Review for 10.1186/s12876-022-02527-z (2022)

Title: Triclosan Exposure Induced Disturbance of Gut Microbiota and Exaggerated
Experimental Colitis in Mice
Version: 1 Date: 23 Jun 2022
Reviewer's report:
In this paper, Jing et al. proposed to determine the effects and mechanisms of triclosan, an
antibiotic heavily used in consumer products, on intestinal inflammation and ulcerative colitis
model. Although the authors use a previously defined model, and assessed parameters to
determine intestinal inflammation, they lacked key experiments to support many of the claims
made. Additionally, some of the statements presented are also unsupported by their results, and
I am concerned by the complete lack of discussion of a previous manuscript published on the
same topic.
Major comments:
1. This work is very similar to a manuscript published in Science Translational Medicine (PMID:
29848663), in which the authors showed that triclosan caused low-grade colonic inflammation,
increased colitis, and exacerbated colitis-associated colon cancer in mice. While this previous
publication doesn’t abolish the merit of the present one, this manuscript should have been
heavily discussed throughout the current work. The discussion should highlight the points of
similarity and differences between both works, and acknowledge the lengthier mechanistic work
performed in the previously published manuscript.
2. This manuscript strongly lacks methodological clarity. Authors mention more details are
provided in supplementary material, although this was not made available for peer review.
Several points that need further description include:
a. Study design: authors mentioned they determined the dose of triclosan was 80ppm, although
it seems it was just chosen based on previous publication. If indeed chosen, what were the other
doses tested? What was the plasma triclosan concentration in each of them? Why was 80ppm
chosen?
b. 16S sequencing: how was microbial DNA isolated/purified from fecal samples? What primers
were used for 16S amplification? What system were the samples run on? What were the steps
of cleaning, filtering, trimming and data analysis? Was sequencing and data analysis done in
house or by a company?
c. Colitis assessment: why were livers chosen for histological examination – line 85? What was
the scoring system used for colitis assessment? What were the primers used for each of the genes
chosen for gene expression assays? Was there a housekeeping gene used for gene expression
calculations?
d. Statistical analysis: did authors use false discovery rate correction for multiple comparisons?
What was the statistical tool used to determine the changes in microbiome taxonomic changes?
How was the data analyzed and plotted?
3. One of the main claims of this manuscript is that triclosan causes intestinal inflammation,
although this is not supported by the data presented. While results show some significant changes
in TLR4 and NF-kB gene expression, the authors report no changes in cytokine levels and
complete absence of mucosal inflammation (Fig 2 and 4, lines 98 and 119). Therefore, this data
does not adequately show that triclosan induces spontaneous colitis in mice.
4. I strongly urge the authors to get familiarized with microbiome data representation and
analysis. All the microbial changes presented in this manuscript seem to be based on generalized
observation from a heat map, which seems to be driven by a few animals in the groups (Fig 3 and
6). With the microbiome response to triclosan treatment being fairly inconsistent, the authors
would have to properly determine the changes in taxonomic community structure by applying
statistical tools to determine the changes in microbial relative abundance for such observations.
5. Still, even if these changes are indeed significant using proper statistical analysis, the places
where authors suggest that pro-inflammatory mechanisms are due to changes in the microbiome
are too speculative. All authors are doing is reporting microbiome changes to triclosan treatment,
and cannot determine causality in this setting. Proper mechanistic work would require use
of gnotobiotic conditions, or other forms of microbiome modulation to confirm the changes
induced by triclosan are causally implicated in the observed changes. All places where this is
suggested should be deleted and only mention to associations and observed changes made.
Minor comments:
6. Authors need to include way more information and details in the figure legends. For the sake
of data transparency, statistical analysis performed, and group sizes should also be reported for
each of the figures presented.
7. Line 30: “established” should be “establish an”.
8. Line 32: “Dextransulfatesodium” should be “Dextran sulfate sodium”.
9. Line 37: “Inflammation” should be “inflammatory”.
10. Line 39: “examples” should be “samples”.
11. Line 41-46: this whole conclusion section and description of potential mechanism is
extremely speculative and should be re-written. This manuscript would require  lengthier
mechanistic work to support these claims of changes in microbiome causing the inflammatory
response. Authors can include some of this as points for discussion, although these should not
be a conclusion of the paper.
12. Line 51: “caused” should be “causes”.
13. Line 51: “As acknowledged” should be deleted.
14. Line 55: “could” should be “can”.
15. Line 56: The reference added after the word “worldwide” does not support this sentence. In
the cited manuscript, authors do not measure triclosan levels in bodily fluids from people at all.
16. Line 57: missing references to triclosan-induced microbiome changes and induced colitis.
17. Line 66: missing a zero in “0.345”.
18. Line 67: again, reference “4” did not measure triclosan levels in humans.
19. Line 73: “age,” should be “aged”.
20. Line 74 (and other instances): no supplementary materials provided.
21. Line 85: Why livers? Should this be colon fragments? Whole colon?
22. Line 89: delete “either”.
23. Line 96 and figure 1 (and all other instances referring to and figures showing similar data):
missing what gene expression was normalized to for reported values. What are the units? Is A
and C cytokine concentration, while B gene expression? Authors keep using both “expression”
and “concentration” interchangeably throughout the manuscript, so this needs to be properly
clarified and revised.
24. Line 96: NF-kB and TLR4 expression showed in Fig 1B, not 1A.
25. Line 98: delete “the” before “intestinal mucosal injury”.
26. Line 105: “alterd” should be “altered”.
27. Line 105-109: It is hard to agree with this paragraph without proper statistical assessment.
While authors mentioned using Wilcoxon-Mann-Whitney, which I assume was for the alpha-
diversity plots, did they apply any statistical analysis to confirm difference in beta-diversity
or bacterial relative abundance? For NMDS ordination on Bray-Curtis dissimilarities, authors
could apply PERMANOVA, or Adonis to confirm community changes. Additionally, to
determine changes in the relative abundance of the bacterial taxa, authors should use non-
parametric Kruskal–Wallis tests followed by post hoc Dunn tests with Benjamin–Holmes with
FDR correction. All these should be included in the statistical analysis section in the methods.
28. Line 108: Lachnosporaceae_UCG006 is an acetate, not butyrate producer (PMID:
31584586).
29. For all microbiome figures (i.e.: Figures 3, 6 and 9), there is no concordant colors between
panels A and B, and C. This could be confusing to some readers. I recommend authors to change
that and have group colors consistent throughout. Similarly, I would also suggest keeping groups
with same names, like control (C) and triclosan-treated (T). having C/S in Figure 3, X/T in figure
6, and DT/DX in figure 9 is confusing.
30. Line 119: There was no increase, please remove this sentence about changes in cytokine
levels. Also, again, when authors say “enhanced the level” assumes cytokine measurement. Was
this gene expression or actually protein quantification?
31. Line 144-146: Here is a clear example of the erroneous interchangeable use of “expression”
and “concentration/protein”. What are authors actually measuring? Was this gene expression or
protein quantification?
32. Line 147: “inflammation” should be “inflammatory”.
33. Line 156: delete “the” before Actinobacteria.
34. Line 173 and 174: “produing” should be “producing”.
35. Line 174: “bateria” should be “bacteria”.
36. Line 175: This reference “10” doesn’t support the suggestion that supplementation
of butyrate-producing bacteria could enrich microbiota diversity. This paper uses a nitrate
treatment to investigate potential protection to DSS-induced changes. While they found no
changes in overall diversity, some changes at genus level were reported.
37. Line 185: missing reference.
38. Line 194-195: this is not what your data show. You reported no changes in intestinal
inflammation in absence of DSS challenge. Based on the data presented, triclosan did not induce
non-specific colitis in a healthy mouse gut.
39. Line 210: authors did not assess intestinal barrier integrity. The claim about reduced occludin
expression can stay, but authors can’t suggest triclosan influences barrier integrity without
properly testing it.
40. Line 217: “accelarated” should be “accelerated”.
41. Line 231: triclosan did not induce colitis, only exacerbated it in the presence of another
stressor.
42. Line 231-232: Authors suggest that pro-inflammatory mechanisms are due to changes in the
microbiome, but that is too speculative. Those are just observations and do not suggest causation
unless properly controlled. Please delete that.
43. Line 234-236: triclosan reduced levels of occluding, but again authors cannot attest
to defective barrier function without testing for it (e.g.: FITC-dextran or FSA for barrier
integrity, or Ussing chamber for tissue permeability). Similarly, authors can’t confirm microbial
translocation to activate/exacerbate TLR4-NF-kB signaling for cytokine release, as this was not
measured. This would require controlled mechanistic work that was not performed.
44. Line 240: separate TLR4 and NF-kB in abbreviation list.