Piper nigrum Fruit Extract as an Antibiotic Resistance Reversal Agent in MDR Bacteria

Round 1

Reviewer 1 Report

Manuscript Title: Piper nigrum fruit extract as an antibiotic resistance reversal agent in MDR bacteria

 

The present manuscript “Piper nigrum fruit extract as an antibiotic resistance reversal agent in MDR bacteria”, has focused on the exploration of P. nigrum potential as a natural antibiotic reversal agent. And it suggests that P. nigrum extracts can be used as natural antibiotic resistance reversal agents without any side effects on the human body and can reverse antibiotic resistance. The study fits within the scope of the Journal and will also contribute to the existing scientific database. However, the authors should address the major concerns raised during the review process stated below and further investigation should be conducted in the future.

 

The following specific comments are observed:

1.     I would like to suggest general editing of the English language especially in introduction and discussion sections, and the authors should consider making reading more agile.

2.     Introduction is too shallow and more information should be added for the justification of the study.

3.     In the Discussion, as a whole, there is little depth and breadth in the discussion, little focus on the theme of this study, and little explanation and comparison of the key results to other references. The parts should be improved the internal logic and highlight the new findings or insights in this study.

4.     The details of the article should also be checked. For example, there should be spaces between the numbers and the parentheses.

5.     If possibly give a separate section for "Quality Control” to provide more details for explaining how to conduct the quality control.

6.     The graph quality should be improved to some degree.

Author Response

Reviewer #1

The present manuscript “Piper nigrum fruit extract as an antibiotic resistance reversal agent in MDR bacteria”, has focused on the exploration of P. nigrum potential as a natural antibiotic reversal agent. And it suggests that P. nigrum extracts can be used as natural antibiotic resistance reversal agents without any side effects on the human body and can reverse antibiotic resistance. The study fits within the scope of the Journal and will also contribute to the existing scientific database. However, the authors should address the major concerns raised during the review process stated below and further investigation should be conducted in the future.

Answer

We would like to thank the Reviewers for taking the necessary time and effort to review the manuscript. Your comments, suggestions, and corrections, helped us greatly improve the manuscript. Corrections in the paper and the responses to the reviewer's comments are as follows:

 

The following specific comments are observed:

1. I would like to suggest general editing of the English language especially in introduction and discussion sections, and the authors should consider making reading more agile.

Answer

A thorough revision of the manuscript has been done.

 

2. Introduction is too shallow and more information should be added for the justification of the study.

Answer

More information has been added mainly in the Introduction section.

 

3. In the Discussion, as a whole, there is little depth and breadth in the discussion, little focus on the theme of this study, and little explanation and comparison of the key results to other references. The parts should be improved the internal logic and highlight the new findings or insights in this study.

Answer

The discussion section has been revised with more details about P. nigrum activity and respective results.

“All antibiotics have a different mechanism of action of the killing of bacteria. P. nigrum extracts have different effects with each antibiotic due to the different compounds present in each extract.  P. nigrum phytochemical analysis explained that flavonoids, glycosides, alkaloids, and tannins are present in them, which have resistance reversal [9] and antioxidant activity [29]. Which helps antibiotics in killing bacteria by blocking efflux pump or curing R-plasmid resistance.”

 

4. The details of the article should also be checked. For example, there should be spaces between the numbers and the parentheses.

Answer

A thorough revision of the manuscript has been done all the writing, space errors and stylistic errors were removed e.g. names of species “S. typhi” and all others.

5. If possibly give a separate section for "Quality Control” to provide more details for explaining how to conduct the quality control.

Answer

In point 2.4. (Antibiotic resistance reversal activity) the reversal activity of plant extracts in combination with antibiotics was compared with the AST (Antibiotic Susceptibility Activity) of plant extracts and antibiotics alone, and their results were used as the control for the combination activity. The treatment of bacteria with plant extracts was for 24hrs after that this mixture was streaked on an agar plate and zones of antibiotic were noted after 24hrs incubation. So they are comparable as no zone of inhibition was seen, by any plant extract alone which acts as a control. And in the case of many antibiotics alone, a Resistant to intermediate zone was shown which goes to a sensitive range after treatment with plant extracts.

6.The graph quality should be improved to some degree.

Answer

Graphs are improved and arranged in the form of panels for easy understanding.

Reviewer 2 Report

Abstract section: the study suggests that P. nigrum extracts can be used as natural antibiotic resistance reversal agents without any side effects on the human body and can reverse antibiotic resistance (lines 28-30). However, the results don’t present evidence to assume that the substance will not present side effects to the human body. And the tests are not enough to determine that the antibiotic resistance will be reversed in vivo. Please revise this statement and use the results presented in the manuscript to conclude the abstract section.

Item 2.3. Bacterial strain and antibiotic susceptibility test: please italicize “S. typhi”. Revise through the text.

Please inform how the plates were made, the growth time for each strain, temperature conditions, and the quantity of each used drug (in the methodology section). The value used is correspondent to the MIC? Indicate the volume used for each extract. How the used volume was determined for each extract? MIC? Please present the experiments made to determine the volume used on plates for each extract. Please inform all the required details to reproduce this experiment.

Since in 3.2. section, the title indicates that the results are related to S. aureus, informing through the subitems that the results belong to S. aureus is redundant.

In 3.2.3. section when the authors consider very significant the increased zone resultant of MPN+Cli combination it mean statistically significant? Please present the statistical analysis of the zones of inhibition through the results section.

The figure correspondent to 3.2.3. and 3.2.6. sections are located distantly from the respective text and in a different pattern from the rest of the manuscript. Positioning the figure just after the results makes the results easier to observe.

Section 3.2.4: CPN+Lvx zone of inhibition increased by 17 mm, and the n-BPN+Lvx zone of inhibition increased just by 1 mm. The authors meant 16 mm in response to n-BPN+Lvx?

Please consider presenting figures 2 to 8 in a single panel. This will facilitate the comparative interpretation of the inhibition zone variation of each used P. nigrum fractionation. The panel must, either, improve the discussion of these results. In the same way, consider presenting the figures in a single panel for S. typhi results.

The 3.3.1 section is entirely italicized.

Discussion section: The discussion section must present and discuss the main results observed by the authors; focusing on describing the class each of the antibiotics used belongs to is not essential to the discussion unless one inhibitory result could be observed for all drugs in a corresponding class. This section must be improved.

Lines 290-292: The authors state that S. aureus growth revealed that the plant extracts increased the zone of inhibition to a significant level. Without statistical analysis, I could assume that only MPN+Cfx, Ery combined with MPN, EAPN, and APN, probably all the Cli and Lvx exposure conditions, EAPN+Cpfx, CPN and APN with Amk, and Gent+MPN, EAPN, and APN are statistically significant. But only after the statistical analysis will these results be confirmed. Anyway, is supposed that MPN, EAPN, and APN could be the most relevant fractions considering S. aureus results? Regarding S. typhi, MPN and APN sustain this relevance as reversal agents. Is this observation appropriate? Additionally, the effects over S. typhi seem to be less significant compared to S. aureus results (the figures in a single panel will improve this analysis). However, the results of S. typhi with Lvx and Tgc are remarkable demanding further attention and discussion. I either suggest including in the discussion the main drugs used to treat each used strain clinically, highlighting how your results must be applicable in the future.

Author Response

Reviewer #2

Abstract section: the study suggests that P. nigrum extracts can be used as natural antibiotic resistance reversal agents without any side effects on the human body and can reverse antibiotic resistance (lines 28-30). However, the results don’t present evidence to assume that the substance will not present side effects to the human body. And the tests are not enough to determine that the antibiotic resistance will be reversed in vivo. Please revise this statement and use the results presented in the manuscript to conclude the abstract section.

 

Dear Reviewer,

Your in-depth comments, suggestions, and corrections, have greatly improved the manuscript. It opened up many new ways of critical thinking for us. Here you can find our response to your comments and corrections. 

 

Answer.

As piper nigrum is a herbal plant used in many natural remedies and has no side effects on the human body. But the statement has been revised as „This study suggests that P. nigrum extracts can be used as natural antibiotic resistance reversal agents that increase the effectiveness of current antibiotics and can reverse antibiotic resistance.”

 

Item 2.3. Bacterial strain and antibiotic susceptibility test: please italicize “S. typhi”. Revise through the text.

Answer: A thorough revision of the manuscript has been done all the writing, space errors and stylistic errors were removed e.g. names of species “S. typhi” and all others.

 

Please inform how the plates were made, the growth time for each strain, temperature conditions, and the quantity of each used drug (in the methodology section). The value used is correspondent to the MIC? Indicate the volume used for each extract. How the used volume was determined for each extract? MIC? Please present the experiments made to determine the volume used on plates for each extract. Please inform all the required details to reproduce this experiment.

Answer

For plate preparation and for antibiotic resistance reversal activity correction of previous section 2.3 and new section 2.4 was added and the Antibiotic susceptibility test (AST) method was used for the above-mentioned procedures.

 

Since in 3.2. section, the title indicates that the results are related to S. aureus, informing through the subitems that the results belong to S. aureus is redundant.

Answer

According to your indication respective portion was removed from the 3.2 section.

In 3.2.3. section when the authors consider very significant the increased zone resultant of MPN+Cli combination it mean statistically significant? Please present the statistical analysis of the zones of inhibition through the results section.

Answer

Yes, the zone of inhibition is clinically significant as the clindamycin (Cli) zone of inhibition was 20mm, it should be ≥ 21 mm to fall in the sensitive zone. By using plant extract MPN and clindamycin zone of inhibition increased from 20mm to 27mm. Which is clinically significant.

 

The figure correspondent to 3.2.3. and 3.2.6. sections are located distantly from the respective text and in a different pattern from the rest of the manuscript. Positioning the figure just after the results makes the results easier to observe.

Answer

Figures corresponding to the above-mentioned sections are relocated next to the respective text portion.

 

Section 3.2.4: CPN+Lvx zone of inhibition increased by 17 mm, and the n-BPN+Lvx zone of inhibition increased just by 1 mm. The authors meant 16 mm in response to n-BPN+Lvx?

Answer

Yes, the CPN+Lvx zone of inhibition increased by 17 mm, and the n-BPN+Lvx zone of inhibition increased just by 1 mm. This means the n-BPN+Lvx zone of inhibition is 16mm and the CPN+Lvx zone of inhibition is 17 mm.

We rewrite it as “CPN+Lvx zone of inhibition increased by 17 mm, and the n-BPN+Lvx zone of inhibition increased by 16 mm.”

 

Please consider presenting figures 2 to 8 in a single panel. This will facilitate the comparative interpretation of the inhibition zone variation of each used P. nigrum fractionation. The panel must, either, improve the discussion of these results. In the same way, consider presenting the figures in a single panel for S. typhi results.

Answer

Figures are placed in a single panel for the purpose of better analysis.

 

The 3.3.1 section is entirely italicized.

Answer

3.3.1 section is justified and the italicization error is removed.

 

Discussion section: The discussion section must present and discuss the main results observed by the authors; focusing on describing the class each of the antibiotics used belongs to is not essential to the discussion unless one inhibitory result could be observed for all drugs in a corresponding class. This section must be improved.

Answer

The discussion section has been revised with more details about P. nigrum activity and respective results.

“All antibiotics have a different mechanism of action of the killing of bacteria. P. nigrum extracts have different effects with each antibiotic due to the different compounds present in each extract.  P. nigrum phytochemical analysis explained that flavonoids, glycosides, alkaloids, and tannins are present in them, which have resistance reversal [9] and antioxidant activity [29]. Which helps antibiotics in killing bacteria by blocking efflux pump or curing R-plasmid resistance.”

 

Lines 290-292: The authors state that S. aureus growth revealed that the plant extracts increased the zone of inhibition to a significant level. Without statistical analysis, I could assume that only MPN+Cfx, Ery combined with MPN, EAPN, and APN, probably all the Cli and Lvx exposure conditions, EAPN+Cpfx, CPN and APN with Amk, and Gent+MPN, EAPN, and APN are statistically significant. But only after the statistical analysis will these results be confirmed. Anyway, is supposed that MPN, EAPN, and APN could be the most relevant fractions considering S. aureus results? Regarding S. typhi, MPN and APN sustain this relevance as reversal agents. Is this observation appropriate? Additionally, the effects over S. typhi seem to be less significant compared to S. aureus results (the figures in a single panel will improve this analysis). However, the results of S. typhi with Lvx and Tgc are remarkable demanding further attention and discussion. I either suggest including in the discussion the main drugs used to treat each used strain clinically, highlighting how your results must be applicable in the future.

Answer

• These results are clinically significant. We rewrite the mentioned points for easy understanding.
• Yes, this observation is appropriate “MPN, EAPN, and APN could be the most relevant fractions considering aureus results. Regarding S. typhi, MPN and APN sustain this relevance as reversal agents”.
• aureus showed more effective results as compared to S. typhi. As Gram-positive and gram-negative bacteria have different structural compositions. But we used specific antibiotics that are used to kill them with a specific mechanism of action. These extracts mainly contain flavonoids, glycosides, alkaloids, and tannins, which help reverse antibiotic resistance.
• Figures are placed in a single panel for the purpose of better analysis.
• The results of typhi with Lvx and Tgc are discussed in detail according to your recommendation.
• Usage of present study results is also discussed in the section of the discussion.

Reviewer 3 Report

The study portrays a current and interesting problem with possible potential for reversing the resistance of Gram-positive and negative bacteria to currently available antibiotics.

However, the article presents weak scientific solidity evidenced by a scarce and rudimentary methodology, where we can enumerate among the main problems:

- Absence of description of the methodology for obtaining the bascterian lineages? Is it ATCC standard? Nothing is reported about.

- Absence of methodology for determining susceptibility tests? Which culture medium? which test? From what I see you only used Inhibition Halo, and didn't do a MIC? In short, a methodology far below to support the proposed results.

- I think you should have added a control group (Bacteria + Extract), to be able to base your findings.

- Absence of statistical methodology to demonstrate how much better the effect of the extracts was. What is the P, what is the confidence interval? What statistical test is used?

- Finally, what is expected in addition to demonstrating the results is to explain the mechanisms involved? It is known that gram-positive bacteria have a different composition from gram-negative ones, and their extracts show very similar results.... Finally, how do you propose this effect? What are the major compounds in your extract? What was the best extract? What mechanism of action is possibly involved to promote this synergism (if any such synergism exists).

Author Response

Reviewer #3

The study portrays a current and interesting problem with possible potential for reversing the resistance of Gram-positive and negative bacteria to currently available antibiotics.

However, the article presents weak scientific solidity evidenced by a scarce and rudimentary methodology, where we can enumerate among the main problems:

- Absence of description of the methodology for obtaining the bascterian lineages? Is it ATCC standard? Nothing is reported about.

We wish to express our appreciation and gratitude for your in-depth comments, suggestions, and corrections, which have greatly improved the manuscript. It opened up many new ways of critical thinking for us. Here you can find our response to your comments and corrections. 

Answer

New section 2.2.3 is added with information related to bacterial strains.

- Absence of methodology for determining susceptibility tests? Which culture medium? which test? From what I see you only used Inhibition Halo, and didn't do a MIC? In short, a methodology far below to support the proposed results.

Answer

For plate preparation and for antibiotic resistance reversal activity correction of previous section 2.3 and new section 2.4 was added and the Antibiotic susceptibility test (AST) method was used for the above-mentioned procedures.

 

- I think you should have added a control group (Bacteria + Extract), to be able to base your findings.

Answer (please indicate the line of the inserted new information)

In point 2.4. (Antibiotic resistance reversal activity) the reversal activity of plant extracts in combination with antibiotics was compared with the AST (Antibiotic Susceptibility Activity) of plant extracts and antibiotics alone, and their results were used as the control for the combination activity. The treatment of bacteria with plant extracts was for 24hrs after that this mixture was streaked on an agar plate and zones of antibiotic were noted after 24hrs incubation. So they are comparable as no zone of inhibition was seen, by any plant extract alone which acts as a control. And in the case of many antibiotics alone, a Resistant to intermediate zone was shown which goes to a sensitive range after treatment with plant extracts.

 

- Absence of statistical methodology to demonstrate how much better the effect of the extracts was. What is the P, what is the confidence interval? What statistical test is used?

Answer (please indicate the line of the inserted new information)

As our study was not survey-based or comparison based. We tried a new experiment for the reversal of antibiotic resistance. We used a Graph pad prism for graphs and we performed all the experiments in triplets. All the values are means of three parallel replicates. Error bars indicate the standard error from the mean.

- Finally, what is expected in addition to demonstrating the results is to explain the mechanisms involved? It is known that gram-positive bacteria have a different composition from gram-negative ones, and their extracts show very similar results.... Finally, how do you propose this effect? What are the major compounds in your extract? What was the best extract? What mechanism of action is possibly involved to promote this synergism (if any such synergism exists).

Answer (please indicate the line of the inserted new information)

Yes, Gram-positive and Gram-negative bacteria have a different compositions. But we used specific antibiotics that are used to kill them. These extracts mainly contain flavonoids, glycosides, alkaloids, and tannins. Which mainly helps in the effect of synergism.

Round 2

Reviewer 1 Report

I have no comments.

Author Response

Dear Reviewer,

Thank you again for your efforts and time.

Best wishes,

Dr. Imre, 

on behalf of the research team

Reviewer 2 Report

Figure 2-8 captions could be resumed in one single sentence (Antibiotic reversal activity of P. nigrum extracts in combination with 2. cefoxitin (30 μg), 3. Erythromycin (15 μg)…). Please consider the same suggestion for figures 9-15.

Please indicate evidence to support the activity of P. nigrum extracts as efflux pump blockers or R-plasmid resistance curers.

 

The sentences included in the discussion section (399-310) are redundant and are already written in other sections of the manuscript (e.g., lines 346 Due to the different mechanisms of action of each antibiotic class, these extracts have a different effect on all antibiotics and lines 399-400 All antibiotics have different mechanisms of action for killing bacteria. P. nigrum extracts have different effects with each antibiotic due to the different compounds present in each extract) 

Author Response

Reviewer #2

Figure 2-8 captions could be resumed in one single sentence (Antibiotic reversal activity of P. nigrum extracts in combination with 2. cefoxitin (30 μg), 3. Erythromycin (15 μg)…). Please consider the same suggestion for figures 9-15.

The authors completely agree the reviewer suggestion. Therefore, the correspondent figure captions were resumed to a single sentence resulting in “Antibiotic reversal activity of P. nigrum extracts in combination with 2. cefoxitin (30 μg), 3. Erythromycin (15 μg), 4. clindamycin (10 µg), 5. levofloxacin (5µg), 6. Ciprofloxacin (5µg), 7. amikacin (30 µg), 8. gentamicin (10 µg)”

Figure 9-15. Antibiotic reversal activity of P. nigrum extracts in combination with 9. amikacin (30 μg), 10. gentamicin (10 µg), 11. ciprofloxacin (5µg), 12. tigecycline (10µg), 13. levofloxacin (5µg), 14. Imipenem (10 µg), 15. cefotaxime (30 µg).

 

Please indicate evidence to support the activity of P. nigrum extracts as efflux pump blockers or R-plasmid resistance curers.

Reference for the P. nigrum extracts as efflux pump blockers or R-plasmid resistance curers was added [34].

The sentences included in the discussion section (399-310) are redundant and are already written in other sections of the manuscript (e.g., lines 346 Due to the different mechanisms of action of each antibiotic class, these extracts have a different effect on all antibiotics and lines 399-400 All antibiotics have different mechanisms of action for killing bacteria. P. nigrum extracts have different effects with each antibiotic due to the different compounds present in each extract) 

The repeated sentences are removed from the discussion portion including line no. 346.

Reviewer 3 Report

Thank you for the effort in trying to solve the questions pointed out in my analysis.

However, my perception of the study still remains: insufficient data study to support the results due to methodological robustness and insufficient discussion of the results.

Author Response

Reviewer #3

 

Thank you for the effort in trying to solve the questions pointed out in my analysis.

Dear Reviewer,

Thank you for your positive feedback, concerning the quality of our revision.

However, my perception of the study still remains: insufficient data study to support the results due to methodological robustness and insufficient discussion of the results.

The studies of (Kumar, V et al 2013), (Reshmi, S.et al 2010), (Scott, I. Et al 2008), and (Das, B et al 1998) show that they have already performed phytochemical analysis and explained that P. nigrum contains flavonoids, glycosides, alkaloids, and tannins that have a resistance reversal activity. In the present study, we correlated our results with the previously known component of P. nigrum. In future, detailed phytochemicals of each extract will be performed and for each component, antibiotic reversal activity will be checked. As each antibiotic has a different mechanism of action that’s why different results were obtained.

Some parts of the discussion are also updated for making the results more clear and more understandable in the revised version.

Thank you agai for your time and valuable comments!