Methanolic Extract of Moringa oleifera Seed Synergizes the Bactericidal Effect of Ampicillin, Cephalexin, and Amoxicillin/Clavulanic Acid Against Multidrug-Resistant Escherichia coli Isolated from Street-Vended Food

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I congratulate the authors for their efforts in conducting this study and also for the relevance of their work. The investigation of multidrug-resistant bacterial isolates, as well as the analysis of M. oleifera seed extract, represent a significant scientific effort, contributing both to the area of health surveillance and food control and to the solution of the problem of bacterial resistance, with the discovery of new therapeutic options. Nevertheless, some improvements are necessary to increase the clarity, consistency, and scientific impact of the manuscript.

ABSTRACT:

Lines 18–21: Although the abstract is clear and descriptive, it is recommended that the introduction elucidate in more detail the importance of the study with the analyzed bacteria, as well as the justification for the selection of the extract and the plant used.

- Line 21: It is important that the authors present the origin and conditions of obtaining the bacterial strains used, in order to ensure methodological transparency and reproducibility of the results.

- Line 23: It is recommended that the authors clearly describe the method used in the phytochemical assay, as well as the origin of the Escherichia coli strains used in the study.

INTRODUCTION:

The introduction is well structured and informative, clearly presenting the relevance of the bacteria selected for the study and providing a pertinent explanation for the choice of extract evaluated.

 

MATERIALS AND METHODS

The authors should review the formatting of scientific names, ensuring the correct use of italics (E. coli, M. oleifera), in accordance with taxonomic standards. E. coli - lines 95, 106, 110, 154, 164; M. oleifera - lines 133, 143, 157, 165, 185

- Lines 92-94: For greater clarity and understanding of the methodological procedures, it is recommended that the Materials and Methods section be restructured. It is suggested that topic 2.1 (lines 91–94) be integrated into topic 2.3 (lines 123–131), and that topic 2.6 (lines 170–176) be presented before topic 2.5 (lines 152–169), in order to give greater coherence to the methodological sequence.

- Lines 102–103: In order to ensure the reproducibility of the experiment, it is recommended that the authors describe in more detail the quantity of samples obtained, as well as how the samples were prepared before being seeded, indicating whether they followed any type of protocol for this. E.g. Among the food samples were: raw vegetables were macerated before being seeded on MacConkey agar.

Characterize in more detail the Escherichia coli isolates used in the sensitivity tests, also specifying the total number of samples analyzed.

 

RESULTS AND DISCUSSION

For better understanding, it is suggested that the authors always refer to the bacterial isolates evaluated, rather than to groups and the years in which the samples were obtained.

- Line 195: It appears that the authors have confused the section cited in the text. The section referred to by the authors is not numbered. In this regard, we suggest revising the numbering of the sections for the necessary consistency.

- Lines 193-197: We recommend that the authors report the number of isolates obtained in each group in order to provide greater transparency and allow for better interpretation of the results.

- Lines 201-202: For a better understanding of the results, we suggest including in the text the number of isolates analyzed, a value obtained by the Raosoft method (as mentioned in the methodology – lines 107 and 108). Although the information is included in Supplementary Table 2, it should also be explained in the body of the text.

- Lines 204-205: It is recommended to provide a more comprehensive description of the observed differential antimicrobial resistance profile, highlighting the main findings and their possible epidemiological or clinical implications.

- Figures 4 and 5: We suggest adding the names of the antibiotics directly to the graphs to facilitate data interpretation. In addition, we recommend clarifying what each column represents in the caption.

Although the authors present a good description and interpretation of the results, the memos do not compare their results with those of other studies. This contextualization is essential to strengthen the discussion.

- Lines 310-312: It is recommended to review the consistency between the results and the discussion. The authors mention the percentage of samples with resistant bacteria and their respective types, but this data is not presented in the results, nor is the method used to obtain it described. The same occurs in lines 314–315.

- Lines 349–350: The data cited requires an appropriate bibliographic reference.

- Figure 6: The chemical structures represented should be described in the caption, allowing the reader to clearly understand which molecules are being presented.

- Lines 361–363: The authors mention that the genetic analysis of the isolates was performed in another study. It is recommended to clarify this information further, indicating the cited work more precisely. For example: In the study conducted by X, which investigated the presence of genes associated with bacterial resistance, no such genes were detected, and resistance to β-lactams was attributed to other mechanisms.

CONCLUSION

- The conclusion should be more concise, responding directly to the proposed objectives, highlighting:

(a) the antimicrobial effects of M. oleifera oil

(b) the adjuvant effects of the oil associated with conventional antibiotics

(c) clinical relevance.

Avoid including concepts already included in the discussion in the conclusion.

Author Response

ABSTRACT:

Lines 18–21: Although the abstract is clear and descriptive, it is recommended that the introduction elucidate in more detail the importance of the study with the analyzed bacteria, as well as the justification for the selection of the extract and the plant used.

Reply: Thank you for your observations. We highlighted that since antibiotics are becoming less effective, we look for alternatives to treat infection diseases that are commonly treated with antibiotics (Lines 19-21).  We are limited by the words established by the journal. Therefore, adding to much information Is not possible.

- Line 21: It is important that the authors present the origin and conditions of obtaining the bacterial strains used, in order to ensure methodological transparency and reproducibility of the results.

Reply: Thank you for your comment. We have added from where the E. coli strains were obtained (Lines 22-23)

- Line 23: It is recommended that the authors clearly describe the method used in the phytochemical assay, as well as the origin of the Escherichia coli strains used in the study.

Reply: Thank you for your observation. We agree with your comment. However, since there is a limit Since the phytochemical assays englobe qualitative procedures that are described in detail on the methodology section, we just added the word “qualitative” to this section (Line 25). Also, considering that there is a word limit and adding more information would surpass the 250 words established by the journal, we cannot add more detailes.

 

MATERIALS AND METHODS

The authors should review the formatting of scientific names, ensuring the correct use of italics (E. coli, M. oleifera), in accordance with taxonomic standards. E. coli - lines 95, 106, 110, 154, 164; M. oleifera - lines 133, 143, 157, 165, 185

Reply: Thank you for your observation. We have correct the formatting of the enlisted words.

- Lines 92-94: For greater clarity and understanding of the methodological procedures, it is recommended that the Materials and Methods section be restructured. It is suggested that topic 2.1 (lines 91–94) be integrated into topic 2.3 (lines 123–131), and that topic 2.6 (lines 170–176) be presented before topic 2.5 (lines 152–169), in order to give greater coherence to the methodological sequence.

Reply: Thank you for this recommendation. We agreed with the observation and have integrated both sections. Know you can find at Line 92-107 section 2.1 Food sampling and E. coli isolation. Line 108-121 section 2.2 Antibiotic sensitivity testing, and 2.3 All referring to M. oleifera processing and extraction of secondary metabolites. Likewise, now section 2.5 (line161) englobes the MIC, MBC, tolerance level and antimicrobial screening of Moringa metabolites.

- Lines 102–103: In order to ensure the reproducibility of the experiment, it is recommended that the authors describe in more detail the quantity of samples obtained, as well as how the samples were prepared before being seeded, indicating whether they followed any type of protocol for this. E.g. Among the food samples were: raw vegetables were macerated before being seeded on MacConkey agar.

Characterize in more detail the Escherichia coli isolates used in the sensitivity tests, also specifying the total number of samples analyzed.

Reply: Thank you for your comment. In order to clarify all related with the origin of the samples, we added the reference at line 95, added the number of samples collected at line 99-100, and detailed the number of samples after grouping by food type in line 104-105.

RESULTS AND DISCUSSION

For better understanding, it is suggested that the authors always refer to the bacterial isolates evaluated, rather than to groups and the years in which the samples were obtained.

Reply: we have moved table S1 to the main text as Table 1 for a better understanding and easy access to samples description and classification.

- Line 195: It appears that the authors have confused the section cited in the text. The section referred to by the authors is not numbered. In this regard, we suggest revising the numbering of the sections for the necessary consistency.

Reply: Thank you for this observation. We have revised the numeration of sections on the methodology and adjusted considering past comments.

- Lines 193-197: We recommend that the authors report the number of isolates obtained in each group in order to provide greater transparency and allow for better interpretation of the results.

Thank you for this comment. We have added more information about the percentages of contamination found in each group on lines 206-211.

- Lines 201-202: For a better understanding of the results, we suggest including in the text the number of isolates analyzed, a value obtained by the Raosoft method (as mentioned in the methodology – lines 107 and 108). Although the information is included in Supplementary Table 2, it should also be explained in the body of the text.

Reply: Thank you for your comment. We added the number of samples collected on line 93 of section 2.1 and line 201 in section 3.1

- Lines 204-205: It is recommended to provide a more comprehensive description of the observed differential antimicrobial resistance profile, highlighting the main findings and their possible epidemiological or clinical implications.

Reply: Thank you for your comment. We agree with your observation and have added more description related to the antibiotic resistance profile of the E. coli strains from line 221-230.

- Figures 4 and 5: We suggest adding the names of the antibiotics directly to the graphs to facilitate data interpretation. In addition, we recommend clarifying what each column represents in the caption.

Reply: Thank you for this observation. We added the names to the graphs and added the legend to each graph of figure 4 and figure 5. We also revised the captions enlisting the correct antibiotics for each figure (lines 304-306 and lines 320-323).

Although the authors present a good description and interpretation of the results, the memos do not compare their results with those of other studies. This contextualization is essential to strengthen the discussion.

- Lines 310-312: It is recommended to review the consistency between the results and the discussion. The authors mention the percentage of samples with resistant bacteria and their respective types, but this data is not presented in the results, nor is the method used to obtain it described. The same occurs in lines 314–315.

Thanks for this observation. We are sorry for the confusion. We have clarified these sections by adding the corresponding information and specifying where the analyses were found (section 3.1 lines 212-214, 3.2 lines 221-230, section 4 lines 331-334, 384-385, 426-428)  

- Lines 349–350: The data cited requires an appropriate bibliographic reference.

Reply: Thank you for your comment. We have added the reference at the end of the sentence.

- Figure 6: The chemical structures represented should be described in the caption, allowing the reader to clearly understand which molecules are being presented.

Thank you for your observation. We agreed with the comment and added the name of the beta-lactam molecule (amoxicillin) and the quinolone molecule (ciprofloxacin). We also edited some typing errors within the figure 6 (lines 404-411).

- Lines 361–363: The authors mention that the genetic analysis of the isolates was performed in another study. It is recommended to clarify this information further, indicating the cited work more precisely. For example: In the study conducted by X, which investigated the presence of genes associated with bacterial resistance, no such genes were detected, and resistance to β-lactams was attributed to other mechanisms.

Thank you for this observation. We clarified this section by adding where the analysis was carried out (lines 384-385).

CONCLUSION

- The conclusion should be more concise, responding directly to the proposed objectives, highlighting:

(a) the antimicrobial effects of M. oleifera oil

(b) the adjuvant effects of the oil associated with conventional antibiotics

(c) clinical relevance.

Avoid including concepts already included in the discussion in the conclusion.

Thank you for your observation. We have modified the conclusion by deleting unnecessary information and highlighting the important parts of our findings (lines 449-457).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents an investigation into the adjuvant potential of Moringa oleifera seed methanolic extract combined with conventional antibiotics against multidrug-resistant Escherichia coli isolated from street-vended foods in Mexico. The research tackles a pressing global health issue by exploring natural adjuvants to restore antibiotic efficacy against resistant pathogens.

Major comments

Despite that the study employs multiple complementary approaches including phytochemical screening, microbroth dilution method, disk diffusion assay, and evaluation of extract-antibiotic combinations across twelve different antibiotics, only qualitative phytochemical screening was performed. The absence of HPLC-MS/MS analysis limits understanding of observed effects. The acknowledged need for "further analysis using HPLC-Mass Spectrometry" highlights this gap.​

 

• Materials and methods section lacks crucial information on seed collection protocol, i.e. when, where the seeds were collected, how the plant was identified and whether a voucher specimen was deposited, which would be in accordance with the current guidelines on studying plant extracts
• In what conditions and with the use of what equipment the seeds were grinded
• no reference bacterial strains were use in any of the experiments (e.g. from the ATCC collection), which makes the experiment impossible to recreate
• for antimicrobial assays usually DMSO is used as an initial solvent for different types of plant extracts, why the authors did use methanol? Why 20%?
• MBC to MIC ratios are basic indicator of mode of action of tested agents, however generally considered ratios for bactericidal effect are MBC/MIC ≤ 4, while for bacteriostatic > 4.
• In this manuscript authors used diffusion assay to determine the relations between tested seed extract and commonly used antibiotics. Generally accepted method for that purpose is to determine fractional inhibitory effect (FIC), why authors did not use that method?
• Figures: all abbreviation used in figures should be explained in footnotes, so the figures explained their content on their own, separately from the text (G1, G2, black bars, white bars in Fig 4 etc).
• The results should be presented and described in more understanding manner, for example authors should rather answer (in word or graphically) the questions whether the observed synergic effect between the seed extract and antibiotic restored their effectiveness against tested E. coli strains
• High MIC values (31.3-125 mg/ml) and MBC values (62-250 mg/ml) suggest very weak antimicrobial activity​, which should be clearly indicated in the discussion section
• Statistical analysis details are minimal; correction for multiple comparisons is not clearly stated

Minor comments

• please use italics in Latin names
• Tolerance Level Calculation: All isolates showed tolerance values ≤4, indicating bactericidal activity, yet the extract alone demonstrated minimal antimicrobial effect with inhibition zones <11.33 mm. This apparent contradiction requires clarification.​
• Table 3 contains extensive numerical data that would benefit from more focused presentation highlighting statistically significant synergistic/antagonistic effects rather than raw measurements.​
• address safety concerns: discuss cytotoxicity in terms that the effective concentrations against E. coli are extremely high

 

 

Conclusion

This manuscript presents valuable preliminary data on M. oleifera seed extract as a potential adjuvant for beta-lactam antibiotics against MDR E. coli. However, the study remains largely descriptive, lacking the mechanistic depth and molecular validation necessary for advancing toward clinical applications.

Major revisions required before publication.

Author Response

• Seed collection protocol, i.e. when, where the seeds were collected, how the plant was identified and whether a voucher specimen was deposited, which would be in accordance with the current guidelines on studying plant extracts

Reply: Thank you for your observation. We added to section 2.3 the specifications of the isolation, highlighting the origin of the seeds and the importance of their analysis (lines 124-129).

• In what conditions and with the use of what equipment the seeds were grinded

Reply: Thank you for this observation. We have specified the equipment and the conditions of the seeds used during this procedure (lines 130-131).

• Clarify were the bacterial strains were obtained

Thank you for this comment. In accordance with the comments of the other reviewer, the sections of the methodology were modified and some of them were unified. You can find at section 2.1 the description of the bacterial strains collection and on section 2.3 all related to collection and secondary metabolites extraction of M. oleifera seeds.

• why the authors did use methanol? Why 20%?

Reply: Thank you for your observation. Methanol is used for extraction of medicinal plants due to its lower boiling point, higher volatility, and higher extraction efficiency. The optimal percentage of methanol for plant extraction varies depending on the plant and the compounds being targeted, but 80% methanol is frequently cited as a good choice for extracting a wide range of phytochemicals due to its polar properties.

• MBC to MIC ratios are basic indicator of mode of action of tested agents, however generally considered ratios for bactericidal effect are MBC/MIC ≤ 4, while for bacteriostatic > 4.

Reply: We agree with this information and has been stated on the methodology, section 2.5 from lines 163-178)

• Generally accepted method for that purpose is to determine fractional inhibitory effect (FIC), why authors did not use that method?

Reply: Thank you for your observations. Considering that we analyzed twelve antibiotics against different samples during a period of three years, the methodology implemented allows us to explore the capacity of M. oleifera as an adjuvant to several antibiotics on a more quickly and cost-effective matter. The protocol was followed based on the study of Saquib and coworkers where the synergic activity of herbal extracts against different pathogens was explored effectively.

• Figures: all abbreviations used in figures should be explained in footnotes, so the figures explained their content on their own, separately from the text (G1, G2, black bars, white bars in Fig 4 etc).

Reply: Thank you for this observation. We have revised the figures 4 and 5 and made the corresponding change of the captions ( lines 304-306 and 320-323)

• The results should be presented and described in more understanding manner, for example authors should rather answer (in word or graphically) the questions whether the observed synergic effect between the seed extract and antibiotic restored their effectiveness against tested E. coli strains

Reply: thank you for this observation. We have made some changes from lines 290-302, 307-318 in order to clarify the observed results.

• High MIC values (31.3-125 mg/ml) and MBC values (62-250 mg/ml) suggest very weak antimicrobial activity​, which should be clearly indicated in the discussion section

Reply: Thank you for your comment. We added the statement within lines 348 and 350.

• Statistical analysis details are minimal; correction for multiple comparisons is not clearly stated

Thank you for your observation. We added more details on section 2.7 lines 192-197.

Minor comments

• please use italics in Latin names

Reply: Thank you for your observation. We have revised all names in accordance with the correct format.

• Tolerance Level Calculation: All isolates showed tolerance values ≤4, indicating bactericidal activity, yet the extract alone demonstrated minimal antimicrobial effect with inhibition zones <11.33 mm. This apparent contradiction requires clarification.​

Reply: We are very grateful for your observation, and your comment is very pertinent. The observed differences are due to the use of different methodologies. In this first intention, we were interested in testing and standardizing metrics to study the phenomenon, which prioritizes costs and accessibility to the techniques. For MIC and MBC determinations, the 96-well plate dilution method was used, while for antimicrobial evaluation, the agar plate disc diffusion method was used. We are aware that several factors could influence the results, including the rate of diffusion of the drug through the agar, its solubility, and the thickness of the agar, among others. Our main objective was to explore the antimicrobial activity of these extracts for the first time. This does not exclude the possibility of standardizing the technique using the methodologies proposed in our following metrics. If the reviewers consider it necessary, we can make the corresponding changes to the manuscript and not present the calculation of the tolerance level in this work.

 

• Table 3 contains extensive numerical data that would benefit from a more focused presentation highlighting statistically significant synergistic/antagonistic effects rather than raw measurements.​

Reply: Thank you for your observations. We have moved this table to supplementary materials, since the processed information is presented in figures 4 and 5.

• address safety concerns: discuss cytotoxicity in terms that the effective concentrations against E. coli are extremely high

Reply: We appreciate your comment. Indeed, any molecule that wants to be directed towards therapeutics in humans requires an assessment of cytotoxicity to consider whether the benefits outweigh the possible effects that can affect homeostasis in human cells, mainly enterocytes for example. In our next methodological steps we want to explore in a relevant model if the dose in an excessive concentration represents a potential risk, the dose response curves will be very enriching in the effects and the safety margins that are required for any potential drug can begin to be projected.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, 
 After reviewing the new version of the manuscript, I find that all the comments and suggestions made during the text correction stage have been duly addressed. I congratulate the authors on their commitment and the quality of the work presented.
 Sincerely,

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript shows noticeable improvement in structure and clarity compared to the previous submission. Nevertheless, the methodological approach has been considerably simplified, which limits the overall scientific rigor and depth of analysis. While the study contributes useful observations, its quality can be regarded as moderate. Publication may be considered at the editor’s discretion.