Peer review of: Bile acids drive the newborn’s gut microbiota maturation (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

This study employed a combination of “omics” approaches to profile gut microbiome, liver 
metabolites and transcriptomics in mice from birth to 56 days of age, which aimed to identify 
intrinsic host factors that help shape the gut microbiome during postnatal development. Bio-
informatics analysis of the multiple data sets showed a strong correlation of hepatic bile acid 
composition and gut microbiome, suggesting that bile acids act as host factors driving the postnatal 
microbiota maturation. Although the experiments are well designed, the study is of descriptive 
nature with major conclusions reached mainly based on correlations between data sets. 

 

Some of the major concerns are listed below: 

 

1. Fig 3b shows that the abundance of hepatic primary bile acids post-weaning is at least an order of 
magnitude larger than that of secondary bile acids, therefore the change in the bile acid pool 
composition was mostly driven by the primary bile acids, which the authors should address on pg 9. 

 

2. Changes in GCA and UDCA in Fig S9 appear to be more related to the initiation of diet, as the data 
variability does not show significant differences with age. It is difficult to determine changes in TCA 
since the d7 values are widely divergent. TMCA does not appear to appreciably change after 
weaning, when comparing between adjacent timepoints. Therefore, there seems to be changes 
between pre-weaning and post-weaning, but not “age-related” as the authors state. 

3.The strong correlation involving UDCA and GCA is puzzling since glycine conjugated bile acids are in 
trace amount in mice and their effect on gut microbiota is likely minimal at such low concentration. 

 

4.Why were only the small intestine OTUs analyzed with the rCCA analysis? Fig 1 shows the colon 
has a much richer bacterial representation. That analysis should be carried out as well or rationale 
for focusing on the small intestine should be added. 

 

5. Why was the oral gavage of bile acids not carried out for 3 days before d7 instead of starting at 
d7? Since the microbiota begin to change significantly after d7, inoculating between d1 and d7 
would seem the best window to impact the baseline bacterial composition. 

 

6.Given the wide spread of the data, the PCoA plots in Fig 4 are not particularly convincing. Perhaps 
another type of analysis could be used to demonstrate similarity between microbiota at d56 and at 
d9 following bile acid treatment. 

 

7. Similarly, the data in Figures 4e-f-g have a lot of variability in bacterial abundance, so the 
Lactobacillus/Escherichia ratio is not significantly different from adult for any of the conditions. 

 

8.The presentation of the data at the bottom of pg 11 is very confusing as both TMCA and UDCA led 
to low Escherichia. 

 

9. Without metagenomics it is impossible to determine whether the microbiome function is altered 
by bile acids. Furthermore, the long-term effect of neonatal bile acid treatment was not determined. 
Therefore, a conclusion that specific bile acids induce development of a microbiome that is 
“beneficial” cannot be truly determined without knowing how function is altered in the developing 
microbiome. 

 

10. On pg. 15, the authors state that TMCA administration fostered growth of Lactobacillus OTUs 
containing BSHs and were closely related to L. reuteri and L. jonsonii. Those relationships were 
inferred in silico and not determined experimentally, which should be noted. 

 

11. How did the authors draw their conclusion that bile acid concentrations and species profiles 
appear to contribute in environmental filtering or niche-based interactions? Which data showed 
that? 

 

12.In the discussion, the authors do not delineate limitations of their study. Lack of metagenomics 
data to determine microbiome function and strain-level correlations with bile acids is a key 
limitation. Metabolomics measurements only encapsulated a small number of metabolites. It is 
possible that untargeted metabolomics could reveal other compound classes which play an 
important role in the development of the microbiome. 

 

Minor Comments: 

Pg 5 lines 21-24 Which figure shows that Mannheimia at 24 h after birth clusters separately? Is it 
driving the clustering at 24 hr in Fig S1a? Why would Corynebacterium not colonize? 

 

Based on the richness graphs in Figs 1 and S1, it seems as if diversification really begins to change 
following d7 rather than d1 as stated by the authors. No increase is observed between d1 and d7. 

 

Sankey plots in Fig S3 seem to show the early proteobacteria contribution is most evident in the 
colon, whereas in the small intestine, early proteobacteria and actinobacteria seem to similarly 
influence later composition. This differential should be noted. 

 

DMM modeling inset in Fig 1d is difficult to interpret. P1 and P7 datapoints cannot be distinguished. 
This figure should be bigger and labeled to aid interpretation. In Fig 1d, what are the clusters with 
the dashed lines referring to vs the solid lines? Are they the different tissues? It in unclear from the 
graph labeling. Suggest Fig 1d should be in supplement. Fig S3 could be included in Fig 1 instead as it 
is more informative. 

 

Lines 11 and 12 on page 7 are difficult to understand. The organs should be grouped with the 
capacity. 

 

Is there a citation supporting the statement that the hepatic metabolome is a proxy for systemic 
metabolism? The metabolomics analyses performed here were limited to 188 compounds, therefore 
it is not likely that systemic metabolism could be effectively modeled with these data. This 
statement should be revised. Furthermore, why was the metabolome not investigated in the feces 
and/or in blood? There may be other factors contributing to microbiome development that circulate 
through the leaky gut in early life. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

Van Best et al present a clear report on a study of the development of neonate mice gut microbiota 
and the concomitant changes in bile acid production and metabolism. The interesting observation 
comes from the oral administration of some of said bile acids, which causes the microbiota to 
mature more quickly, partially recapitulating developments that would usually happen later. While 
the results are not overly surprising, they are for sure interesting and the study is performed using 
standard tools and without noticeable flaws. The language is clear and the figures informative. I have 
listed a few instances of missing details in the report and have some minor issues with the 
presentations, as detailed below. 

 

missing methodological/statistical detail: 

- abstract: mention N 

- p5/l4-6: mention N; also, what's the difference between 24h and 1 day? from the fact that the 
profile in 24h looks different to the one on d1, I guess these are different sets of studies, but this is 
pretty unclear; throughout the manuscript PND1 is referred to as "at birth"- is it the within 24h after 
birth or on the second day? 

- p5/l7-9: the text suggests that 10 time points were studied (10 mice per litter?) but figure 1a 
suggests 6 time points - which is it?; also the text is missing an indication of numbers of litters and 
ergo total number of samples 

-p8/l20-22: the GF mice were not introduced before, maybe say more about this treatment? 

-p10/l25: which adult microbiota samples are these? 

-p11/l1-2: is there a statistical basis for the statement, or is this based purely on the impression of 
the first two PCs? 

-p20/l4: add url and/or reference for the platform; what's the rationale for the amplicon length 
settings? for the used fragments, I'd expect most OTUs to have a length of 294, some taxonomic 
groups slightly above 300 bases (if the primers were kept- no removal is indicated), so the 200 seems 
oddly loose and the 300 oddly stringent? what's the rationale for the 97% clustering - as one of the 
aims of the study was to trace OTUs over time, wouldn't an exact sequence variant approach be 
more suitable? 

 

-methods: 

p25: it's good to analyse absolute bacterial abundances (or approximations thereof) by qPCR. 
However, relative numbers of reads per OTU from amplicon sequencing are not reflective of real 
proportions of organisms in a sample (see eg https://elifesciences.org/articles/46923). Therefore, 
this analysis is not valid. As it is not a major point of the manuscript, I'd be not too worried, but 
would like to see if the conclusions hold if the normalisation to absolute numbers are not performed 
or if the analysis is performed on presence/absence data or a different normalisation (e.g. as 
suggested in the linked article). 

 

minor: 

-discussion: 

general: it's not always clear which statements refer to humans, mice or both 

p13/l17-19: high richness in meconium has been observed repeatedly so it's not surprising, e.g. 
Mueller et al, Genes 2017, Wampach et al, Frontiers in Microbiology 2017, Palmer et al, Plos1 2007 
(you also cite this one) 

-terminology: 

p2/l4: most of the develop is succession rather than evolution 

p2/l15: it's unclear what about the maturation is "enhanced" - is it just quicker? 

p3/l3: I'd suggest to add "mammalian" or "human" or "murine" before host. 

p6/l5: the intestinal content is not a tissue 

p7/l21: while I've seen the term in literature, I find "breast milk" inappropriate in mice and would 
suggest to call it just "milk"; or replace "switch in diet .... " by "weaning" 

p9/l5: If this statement is based on the Picrust analysis, it should read "the predicted abundance of 
genes for the key enzyme..." 

p9/l7: introduce "predicted" before "BSH" 

p9/l8: the second part of this sentence "expression of proteins" refers to liver transcriptomes, 
correct? this should be clarified (at the moment it reads more like either Picrust microbiome genes 
or liver proteins). 

- language: 

p3/l9-10: move "human" from before "disease" to before "intestinal" 

p4/l17: replace "identical" with "same" 

legend Sfig 3 - should be kinetics rather than kinetic, although I am not convinced by the term in this 
context 

p11/l3: replace "enhance" with "increase" 

 

figures: 

- Fig 1: what kind of smoothing is applied in figure 1e/f and Sfig 1 d/e? are these mean values? it's 
unclear how the ellipses for the DMM-clusters are chosen, since they hardly include any points? 
something's wrong with figure legend "(G) and (G)" 

- SFig 3 - why are there these shapes going from nowhere to nowhere around the rectangles on 
PND1 (e.g Actinobacteria, Proteobacteria)? the legend claims that the height reflects relative 
number of OTUs - relative to what? the text describes that the plots visualize the increase in 
diversity, isn't that contradictory to the legend? 

Fig 2: y-axis labels are incomplete 

Fig 4: a: the pink and orange dots are really difficult to tell apart. I'd suggest adding differentiating 
symbols, too; d: the y-axis should only go up to 1; the title is redundant with the y-axis label 

Fig 5: missing n in Dunn's name; legend should contain more detail regarding the way the tree was 
built. 

 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

This is a well-designed and performed study, data presented are novel and interesting.Some 
correlations between bile acid species and hepatic metabolism are speculative and difficult to 
explain. 

 

Major comment: 

Bile salt hydrolase (BSH) deconjugation of bile acids mainly occurs in the colon, not small intestine. 
Small intestine, mainly in ileum, reabsorbs 95% conjugated bile acids. Also bacterial contents 
increase and microbiota change from upper intestine to small intestine to colon. Bile acid 
compositions also varied from indium (mostly conjugated bile acids) to cecum (mostly deconjugated 
secondary bile acids) to feces (mostly DCA). Correlation of bile acid species and BSH activity in the 
small intestine to hepatobiliary transport maturation does not make sense and need to be clarified. 
Overall, this is a very nice study of bile acids and gut microbiome in development from neonate to 
maturity in mice. Most data are consistent to current understanding of bile acid metabolism in 
development. 

 

In Fig. 1e, what are the color codes? Same as in 1f? some colors and symbols are difficult to discern 
in manny figures. 

Page 8, lines 6-17 need modification. “BSH increased in small intestine”?. De-conjugation of bile 
acids mainly occur in the colon, where bacteria enriched with BSH are located. Most conjugated bile 
acids are reabsorbed in ileum. “Increased BSH was paralleled by enhanced expression of proteins 
involved in hepatobiliary transport of bile acids. ..”. Conjugated bile acids efflux from liver (Abcb11) 
and up taken into intestinal (Asbt) in the ileum, thus the increased intestine BSH parallels bile acid 
transporters is not consistent with enhanced BSH and maturation of the hepatobiliary transport 
system as stated in these sentences. In the colon, most bile acids are un-conjugated (by BSH) and are 
transported by passive transport/diffusion to portal circulation or excreted into feces.