 
this done in the same way for all subjects and both study groups?  
 
4) Terminology.  
The authors use both the terms gut microbiota and gut flora in the manuscript. While the term gut 
flora has been used in previous literature , there is widespread consensus that this term should not 
be used in scientific papers. I suggest to replace flora throughout the manuscript by microbiota.  
 
5) Results, Table 1  
Could the normal value for each parameter be added to the table? As these can differ between 
countries, and furthermore, in many countries different units are used.  
 
6) Results, Table 1  
The proportion of patients with hepatitis B and C as aetiology of cirrhosis, as well as 
cryptogenic, is quite high, compared to European or the US studies, where alcohol and fatty liver 
are much more frequent causes of liver cirrhosis. Is this due to demographic differences or due to 
the used inclusion criteria, in particular the criterion for 'no co -existing disease', which could 
have led to exclusi on of NAFLD patients with diabetes mellitus by example?  
This should be discussed by the authors in the discussion section.  
 
7) Identified grammatical/spelling errors and small remarks:  
- Line 217: At the end of the sentence the parentheses should be removed.  
- Line 293: "mixture of bacteria present in an individual's gut - collectively referred to as the 
'gut microbiota".  
Actually, bacteria are the largest part of gut microbiota, but t here are other micro -organisms as 
well. This should be stated.  
- Line 297: It should state 'among others', as gut microbiota is shown to be involved in more 
diseases and disorders than listed here.  
- Line 304: also here 'among others'.  
 
8) Discussion.  
The discussion section is well written and a thorough comparison to relevant literature is 
provided. Furthermore, the authors show insight in the limitations of the study and address this 
well. I do have one small note on this point. Although the authors do  acknowledge the small 
sample number as a possible limitation, they argue that the paired sample design reduces a 
possible selection bias due to the small study population. I do agree partly with the authors on 
this point.  
Liver cirrhosis, as the authors show in table 1, is based on very different etiological factors and is 
characterised by a range of complications and scales of severity. This indicates that the patient 
population is very heterogeneous. Therefore, to provide robust conclusions with regard to 
differences in microbiota composition and diversity between patients and controls, large sample 
numbers are required. This should be included in the discussion section. Also as advise for future 
studies.  
 
9) Discussion.  
The authors have used the PICRUS t analysis to investigate possible metabolic function of the 
microbiota. In the discussion, it should be indicated clearly that based on the 16s rRNA gene 
sequencing data only an estimate of function can be provided, as it is not measured by this 
method. F urthermore, it should be stated that more useful techniques, including metagenomics 
sequencing and metabolic profiling of faecal water and urine, are available, and should be used 
in future studies that focus on the current patient population to study the metabolic functionality 
and activity of gut microbiota.  
 
10) Discussion.  
The authors should discuss the point of hepatic encephalopathy. How many of the subjects 
included developed HE during the study period, if any. And should the main research question 
be (possible in future studies) to study gut microbiota of subjects pre and post HE who are 
treated with lactulose, possibly with a subgroup analysis that includes a group of patients well 
responsive to lactulose treatment compared to those who do not resp ond to therapy. Such an 
approach would lead to better understanding of the role of gut microbiota in development of HE 
in cirrhotic patients and could provide leads for patient selection with regard to therapy.  
This should be discussed in some degree in t he discussion section as the authors should include a 
section with their advice for future studies.  
 
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 adequa tely supported by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommende d, 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 interest s 
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 pub lication of this  
manuscript, either now 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 cu rrently 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 de tails below.  
 
No competing interests of any kind  
 
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 a ttachments 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 comment s 
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: Faecal bacterial microbiota in patients with cirrhosis a nd the effect of lactulose  
administration  
 
Version: 0 Date: 01 Aug 2017  
 
Reviewer: Zlatan Mujagic  
 
Reviewer's report:  
The study deals with the important topic of the role of microbiota in patients with liver cirrhosis 
and the effects of lactulose on faecal microbiota composition, assessed using 16S rRNA gene 
sequencing. The research question is very relevant, as hepatic e ncephalopathy is one of the major 
complications of liver cirrhosis and is treated using lactulose or rifaximin, both targeting gut 
microbiota. On the other hand, the mechanisms involved and the exact role of the gut microbiota 
is very poorly understood. Th erefore, better understanding of this role, which is the aim of the 
study, could lead to improvement of the treatment strategies.  
 
The article is well written and backgrounds, methods and results are well presented. 
Furthermore, the results are well discu ssed including a fair approach to available literature. 
Overall, I am positive about the study and support the publication of this manuscript. However, I 
do have several remarks and suggestions for revision.  
 
1) Research question.  
The last sentence of th e introduction does describe what is done. But could the authors state a 
clear research question and possibly include a hypothesis in the introduction section. As I see it, 
there are 2 research questions in this paper; regarding the difference in faecal mi crobiota 
composition between cirrhosis patients and healthy controls, and the possible change of gut 
microbiota composition in cirrhosis patients after treatment with lactulose.  
 
2) Methods.  
The inclusion and exclusion criteria are described by the author s. But, could the authors provide 
more information on the following points:  
- Via which route were the patients included? Out -patient clinics, hospital or GP patients, 
inpatients, one or more centres?  
- In which time period were the subjects included?  
- How were the controls included? Public advertisements? Via the included patients (family 
members)?   
- How were the healthy controls defined as healthy? Did they use any medication, or did they 
have any current or previous diseases or disorders? Was a physi cal examination conducted or 
did the researchers include oral medical history taking?  
 
3) Methods.  
Stool sample collection was just shortly mentioned. Could the authors indicate how the samples 
were collected, transported and stored until analyses? When w ere the samples frozen? And was 
this done in the same way for all subjects and both study groups?  
 
4) Terminology.  
The authors use both the terms gut microbiota and gut flora in the manuscript. While the term gut 
flora has been used in previous literature , there is widespread consensus that this term should not 
be used in scientific papers. I suggest to replace flora throughout the manuscript by microbiota.  
 
5) Results, Table 1  
Could the normal value for each parameter be added to the table? As these can differ between 
countries, and furthermore, in many countries different units are used.  
 
6) Results, Table 1  
The proportion of patients with hepatitis B and C as aetiology of cirrhosis, as well as 
cryptogenic, is quite high, compared to European or the US studies, where alcohol and fatty liver 
are much more frequent causes of liver cirrhosis. Is this due to demographic differences or due to 
the used inclusion criteria, in particular the criterion for 'no co -existing disease', which could 
have led to exclusi on of NAFLD patients with diabetes mellitus by example?  
This should be discussed by the authors in the discussion section.  
 
7) Identified grammatical/spelling errors and small remarks:  
- Line 217: At the end of the sentence the parentheses should be removed.  
- Line 293: "mixture of bacteria present in an individual's gut - collectively referred to as the 
'gut microbiota".  
Actually, bacteria are the largest part of gut microbiota, but t here are other micro -organisms as 
well. This should be stated.  
- Line 297: It should state 'among others', as gut microbiota is shown to be involved in more 
diseases and disorders than listed here.  
- Line 304: also here 'among others'.  
 
8) Discussion.  
The discussion section is well written and a thorough comparison to relevant literature is 
provided. Furthermore, the authors show insight in the limitations of the study and address this 
well. I do have one small note on this point. Although the authors do  acknowledge the small 
sample number as a possible limitation, they argue that the paired sample design reduces a 
possible selection bias due to the small study population. I do agree partly with the authors on 
this point.  
Liver cirrhosis, as the authors show in table 1, is based on very different etiological factors and is 
characterised by a range of complications and scales of severity. This indicates that the patient 
population is very heterogeneous. Therefore, to provide robust conclusions with regard to 
differences in microbiota composition and diversity between patients and controls, large sample 
numbers are required. This should be included in the discussion section. Also as advise for future 
studies.  
 
9) Discussion.  
The authors have used the PICRUS t analysis to investigate possible metabolic function of the 
microbiota. In the discussion, it should be indicated clearly that based on the 16s rRNA gene 
sequencing data only an estimate of function can be provided, as it is not measured by this 
method. F urthermore, it should be stated that more useful techniques, including metagenomics 
sequencing and metabolic profiling of faecal water and urine, are available, and should be used 
in future studies that focus on the current patient population to study the metabolic functionality 
and activity of gut microbiota.  
 
10) Discussion.  
The authors should discuss the point of hepatic encephalopathy. How many of the subjects 
included developed HE during the study period, if any. And should the main research question 
be (possible in future studies) to study gut microbiota of subjects pre and post HE who are 
treated with lactulose, possibly with a subgroup analysis that includes a group of patients well 
responsive to lactulose treatment compared to those who do not resp ond to therapy. Such an 
approach would lead to better understanding of the role of gut microbiota in development of HE 
in cirrhotic patients and could provide leads for patient selection with regard to therapy.  
This should be discussed in some degree in t he discussion section as the authors should include a 
section with their advice for future studies.  
 
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 adequa tely supported by the data shown?  
If not, please explain in your comments to the authors.  
Yes 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommende d, 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 interest s 
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 pub lication of this  
manuscript, either now 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 cu rrently 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 de tails below.  
 
No competing interests of any kind  
 
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 a ttachments 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 comment s 
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