Reviewer ’s report  
Title: Faecal bacterial microbiota in patients with cirrhosis and the effect of lactulose  
administration  
 
Version: 0 Date: 24 Jul 2017  
 
Reviewer: Alexandra Zhernakova  
 
Reviewer's report:  
The paper by Sarangi and co -authors "Faecal bacterial microbiota in patients wit h cirrhosis and 
the effect of lactulose administration" Investigates the effect of administration of lactulose on 
microbiome of patients with cirrhosis. Based on the analysis in 35 individuals, the authors 
conclude that administration of lactulose does not  have an effect on microbiome in patients with 
cirrhosis.  
The microbiome analysis is performed well and described in details. The authors excluded 
patients with microbiome -modifier medications. The major criticism of this paper is the small 
sample size: 3 5 patients, of them 21 in the longitudinal study, and 18 controls.  
The authors observed the difference between patients and controls (which is also known from 
other studies) but did not see the effect of lactulose on the gut microbiome. Although the study 
cohort is small, it is the first study investigating the effect of lactulose on gut microbiome, and 
lack of effect of treatment on microbiome in paired samples is an informative message.  
There are several major and minor comments for this paper  
 
Major com ments:  
1. It is unclear if cirrhosis patients at the baseline were treatment -naïve since the authors say: 
Thirty of them received regular lactulose administration as a part of a standard of care. Does 
it mean that 30 out of 35 "lactulose -free" patients have recei ved lactulose treatment earlier? 
How long was the end of previous treatment before sample collection? Could the previous 
lactulose intake influence the microbiome?  
 
2. The difference in phylum abundance was mainly observed for rare phyla. I wonder if the 
DNA collection and isolation methods were exactly the same in cases and controls since I 
expect that that might influence the results. The stool collection protocols should be briefly 
described in the methods.  
 
3. The authors say: "Cirrhosis -dysbiosis ratio  was significantly lower in patients with cirrhosis 
than in controls (1.55±1.86 versus 2.71±1.48; p=0.019, Mann -Whitney U test)." Do I 
understand correctly that this is counterintuitive: the cirrhosis index is lower in cirrhosis 
patients?  
 
4. Did the autho rs correct for medication, diet and lifestyle differences when comparing 
cohorts with and without cirrhosis?  Did they correct for age and gender in their analysis? 
This should be added to the discussion.  
 
5. In the discussion, the authors compare their s tudy with other microbiome studies in cirrhosis 
and lactulose intake. The authors should mention the number of samples in other studies, to 
allow the readers of better comparing their and others results.  
 
Minor comments:  
1. "The number of bacterial cells in the intestinal lumen of an individual far exceeds that of 
human cells in the body" Recent estimations suggest that the number of host and bacterial cells is 
about the same.  
 
2. Child -Turcotte -Pugh (CTP) class - should be briefly explained when first use d (at least 
mention that it is assessment of the severity of the disease)  
 
3. Add the primer sequence for the methods.  
 
4. The authors sometimes mix up information between methods and results sections. For 
example, this part of results should be in the met hods section: The 74 stool specimens studied 
(18 controls; 35 pre -lactulose and 21 post -lactulose) yielded a total of 40,023,099 high -quality 
reads (median [range] = 474,267 [131,802 -1,267,206]), their rarefaction curves are shown in 
Additional File 3: Fig  S1. 
The same applies for the other part of results, for example, part of the paragraph starting with 
"Median [range] number of high -quality reads for 18 controls and 35 baseline cirrhosis 
specimens were 407,482 (316,749 -1,139,360; total reads 9,388,696) a nd 504,182 (131,802 -
1,267,206; total 19,466,867), respectively.  
 
5. English should be edited for easier reading. For example:  
a. "Those patients were selectively enrolled whose physicians were planning to start them on 
lactulose."  
 
b. "For patients who we re started on lactulose, a second specimen was collected after 6 weeks of 
lactulose administration (in a dose of 30 -60 ml/day, adjusted to obtain 2 -3 semisolid stools 
daily), except in those who had worsening of clinical condition, or required another medi cation 
or hospitalization."  
 
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:  
Needs some language corrections before being published  
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 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  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 wi sh 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 and the effect of lactulose  
administration  
 
Version: 0 Date: 24 Jul 2017  
 
Reviewer: Alexandra Zhernakova  
 
Reviewer's report:  
The paper by Sarangi and co -authors "Faecal bacterial microbiota in patients wit h cirrhosis and 
the effect of lactulose administration" Investigates the effect of administration of lactulose on 
microbiome of patients with cirrhosis. Based on the analysis in 35 individuals, the authors 
conclude that administration of lactulose does not  have an effect on microbiome in patients with 
cirrhosis.  
The microbiome analysis is performed well and described in details. The authors excluded 
patients with microbiome -modifier medications. The major criticism of this paper is the small 
sample size: 3 5 patients, of them 21 in the longitudinal study, and 18 controls.  
The authors observed the difference between patients and controls (which is also known from 
other studies) but did not see the effect of lactulose on the gut microbiome. Although the study 
cohort is small, it is the first study investigating the effect of lactulose on gut microbiome, and 
lack of effect of treatment on microbiome in paired samples is an informative message.  
There are several major and minor comments for this paper  
 
Major com ments:  
1. It is unclear if cirrhosis patients at the baseline were treatment -naïve since the authors say: 
Thirty of them received regular lactulose administration as a part of a standard of care. Does 
it mean that 30 out of 35 "lactulose -free" patients have recei ved lactulose treatment earlier? 
How long was the end of previous treatment before sample collection? Could the previous 
lactulose intake influence the microbiome?  
 
2. The difference in phylum abundance was mainly observed for rare phyla. I wonder if the 
DNA collection and isolation methods were exactly the same in cases and controls since I 
expect that that might influence the results. The stool collection protocols should be briefly 
described in the methods.  
 
3. The authors say: "Cirrhosis -dysbiosis ratio  was significantly lower in patients with cirrhosis 
than in controls (1.55±1.86 versus 2.71±1.48; p=0.019, Mann -Whitney U test)." Do I 
understand correctly that this is counterintuitive: the cirrhosis index is lower in cirrhosis 
patients?  
 
4. Did the autho rs correct for medication, diet and lifestyle differences when comparing 
cohorts with and without cirrhosis?  Did they correct for age and gender in their analysis? 
This should be added to the discussion.  
 
5. In the discussion, the authors compare their s tudy with other microbiome studies in cirrhosis 
and lactulose intake. The authors should mention the number of samples in other studies, to 
allow the readers of better comparing their and others results.  
 
Minor comments:  
1. "The number of bacterial cells in the intestinal lumen of an individual far exceeds that of 
human cells in the body" Recent estimations suggest that the number of host and bacterial cells is 
about the same.  
 
2. Child -Turcotte -Pugh (CTP) class - should be briefly explained when first use d (at least 
mention that it is assessment of the severity of the disease)  
 
3. Add the primer sequence for the methods.  
 
4. The authors sometimes mix up information between methods and results sections. For 
example, this part of results should be in the met hods section: The 74 stool specimens studied 
(18 controls; 35 pre -lactulose and 21 post -lactulose) yielded a total of 40,023,099 high -quality 
reads (median [range] = 474,267 [131,802 -1,267,206]), their rarefaction curves are shown in 
Additional File 3: Fig  S1. 
The same applies for the other part of results, for example, part of the paragraph starting with 
"Median [range] number of high -quality reads for 18 controls and 35 baseline cirrhosis 
specimens were 407,482 (316,749 -1,139,360; total reads 9,388,696) a nd 504,182 (131,802 -
1,267,206; total 19,466,867), respectively.  
 
5. English should be edited for easier reading. For example:  
a. "Those patients were selectively enrolled whose physicians were planning to start them on 
lactulose."  
 
b. "For patients who we re started on lactulose, a second specimen was collected after 6 weeks of 
lactulose administration (in a dose of 30 -60 ml/day, adjusted to obtain 2 -3 semisolid stools 
daily), except in those who had worsening of clinical condition, or required another medi cation 
or hospitalization."  
 
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:  
Needs some language corrections before being published  
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 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  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 wi sh 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 and the effect of lactulose  
administration  
 
Version: 0 Date: 24 Jul 2017  
 
Reviewer: Alexandra Zhernakova  
 
Reviewer's report:  
The paper by Sarangi and co -authors "Faecal bacterial microbiota in patients wit h cirrhosis and 
the effect of lactulose administration" Investigates the effect of administration of lactulose on 
microbiome of patients with cirrhosis. Based on the analysis in 35 individuals, the authors 
conclude that administration of lactulose does not  have an effect on microbiome in patients with 
cirrhosis.  
The microbiome analysis is performed well and described in details. The authors excluded 
patients with microbiome -modifier medications. The major criticism of this paper is the small 
sample size: 3 5 patients, of them 21 in the longitudinal study, and 18 controls.  
The authors observed the difference between patients and controls (which is also known from 
other studies) but did not see the effect of lactulose on the gut microbiome. Although the study 
cohort is small, it is the first study investigating the effect of lactulose on gut microbiome, and 
lack of effect of treatment on microbiome in paired samples is an informative message.  
There are several major and minor comments for this paper  
 
Major com ments:  
1. It is unclear if cirrhosis patients at the baseline were treatment -naïve since the authors say: 
Thirty of them received regular lactulose administration as a part of a standard of care. Does 
it mean that 30 out of 35 "lactulose -free" patients have recei ved lactulose treatment earlier? 
How long was the end of previous treatment before sample collection? Could the previous 
lactulose intake influence the microbiome?  
 
2. The difference in phylum abundance was mainly observed for rare phyla. I wonder if the 
DNA collection and isolation methods were exactly the same in cases and controls since I 
expect that that might influence the results. The stool collection protocols should be briefly 
described in the methods.  
 
3. The authors say: "Cirrhosis -dysbiosis ratio  was significantly lower in patients with cirrhosis 
than in controls (1.55±1.86 versus 2.71±1.48; p=0.019, Mann -Whitney U test)." Do I 
understand correctly that this is counterintuitive: the cirrhosis index is lower in cirrhosis 
patients?  
 
4. Did the autho rs correct for medication, diet and lifestyle differences when comparing 
cohorts with and without cirrhosis?  Did they correct for age and gender in their analysis? 
This should be added to the discussion.  
 
5. In the discussion, the authors compare their s tudy with other microbiome studies in cirrhosis 
and lactulose intake. The authors should mention the number of samples in other studies, to 
allow the readers of better comparing their and others results.  
 
Minor comments:  
1. "The number of bacterial cells in the intestinal lumen of an individual far exceeds that of 
human cells in the body" Recent estimations suggest that the number of host and bacterial cells is 
about the same.  
 
2. Child -Turcotte -Pugh (CTP) class - should be briefly explained when first use d (at least 
mention that it is assessment of the severity of the disease)  
 
3. Add the primer sequence for the methods.  
 
4. The authors sometimes mix up information between methods and results sections. For 
example, this part of results should be in the met hods section: The 74 stool specimens studied 
(18 controls; 35 pre -lactulose and 21 post -lactulose) yielded a total of 40,023,099 high -quality 
reads (median [range] = 474,267 [131,802 -1,267,206]), their rarefaction curves are shown in 
Additional File 3: Fig  S1. 
The same applies for the other part of results, for example, part of the paragraph starting with 
"Median [range] number of high -quality reads for 18 controls and 35 baseline cirrhosis 
specimens were 407,482 (316,749 -1,139,360; total reads 9,388,696) a nd 504,182 (131,802 -
1,267,206; total 19,466,867), respectively.  
 
5. English should be edited for easier reading. For example:  
a. "Those patients were selectively enrolled whose physicians were planning to start them on 
lactulose."  
 
b. "For patients who we re started on lactulose, a second specimen was collected after 6 weeks of 
lactulose administration (in a dose of 30 -60 ml/day, adjusted to obtain 2 -3 semisolid stools 
daily), except in those who had worsening of clinical condition, or required another medi cation 
or hospitalization."  
 
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:  
Needs some language corrections before being published  
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 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  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 wi sh 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