In Vitro Efficacy of Hungarian Propolis against Bacteria, Yeast, and Trichomonas gallinae Isolated from Pigeons—A Possible Antibiotic Alternative?

Round 1

Reviewer 1 Report

Dear author

In your manuscript you described the antibactirial properties of propolis. You had an extensive literature research and you described well your experimental part. In my opinion you have to add some more information about the collection of propolis and its production. Please fix the tables and their legends to look and to read better. There are legends that are in different pages. 

In lines 188-193 correct your English language because it is difficult to make sense. Also in line 192 what is thermostat? Probably it is an incubator? or an oven? Thermostat is a device that automatically regulates the temperature or activates a device when the temperature reaches a certain point. 

In lines 210-214 please edit your English language to make sense

Please explain what is MIC and MEC values at the first time that appears in the text, after that you can use the abbraviations. You explained that only in your abstract 

You have so many tables and figures. Please decide which one you will provied as supplementary material 

In line 492 Trichomonas gallinae must be with italics 

Lines 486- 504 edit your English 

Line 597 the names of the authors are not provided 

Please edit some parts of your manuscript that are mention above 

Author Response

Dear Reviewer,

Thank you very much for your comments and constructive suggestions.
I have resized the figures and tables to make them easier to read and so that they do not overlap on a page.
Lines 188-193 have been reworded as requested to make them easier to understand.
Lines 210-201 have also been reworded to make them clearer.
The concepts of MIC and MEC have been defined (lines 167-172).
If you think that there are too many illustrations of the data, in this case I would like to ask you to move the tables containing the statistics as a substitute for the others, because they are less important than the others.
Thank you for pointing out that there is no italics in line 492, this has since slipped to line 505, which I have corrected.
Thank you for pointing out lines 486-504, this has been reworded to make it clearer and moved to lines 499-518.
Thank you for noting the inaccuracy in the author names on line 597, which I have corrected (line 611).
I have highlighted the changes in yellow in the manuscript.

Thank you very much for your thorough review, which helped me to improve the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

 One limitation might be the ethanol concentration, allergy-please insert it in the discussion

It is an excellent idea; I think you can support it at all. The study design is excellent and the methodology is clear.

Author Response

Dear Reviewer,
Thank you very much for your supportive suggestions.
As requested, I have included the potential limiting role of ethanol extractant in usability.
I have highlighted the change in green (lines 520-521).
Thank you very much for taking the time to improve the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

In the present study, the authors investigated the efficacy of propolis against Staphylococcus spp., Enterococcus spp., Escherichia coli and Salmonella enterica, Candida albicans fungi and Trichomonas gallinae isolated from pigeons. For each pathogen, minimum inhibitory concentration (MIC) and minimum eradication concentration (MEC) of 8 isolates were determined for 96%, 90%, 80%, 70% and 60% ethanolic extracts of propolis from the region of Észak-Alföld. The results suggest that propolis could be an effective alternative treatment for external infections caused by Gram-positive bacteria (e.g., skin infections), but further in vivo studies are needed. The efficacy against C. albicans is also outstanding but it would be worthwhile to conduct studies on more fungal species. The efficacy of propolis against Trichomonas gallinae is less pronounced compared to human Trichomonas vaginalis studies, and no other comparative studies are available for these.

The manuscript is interesting, has merit and novelty. However, prior to suggest its acceptance, additional analysis and modifications are necessary as follow:

-          The authors should describe the final inoculum value of MIC analysis.

-          How exactly was the MIC determined? Visually? Reading at spectrophotometer?

-          Table 2: The authors need to explain MIC 50 and MIC 90. What is the unit of the results? mg/mL? ug/mL?

-          How many distinct experiments were performed? What is the n of analysis? Since we need to perform multiple experiments in microbiology, should Figure 1 present an error bar indicating the variation between the distinct experiments? The same applies to all bars of Figure 2, not only the untreated bar.

-          The first paragraph of the discussion presents too much information. The authors should rephrase it to make it easy to read. Also, the authors should try to explain the reason for so many different results. Chemical composition?

-          A Chemical analysis of samples that identifies some compounds are necessary. The authors well-describe the chemical composition of propolis samples, indicating its mechanisms of action in the introduction. However, in their manuscript, they did not identify the main compounds of their samples. They need to do that. In addition, in the discussion, they need to describe better the chemical composition of propolis types cited in the discussion.

Author Response

Dear Reviewer,
Thank you very much for the helpful comments you have brought to our attention.
The final inoculum volume was indeed omitted, I have corrected it (lines 167-168).
The MIC value was determined by visual method based on the presence/extent of turbidity (lines 182-183).
The meaning of the MIC50 and MIC90 values was defined: the MIC50 value is the minimum drug concentration to which at least 50% of the microbial population under study is sensitive, and at least 90% for the MIC90 value (lines 246-247). And the unit of measurement is included in the table (Table 2).
Thank you very much for your comment on the sample element number. For bacteria and fungi, the sample element number was 8 in all cases, these tests were performed in parallel, the sample element number was indicated in the table. In the case of parasites, three (Strain 1, Strain 2, Strain 3) were tested in parallel and marked as in Figures 2 and 3. As it is extremely time consuming and cumbersome to test for the parasite, a minimum of three parallel tests was set. We have plotted these as separate columns, so we cannot (because there is no) error bars for individual plots. In Figure 2, the only time we saw sense to calculate an average was for the control (untreated) and the error bar was added to that. Does the reviewer see the need to combine the 3 parallel tests for each parasite and put an error bar on that?
Thank you very much for pointing out that the first chapter of the discussion was too concise and less understandable. As requested, I have reworded and re-structured it to make it more followable, understandable and transparent (lines 386-417).
Furthermore, the main reason for the different results compared to the literature is the geographically different chemical composition of propolis (lines 421-422).
Thank you very much for the suggestion to identify the main components of propolis. This was not part of our basic analysis, as we did not aim to investigate the more than 200 components described in the literature. Our main aim was to make a first round of informative investigation of the scarce domestic studies to see the differences in the international literature compared to other countries. We could select components based on literature data for the main components, but we do not have the laboratory facilities to measure them. If the reviewer insists, we can select some potential ingredients. In this case, we would have to find funding for very expensive measurements and there is uncertainty as to when we will get these results. As our study was primarily not aimed at analytical composition, we would like the reviewer to be able to waive this.

I have highlighted the changes in dark green in the manuscript.
Thank you very much for drawing our attention to the elements that we could improve the value of the manuscript by correcting.

Author Response File: Author Response.docx

Reviewer 4 Report

This study evaluated propolis as an antibacterial efficacy against various pathogenic bacteria from pigeons including Staphylococcus spp., Enterococcus spp., Escherichia coli, Salmonella enterica, Candida albicans fungi, and Trichomonas gallinae. And authors demonstrated that propolis could restrict Gram-positive bacteria's growth more efficiently than Gram-negative bacteria.  It is a very interesting experimental hypothesis and results, however, there are some critical and crucial concerns.

1. Please add the exact strain name of the pathogenic bacteria listed in Table 1.

2. I have curious about the ethanol extract. the anti-bacterial effect of this study is derived from propolis or remaining ethanol?  Please define the exact internal metabolite validation for propolis extract.

3. Please add an error bar in all figures. And please use the figure-specific program, not Excel.

4. There is numerous confusion in Tables 3, 4. Please change the more acceptable form for understanding readers. 

There were no language problem throughout the entire manuscript.

Author Response

Dear Reviewer,

Thank you very much for pointing out the inaccuracies and corrections.
The asterisked additions in Table 1 refer to clinical isolates of poultry origin and the single ATCC strain number has been provided. We apologise for the inaccuracy and the omitted strain number.
We also prepared the same dilution series of ethanol used as solvent under each strain tested. This allowed us to see visually exactly how far ethanol had an inhibitory effect and how far only propolis had an inhibitory effect (lines 184-186).
For parasitological studies, three strains were tested (Strain 1, Strain 2, Strain 3) as indicated. In this case it was considered better to plot the strains separately and not to take averages. Therefore, there are no error bars because they do not belong to one data. We can consider the three strains as three replicates. If the reviewer insists, we can average the results and then we can put error bars on the three replicate measurements.
In the case of Tables 3-4, the p-values are plotted, in a matrix, comparing the results of the different ethanol percentage extractions. In terms of space and what we were comparing, this was the most transparent representation. You can edit it into a plain table, but in that format you get a table of at least 2-3 pages. It was raised by another reviewer that these are not really that important tables, so we could attach them as supplementary material. What is your opinion on this? Anyway, I have simplified the tables by taking out all the p-letters indicating p-values, as it is clearly stated in the explanation of the table that it contains p-values.

I have highlighted the changes in red in the manuscript.
Thank you very much for your helpful advice, we trust that these changes will increase the value of the manuscript and make it even clearer.

Author Response File: Author Response.docx

Reviewer 5 Report

The current study entitled "In vitro efficacy of Hungarian propolis against bacteria, yeast and Trichomonas gallinae isolated from pigeons – a possible antibiotic alternative?" presents the in vitro activity of ethanolic extracts of propolis against bacteria, fungi and protozoa. The study is interesting, but the more extensive study is required in order to make it acceptable.

1. Introduction: line 53-54, What do the authors mean C. albicans from fungi? The sentence should be edited. 

2. The authors need to include a table showing various active constituents in propolis.

3. How did the authors remove the ethanol from the extract?? Need to clearly mention in the methodology section.

4. Why did the authors, not perform the agar well diffusion assay to determine the antibacterial and antifungal activity of propolis extract?

5. Minimum bactericidal and fungicidal concentration of propolis extracts should be determined.

There are minor English language mistakes that should be corrected.

Author Response

Dear Reviewer,
Thank you very much for your constructive comments.
Lines 53-54 were indeed ambiguously worded and have been reworded.
This was discussed in detail in the literature review on the active components of propolis. It is not entirely clear whether the literature review section would need an overview table on this, or our propolis extract from the study as a result. For the latter, we did not perform a precise ingredient definition, we only looked at total flavanoid content, which is included in the manuscript. The search for individual components is extremely costly by chromatographic analysis. If the reviewer insists, we can try to test for some of the components often mentioned in the literature, but we need to raise funds and we do not know how long this will take. Please be sure to let us know whether you insist on this or whether you can dispense with it.
Ethanol has not been removed from the extract. It is clearly indicated in the methodology that we also prepared a dilution series with pure ethanol as solvent as a control in each assay to exclude its effect. This was done in all cases and, as described, at low dilution levels of ethanol, where MIC/MEC values were shown, it no longer had any effect. We do not fully understand what the reviewer concluded that we removed the ethanol. The control placed next to the mnden excluded its effect with absolute certainty, so our results at these dilution levels were not affected. I hope I have dispelled any doubts with this explanation.
Although international standards recognize the agar well diffusion assay method, the broth dilution method is a more reliable and quantitative method. It was therefore chosen because it gives much more reliable and accurate results.
MIC values for propolis have been determined and are listed in Table 2 by species. MIC50 and MIC90 values were calculated based on the results of 8 parallel studies.
Inter-text modifications are indicated by blue highlighting (lines 54-55).

Thank you very much for the excellent feedback and constructive suggestions. We hope that we have been able to address any remaining uncertainties.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors answered all of my previous concerns.

Author Response

Dear Reviewer,

Thank you very much for agreeing with the importance of the research topic and for your constructive suggestions to improve our manuscript.

Reviewer 5 Report

They have responded to my comments, but their response is unsatisfactory. 

The authors need to provide the data of agar well diffusion assay as an evidence to show the activity of propolis extract.

The authors need to provide that shows important active constituents in propolis extract.

English language seems to be fine.

Author Response

Dear Reviewer,

Thank you very much for your constructive comments, which we will certainly consider incorporating into similar future studies of this kind.

The first suggestion is to conduct agar well diffusion assay method studies on the efficacy of propolis extract. While this methodology has long been used to test the antimicrobial efficacy of various plant extracts, the methodology itself is a much older method compared to the microdilution method. Several comparative studies have shown that agar diffusion methods, in particular disk diffusion methods, do not provide as reliable results as the microdilution method, which is more practical and also provides quantitative results. In the case of the former, only a yes-no answer is obtained and the diffusion itself can be affected by many factors. The more accepted method of the international standard is the microdilution method, which we have used ourselves to determine the efficacy of our propolis extract. We believe that this is a more forward-looking, expedient and forward-looking methodology.

The second suggestion that the reviewer insists on is to determine the exact composition of the propolis extract that we use, what the main active ingredients might be that are responsible for the effect. Although the composition of propolis is geographically very diverse and many factors influence its exact composition, the international literature has examined its composition in great detail. These studies have focused specifically on analytical studies and are extremely expensive. With more than 200 possible components, testing all of them would be impossible, at least at a cost of tens of thousands of euros, for which we certainly do not have the resources. Based on the literature, we can select a few potential main active substance candidates, but we do not have a laboratory available for the study. We have already enquired with an external company and were informed that they can undertake such a study with a lead time of about 2-3 months due to the workload of the laboratories.

Dear Reviewer, we do not wish to have such a long delay in the publication of our manuscript, so if there is a way we would respectfully ask you to waive this request. We would like to emphasize again that our research was not aimed at analytics, there are many manuscripts on propolis in the literature which also do not investigate its composition. The aim of our research was that, since no one in our country has published the efficacy of the domestic extract, we would like to provide a basis for comparison with our propolis for the international literature. Just as the other four reviewers accepted that this research is not about analytical testing, we are confident that the final reviewer can dispense with an extremely costly and currently time-consuming additional study.

Round 3

Reviewer 5 Report

The authors did not respond to the comments. 

Minor editing is needed.

Author Response

Dear Reviewer,

Thank you very much for your constructive comments, which we will certainly consider incorporating into similar future studies of this kind.

The first suggestion is to conduct agar well diffusion assay method studies on the efficacy of propolis extract. While this methodology has long been used to test the antimicrobial efficacy of various plant extracts, the methodology itself is a much older method compared to the microdilution method. Several comparative studies have shown that agar diffusion methods, in particular disk diffusion methods, do not provide as reliable results as the microdilution method, which is more practical and also provides quantitative results. In the case of the former, only a yes-no answer is obtained and the diffusion itself can be affected by many factors. The more accepted method of the international standard is the microdilution method, which we have used ourselves to determine the efficacy of our propolis extract. We believe that this is a more forward-looking, expedient and forward-looking methodology.

The second suggestion that the reviewer insists on is to determine the exact composition of the propolis extract that we use, what the main active ingredients might be that are responsible for the effect. Although the composition of propolis is geographically very diverse and many factors influence its exact composition, the international literature has examined its composition in great detail. These studies have focused specifically on analytical studies and are extremely expensive. With more than 200 possible components, testing all of them would be impossible, at least at a cost of tens of thousands of euros, for which we certainly do not have the resources. Based on the literature, we can select a few potential main active substance candidates, but we do not have a laboratory available for the study. We have already enquired with an external company and were informed that they can undertake such a study with a lead time of about 2-3 months due to the workload of the laboratories.

Dear Reviewer, we do not wish to have such a long delay in the publication of our manuscript, so if there is a way we would respectfully ask you to waive this request. We would like to emphasize again that our research was not aimed at analytics, there are many manuscripts on propolis in the literature which also do not investigate its composition. The aim of our research was that, since no one in our country has published the efficacy of the domestic extract, we would like to provide a basis for comparison with our propolis for the international literature. Just as the other four reviewers accepted that this research is not about analytical testing, we are confident that the final reviewer can dispense with an extremely costly and currently time-consuming additional study.