Reviewer ’s report  
 
Title:  Changes in microbiota composition, bile and fatty acid metabo lism, in successful faecal 
microbiota transplantation for Clostridioides difficile infection.  
 
Version: 0 Date: 14 May 2018 
 
Reviewer:  Jennifer  Auchtung  
 
Reviewer’s report:  
 
In their study, Brown and colleagues describe the work that they have done to characterize the 
microbial and metabolomic changes that accompany recovery in patients who have received 
fecal transplants. Similar to several previous studies, their work find s correlations between 
increased microbial diversity restoration of secondary/primary bile acid ratios in the feces of 
FMT recipients to levels more similar to healthy donors. One unique contribution of this work is 
the identification of distinct bacterial  co-abundance groups that correlate primarily with healthy 
donors or C. difficile -infected patients. The authors identified correlations between the 
abundance of different co -abundance groups in donor samples with their ability to restore 
microbial diversi ty in C. difficile infected patients. The authors propose that these CAGs could 
be a useful tool to screen FMT donors for potential efficacy prior to transplant. A second unique 
contribution of this work is the characterization of the abundance of fecal me dium and long 
chain fatty acids in donors, C. difficile infected patients, and those that have undergone 
transplants. Their data presented clear differences between healthy donors and C. difficile 
infected patients. Because fecal fatty acid profiles showed  only a slight shift toward the donor 
profile following transplant, it is unclear what role fecal fatty acids may play in recovery from 
infection.  
 
In spite of these strengths, there are significant weaknesses throughout the paper that need to be 
remedied . Descriptions of bile acids as primary or secondary and their relative impacts on C. 
difficile germination and growth inhibition are described inconsistently throughout the paper. 
The current literature demonstrates that the primary bile salt cholate, and  the conjugated primary 
bile salts taurocholate and glycocholate, as well as the secondary bile salt deoxycholate promote 
germination of C. difficile. The primary bile salt chenodeoxycholate, its conjugated primary bile 
salts tauorchenodeoxycholate and gly cochenodeoxycholate, and its secondary bile salt 
lithocholate inhibit germination. Secondary bile salts (deoxycholate and lithocholate) also inhibit 
vegetative growth of C. difficile. Other secondary bile salts may inhibit germination as described 
by Winst on and Theriot (reference 66), although their assays do not separate inhibition of 
germination from outgrowth. Further, Hoffman et al. (1992, Journal of Lipid Research 33:599 -
604) recommended in their paper proposing a standard nomenclature for bile acids that the term 
"tertiary" bile salts no longer be used. If the authors wish to use this term here, they should 
define it as well as those bile acids that they classify as tertiary bile salts.  
 
Several observations from the literature are stated as declarati ve fact when they are either 1) not 
supported by the literature reference provided or 2) still an area of active debate in the field. 
These sections should be revised to more carefully state the current knowledge in the field.  
Because the number of sample s that you analyzed is relatively small, it would be interesting to 
see supplemental figures that track responses across different donor:pre -FMT recipient:post -
FMT recipient triads. Specifically, examining CAG abundances and and fecal bile profiles.  
 
Line/ figure specific comments below.  
 
Line 39 -40. Your work did not demonstrate that these bacterial genera confer resistance to 
growth and germination to C. difficile only that they have been correlated with this in the past. 
Further, as bacterial physiology c an differ significantly even at the strain -level, it is hard to draw 
conclusions based upon the abundance of genera (or higher) level classification of partial 16S 
rRNA gene sequences.  
 
Line 42. Only specific SCFAs (pyruvic and lactic acid) increased in CDI  patients. Decreases in 
other SCFAs (butyrate, propionate, acetate) have been reported with CDI in other studies (most 
recently, Seekatz et al. 2018. Anaerobe. https://doi.org/10.1016/j.anaerobe.2018.04.001) This 
should be described more clearly. Further, changes in fecal fatty acid levels may be changed by 
factors other than inflammation so cannot be used as declarative evidence of inflammatory cell 
recruitment.  
 
Line 50 -51. You failed to detect significant changes in fatty acid levels following FMT so th is 
sentence should be reworded.  
 
Line 59. "requires secondary bile acids for spore germination" as stated above this is an 
inaccurate description of the literature as well as the citations given in the text.  
 
Line 62 -63: Sentence should be clarified to indicate that studies with fecal IgA were from 
primarily asymptomatic infants and may not extend to adult populations. E.g. (C. difficile 
colonization is inversely correlated with immunoglobulin A levels [in infa nt] and [may] 
determine microbial colonization.) If you want to describe additional role(s) for the immune 
system, there is stronger support for the role of an IgG response in limiting C. difficile recurrence 
in adult patients that you could cite.  
 
1. Kyne et al 2000. N Engl J Med 342:390 -397  
 
2. Sánchez -Hurtado et al. 2008. J Med Microbiol 57:717 -724  
 
3. Solomon K. 2013. Ther Adv Infect Dis 1:19 -35  
 
Line 71. Reference 9 does not describe the microbial dynamics in control of C. difficile infection 
nor do I think this specific dynamic pattern is accepted as fact of C. difficile susceptibility.  
 
Line 79. Although bile acids are an important factor, it is hyperbolic to call them the most 
important factor based upon the current literature. For a good summary o f current literature -
based evidence that other factors are important for colonization resistance see the introduction in 
Seekatz et al. 2018. Anaerobe. https://doi.org/10.1016/j.anaerobe.2018.04.001).  
 
Line 80. "acid" should be "acids"  
 
Line 81 -82. Althoug h data is consistent that secondary bile acids suppress C. difficile in the 
colon, stating this as declarative fact is an over -interpretation of the data.  
 
Line 98. "interaction" should be "interactions"; "enhances" should be "enhance"  
 
Line 252. Why were alpha -diversity measures evaluated with a student's t -test rather than a non -
parametric test? Did you test for normal distributions? If the data was normal, was it a one or 
two-tailed test? Please provide more detail or perform a non -parametric test.  
 
Supp lementary Figure 1 is more informative than Figure 1 and should replace it in the main text. 
As the groups identified in Figure 1 are not discussed in more detail later, it would be sufficient 
to include this as a Supplementary Figure.  
 
In the figure legen d for Supplementary Fig. 1, the F -statistic as well as the p -value should be 
reported for PERMANOVA to provide more information regarding the magnitude of the 
differences.  
 
In addition to examining the changes in microbial composition (unweighted UniFrac) , you 
should also examine changes in microbial community structure (weighted UniFrac or other 
compositional measure) as this is potentially more informative regarding the success of microbial 
engraftment.  
 
Line 327. Provide a simple definition of co -abunda nce groups  
 
Line 356. The phrase "overall bile acid levels" in this sentence is unclear. Did you compare 
differences in the amount of total aggregate bile acids or did you compare changes in the relative 
proportions across samples. Based upon later section s of the text, I think that it is the latter, but it 
is unclear here.  
 
Because Line 358 states that the total levels of fecal bile acids are increased, it is important to 
include that panel in figure 3B. Although one could roughly calculate levels based upon "P" and 
"ST", it would be easier to have it plotted. Further if tota l glycol -conjugated acids are plotted, 
total tauro -conjugated acids should be plotted, as should CDCA levels as you make statements 
about both of these levels in lines 362 -368. 
 
Line 359 & 363. It is more accurate to call these measure of bile acids in the  feces rather than the 
lower GI tract.  
 
Line 365. In text citations/references should be provided to support hypothesis of CDCA 
regulation of bile acid pool through FXR/Fgf19. Further, is the ~2 -fold change in total bile acids 
statistically significant?  
 
Line 376: "glycol -" should be "glyco -" 
 
Lines 411 -419 has no in -text citations/references and is also inaccurately described. Arachidonic 
acid is an -6 PUFA. DHA and EPA are -3 PUFA. The sentence on lines 417 -419 does not make 
much sense. Did you mean to sa y that anti -inflammatory resolvins, docosatrienes, 
neuroprotectins (derived from -e PUFA, DHA) and E -series resolvins (derived from -3 PUFA, 
EPA) dilute the effect of inflammatory -6 PUFA arachidonic acid? This section should be 
reworked.  
 
Lines 428 -433. As the data presented indicates very modest changes and is not statistically 
significant, the authors should be extremely cautious in drawing any conclusions from this data 
in FMT recipients.  
 
Line 434 -438: What is the evidence that changes in FA levels ar e due to inflammation rather 
than some diet -host-microbiome interaction? Do changes in levels of FA in CDI patients 
correlate with data from patients with other types of inflammation? Are other interpretations 
possible? This is partially addressed in the d iscussion (lines 504 -510), but should not be stated 
only in favor of inflammation in this section of the text.  
 
Line 460: spell out "co -abundance groups" rather than CAGS  
 
Line 462: Although 4 of the patients were over 65, the other 6 patients were not and  all had been 
on antibiotics. Unless you saw specific patterns related to age in your samples that you did not 
report, it is not clear the relevance that age has to your study results.  
 
Line 474 -478: As Lactobacillus, Streptococcus, Ruminococcus and Oscill ibacter are all 
Firmicutes, it might be better to use the stratifying Class designations - Clostridia 
(Ruminococcus/Oscillibacter) and Bacilli (Lactobacillus, Streptococcus)  
 
Line 486: Chenodeoxycholic acid is a primary bile acid, not a secondary bile acid  
 
Line 494 -495: Glyco - and Tauro -conjugates of CA induce germination, with taurocholate having 
a much more pronounced affect in vitro. CDCA does not induce germination, it inhibits 
germination.  
 
Line 518 -522: The introduction of the statin hypothesis was s omewhat unusual as several of your 
FMT recipients are under 35 and potentially less -likely to be taking statins.  
 
Line 529 -530: Although your results are consistent with the observation that restoration of bile 
acid metabolism correlates with successful er adication of recurrent CDI, they do not demonstrate 
that the "efficacy of FMT is bile -mediated".  
 
Line 547 -552: Many more abbreviations for bile acids were used throughout the paper than are 
described here. All abbreviations used more than once should be described here.  
 
Line 550: "rRna" should be "rRNA"  
 
As Figure 7 is not discussed in the text, it cou ld be included in the supplement rather than the 
main paper.  
 
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 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 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 will be posted on the website along with the au thors' 
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:  Changes in microbiota composition, bile and fatty acid metabo lism, in successful faecal 
microbiota transplantation for Clostridioides difficile infection.  
 
Version: 0 Date: 14 May 2018 
 
Reviewer:  Jennifer  Auchtung  
 
Reviewer’s report:  
 
In their study, Brown and colleagues describe the work that they have done to characterize the 
microbial and metabolomic changes that accompany recovery in patients who have received 
fecal transplants. Similar to several previous studies, their work find s correlations between 
increased microbial diversity restoration of secondary/primary bile acid ratios in the feces of 
FMT recipients to levels more similar to healthy donors. One unique contribution of this work is 
the identification of distinct bacterial  co-abundance groups that correlate primarily with healthy 
donors or C. difficile -infected patients. The authors identified correlations between the 
abundance of different co -abundance groups in donor samples with their ability to restore 
microbial diversi ty in C. difficile infected patients. The authors propose that these CAGs could 
be a useful tool to screen FMT donors for potential efficacy prior to transplant. A second unique 
contribution of this work is the characterization of the abundance of fecal me dium and long 
chain fatty acids in donors, C. difficile infected patients, and those that have undergone 
transplants. Their data presented clear differences between healthy donors and C. difficile 
infected patients. Because fecal fatty acid profiles showed  only a slight shift toward the donor 
profile following transplant, it is unclear what role fecal fatty acids may play in recovery from 
infection.  
 
In spite of these strengths, there are significant weaknesses throughout the paper that need to be 
remedied . Descriptions of bile acids as primary or secondary and their relative impacts on C. 
difficile germination and growth inhibition are described inconsistently throughout the paper. 
The current literature demonstrates that the primary bile salt cholate, and  the conjugated primary 
bile salts taurocholate and glycocholate, as well as the secondary bile salt deoxycholate promote 
germination of C. difficile. The primary bile salt chenodeoxycholate, its conjugated primary bile 
salts tauorchenodeoxycholate and gly cochenodeoxycholate, and its secondary bile salt 
lithocholate inhibit germination. Secondary bile salts (deoxycholate and lithocholate) also inhibit 
vegetative growth of C. difficile. Other secondary bile salts may inhibit germination as described 
by Winst on and Theriot (reference 66), although their assays do not separate inhibition of 
germination from outgrowth. Further, Hoffman et al. (1992, Journal of Lipid Research 33:599 -
604) recommended in their paper proposing a standard nomenclature for bile acids that the term 
"tertiary" bile salts no longer be used. If the authors wish to use this term here, they should 
define it as well as those bile acids that they classify as tertiary bile salts.  
 
Several observations from the literature are stated as declarati ve fact when they are either 1) not 
supported by the literature reference provided or 2) still an area of active debate in the field. 
These sections should be revised to more carefully state the current knowledge in the field.  
Because the number of sample s that you analyzed is relatively small, it would be interesting to 
see supplemental figures that track responses across different donor:pre -FMT recipient:post -
FMT recipient triads. Specifically, examining CAG abundances and and fecal bile profiles.  
 
Line/ figure specific comments below.  
 
Line 39 -40. Your work did not demonstrate that these bacterial genera confer resistance to 
growth and germination to C. difficile only that they have been correlated with this in the past. 
Further, as bacterial physiology c an differ significantly even at the strain -level, it is hard to draw 
conclusions based upon the abundance of genera (or higher) level classification of partial 16S 
rRNA gene sequences.  
 
Line 42. Only specific SCFAs (pyruvic and lactic acid) increased in CDI  patients. Decreases in 
other SCFAs (butyrate, propionate, acetate) have been reported with CDI in other studies (most 
recently, Seekatz et al. 2018. Anaerobe. https://doi.org/10.1016/j.anaerobe.2018.04.001) This 
should be described more clearly. Further, changes in fecal fatty acid levels may be changed by 
factors other than inflammation so cannot be used as declarative evidence of inflammatory cell 
recruitment.  
 
Line 50 -51. You failed to detect significant changes in fatty acid levels following FMT so th is 
sentence should be reworded.  
 
Line 59. "requires secondary bile acids for spore germination" as stated above this is an 
inaccurate description of the literature as well as the citations given in the text.  
 
Line 62 -63: Sentence should be clarified to indicate that studies with fecal IgA were from 
primarily asymptomatic infants and may not extend to adult populations. E.g. (C. difficile 
colonization is inversely correlated with immunoglobulin A levels [in infa nt] and [may] 
determine microbial colonization.) If you want to describe additional role(s) for the immune 
system, there is stronger support for the role of an IgG response in limiting C. difficile recurrence 
in adult patients that you could cite.  
 
1. Kyne et al 2000. N Engl J Med 342:390 -397  
 
2. Sánchez -Hurtado et al. 2008. J Med Microbiol 57:717 -724  
 
3. Solomon K. 2013. Ther Adv Infect Dis 1:19 -35  
 
Line 71. Reference 9 does not describe the microbial dynamics in control of C. difficile infection 
nor do I think this specific dynamic pattern is accepted as fact of C. difficile susceptibility.  
 
Line 79. Although bile acids are an important factor, it is hyperbolic to call them the most 
important factor based upon the current literature. For a good summary o f current literature -
based evidence that other factors are important for colonization resistance see the introduction in 
Seekatz et al. 2018. Anaerobe. https://doi.org/10.1016/j.anaerobe.2018.04.001).  
 
Line 80. "acid" should be "acids"  
 
Line 81 -82. Althoug h data is consistent that secondary bile acids suppress C. difficile in the 
colon, stating this as declarative fact is an over -interpretation of the data.  
 
Line 98. "interaction" should be "interactions"; "enhances" should be "enhance"  
 
Line 252. Why were alpha -diversity measures evaluated with a student's t -test rather than a non -
parametric test? Did you test for normal distributions? If the data was normal, was it a one or 
two-tailed test? Please provide more detail or perform a non -parametric test.  
 
Supp lementary Figure 1 is more informative than Figure 1 and should replace it in the main text. 
As the groups identified in Figure 1 are not discussed in more detail later, it would be sufficient 
to include this as a Supplementary Figure.  
 
In the figure legen d for Supplementary Fig. 1, the F -statistic as well as the p -value should be 
reported for PERMANOVA to provide more information regarding the magnitude of the 
differences.  
 
In addition to examining the changes in microbial composition (unweighted UniFrac) , you 
should also examine changes in microbial community structure (weighted UniFrac or other 
compositional measure) as this is potentially more informative regarding the success of microbial 
engraftment.  
 
Line 327. Provide a simple definition of co -abunda nce groups  
 
Line 356. The phrase "overall bile acid levels" in this sentence is unclear. Did you compare 
differences in the amount of total aggregate bile acids or did you compare changes in the relative 
proportions across samples. Based upon later section s of the text, I think that it is the latter, but it 
is unclear here.  
 
Because Line 358 states that the total levels of fecal bile acids are increased, it is important to 
include that panel in figure 3B. Although one could roughly calculate levels based upon "P" and 
"ST", it would be easier to have it plotted. Further if tota l glycol -conjugated acids are plotted, 
total tauro -conjugated acids should be plotted, as should CDCA levels as you make statements 
about both of these levels in lines 362 -368. 
 
Line 359 & 363. It is more accurate to call these measure of bile acids in the  feces rather than the 
lower GI tract.  
 
Line 365. In text citations/references should be provided to support hypothesis of CDCA 
regulation of bile acid pool through FXR/Fgf19. Further, is the ~2 -fold change in total bile acids 
statistically significant?  
 
Line 376: "glycol -" should be "glyco -" 
 
Lines 411 -419 has no in -text citations/references and is also inaccurately described. Arachidonic 
acid is an -6 PUFA. DHA and EPA are -3 PUFA. The sentence on lines 417 -419 does not make 
much sense. Did you mean to sa y that anti -inflammatory resolvins, docosatrienes, 
neuroprotectins (derived from -e PUFA, DHA) and E -series resolvins (derived from -3 PUFA, 
EPA) dilute the effect of inflammatory -6 PUFA arachidonic acid? This section should be 
reworked.  
 
Lines 428 -433. As the data presented indicates very modest changes and is not statistically 
significant, the authors should be extremely cautious in drawing any conclusions from this data 
in FMT recipients.  
 
Line 434 -438: What is the evidence that changes in FA levels ar e due to inflammation rather 
than some diet -host-microbiome interaction? Do changes in levels of FA in CDI patients 
correlate with data from patients with other types of inflammation? Are other interpretations 
possible? This is partially addressed in the d iscussion (lines 504 -510), but should not be stated 
only in favor of inflammation in this section of the text.  
 
Line 460: spell out "co -abundance groups" rather than CAGS  
 
Line 462: Although 4 of the patients were over 65, the other 6 patients were not and  all had been 
on antibiotics. Unless you saw specific patterns related to age in your samples that you did not 
report, it is not clear the relevance that age has to your study results.  
 
Line 474 -478: As Lactobacillus, Streptococcus, Ruminococcus and Oscill ibacter are all 
Firmicutes, it might be better to use the stratifying Class designations - Clostridia 
(Ruminococcus/Oscillibacter) and Bacilli (Lactobacillus, Streptococcus)  
 
Line 486: Chenodeoxycholic acid is a primary bile acid, not a secondary bile acid  
 
Line 494 -495: Glyco - and Tauro -conjugates of CA induce germination, with taurocholate having 
a much more pronounced affect in vitro. CDCA does not induce germination, it inhibits 
germination.  
 
Line 518 -522: The introduction of the statin hypothesis was s omewhat unusual as several of your 
FMT recipients are under 35 and potentially less -likely to be taking statins.  
 
Line 529 -530: Although your results are consistent with the observation that restoration of bile 
acid metabolism correlates with successful er adication of recurrent CDI, they do not demonstrate 
that the "efficacy of FMT is bile -mediated".  
 
Line 547 -552: Many more abbreviations for bile acids were used throughout the paper than are 
described here. All abbreviations used more than once should be described here.  
 
Line 550: "rRna" should be "rRNA"  
 
As Figure 7 is not discussed in the text, it cou ld be included in the supplement rather than the 
main paper.  
 
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 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 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 will be posted on the website along with the au thors' 
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:  Changes in microbiota composition, bile and fatty acid metabo lism, in successful faecal 
microbiota transplantation for Clostridioides difficile infection.  
 
Version: 0 Date: 14 May 2018 
 
Reviewer:  Jennifer  Auchtung  
 
Reviewer’s report:  
 
In their study, Brown and colleagues describe the work that they have done to characterize the 
microbial and metabolomic changes that accompany recovery in patients who have received 
fecal transplants. Similar to several previous studies, their work find s correlations between 
increased microbial diversity restoration of secondary/primary bile acid ratios in the feces of 
FMT recipients to levels more similar to healthy donors. One unique contribution of this work is 
the identification of distinct bacterial  co-abundance groups that correlate primarily with healthy 
donors or C. difficile -infected patients. The authors identified correlations between the 
abundance of different co -abundance groups in donor samples with their ability to restore 
microbial diversi ty in C. difficile infected patients. The authors propose that these CAGs could 
be a useful tool to screen FMT donors for potential efficacy prior to transplant. A second unique 
contribution of this work is the characterization of the abundance of fecal me dium and long 
chain fatty acids in donors, C. difficile infected patients, and those that have undergone 
transplants. Their data presented clear differences between healthy donors and C. difficile 
infected patients. Because fecal fatty acid profiles showed  only a slight shift toward the donor 
profile following transplant, it is unclear what role fecal fatty acids may play in recovery from 
infection.  
 
In spite of these strengths, there are significant weaknesses throughout the paper that need to be 
remedied . Descriptions of bile acids as primary or secondary and their relative impacts on C. 
difficile germination and growth inhibition are described inconsistently throughout the paper. 
The current literature demonstrates that the primary bile salt cholate, and  the conjugated primary 
bile salts taurocholate and glycocholate, as well as the secondary bile salt deoxycholate promote 
germination of C. difficile. The primary bile salt chenodeoxycholate, its conjugated primary bile 
salts tauorchenodeoxycholate and gly cochenodeoxycholate, and its secondary bile salt 
lithocholate inhibit germination. Secondary bile salts (deoxycholate and lithocholate) also inhibit 
vegetative growth of C. difficile. Other secondary bile salts may inhibit germination as described 
by Winst on and Theriot (reference 66), although their assays do not separate inhibition of 
germination from outgrowth. Further, Hoffman et al. (1992, Journal of Lipid Research 33:599 -
604) recommended in their paper proposing a standard nomenclature for bile acids that the term 
"tertiary" bile salts no longer be used. If the authors wish to use this term here, they should 
define it as well as those bile acids that they classify as tertiary bile salts.  
 
Several observations from the literature are stated as declarati ve fact when they are either 1) not 
supported by the literature reference provided or 2) still an area of active debate in the field. 
These sections should be revised to more carefully state the current knowledge in the field.  
Because the number of sample s that you analyzed is relatively small, it would be interesting to 
see supplemental figures that track responses across different donor:pre -FMT recipient:post -
FMT recipient triads. Specifically, examining CAG abundances and and fecal bile profiles.  
 
Line/ figure specific comments below.  
 
Line 39 -40. Your work did not demonstrate that these bacterial genera confer resistance to 
growth and germination to C. difficile only that they have been correlated with this in the past. 
Further, as bacterial physiology c an differ significantly even at the strain -level, it is hard to draw 
conclusions based upon the abundance of genera (or higher) level classification of partial 16S 
rRNA gene sequences.  
 
Line 42. Only specific SCFAs (pyruvic and lactic acid) increased in CDI  patients. Decreases in 
other SCFAs (butyrate, propionate, acetate) have been reported with CDI in other studies (most 
recently, Seekatz et al. 2018. Anaerobe. https://doi.org/10.1016/j.anaerobe.2018.04.001) This 
should be described more clearly. Further, changes in fecal fatty acid levels may be changed by 
factors other than inflammation so cannot be used as declarative evidence of inflammatory cell 
recruitment.  
 
Line 50 -51. You failed to detect significant changes in fatty acid levels following FMT so th is 
sentence should be reworded.  
 
Line 59. "requires secondary bile acids for spore germination" as stated above this is an 
inaccurate description of the literature as well as the citations given in the text.  
 
Line 62 -63: Sentence should be clarified to indicate that studies with fecal IgA were from 
primarily asymptomatic infants and may not extend to adult populations. E.g. (C. difficile 
colonization is inversely correlated with immunoglobulin A levels [in infa nt] and [may] 
determine microbial colonization.) If you want to describe additional role(s) for the immune 
system, there is stronger support for the role of an IgG response in limiting C. difficile recurrence 
in adult patients that you could cite.  
 
1. Kyne et al 2000. N Engl J Med 342:390 -397  
 
2. Sánchez -Hurtado et al. 2008. J Med Microbiol 57:717 -724  
 
3. Solomon K. 2013. Ther Adv Infect Dis 1:19 -35  
 
Line 71. Reference 9 does not describe the microbial dynamics in control of C. difficile infection 
nor do I think this specific dynamic pattern is accepted as fact of C. difficile susceptibility.  
 
Line 79. Although bile acids are an important factor, it is hyperbolic to call them the most 
important factor based upon the current literature. For a good summary o f current literature -
based evidence that other factors are important for colonization resistance see the introduction in 
Seekatz et al. 2018. Anaerobe. https://doi.org/10.1016/j.anaerobe.2018.04.001).  
 
Line 80. "acid" should be "acids"  
 
Line 81 -82. Althoug h data is consistent that secondary bile acids suppress C. difficile in the 
colon, stating this as declarative fact is an over -interpretation of the data.  
 
Line 98. "interaction" should be "interactions"; "enhances" should be "enhance"  
 
Line 252. Why were alpha -diversity measures evaluated with a student's t -test rather than a non -
parametric test? Did you test for normal distributions? If the data was normal, was it a one or 
two-tailed test? Please provide more detail or perform a non -parametric test.  
 
Supp lementary Figure 1 is more informative than Figure 1 and should replace it in the main text. 
As the groups identified in Figure 1 are not discussed in more detail later, it would be sufficient 
to include this as a Supplementary Figure.  
 
In the figure legen d for Supplementary Fig. 1, the F -statistic as well as the p -value should be 
reported for PERMANOVA to provide more information regarding the magnitude of the 
differences.  
 
In addition to examining the changes in microbial composition (unweighted UniFrac) , you 
should also examine changes in microbial community structure (weighted UniFrac or other 
compositional measure) as this is potentially more informative regarding the success of microbial 
engraftment.  
 
Line 327. Provide a simple definition of co -abunda nce groups  
 
Line 356. The phrase "overall bile acid levels" in this sentence is unclear. Did you compare 
differences in the amount of total aggregate bile acids or did you compare changes in the relative 
proportions across samples. Based upon later section s of the text, I think that it is the latter, but it 
is unclear here.  
 
Because Line 358 states that the total levels of fecal bile acids are increased, it is important to 
include that panel in figure 3B. Although one could roughly calculate levels based upon "P" and 
"ST", it would be easier to have it plotted. Further if tota l glycol -conjugated acids are plotted, 
total tauro -conjugated acids should be plotted, as should CDCA levels as you make statements 
about both of these levels in lines 362 -368. 
 
Line 359 & 363. It is more accurate to call these measure of bile acids in the  feces rather than the 
lower GI tract.  
 
Line 365. In text citations/references should be provided to support hypothesis of CDCA 
regulation of bile acid pool through FXR/Fgf19. Further, is the ~2 -fold change in total bile acids 
statistically significant?  
 
Line 376: "glycol -" should be "glyco -" 
 
Lines 411 -419 has no in -text citations/references and is also inaccurately described. Arachidonic 
acid is an -6 PUFA. DHA and EPA are -3 PUFA. The sentence on lines 417 -419 does not make 
much sense. Did you mean to sa y that anti -inflammatory resolvins, docosatrienes, 
neuroprotectins (derived from -e PUFA, DHA) and E -series resolvins (derived from -3 PUFA, 
EPA) dilute the effect of inflammatory -6 PUFA arachidonic acid? This section should be 
reworked.  
 
Lines 428 -433. As the data presented indicates very modest changes and is not statistically 
significant, the authors should be extremely cautious in drawing any conclusions from this data 
in FMT recipients.  
 
Line 434 -438: What is the evidence that changes in FA levels ar e due to inflammation rather 
than some diet -host-microbiome interaction? Do changes in levels of FA in CDI patients 
correlate with data from patients with other types of inflammation? Are other interpretations 
possible? This is partially addressed in the d iscussion (lines 504 -510), but should not be stated 
only in favor of inflammation in this section of the text.  
 
Line 460: spell out "co -abundance groups" rather than CAGS  
 
Line 462: Although 4 of the patients were over 65, the other 6 patients were not and  all had been 
on antibiotics. Unless you saw specific patterns related to age in your samples that you did not 
report, it is not clear the relevance that age has to your study results.  
 
Line 474 -478: As Lactobacillus, Streptococcus, Ruminococcus and Oscill ibacter are all 
Firmicutes, it might be better to use the stratifying Class designations - Clostridia 
(Ruminococcus/Oscillibacter) and Bacilli (Lactobacillus, Streptococcus)  
 
Line 486: Chenodeoxycholic acid is a primary bile acid, not a secondary bile acid  
 
Line 494 -495: Glyco - and Tauro -conjugates of CA induce germination, with taurocholate having 
a much more pronounced affect in vitro. CDCA does not induce germination, it inhibits 
germination.  
 
Line 518 -522: The introduction of the statin hypothesis was s omewhat unusual as several of your 
FMT recipients are under 35 and potentially less -likely to be taking statins.  
 
Line 529 -530: Although your results are consistent with the observation that restoration of bile 
acid metabolism correlates with successful er adication of recurrent CDI, they do not demonstrate 
that the "efficacy of FMT is bile -mediated".  
 
Line 547 -552: Many more abbreviations for bile acids were used throughout the paper than are 
described here. All abbreviations used more than once should be described here.  
 
Line 550: "rRna" should be "rRNA"  
 
As Figure 7 is not discussed in the text, it cou ld be included in the supplement rather than the 
main paper.  
 
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 
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3. Do you hold or are you currently applying for any patents relating to the content of the 
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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  
 
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