Combined Application of Juniperus communis Essential Oil and Amikacin, Clarithromycin and Rifampicin against Mycobacterium avium and Mycobacterium intracellulare

Round 1

Reviewer 1 Report

In my opinion, the manuscript might be suitable for publication after major revisions. The authors have carried out the analysis of the synergistic effect of essential oils of Juniperus communis combined with traditional medicines to combat nontuberculous mycobacteria.

Ø  The title should include the scientific name of the plant, which should be in italics.

Ø  I recommend using the scientific name of the plant studied as a keyword.

Ø  Correct the use of quotes in lines 89 and 90.

Ø  It is not appropriate to use concentrations with such many zeros, please change from mg/mL to μg/mL lines 129 and 130 in section 2.5. But in general, changes the concentration units in all document.

Ø  In the tables change the color of the words to black in all sections.

Not commet

Author Response

1. The title should include the scientific name of the plant, which should be in italics.

Thanks for the suggestion. We have changed the name of the plant used to the scientific name in italics.

2. I recommend using the scientific name of the plant studied as a keyword.

Thanks for the suggestion. We included Juniperus communis in the keywords.

3. Correct the use of quotes in lines 89 and 90.

Apologies for the omission. We have corrected in the text.

4. It is not appropriate to use concentrations with such many zeros, please change from mg/mL to μg/mL lines 129 and 130 in section 2.5. But in general, changes the concentration units in all document.

Thank you for the suggestion. We have changed the concentration units from mg/mL to μg/mL throughout the document.

5. In the tables change the color of the words to black in all sections.

Thank you for your comment. In all tables, the words have been changed to black.

Author Response File: Author Response.docx

Reviewer 2 Report

In M. avium complex (MAC) infection, the most crucial antibiotics are macrolides such as clarithromycin or azithromycin. On the other hand, antibiotics like rifampicin alone are insufficient in achieving clinical therapeutic effects. The synergistic effect demonstrated in this study between J. communis EO and rifampicin is noteworthy. I believe that if additional drugs or natural substances that can enhance the effectiveness of rifampicin are identified, it could contribute to improving the treatment outcomes for MAC infection patients. There are no disagreements regarding the experimental content or the process of reaching conclusions. However, I have a minor comment to make. In this study, only standard strains were used, while MAC strains isolated from clinical patients may have various MICs for several antibiotics. It would be advisable to briefly mention this as a limitation. Do you have any plans to conduct new research in the future using clinical isolates.

Author Response

In M. avium complex (MAC) infection, the most crucial antibiotics are macrolides such as clarithromycin or azithromycin. On the other hand, antibiotics like rifampicin alone are insufficient in achieving clinical therapeutic effects. The synergistic effect demonstrated in this study between J. communis EO and rifampicin is noteworthy. I believe that if additional drugs or natural substances that can enhance the effectiveness of rifampicin are identified, it could contribute to improving the treatment outcomes for MAC infection patients. There are no disagreements regarding the experimental content or the process of reaching conclusions. However, I have a minor comment to make. In this study, only standard strains were used, while MAC strains isolated from clinical patients may have various MICs for several antibiotics. It would be advisable to briefly mention this as a limitation. Do you have any plans to conduct new research in the future using clinical isolates.

Thank you for the interesting proposal. In earlier research, we examined the synergistic effect of different essential oils of Mediterranean plants, so it would be really interesting to try, in addition to combining two components, to test the effect of three or more different components, including antimicrobial combinations. We will certainly try to expand the methodology.

Thank you for reminding us of the objective possibility of a limiting factor in using only standard strains. We emphasized this in the discussion (in lines 412 to 414). Since we have been researching the impact of different essential oils on standard strains of environmental mycobacteria for a long time, which we have now extended to their combinations with antibiotics, we are definitely considering extending all research to testing clinical isolates as well.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors investigated the interaction of common juniper (Juniperus communis) EO and the antimicrobials amikacin, clarithromycin and rifampicin against Mycobacterium avium and M. intracellulare using the checkerboard synergy method.

 Minor revision is required.

1.

The reviewer would like a detailed discussion of the mechanism by which this oil has an antimicrobial effect on NTM bacteria. 　

Is it related to the high lipid contents of NTM bacteria? 

2.

The reviewer asks a detailed discussion about the reason why there is no synergistic effect with clarithromycin in M. intracellulare. Similarly, please discuss why there is synergistic effects with rifampicin and amikacin in both NTMs.

Author Response

1. The reviewer would like a detailed discussion of the mechanism by which this oil has an antimicrobial effect on NTM bacteria. 　

Is it related to the high lipid contents of NTM bacteria? 

We did not want to repeat in this paper what we have already presented in several previous papers in which we determined, among others, the chemical composition of common juniper essential  oil (references 28, 29, 31 and 49). We found that the dominant ingredient is α-pinene, which chemically belongs to the group of monoterpenes. More than two-thirds of the chemical composition of J. communis EO consisted of monoterpenes. Monoterpene hydrocarbons can easily pass through the cell membrane lipid bilayer, most probably by diffusion. We believe that the large amount of lipids in the cell wall of mycobacteria is a factor that enables and facilitates the effect of J. communis EO on these bacteria. In the discussion, we emphasized our finding that J. communis EO leads to cell wall damage that facilitates the entry of antibiotics into the mycobacterial cell (line 477 to 486).

2. The reviewer asks a detailed discussion about the reason why there is no synergistic effect with clarithromycin in  intracellulare. Similarly, please discuss why there is synergistic effects with rifampicin and amikacin in both NTMs.

We thank you for noting the insufficient explanation of the synergy mechanism of the used EO and the tested antibiotics. As we explained in the previous point, we observed that the main mechanism of action of J. communis EO on mycobacteria is damage to the cell wall, which is facilitated by the lipophilic structure of the wall and the content of lipophilic components in the EO. We also visualized the mentioned damage with the help of TEM. In the discussion, we additionally emphasized the assumed mechanisms of synergistic action of this EO and amikacin and rifampicin from line 432 to 438 and EO and clarithromycin from line 466 to 476.

We also stated that clarithromycin did not show a synergistic effect with J. communis EO against M. intracellulare, which may be the result of the unfavorable MBC/MIC ratio of this antibiotic in the used ATCC strain of M. intracellulare, but also a possible drop in the pH value of the medium, which then adversely affects activity of clarithromycin. We will definitely try to investigate these mechanisms of interaction between antibiotics and EO on clinical isolates of non-tuberculous mycobacteria in the future.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been adjusted according to the comments made in the initial review. Just one last comment. It is appropriate and necessary to include the following information in the discussion section:

The discussion between lines 432 to 438 can possibly be explained by the presence of phthiocerol dimycocerosate in mycobacterial cell wall. A recent study evidenced the change in the concentration of this lipid in in vitro studies of rifampicin monoresistant mucobacterial cells. Please supplement your discussion (which has no citations in this paragraph) by reviewing this recent publication. Consult:

Heterogeneous fitness landscape cues, pknG low expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth

DOI: https://doi.org/10.1016/j.tube.2021.102156

Author Response

Thank you for your suggestion. We included the suggested literary reference in the discussion from lines 432 to 438 under reference number 51.

Author Response File: Author Response.docx