Characterizing Aqueous Extracts of Native Plants in Northeastern Mexico: Prospects for Quorum-Sensing Inhibition Against Gram-Negative Bacteria

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors tried to study the inhibitory effects of native plants in northeast Mexico against quorum sensing of gram negative bacteria. The results from the authors provided some novel information developing drugs against antibiotic-resistant bacteria. But there are several issue need to be dissolve.

1 The third paragraph and the 6^th paragraph can be merged together.

2 The source of S. typhimurium should be addressed.

3 The authors should explain why use S. typhimurium in the study.

4 The time of biofilm formation should be verified by the authors. I do not think 3 h can form biofilm.

5 The error bars should add in Figure 1.

6 The descriptions of Table 2 should be revised. The MICs are important for the study.

7 The relationship between dose and inhibitory effects in needed in the study.

8 The inhibitory effects of compounds such as Chlorogenic acid, Kaempferol and Chlorogenic acid isomer should be clarified to better known the main compounds inhibiting QS in the tested plants.

9 The inhibitory zones of gentamincin should be added in Table 2.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The language must be improved.

Author Response

Dear Reviewer,

We sincerely appreciate the time and effort you dedicated to reviewing our manuscript entitled “[Title of the manuscript]”. We are grateful for your constructive and insightful comments, which have significantly contributed to improving the quality and clarity of our work. Your recognition of the novelty and relevance of our findings in the context of developing strategies against antibiotic-resistant bacteria is particularly encouraging.

Below, we provide a point-by-point response to your comments. We have revised the manuscript accordingly and highlighted the changes in the revised version. We hope that our responses and modifications meet your expectations.

Comment 1: The third paragraph and the 6th paragraph can be merged together.
Response: We appreciate this suggestion. We have revised the Introduction section by merging the third and sixth paragraphs to improve the logical flow while preserving all original references and ideas. This revised paragraph integrates the rationale for targeting quorum sensing (QS) with an overview of its mechanisms in Gram-negative bacteria.

Comment 2: The source of S. typhimurium should be addressed.
Response: The strain used in this study was Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028. This information has now been added in the “Bacterial Strains” subsection of the Materials and Methods (Section 2.1).

Comment 3: The authors should explain why use S. typhimurium in the study.
Response: We have included a justification for the use of S. Typhimurium based on its clinical relevance as a Gram-negative pathogen, its ability to form biofilms, and its reliance on AI-2 quorum sensing systems. This explanation is included in Section 2.1, with appropriate references: Jesudhasan et al. (2010) and Parker & Sperandio (2009).

Comment 4: The time of biofilm formation should be verified by the authors. I do not think 3 h can form biofilm.
Response: Thank you for the observation. We have carefully reviewed the manuscript and confirm that the incubation period used for biofilm formation in our assays was 8 hours, not 3 hours. We believe the misunderstanding may stem from a misreading or a previous draft. No reference to 3 hours remains in the current version.

Comment 5: The error bars should add in Figure 1.
Response: We confirm that standard deviation error bars (n = 5) have been added to Figure 1, which has been updated accordingly for improved clarity and presentation quality.

Comment 6: The descriptions of Table 2 should be revised. The MICs are important for the study.
Response: We agree with the reviewer. The descriptions and content of Table 2 have been revised to highlight the importance of the MIC data. A clear explanation of the inhibitory profiles and limitations of zone-based assays is now provided in Section 3.3.

Comment 7: The relationship between dose and inhibitory effects in needed in the study.
Response: This relationship is now more clearly discussed in the Results section (Section 3.3), emphasizing the contrast between MIC values and lack of inhibition zones for some extracts. We also note in the text that high MICs do not preclude anti-QS activity, as some extracts act through mechanisms unrelated to bactericidal effects.

Comment 8: The inhibitory effects of compounds such as Chlorogenic acid, Kaempferol and Chlorogenic acid isomer should be clarified to better known the main compounds inhibiting QS in the tested plants.
Response: We have expanded the discussion regarding the potential anti-QS effects of chlorogenic acid and kaempferol, compounds identified in our UPLC analysis. We now include recent literature that supports their activity against QS-regulated pathways in Pseudomonas aeruginosa, with updated references:

• Xu et al., 2022 [https://doi.org/10.1111/jam.15275]
• Majumdar & Mandal, 2025 [https://doi.org/10.1016/j.microb.2025.100259]
• Wang et al., 2019 [https://doi.org/10.1007/s00253-018-9482-7]

Comment 9: The inhibitory zones of gentamicin should be added in Table 2.
Response: We have added gentamicin as a positive control to Table 2, including its inhibition zone diameter (22.4 ± 0.85 mm, 1.6 µg) based on our experimental data. The table and corresponding description in the Results section (Section 3.3) have been updated.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Quiroz-Hernandez et al. investigate the use of aqueous extract derived from five different plant species originating from northeastern Mexico as antimicrobial agents for Gram negative bacteria. The authors assessed the inhibition of antimicrobial activity as well as antibiofilm properties. The presented information is interesting and relevant to the readers of Applied Microbiology. The approach of the authors is well executed and the applied methodology merits publication. However, before considering this article for publication, there are some minor and major comments that need to be addressed:

• The authors propose five different native plants present in northeastern Mexico. Based on what is this selection made? Can the authors justify why why these specific plants were chosen? Is it based on scientific diversity (i.e., different chemical composition of the species), based on abundance and availability, or economic relevance? Please elaborate in the introduction section. In lines 89-90 a basic explanation is provided. However, this should be expanded and included in the introduction section.
• The novelty of this study seems to be the selection of the five plant species. Is this correct? Is there other novelty that will be addressed in the manuscript? The authors should emphasize the different novel aspects in the introduction section.
• The authors present yields of aqueous extracts in the materials and methods section before explaining the extraction process. Please move experimental results to the results section.
• What was the particle size of the plant materials after grinding? What was the average particle size (d50) or particle size distribution? Did the authors sieve the plant material after grinding? If so, what mesh size was used?
• Table 1: please italicize species’ names.
• Line 122 and Line 137: capitalize c and t.
• Please – briefly – add more details regarding the UPLC method used instead of referring to a previous work (i.e., detector type, gradient method, etc).
• The authors obtained aqueous extracts of five different plant species. The current methodology is somewhat black box. The authors assess the inhibition of antimicrobial activity of the extracts. What compounds in the extracts are responsible for the inhibition of antimicrobial activity? Did the authors perform analyses to go more in depth regarding the fundamentals of inhibited microbial activity? In Table 3 the authors identify five potential compounds based on the UPLC method provided. Do the authors envisage other compounds that might be present but are not detectable via UPLC (based on its detection technique/method used)?
• Figure 1: please italicize species’ names. It looks as if the error bars on the red bar plots are missing. Is this correct?
• Line 160: please italicize species’ names. Also Line 169, 172 and 173.
• Figure 3 (both caption and Figure): please italicize species’ names.
• Please improve the quality of the Figures (both Figure 1 and Figure 2). Remove the rectangle surrounding the Figure, and use different colors. Both text, bars, standard errors and numbers are blue which does not facilitate reading.
• Line 192: please italicize species’ names.
• Lines 209-211: please provide a reference.
• Lines 213-215: please provide a reference.
• Line 218 and 219: please italicize species’ names.
• The reviewer is not really convinced regarding the added value of the discussion section. This should be integrated in the results and conclusion section as not much new information is presented to the reader.
• Please avoid the use of references in the conclusion section. Please shorten the conclusion section and keep it strictly to the key (quantitative) results and insights of this study.

Author Response

We thank the reviewer for the thorough evaluation and constructive comments provided on our manuscript entitled “Quorum sensing and biofilm inhibition by aqueous extracts of five medicinal plants from northeastern Mexico”. We have carefully considered all suggestions and implemented the requested modifications to improve the clarity, scientific quality, and presentation of the manuscript. Below, we provide a detailed point-by-point response to each comment.

Comment 1:
The authors propose five different native plants present in northeastern Mexico. Based on what is this selection made? Can the authors justify why these specific plants were chosen? Is it based on scientific diversity (i.e., different chemical composition of the species), based on abundance and availability, or economic relevance? Please elaborate in the introduction section. In lines 89–90 a basic explanation is provided. However, this should be expanded and included in the introduction section.

Response 1:
We thank the reviewer for this insightful comment. In response, we have expanded the introduction section to provide a more comprehensive explanation of the criteria used for plant selection. Specifically, we now clarify that the selection was based on ethnobotanical relevance, historical use in traditional medicine, prior reports of biological activity in northern Mexico, and the potential for anti-virulence rather than purely bactericidal mechanisms. This information is now included in the revised paragraph in the Introduction section (lines 77–91 of the revised manuscript).

Comment 2:
The novelty of this study seems to be the selection of the five plant species. Is this correct? Is there other novelty that will be addressed in the manuscript? The authors should emphasize the different novel aspects in the introduction section.

Response 2:
We appreciate the reviewer’s suggestion and agree that clarifying the novelty of the work is essential. We have updated the Introduction to emphasize that, beyond the selection of these five underexplored native plants, the novelty lies in their comprehensive evaluation across three complementary assays: quorum sensing inhibition, antimicrobial activity, and anti-biofilm formation. Additionally, we highlight that this is the first report of violacein inhibition by Gymnosperma glutinosum and Tecoma stans, and the first identification of specific phytochemicals in these species through UPLC-MS related to QS inhibition. These contributions are now clearly stated in the revised Introduction (lines 92–104).

Comment 3:
The authors present yields of aqueous extracts in the materials and methods section before explaining the extraction process. Please move experimental results to the results section.

Response 3:
Thank you for pointing this out. As recommended, we have moved the extract yield data from the Materials and Methods section (previously in Table 1) to a newly created subsection in the Results section (Section 3.1: Extract yields). The Materials and Methods section now focuses solely on the experimental procedures, and all quantitative outcomes are reported and interpreted within the Results section (see lines 248–255).

Comment 4:
What was the particle size of the plant materials after grinding? What was the average particle size (d50) or particle size distribution? Did the authors sieve the plant material after grinding? If so, what mesh size was used?

Response 4:
Thank you for this pertinent question. We have now clarified in the Materials and Methods section that the dried plant materials were ground using a stainless-steel knife mill and subsequently sieved through a 60-mesh screen, which corresponds to a particle size of ≤250 µm. Although we did not perform a full particle size distribution analysis (e.g., d50), this mesh size ensured a uniform and reproducible extraction process. The updated information is included in Section 2.3 (lines 167–169).

Comment 5:
Table 1: please italicize species’ names.

Response 5:
Thank you for noting this formatting issue. We have revised Table 1 to ensure that all species names are properly italicized according to scientific conventions.

Comment 6:
Line 122 and Line 137: capitalize c and t.

Response 6:
We appreciate the reviewer’s attention to detail. We have corrected the typographical inconsistencies by capitalizing the genus abbreviations (“Chromobacterium violaceum” as C. violaceum and “Salmonella typhimurium” as S. Typhimurium) in both Line 122 and Line 137 of the revised manuscript.

Comment 7:
Please – briefly – add more details regarding the UPLC method used instead of referring to a previous work (i.e., detector type, gradient method, etc).

Response 7:
Thank you for this suggestion. We have expanded the description of the UPLC-MS method in Section 2.6 to include specific details such as the equipment used (ACQUITY UPLC coupled to a QDa mass detector), the reversed-phase column (BEH C18), the gradient program with mobile phases A (0.1% formic acid in water) and B (acetonitrile), flow rate, and detection settings in negative ESI mode. This modification improves the methodological transparency of our phytochemical profiling. Please refer to lines 210–218 in the revised manuscript.

Comment 8:
The authors obtained aqueous extracts of five different plant species. The current methodology is somewhat black box. The authors assess the inhibition of antimicrobial activity of the extracts. What compounds in the extracts are responsible for the inhibition of antimicrobial activity? Did the authors perform analyses to go more in depth regarding the fundamentals of inhibited microbial activity? In Table 3 the authors identify five potential compounds based on the UPLC method provided. Do the authors envisage other compounds that might be present but are not detectable via UPLC (based on its detection technique/method used)?

Response 8:
We agree that it is essential to address the potential bioactive compounds underlying the observed antimicrobial effects. To that end, we have significantly expanded the discussion section to interpret our UPLC-MS data, highlighting kaempferol, chlorogenic acid, and quinic acid as key phytochemicals associated with QS inhibition and antimicrobial activity. Furthermore, we acknowledge the methodological limitations of UPLC-MS, particularly under the chosen negative ESI mode, which may not detect highly polar or glycosylated metabolites, or those present at low abundance. We have now explicitly discussed the possibility of undetected compounds and proposed future metabolomic studies and bioassay-guided fractionation to address this gap. Please refer to lines 365–386 and 396–405 in the revised discussion section.

omment 9:
Figure 1: please italicize species’ names. It looks as if the error bars on the red bar plots are missing. Is this correct?

Response 9:
We appreciate this observation. The species names in Figure 1 have been corrected to appear in italics. Regarding the error bars, they were indeed present but not clearly visible due to the choice of color and formatting. We have now improved the figure by adjusting the contrast, colors, and visibility of error bars to enhance readability. The revised figure has been uploaded with the updated manuscript.

Comment 10:
Line 160: please italicize species’ names. Also Line 169, 172 and 173.

Response 10:
Thank you for your careful attention to formatting. We have revised the manuscript to italicize all species names mentioned in Lines 160, 169, 172, and 173, following the appropriate scientific conventions.

Comment 11:
Figure 3 (both caption and Figure): please italicize species’ names.

Response 11:
We have updated Figure 3 and its caption to ensure that all scientific names are properly italicized. This formatting change has been applied consistently across all figures and captions in the manuscript.

Comment 12:
Please improve the quality of the Figures (both Figure 1 and Figure 2). Remove the rectangle surrounding the Figure, and use different colors. Both text, bars, standard errors and numbers are blue which does not facilitate reading.

Response 12:
Thank you for this valuable feedback. We have re-designed Figures 1 and 2 to enhance clarity and visual quality. The background boxes have been removed, and distinct color schemes have been applied to differentiate bars, error bars, and annotations. This updated version improves both visual appeal and interpretability. Revised figures are included in the new version of the manuscript.

Comment 13:
Line 192: please italicize species’ names.

Response 13:
This typographical issue has been corrected. The species names in Line 192 have now been italicized in accordance with scientific standards.

Comment 14:
Lines 209-211: please provide a reference.

Response 14:
We agree with the reviewer’s suggestion and have added an appropriate reference to support the observation of multiple peaks likely corresponding to phenolic acids and flavonoid glycosides. The following reference has been included in the revised manuscript:
Pires et al., 2017. Rev. Bras. Farmacogn. 27, 426–433. https://doi.org/10.1016/j.bjp.2017.03.004
Please refer to Line 219 in the updated manuscript.

Comment 15:
Lines 213-215: please provide a reference.

Response 15:
Thank you for pointing this out. We have now added supporting literature to substantiate the claim that the biological activities observed are likely mediated by synergistic interactions among multiple compounds. The following reference has been added:
Nazzaro, F.; Fratianni, F.; Coppola, R. Quorum sensing and phytochemicals. Int. J. Mol. Sci. 2013, 14, 12607–12619. https://doi.org/10.3390/ijms14061
This has been integrated at Line 223 in the revised discussion section.

Comment 16:
Line 218 and 219: please italicize species’ names.

Response 16:
Thank you for noting this formatting issue. We have corrected the text in Lines 218 and 219 to ensure all species names are italicized in accordance with scientific writing conventions.

Comment 17:
The reviewer is not really convinced regarding the added value of the discussion section. This should be integrated in the results and conclusion section as not much new information is presented to the reader.

Response 17:
We appreciate the reviewer’s feedback regarding the structure and contribution of the discussion section. In response, we have restructured the manuscript by merging the discussion with the results and removing repetitive or non-essential commentary. The new integrated section offers a clearer, evidence-based interpretation of the findings, ensuring that each discussion point is directly tied to the experimental outcomes. This revision highlights the biological relevance and mechanistic insights while maintaining coherence throughout the manuscript.

Comment 18:
Please avoid the use of references in the conclusion section. Please shorten the conclusion section and keep it strictly to the key (quantitative) results and insights of this study.

Response 18:
As recommended, we have revised the conclusion section by removing all references and shortening the text to focus exclusively on the main quantitative findings and insights. The revised conclusion now emphasizes the principal outcomes of the study, such as the degree of violacein inhibition, biofilm disruption percentages, and phytochemicals identified, in a concise and impactful summary.

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed the concerns. The manuscript can be accepted for publiaction.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed most of the reviewer's comments, and therefore, the reviewer acceptis this manuscript for publication in Applied Microbiology.