Title: Triclosan Exposure Induced Disturbance of Gut Microbiota and Exaggerated
Experimental Colitis in Mice
Version: 5 Date: 05 Aug 2022
Reviewer's report:
In this paper, Jing et al. proposed to determine the effects and mechanisms of triclosan (TCS), an
antibiotic heavily used in consumer products, on intestinal inflammation and ulcerative colitis
model. While this manuscript still doesn’t present mechanistic work, I would like to compliment
the authors for heavily changing the method and discussion sections, which make this manuscript
much nicer to read. The authors have addressed most of my comments, although some things
still need to be reviewed before this work can be published.
Minor comments:
1. Line 22: Authors do not investigate mechanisms of TCS induced microbiome changes or
immune activation. Therefore, the sentence “This animal experiment aimed to clarify effects
and mechanisms of triclosan” is misleading. Please change to “This animal experiment aimed
to clarify the effects of triclosan exposure on the microbiome and intestinal immune functions
in healthy and ulcerative colitis model”.
2. Line 34 (and every other instance it appears – including lines 36, 161, 162, etc and figure
legends): in response to previous review document, authors did not determine gene expression,
but rather protein concentration via ELISA or Western Blot. Therefore, the word “expression”
cannot be used to describe any of the results presented in this report. Please correct that
throughout and have it replaced by “protein levels”, “protein concentration”, or equivalent.
3. Line 73: Same as comment number 1. Please change sentence “This study aimed to clarify
effects and mechanisms of TCS on UC in mice.” by “This study aimed to clarify the effects of
TCS exposure on the microbiome and intestinal immune functions in healthy and UC model”.
4. Line 79: again, authors did not determine the dose of TCS, rather used a previously published
concentration. Where is says “Firstly, we determined the dose of TCS was 80ppm”, should be
replaced by “We treated mice with 80ppm of TCS, as previously published [4]”.
5. Line 79-80: replace “when mice exposed…” and “TCS in mice was” to “when mice were
exposed…” and “TCS was”.
6. Table line 87: The three instances the word “maintain” is shown, should be replaced by
“maintained”.
7. Line 92-118: The 16S rRNA sequencing methods described here are very confusing,
repetitive, and sometimes out of order. To my experience in amplicon sequencing techniques,
this methods section should read (please correct if not): Please also note authors are still missing
to include key information that would allow for methodological transparency (eg: DNA isolation
kit, sequencer used, preparation performed in house or by a company, etc)
“Genomic DNA was extracted from fecal samples using XXX kit (VENDOR) following
vendor’s instructions. NanoDrop and agarose gel electrophoresis were used to determine
concentration and purity of gDNA, respectively. Extracted DNA was diluted to a concentration
of 1 ng/µl and stored at -20# until further processing. The V3-V4 variable region of the 16S
gene was amplified using barcoded PCR with universal primers 343 F and 798 R. Using the
V3-V4 front-end primer: 343F - 5'-TACGGRAGGCAGCAG-3'; and  back-end primer: 798R
- 5'-AGGGTATCTAATCCT-3', the PCR was performed using Takara Ex Taq high-fidelity
enzyme (Takara Corporation). PCR products were detected by electrophoresis and then purified
by magnetic beads (kit? Vendor?). The purified products were used in a second PCR round,
and again detected by electrophoresis and purified by magnetic beads. Secondary PCR products
were quantified with Qubit. Samples were further processed (please add more details as needed)
and sequenced at XXX system at XXX institute/company/in-house/etc. Sequencing analysis was
performed by XXX company using the following protocol. Raw sequencing data were obtained
in FASTQ format. Paired-end reads were preprocessed using Trimmomatic software to detect
and cut off ambiguous bases (N). It also cut off low-quality sequences with average quality
score below 20 using sliding window trimming approach. After trimming, paired-end reads were
assembled using FLASH software. Parameters of assembly were: 10bp of minimal overlapping,
200bp of maximum overlapping, and 20% of maximum mismatch rate. Sequences were further
denoised using QIIME (version 1.8.0) as follows: reads with ambiguous, homologous sequences
or below 200bp were abandoned. Reads with 75% of bases above Q20 were retained. Then,
reads with chimera were detected and removed. After processing and generating high-quality
sequences, Vsearch software was used to cluster the sequences into operational taxonomic units
(OTUs) according to sequence similarity. For 16S, Greengenes or Silva (Version123) database
were used for comparison and annotation of species by RDP Classifier software or Blast software
[11-15].”
8. Line 120: For the performed ELISA assays: what kits were used? What was the company/
vendor for each? If not used commercial kits, what were the antibodies/clones used? What was
the company/vendor each were purchased from?
9. Line 131-132: For the antibodies used for Western-blot assays: what were the clones for each
of them? What was the company/vendor each were purchased from?
10. Line 136-137: colon processing steps should read “colon fragments were fixed in 10%
formalin (phosphate buffered), paraffin embedded, and histological cuts were stained with
hematoxylin and eosin (H&E).”
11. Fig 1, 4 and 7: missing concentration unit to panel B, which I believe is “Protein/#-actin
concentration”, but correct if not.
12. Legend for Fig 1, 4 and 7: replace “expression” by appropriate terms (as per comment 2).
Also, please clarify which student’s t test or Wilcoxon-Mann-Whitney test were used for each
panel presented. For letter “C” in Fig 1, it should read “Representative pictures for Western blot
runs”.
13. Legend for Fig 3, 6 and 9: boxplot and NMDS plots are forms of representation, not analysis,
please correct that. Also, please clarify which student’s t test or Wilcoxon-Mann-Whitney test
were used for each panel presented.
14. Line 225: I still urge the authors to confirm ability of Lachnospiraceae_UCG_006 to produce
butyrate, previous reports suggest otherwise. While belonging to Firmicutes, we know not all
members of this phylum are able to produce butyrate (e.g., Blautia spp., Eubacterium eligens,
Ruminococcus spp. – PMID 27928878).
15. Line 272-274: As per my previous comment, please delete “TCS exposure enhanced the
level of IL-13, IL-17 and TNF-# in plasma, the protein levels of IL-6, IL-10, IL-17, TNF- #, IFN-
#, p-NF-#b and TLR9 in colon tissues, but there was no statistical significance (all the P>0.05)
(Fig.4).”, as there is no increase.
16. Figure 8: please clarify if student’s t-test or Wilcoxon-Mann-Whitney test were used for this
panel. One is used for parametric, with the other for non-parametric analysis. Therefore, you
can’t use one or the other for checking for the significance in this dataset.
17. Line 375: this comment is still very speculative and not supported by the data. Please replace
“our results suggested that TCS exposure attenuated the protective effect of probiotics on gut
health”, by “our results suggest that TCS exposure could potentially impact DSS severity by
attenuating the protective effect of probiotics on gut health.”
18. Line 409-410: it should read: “Similarly, our study showed that TCS exposure reduced the
levels of Occludin and increased TLR4, p-NF-#b and IFN-# in gut tissues”.
19. Line 411: Where it reads ‘innate immunity receptor” please change to “innate immune
receptor”.
20. Line 412: Missing reference after “which is physically separated from the microbiome”.
21. Line 435: Those are not “pathological smears” but “histological sections of gut tissue”.
22. Microbial scientific names should be italicized throughout the manuscript.
Title: Triclosan Exposure Induced Disturbance of Gut Microbiota and Exaggerated
Experimental Colitis in Mice
Version: 5 Date: 05 Aug 2022
Reviewer's report:
In this paper, Jing et al. proposed to determine the effects and mechanisms of triclosan (TCS), an
antibiotic heavily used in consumer products, on intestinal inflammation and ulcerative colitis
model. While this manuscript still doesn’t present mechanistic work, I would like to compliment
the authors for heavily changing the method and discussion sections, which make this manuscript
much nicer to read. The authors have addressed most of my comments, although some things
still need to be reviewed before this work can be published.
Minor comments:
1. Line 22: Authors do not investigate mechanisms of TCS induced microbiome changes or
immune activation. Therefore, the sentence “This animal experiment aimed to clarify effects
and mechanisms of triclosan” is misleading. Please change to “This animal experiment aimed
to clarify the effects of triclosan exposure on the microbiome and intestinal immune functions
in healthy and ulcerative colitis model”.
2. Line 34 (and every other instance it appears – including lines 36, 161, 162, etc and figure
legends): in response to previous review document, authors did not determine gene expression,
but rather protein concentration via ELISA or Western Blot. Therefore, the word “expression”
cannot be used to describe any of the results presented in this report. Please correct that
throughout and have it replaced by “protein levels”, “protein concentration”, or equivalent.
3. Line 73: Same as comment number 1. Please change sentence “This study aimed to clarify
effects and mechanisms of TCS on UC in mice.” by “This study aimed to clarify the effects of
TCS exposure on the microbiome and intestinal immune functions in healthy and UC model”.
4. Line 79: again, authors did not determine the dose of TCS, rather used a previously published
concentration. Where is says “Firstly, we determined the dose of TCS was 80ppm”, should be
replaced by “We treated mice with 80ppm of TCS, as previously published [4]”.
5. Line 79-80: replace “when mice exposed…” and “TCS in mice was” to “when mice were
exposed…” and “TCS was”.
6. Table line 87: The three instances the word “maintain” is shown, should be replaced by
“maintained”.
7. Line 92-118: The 16S rRNA sequencing methods described here are very confusing,
repetitive, and sometimes out of order. To my experience in amplicon sequencing techniques,
this methods section should read (please correct if not): Please also note authors are still missing
to include key information that would allow for methodological transparency (eg: DNA isolation
kit, sequencer used, preparation performed in house or by a company, etc)
“Genomic DNA was extracted from fecal samples using XXX kit (VENDOR) following
vendor’s instructions. NanoDrop and agarose gel electrophoresis were used to determine
concentration and purity of gDNA, respectively. Extracted DNA was diluted to a concentration
of 1 ng/µl and stored at -20# until further processing. The V3-V4 variable region of the 16S
gene was amplified using barcoded PCR with universal primers 343 F and 798 R. Using the
V3-V4 front-end primer: 343F - 5'-TACGGRAGGCAGCAG-3'; and  back-end primer: 798R
- 5'-AGGGTATCTAATCCT-3', the PCR was performed using Takara Ex Taq high-fidelity
enzyme (Takara Corporation). PCR products were detected by electrophoresis and then purified
by magnetic beads (kit? Vendor?). The purified products were used in a second PCR round,
and again detected by electrophoresis and purified by magnetic beads. Secondary PCR products
were quantified with Qubit. Samples were further processed (please add more details as needed)
and sequenced at XXX system at XXX institute/company/in-house/etc. Sequencing analysis was
performed by XXX company using the following protocol. Raw sequencing data were obtained
in FASTQ format. Paired-end reads were preprocessed using Trimmomatic software to detect
and cut off ambiguous bases (N). It also cut off low-quality sequences with average quality
score below 20 using sliding window trimming approach. After trimming, paired-end reads were
assembled using FLASH software. Parameters of assembly were: 10bp of minimal overlapping,
200bp of maximum overlapping, and 20% of maximum mismatch rate. Sequences were further
denoised using QIIME (version 1.8.0) as follows: reads with ambiguous, homologous sequences
or below 200bp were abandoned. Reads with 75% of bases above Q20 were retained. Then,
reads with chimera were detected and removed. After processing and generating high-quality
sequences, Vsearch software was used to cluster the sequences into operational taxonomic units
(OTUs) according to sequence similarity. For 16S, Greengenes or Silva (Version123) database
were used for comparison and annotation of species by RDP Classifier software or Blast software
[11-15].”
8. Line 120: For the performed ELISA assays: what kits were used? What was the company/
vendor for each? If not used commercial kits, what were the antibodies/clones used? What was
the company/vendor each were purchased from?
9. Line 131-132: For the antibodies used for Western-blot assays: what were the clones for each
of them? What was the company/vendor each were purchased from?
10. Line 136-137: colon processing steps should read “colon fragments were fixed in 10%
formalin (phosphate buffered), paraffin embedded, and histological cuts were stained with
hematoxylin and eosin (H&E).”
11. Fig 1, 4 and 7: missing concentration unit to panel B, which I believe is “Protein/#-actin
concentration”, but correct if not.
12. Legend for Fig 1, 4 and 7: replace “expression” by appropriate terms (as per comment 2).
Also, please clarify which student’s t test or Wilcoxon-Mann-Whitney test were used for each
panel presented. For letter “C” in Fig 1, it should read “Representative pictures for Western blot
runs”.
13. Legend for Fig 3, 6 and 9: boxplot and NMDS plots are forms of representation, not analysis,
please correct that. Also, please clarify which student’s t test or Wilcoxon-Mann-Whitney test
were used for each panel presented.
14. Line 225: I still urge the authors to confirm ability of Lachnospiraceae_UCG_006 to produce
butyrate, previous reports suggest otherwise. While belonging to Firmicutes, we know not all
members of this phylum are able to produce butyrate (e.g., Blautia spp., Eubacterium eligens,
Ruminococcus spp. – PMID 27928878).
15. Line 272-274: As per my previous comment, please delete “TCS exposure enhanced the
level of IL-13, IL-17 and TNF-# in plasma, the protein levels of IL-6, IL-10, IL-17, TNF- #, IFN-
#, p-NF-#b and TLR9 in colon tissues, but there was no statistical significance (all the P>0.05)
(Fig.4).”, as there is no increase.
16. Figure 8: please clarify if student’s t-test or Wilcoxon-Mann-Whitney test were used for this
panel. One is used for parametric, with the other for non-parametric analysis. Therefore, you
can’t use one or the other for checking for the significance in this dataset.
17. Line 375: this comment is still very speculative and not supported by the data. Please replace
“our results suggested that TCS exposure attenuated the protective effect of probiotics on gut
health”, by “our results suggest that TCS exposure could potentially impact DSS severity by
attenuating the protective effect of probiotics on gut health.”
18. Line 409-410: it should read: “Similarly, our study showed that TCS exposure reduced the
levels of Occludin and increased TLR4, p-NF-#b and IFN-# in gut tissues”.
19. Line 411: Where it reads ‘innate immunity receptor” please change to “innate immune
receptor”.
20. Line 412: Missing reference after “which is physically separated from the microbiome”.
21. Line 435: Those are not “pathological smears” but “histological sections of gut tissue”.
22. Microbial scientific names should be italicized throughout the manuscript.
Please review this paper.
user
Title: Triclosan Exposure Induced Disturbance of Gut Microbiota and Exaggerated
Experimental Colitis in Mice
Version: 5 Date: 05 Aug 2022
Reviewer's report:
In this paper, Jing et al. proposed to determine the effects and mechanisms of triclosan (TCS), an
antibiotic heavily used in consumer products, on intestinal inflammation and ulcerative colitis
model. While this manuscript still doesn’t present mechanistic work, I would like to compliment
the authors for heavily changing the method and discussion sections, which make this manuscript
much nicer to read. The authors have addressed most of my comments, although some things
still need to be reviewed before this work can be published.
Minor comments:
1. Line 22: Authors do not investigate mechanisms of TCS induced microbiome changes or
immune activation. Therefore, the sentence “This animal experiment aimed to clarify effects
and mechanisms of triclosan” is misleading. Please change to “This animal experiment aimed
to clarify the effects of triclosan exposure on the microbiome and intestinal immune functions
in healthy and ulcerative colitis model”.
2. Line 34 (and every other instance it appears – including lines 36, 161, 162, etc and figure
legends): in response to previous review document, authors did not determine gene expression,
but rather protein concentration via ELISA or Western Blot. Therefore, the word “expression”
cannot be used to describe any of the results presented in this report. Please correct that
throughout and have it replaced by “protein levels”, “protein concentration”, or equivalent.
3. Line 73: Same as comment number 1. Please change sentence “This study aimed to clarify
effects and mechanisms of TCS on UC in mice.” by “This study aimed to clarify the effects of
TCS exposure on the microbiome and intestinal immune functions in healthy and UC model”.
4. Line 79: again, authors did not determine the dose of TCS, rather used a previously published
concentration. Where is says “Firstly, we determined the dose of TCS was 80ppm”, should be
replaced by “We treated mice with 80ppm of TCS, as previously published [4]”.
5. Line 79-80: replace “when mice exposed…” and “TCS in mice was” to “when mice were
exposed…” and “TCS was”.
6. Table line 87: The three instances the word “maintain” is shown, should be replaced by
“maintained”.
7. Line 92-118: The 16S rRNA sequencing methods described here are very confusing,
repetitive, and sometimes out of order. To my experience in amplicon sequencing techniques,
this methods section should read (please correct if not): Please also note authors are still missing
to include key information that would allow for methodological transparency (eg: DNA isolation
kit, sequencer used, preparation performed in house or by a company, etc)
“Genomic DNA was extracted from fecal samples using XXX kit (VENDOR) following
vendor’s instructions. NanoDrop and agarose gel electrophoresis were used to determine
concentration and purity of gDNA, respectively. Extracted DNA was diluted to a concentration
of 1 ng/µl and stored at -20# until further processing. The V3-V4 variable region of the 16S
gene was amplified using barcoded PCR with universal primers 343 F and 798 R. Using the
V3-V4 front-end primer: 343F - 5'-TACGGRAGGCAGCAG-3'; and  back-end primer: 798R
- 5'-AGGGTATCTAATCCT-3', the PCR was performed using Takara Ex Taq high-fidelity
enzyme (Takara Corporation). PCR products were detected by electrophoresis and then purified
by magnetic beads (kit? Vendor?). The purified products were used in a second PCR round,
and again detected by electrophoresis and purified by magnetic beads. Secondary PCR products
were quantified with Qubit. Samples were further processed (please add more details as needed)
and sequenced at XXX system at XXX institute/company/in-house/etc. Sequencing analysis was
performed by XXX company using the following protocol. Raw sequencing data were obtained
in FASTQ format. Paired-end reads were preprocessed using Trimmomatic software to detect
and cut off ambiguous bases (N). It also cut off low-quality sequences with average quality
score below 20 using sliding window trimming approach. After trimming, paired-end reads were
assembled using FLASH software. Parameters of assembly were: 10bp of minimal overlapping,
200bp of maximum overlapping, and 20% of maximum mismatch rate. Sequences were further
denoised using QIIME (version 1.8.0) as follows: reads with ambiguous, homologous sequences
or below 200bp were abandoned. Reads with 75% of bases above Q20 were retained. Then,
reads with chimera were detected and removed. After processing and generating high-quality
sequences, Vsearch software was used to cluster the sequences into operational taxonomic units
(OTUs) according to sequence similarity. For 16S, Greengenes or Silva (Version123) database
were used for comparison and annotation of species by RDP Classifier software or Blast software
[11-15].”
8. Line 120: For the performed ELISA assays: what kits were used? What was the company/
vendor for each? If not used commercial kits, what were the antibodies/clones used? What was
the company/vendor each were purchased from?
9. Line 131-132: For the antibodies used for Western-blot assays: what were the clones for each
of them? What was the company/vendor each were purchased from?
10. Line 136-137: colon processing steps should read “colon fragments were fixed in 10%
formalin (phosphate buffered), paraffin embedded, and histological cuts were stained with
hematoxylin and eosin (H&E).”
11. Fig 1, 4 and 7: missing concentration unit to panel B, which I believe is “Protein/#-actin
concentration”, but correct if not.
12. Legend for Fig 1, 4 and 7: replace “expression” by appropriate terms (as per comment 2).
Also, please clarify which student’s t test or Wilcoxon-Mann-Whitney test were used for each
panel presented. For letter “C” in Fig 1, it should read “Representative pictures for Western blot
runs”.
13. Legend for Fig 3, 6 and 9: boxplot and NMDS plots are forms of representation, not analysis,
please correct that. Also, please clarify which student’s t test or Wilcoxon-Mann-Whitney test
were used for each panel presented.
14. Line 225: I still urge the authors to confirm ability of Lachnospiraceae_UCG_006 to produce
butyrate, previous reports suggest otherwise. While belonging to Firmicutes, we know not all
members of this phylum are able to produce butyrate (e.g., Blautia spp., Eubacterium eligens,
Ruminococcus spp. – PMID 27928878).
15. Line 272-274: As per my previous comment, please delete “TCS exposure enhanced the
level of IL-13, IL-17 and TNF-# in plasma, the protein levels of IL-6, IL-10, IL-17, TNF- #, IFN-
#, p-NF-#b and TLR9 in colon tissues, but there was no statistical significance (all the P>0.05)
(Fig.4).”, as there is no increase.
16. Figure 8: please clarify if student’s t-test or Wilcoxon-Mann-Whitney test were used for this
panel. One is used for parametric, with the other for non-parametric analysis. Therefore, you
can’t use one or the other for checking for the significance in this dataset.
17. Line 375: this comment is still very speculative and not supported by the data. Please replace
“our results suggested that TCS exposure attenuated the protective effect of probiotics on gut
health”, by “our results suggest that TCS exposure could potentially impact DSS severity by
attenuating the protective effect of probiotics on gut health.”
18. Line 409-410: it should read: “Similarly, our study showed that TCS exposure reduced the
levels of Occludin and increased TLR4, p-NF-#b and IFN-# in gut tissues”.
19. Line 411: Where it reads ‘innate immunity receptor” please change to “innate immune
receptor”.
20. Line 412: Missing reference after “which is physically separated from the microbiome”.
21. Line 435: Those are not “pathological smears” but “histological sections of gut tissue”.
22. Microbial scientific names should be italicized throughout the manuscript.
assistant