Identification and Characterization of Two Antibacterial Compounds Extracted from Thuja arborvitae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A valuable article with new information on plant-derived antibacterial compounds from thuja (Thuja arborvitae). Isolation of individual compounds was carried out and their effect on bacterial growth and viability was determined. Insights into the mechanisms of antibacterial action of promising compounds are provided.

The work contains several inaccuracies and/or careless errors:

• Rows 21, 82, 197, 200, 247, 330, 331 – g/ml, cannot be grams, must be corrected
• Row 990 – 50 l - they are not liters
• Rows 140, 147, 151, 189, 214, 290, 294, 296, 313 – aureus should be written in italics everywhere
• Row 107 – 600 nm instead of 600nm
• Row 153 – BrdU (20 M), there are not M
• Row 158, 172 – grams?!
• Rows 159, 160, 174, 175, 176 – liters?!
• Rows 171, 327, 341, 408 – in vitro instead of in vitro
• Row 189 – I did not find sTable 1
• Row 193, 222 – radicans in italics
• Rows 214, 216, 222, 230, 246 – arborvitae in italics
• Figure 2 – arborvitae instead of T. arbovitae
• Row 248 – strain instead of stain
• Row 248, 262, 267, 271 – I did not find Table 1
• Rows 253, 254, 255, 247 – I did not find 1 and sFig. 2
• Row 321 – SDS-polyacrylamide instead of DSD-polyacrylamide
• Row 342 – coli in italics
• Rows 374-378 – add a reference to the literature

Author Response

We are sorry for so many typos and errors, which were generated by the font changes and  were corrected in the revised manuscript as suggested.

The work contains several inaccuracies and/or careless errors:

• Rows 21, 82, 197, 200, 247, 330, 331 – g/ml, cannot be grams, must be corrected:

 Changed to mg/ml.

• Row 990 – 50 l - they are not liters:  

Changed to ml.

• Rows 140, 147, 151, 189, 214, 290, 294, 296, 313 – aureus should be written in italics everywhere: 

Changed as suggested.

• Row 107 – 600 nm instead of 600nm

Changed as suggested.

• Row 153 – BrdU (20 M), there are not M

Changed to mM.

• Row 158, 172 – grams?!

Changed to mg.

• Rows 159, 160, 174, 175, 176 – liters?!

Changed to ml.

• Rows 171, 327, 341, 408 – in vitro instead of in vitro

Changed as suggested.

• Row 189 – I did not find sTable 1

sTable 1 is in the supplementary materials.

• Row 193, 222 – radicans in italics

Changed as suggested.

• Rows 214, 216, 222, 230, 246 – arborvitae in italics

Changed as suggested.

• Figure 2 – arborvitae instead of T. arborvitae

Changed as suggested.

• Row 248 – strain instead of stain

Changed as suggested.

• Row 248, 262, 267, 271 – I did not find Table 1

Table 1 was included.

• Rows 253, 254, 255, 247 – I did not find sFig.1 and sFig. 2

sFig. 1 and sFig. 2 are in the supplementary materials.

• Row 321 – SDS-polyacrylamide instead of DSD-polyacrylamide

Changed as suggested.

• Row 342 – coli in italics

Changed as suggested.

• Rows 374-378 – add a reference to the literature

A reference was added.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

After Reviewing your original research article entitled "Identification and Characterization of Antibacterial Compounds from Thuja arborvitae"

This work is very important since it highlights an important medical topic in the research aiming to treat bacterial infection mainly by discovering (identification and characterization) of new antibacterial compounds extracted from the leave of the plant "Thuja arbovitae"

The article is well written in English Language, well presented and well designed. Figures in the article are very clear and are free from any unnecessary modifications.

Kindly find below my list of comments concerning the present work:

01- You are kindly invited to change the title from "Identification and Characterization of Antibacterial Compounds from Thuja arborvitae" to "Identification and Characterization of two Antibacterial Compounds extracted from Thuja arborvitae leave".

02- In the Introduction section, you are kindly invited to talk about the importance of finding new strategies to fight bacterial infections, mainly those caused by multidrug resistance bacteria. Regarding this point I invite you to talk about two recent strategies that may be used to fight bacterial infections such as using "Trojan Horse Technique" to introduce an antibiotic inside a bacterial cell, or targeting specific ultra-structures in bacteria such as targeting "Bacterial Metallophores". You can use the following papers as references for this point:

XX Trojan Horse Antibiotics—A Novel Way to Circumvent Gram-Negative Bacterial Resistance?

XX Towards new antibiotics classes targeting bacterial metallophores.

03- In the Whole manuscript (text, figures), Authors are invited to wrote the names of bacterial species in italic.

04- I have a major point regarding the selection of the four bacterial strains, why did you select these four bacteria and not others?

05- Concerning Figure 1, it is not clear for readers, and its legends are not very clear also. For example concerning the figure 1.a: Why S. aureus is represented in two figures (A and C) and what is the difference between these two figures 1.a.A and 1.a.C??

06- Concerning Figure 1, it is not clear for readers, and its legends are not very clear also. For example concerning the figure 1.a: In the Figure it is mentioned that 1.a.B is for A. baumanni while in the legends it is mentioned P. aeruginosa.

07- Concerning Figure 1, it is not clear for readers, and its legends are not very clear also. For example concerning the figure 1.c. there is 4 molecules A, B, C and D while in the legends you are just listing the names of 3 molecules A, B and C. What about D?

08- Concerning Figure 4, Gel d, It is not clear at all where are the proteins portions!!

09- The Article lacks a Conclusion, you are kindly invited to add a Conclusion to this work.

Best Regards,

Author Response

We thank the reviewer’s appreciation for our manuscript.  The concerns raised by the reviewer are addressed as the following:

• You are kindly invited to change the title from "Identification and Characterization of Antibacterial Compounds from Thuja arborvitae" to "Identification and Characterization of two Antibacterial Compounds extracted from Thuja arborvitae leave".

Changed as suggested.

 

• In the Introduction section, you are kindly invited to talk about the importance of finding new strategies to fight bacterial infections, mainly those caused by multidrug resistance bacteria. Regarding this point I invite you to talk about two recent strategies that may be used to fight bacterial infections such as using "Trojan Horse Technique" to introduce an antibiotic inside a bacterial cell, or targeting specific ultra-structures in bacteria such as targeting "Bacterial Metallophores". You can use the following papers as references for this point:

This point and two references were added in the revised manuscript (lines 52-56).

 

• In the Whole manuscript (text, figures), Authors are invited to wrote the names of bacterial species in italic.

Changed as suggested.

 

• I have a major point regarding the selection of the four bacterial strains, why did you select these four bacteria and not others?

The reasons for select four bacterial strains were that they are commercially available from ATCC and are mostly involved in common infections.  This information was added in the revised manuscript (lines 197-198).

 

• Concerning Figure 1, it is not clear for readers, and its legends are not very clear also. For example concerning the figure 1.a: Why aureus is represented in two figures (A and C) and what is the difference between these two figures 1.a.A and 1.a.C??

Experiments shown in Fig.1a, panels A and C were performed at different time.

 

• Concerning Figure 1, it is not clear for readers, and its legends are not very clear also. For example concerning the figure 1.a: In the Figure it is mentioned that 1.a.B is for baumanni while in the legends it is mentioned P. aeruginosa.

It should be A. baumanni not P. aeruginosa.  The error was corrected.

 

• Concerning Figure 1, it is not clear for readers, and its legends are not very clear also. For example concerning the figure 1.c. there is 4 molecules A, B, C and D while in the legends you are just listing the names of 3 molecules A, B and C. What about D?

Panel D was included.

 

• Concerning Figure 4, Gel d, It is not clear at all where are the proteins portions!!

The proteins were stained with Coomassie blue and revealed as distinct bands in the gel which were marked in the revised figures.

 

• The Article lacks a Conclusion, you are kindly invited to add a Conclusion to this work.

Conclusions were included in the revised manuscript (lines 366-372).

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes the isolation and characterization of two antibacterial compounds (apigenin-7-di-p-coumarylglucoside and eicosapentaenoic acid) from Thuja arborvitae, with activity against Gram-positive and Gram-negative bacteria, including a multidrug-resistant strain. The study is well-structured, and the findings are of potential interest in the context of antimicrobial resistance. However, several significant issues require attention before the manuscript can be considered for publication.

General Comments

1. Novelty and Significance:

- However, eicosapentanoic acid’s antibacterial activity is already documented in the literature (reference 24). The authors should clarify the novelty of this finding in the context of Thuja arborvitae. 

- The activity against multidrug-resistant Acinetobacter baumannii is strong, but deeper mechanistic insights are needed.

2. Clarity and Consistency:

- Several typographical errors and inconsistencies in species names (e.g., Thuja arborvitae should be italic in all the manuscript, and do this with all bacterial names) and units (µg/ml vs. g/ml).

- Language should be improved (trees in North America and in East Asia, from Thuja leaves, bacterial infections, eicosapentaenoic acid, was distributed and so on). 

- The manuscript requires thorough proofreading and formatting adjustments to align with journal guidelines.

- Plagiarism Percent match: 29%, which is too much; it should be less than 25%.

Specific Comments

1. Title and Abstract

- The abstract should clearly state the MIC/IC50 values in µg/ml (currently written as “g/ml,” which is ambiguous). 

- Specify that the activity against A. baumannii includes a multidrug-resistant strain.

2. Introduction

- Consider adding a sentence on the ethnobotanical use of Thuja species to strengthen the rationale.

3. Materials and Methods

- Section 2.1: Clarify the plant source(s)—were leaves, seeds, or both used for extraction? 

- Section 2.4–2.5: Specify the solvent used for negative controls in MIC assays.

- Line 90: Wells was then filled with 50 l of the tested samples. Filled with what?

- Section 2.6: The purification protocol is well-described, but yield calculations (mg compound per g dry weight) should be included for reproducibility. 

- Section 2.10: The in vitro translation assay uses E. coli extracts; justify its relevance to the tested bacterial strains.

4. Results

- Figures 1–2: Ensure axis labels, units, and legends are consistent and clearly described in captions. 

- Table 1: Where is table 1? 

- Figure 3: The LIVE/DEAD assay is convincing but includes scale bars in microscopy images. 

- Figure 4: The membrane permeability data for P4 is compelling. However, the lack of effect by P3 and chloramphenicol should be discussed in the context of their modes of action. 

- Figure 5: The dual effect of P3 on translation (enhancement at 1× MIC, inhibition at 2× MIC) is unusual and warrants deeper discussion or validation.

5. Discussion

- The discussion should more critically address the following: 

- Why does P3 enhance translation at low concentrations? Is this an artifact or a biologically relevant phenomenon? 

- The mechanism of DNA synthesis inhibition by P3 is speculative. Suggest experiments (e.g., gyrase/topoisomerase assays) to validate this claim. 

- Compare and contrast the membrane-disrupting activity of eicosapentaenoic acid with known polymyxins.

6. Conclusion

- Where is the conclusion?

Technical Corrections

1. Units: Correct “g/ml” to “µg/ml” throughout (e.g., lines 21, 247).
2. Species Names: Use italics consistently for all binomial names (e.g., Thuja arborvitae).
3. Abbreviations: Define all abbreviations at first use (e.g., EPA, OA, MIC).

Ethics and Data Availability

- The statements are adequate but confirm that no ethical approvals were required for the bacterial strains used.

Author Response

General Comments

1. Novelty and Significance:

- However, eicosapentanoic acid’s antibacterial activity is already documented in the literature (reference 24). The authors should clarify the novelty of this finding in the context of Thuja arborvitae. 

This point was added in the revised manuscript (278-281).

- The activity against multidrug-resistant Acinetobacter baumannii is strong, but deeper mechanistic insights are needed.

The purified compounds demonstrated potent activity against multidrug-resistant A. baumannii, but the mechanisms are unknown.   This point was added to Discussion part (lines 395-396)

1. Clarity and Consistency:

- Several typographical errors and inconsistencies in species names (e.g., Thuja arborvitae should be italic in all the manuscript, and do this with all bacterial names) and units (µg/ml vs. g/ml).

Typos were corrected.

- Language should be improved (trees in North America and in East Asia, from Thuja leaves, bacterial infections, eicosapentaenoic acid, was distributed and so on).

“Thuja leaves” changed to“Thuja leaf” ; “bacterial infections” changed to “bacteria infections”; “eicosapentaenoic acid” changed to “EPA”.

- The manuscript requires thorough proofreading and formatting adjustments to align with journal guidelines.

Drs. Ethan Bowen and D’Netria Bledsoe proofread the manuscript.

- Plagiarism Percent match: 29%, which is too much; it should be less than 25%.

Plagiarism checked by JustDone percent identical match was 12% for the revised manuscript.

Specific Comments

1. Title and Abstract

- The abstract should clearly state the MIC/IC50 values in µg/ml (currently written as “g/ml,” which is ambiguous).

Changed as suggested. 

- Specify that the activity against A. baumannii includes a multidrug-resistant strain.

Included the IC50 to A. baumannii (line 22).

1. Introduction

- Consider adding a sentence on the ethnobotanical use of Thuja species to strengthen the rationale.

Added (lines 379-380).

1. Materials and Methods

- Section 2.1: Clarify the plant source(s)—were leaves, seeds, or both used for extraction? 

Described in the revised manuscript (73-77).

- Section 2.4–2.5: Specify the solvent used for negative controls in MIC assays.

The solvent is methanol and this information was added in the revised manuscript (line 100).

- Line 90: Wells was then filled with 50 l of the tested samples. Filled with what?

It should be 50 ml.  The error was corrected.

- Section 2.6: The purification protocol is well-described, but yield calculations (mg compound per g dry weight) should be included for reproducibility.

The information was added (lines 141-142). 

- Section 2.10: The in vitro translation assay uses E. coli extracts; justify its relevance to the tested bacterial strains.

Since the purified compounds also inhibited growth of E. coli so the in vitro translation with E. coli extract was used.  This information was added in the revised manuscript (lines 182-183).

1. Results

- Figures 1–2: Ensure axis labels, units, and legends are consistent and clearly described in captions. 

- Table 1: Where is table 1?  Added. 

- Figure 3: The LIVE/DEAD assay is convincing but includes scale bars in microscopy images.

Scale bars were added in the revised figures. 

- Figure 4: The membrane permeability data for P4 is compelling. However, the lack of effect by P3 and chloramphenicol should be discussed in the context of their modes of action. 

This point was discussed in the revised manuscript (435-436).

- Figure 5: The dual effect of P3 on translation (enhancement at 1× MIC, inhibition at 2× MIC) is unusual and warrants deeper discussion or validation.

This point was discussed in the revised manuscript (422-426).

1. Discussion

- The discussion should more critically address the following: 

- Why does P3 enhance translation at low concentrations? Is this an artifact or a biologically relevant phenomenon? 

This experiment was repeated several times.  We also observed that ampicillin also enhanced translation in this system.  But the reason is not clear.  This point was mentioned in the revised manuscript (422-426).

- The mechanism of DNA synthesis inhibition by P3 is speculative. Suggest experiments (e.g., gyrase/topoisomerase assays) to validate this claim. 

The current research in the PI lab is focused on identification of the targets of P3 compound.  These include DNA gyrase and topoisomerase.

- Compare and contrast the membrane-disrupting activity of eicosapentaenoic acid with known polymyxins.

This was discussed in the revised manuscript (lines 397-411).

1. Conclusion

- Where is the conclusion?  Added (lines 366-372).

Technical Corrections

1. Units: Correct “g/ml” to “µg/ml” throughout (e.g., lines 21, 247).  Changed as suggested.
2. Species Names: Use italics consistently for all binomial names (e.g., Thuja arborvitae).  Changed as suggested.
3. Abbreviations: Define all abbreviations at first use (e.g., EPA, OA, MIC).

Abbreviation list was added (Lines 27-28).

Ethics and Data Availability

- The statements are adequate but confirm that no ethical approvals were required for the bacterial strains used.

Added as suggested (line 448).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

After Reviewing the revised version of your original research article entitled "Identification and Characterization of Antibacterial Compounds from Thuja arborvitae"

This work is more suitable for publication in its present version. Thanks to the modifications you made.

Best Regards,

Author Response

Thank you for your time and efforts.

Reviewer 3 Report

Comments and Suggestions for Authors

The author should move Table 1 from the Supplementary Material into the main text.

Author Response

The sTable1 was moved from the Supplementary Material into the main text as suggested.