The Intestinal Biofilm of Pseudomonas aeruginosa and Staphylococcus aureus Is Inhibited by Antimicrobial Peptides HBD-2 and HBD-3

Round 1

Reviewer 1 Report

In this manuscript " The Intestinal Biofilm of Pseudomonas aeruginosa and Staphylococcus aureus is Inhibited by Antimicrobial Peptides HBD-2 and HBD-3

” by Alessandra Fusco et.al., examined a new approach in the prevention of and therapy for biofilm-borne infections of P. aeruginosa and S. aureus, based on the increased intake of antimicrobial peptides HBD-2 and HBD-3

The comments and suggestions for this manuscript are as follows-

1. The author should provide a more comprehensive introduction with some more recent published data and appropriate references. Likewise, the separate conclusion portion can be added to this manuscript.

 

2. Page 1, lines 22-24. The statement “The possibility of using endogenous antimicrobial peptides as new anti-biofilm therapy, in isolation or combination with conventional antibiotics, can be an interesting prospect in the treatment of chronic and multi-drug resistant infections”. Did the author try the peptides HBD-2 and HBD-3 with other conventional antibiotics?

 

3. Page-2, line 45. The author should provide standard Greek symbols for the peptide names.
4. The author should provide the details of appropriate statistical tests, used in figure analysis.
5. The author should follow the uniform format for the bacterial name.

Author Response

Dear Editor, Dear Reviewers,

we thank so much the referee for your valuable comments. As reported in the following, we answered all your questions and we've made all the suggested changes (highlighted in the manuscript) for re-submission of our paper.

 

REVIEWER 1:

 

• The author should provide a more comprehensive introduction with some more recent published data and appropriate references. Likewise, the separate conclusion portion can be added to this manuscript.

 

The references in the paper have been replaced, where possible, with more recent ones, to give greater significance to the state of the art.

Furthermore, as requested, the final part of the Conclusions has been separated from the Discussion.

 

• Page 1, lines 22-24. The statement “The possibility of using endogenous antimicrobial peptides as new anti-biofilm therapy, in isolation or combination with conventional antibiotics, can be an interesting prospect in the treatment of chronic and multi-drug resistant infections”. Did the author try the peptides HBD-2 and HBD-3 with other conventional antibiotics?

 

Now we have not yet tested the association, but it is certainly our future prospect. Thanks for the great suggestion.

 

 

• Page-2, line 45. The author should provide standard Greek symbols for the peptide names.

 

Done.

 

• The author should provide the details of appropriate statistical tests, used in figure analysis.

 

We apologize for the oversight. The paragraph on statistical analysis has been added to the Materials and Methods section.

 

• The author should follow the uniform format for the bacterial name.

 

After writing the complete name of a microorganism in the first mention (both in the Abstract and in the paper), the genus name can be shortened to just the capital letter.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors provide a study evaluating the abilites of two antimicrobial peptides to inhibit Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation on Caco-2 cells. 

The concepts and experiments are appropriate.

As a non-native English speaker myself, I would like to make a friendly recommendation to the writers, since I know reviewers who would reject a paper like this. Many sentences are just a translation to English, but that does not make it appropriate. I am not qualified to go through everything, but I have amended the things that I was certain about. I have suggested other comments to make the reading more fluid. In South Europe, we use many words to express an idea, with literary constructions, but you need to keep it to a minimum here. If you are unsure how to construct a long sentence, make short sentences. Some paragraphs had more than 4-5 lines without a dot, with lots of "comments" within the sentence.  If it is not well constructed, it will look poor on your study, so just keep it simple and safe. 

A few comments:

-Line 14. What do you mean by "considerably significant"? Please, rephrase for clarity.

-Line 21. "...showed differentiated antibiofilm activity, but they acted in a differentiated manner against P. aeruginosa and S. aureus".

-Line 30. You state "bacteria (about 99%)". I very much doubt that the 99% of the microorganisms in the gut are bacteria. I have checked the reference that you provide, but it does not say anything about 99% bacteria. I would suggest to clarify this statement, either saying that 99% of the reported microorganisms are bacteria, and provide a reference for it, or by removing that 99%. Even if you choose to include it and can find a reference, have in mind that there could be a bias in that report, as culturing or sequencing faecal samples have methodological limitations that are constantly being improved. My suggestion is to lose that 99%. 

-Line 34. The expression " amplify the metabolic potential of the host" is a bit confusing. Please, elaborate or remove for clarity. The following sentence says that it is a "property" and "an evolution of bacteria".  I understand what you mean and the idea that you want to express but, as it is now, it is not fully appropriate. Maybe link it to the previous sentence and say something along the lines of "which could be considered a trait for coexistence with a human host" or something similar. 

-Line 38. Remove "made".

-Line 52. Substitute "in which" with "where"

-Line 53. Remove the "with the" and the "of".

-Lines 60, 242. "Microorganisms" do not need hyphen and replace 

-Line 60-63. Replace sinde "pathogenic ... until end of sentence" with "pathogenic environmental microorganisms and opportunistic members of the microbiota". 

-Line 65. Remove the paragraph. 

-Line 66. Remove "In this scenario".

-Line 67. These two infections are frequently found? How frequently? Include a reference or provide some context, as probably these are not the most frequent gut infections, unless you mean "biofilm infections", which I suspect. Please, amend for clarity to the reader. 

-Line 76. Include a dot after the reference and separate in two senteces. 

-Line 84. Remove "in fact".

-Lines 85 and 87. Replace "such as with" with "like".

-Line 96. Gene cluster in italics.

-Line 98. Replace "that can" with "to"

-Line 104. As it is the first time that you mention it, include the full name Salmonella enterica subsp. enterica serovar Typhimurium,  and later on you can write S. Typhimurium, where the serovar name is not in italics. 

-Line 106. You mention "based on the increase intake of antimicrobial peptides", but I understand that it is the intestinal cells the ones that are producing them, right? Please, modify it for clarity.

-Blasticidin is used with and without capital B. Choose one.

-Lines 143-148. Not a single dot. You can separate two sentences after "...CFU/mL.  Aliquots...".

-Line 159. Remove "then".

-Line 163. Replace "by" with "using".

-Table 1. Gene names in italics.

-Lines 193, 197, 200, 206, 209. Bacterial names in italics.

-Figures 1 and 2. I believe you can abbreviate the names.

-Line 217. Replace "The analysis of biofilms..." with "the biofilm analyses"... following line "show". "an extended P. aeruginosa biofilm".

-Line 220. Comma after "and", not before.

-Line 222. Replace "For S. aureus, the biofilm, present..." with "S. aureus biofilm, present....".

-Line 223. Remove "in fact".

-Line 231. "The evaluation of expression of the P. aeruginosa biofilm-associated genes conducted by Real-Time 230 PCR (Figure 4), shows in P. aeruginosa a marked downregulation".

-Figure 4.  I think a space might be missing after the dot in the bacterial names. Genes in italics. Why some genes have capital first letter (also in the text)? Please amend.

-Line 249. "Evidence is accumulating showing..." is an idiomatic expression. Replace with "Increasing evidence suggests...".

-Line 250. Quorum does not require capital Q.

-Line 256. Remove the comma after "genes".

-Line 266. "...over the past decade to develop strategies.."

-Line 289. Remove "mainly".

-The final paragraph, you go over 5 lines without a dot, trying to connect several sentences. Rearrange the text. 

-Comment on the figures. Figure 2 title is in capital letters. Titles are not very informative. Please elaborate a bit more. A suggestion on Figure 1 might be "HBD-2 and HBD-3 effects on P. aeruginosa and S. aureus biofilm formation". Something along those lines. 

 

 

 

 

 

 

 

Author Response

REVIEWER 2:

 

As a non-native English speaker, myself, I would like to make a friendly recommendation to the writers, since I know reviewers who would reject a paper like this. Many sentences are just a translation to English, but that does not make it appropriate. I am not qualified to go through everything, but I have amended the things that I was certain about. I have suggested other comments to make the reading more fluid. In South Europe, we use many words to express an idea, with literary constructions, but you need to keep it to a minimum here. If you are unsure how to construct a long sentence, make short sentences. Some paragraphs had more than 4-5 lines without a dot, with lots of "comments" within the sentence.  If it is not well constructed, it will look poor on your study, so just keep it simple and safe.

 

First, thank you very much for your suggestion which is always welcome to help us improve. We have covered all the corrections you indicated, as regards the editing of the English language, we had already made use of this service, the certificate of which is attached, via papertrue.com.

 

• Line 14. What do you mean by "considerably significant"? Please, rephrase for clarity.

 

Done

 

• Line 21. “…showed differentiated antibiofilm activity, manner against P. aeruginosa and S. aureus”.

 

Done

 

• Line 30. You state "bacteria (about 99%)". I very much doubt that the 99% of the microorganisms in the gut are bacteria. I have checked the reference that you provide, but it does not say anything about 99% bacteria. I would suggest to clarify this statement, either saying that 99% of the reported microorganisms are bacteria, and provide a reference for it, or by removing that 99%. Even if you choose to include it and can find a reference, have in mind that there could be a bias in that report, as culturing or sequencing faecal samples have methodological limitations that are constantly being improved. My suggestion is to lose that 99%.

 

We followed your suggestion and eliminated the percentage figure.

 

 

• Line 34. The expression " amplify the metabolic potential of the host" is a bit confusing. Please, elaborate or remove for clarity. The following sentence says that it is a "property" and "an evolution of bacteria".  I understand what you mean and the idea that you want to express but, as it is now, it is not fully appropriate. Maybe link it to the previous sentence and say something along the lines of "which could be considered a trait for coexistence with a human host" or something similar. 

 

As suggested, we have corrected the sentence to make it more understandable.

 

• Line 38. Remove "made".

Done

 

• Line 52. Substitute "in which" with "where"

 

Done

 

• Line 53. Remove the "with the" and the "of".

 

Done

 

• Lines 60, 242. "Microorganisms" do not need hyphen and replace 

 

Done

 

• Line 60-63. Replace sinde "pathogenic ... until end of sentence" with "pathogenic environmental microorganisms and opportunistic members of the microbiota". 

 

Done

 

• Line 65. Remove the paragraph. 

 

Done

 

• Line 66. Remove "In this scenario".

 

Done

 

• Line 67. These two infections are frequently found. How frequently? Include a reference or provide some context, as probably these are not the most frequent gut infections, unless you mean "biofilm infections", which I suspect. Please, amend for clarity to the reader. 

 

In accordance with your and Reviewer's 3 suggestion, we have included the required references

 

• Line 76. Include a dot after the reference and separate in two sentences. 

 

Done

 

• Line 84. Remove "in fact".

 

Done

 

• Lines 85 and 87. Replace "such as with" with "like".

 

Done

 

• Line 96. Gene cluster in italics.

 

Done

 

• Line 98. Replace "that can" with "to"

 

Done

 

• Line 104. As it is the first time that you mention it, include the full name Salmonella entericaenterica serovar Typhimurium, and later you can write S. Typhimurium, where the serovar name is not in italics. 

 

Done

 

• Line 106. You mention "based on the increase intake of antimicrobial peptides", but I understand that it is the intestinal cells the ones that are producing them, right? Please, modify it for clarity.

 

Right observation, we corrected with "increase production".

 

• Blasticidin is used with and without capital B. Choose one.

 

Done

 

• Lines 143-148. Not a single dot. You can separate two sentences after "...CFU/mL.  Aliquots...".

 

Done

 

• Line 159. Remove "then".

 

Done

 

• -Line 163. Replace "by" with "using".

 

Done

 

• Table 1. Gene names in italics.

 

Done

 

• Lines 193, 197, 200, 206, 209. Bacterial names in italics.

 

We apologize if we missed something, but it seems to us that all the names of the bacteria are in italics

• Figures 1 and 2. I believe you can abbreviate the names.

 

Done

 

• -Line 217. Replace "The analysis of biofilms..." with "the biofilm analyses"... following line "show". "an extended aeruginosa biofilm".

 

Done

 

• Line 220. Comma after "and", not before.

 

Done

 

• Line 222. Replace "For aureus, the biofilm, present..." with "S. aureus biofilm, present....".

 

Done

 

• Line 223. Remove "in fact".

 

Done

 

• Line 231. "The evaluation of expression of the aeruginosa biofilm-associated genes conducted by Real-Time 230 PCR (Figure 4), shows in P. aeruginosa a marked downregulation".

 

Done

 

• Figure 4.  I think a space might be missing after the dot in the bacterial names. Genes in italics. Why some genes have capital first letter (also in the text)? Please amend.

 

Errors have been corrected in the figure and in the text.

 

• Line 249. "Evidence is accumulating showing..." is an idiomatic expression. Replace with "Increasing evidence suggests...".

 

Done

 

• Line 250. Quorum does not require capital Q.

 

Done

 

• Line 256. Remove the comma after “genes”.

 

Done

 

• Line 266. "...over the past decade to develop strategies.."

 

Done

 

• Line 289. Remove "mainly".

 

Done

 

• The final paragraph, you go over 5 lines without a dot, trying to connect several sentences. Rearrange the text.

 

Done 

 

• Comment on the figures. Figure 2 title is in capital letters. Titles are not very informative. Please elaborate a bit more. A suggestion on Figure 1 might be "HBD-2 and HBD-3 effects on aeruginosa and S. aureus biofilm formation". Something along those lines. 

 

The suggested changes have been made to both figures

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors, I found your study quite well built and enjoyed reading it. However, there are some parts needed more attention. 

Here are my comments to help publishing your manuscript.

1. Line 67-70; This sentence could use a reference.
2. Line 94-96; When you read this sentence, you got a meaning that PIA alone could buid the whole biofilm structure. Biofilm formation is a complex phenomenon including many metabolic pathways and physiological activities. Instead, you can say sth. like “PIA is one of the most important biofilm compenent ….”
3. Line 104; “Typhimurium” should be decapitalized.
4. Line 125-127; You mentioned that you have tested the Cell-free supernatants for their HBD-2 and 3 content with ELISA. Showing your results with a graph will help your audience. This will also prove that the HBD-2 expressed in transfected Caco-2 cell lines can be secreted out of the cells. Or these antimicrobial peptides comes from the dead cells? You might want to add an experiment by bursting the cells and check the HBD levels with supernatant samples, if applicable. This is not required for your manuscript to be published but it will be a leader study for the other researchers.
5. Line 143-148; Too long to follow. Please break this sentence into smaller one. The meaning is not clear. Please rephrase.
6. Line 148; Please define how you created the anaerobic conditions. Did you use a jar and a pouch system or did you burn the oxygen in a desiccator? Or any other method.
7. Line 158; “thrice” did you mean “twice”.
8. Line 157-161; You could use a reference for the method.
9. Line 161; Please put a space before the sentence.
10. Line 162; “Fluorescens” should be corrected as “Fluorescence”
11. Line 183; “Primers sequences” should be corrected as “Primer sequences”. Same thing can be applied to head line of the table. Inside the table please remove the spaces before “˚” signs. Please be consistent. Also consider typing those gene names in italic characters.
12. Line 193; Species names should be italicized. Same thing goes for the line 200, 206, and 209 too. Please also check the rest of the paper.
13. In figure 3. “P. aeruginosa” and “S. aureus” first letters should be italicized.
14. In figure 3, it seems that the differentiation of live and dead cells are not distinctive enough (or most the cells are already dead). In other words, the color differences can not be easily identified. To be frank, these results are confusing. Live and dead staining should not overlap that much red and green signals in the mock samples assuming most of the bacterial cells are still alive. In the red filter of the mock samples of aeruginosa cells, there are many dead cells. This is a biofilm only treated with Caco-2 cell line supernatant. This means the supernatant of Caco-2 cells (with no HBD2 or 3 expression) can kill almost all bacterial cells in these biofilms. The case is more like a biofilm removal in HBD-2 and HBD-3 samples. You might want to try to optimize the staining or treatment time or even the concentration of the dyes within the limits of manufacturer suggestion. You can also check the exposure time when you are capturing the florescent images.

 

Fluorescence analysis of live and dead cells from your treatments should include more details in material and methods section. There is no details about incubation time and conditions of the biofilms with cell culture supernatants for this experiment. It is clear to follow biofilm experiment with crystal violet experiment but not with fluorescent one. Please define the discs and other experimental details. For example; how old were the supernatant of cell culture, did you just use culture media to kill the already grown biofilms or did you grow the biofilms with the supernatants. How old were the biofilms on the discs. Were the discs glass or steel?

Line 218-219; Is is said that “clear majority of live cells in the supernatants of untransfected Caco-2 cells.” Assuming these samples were shown under the name of “Mock” in Figure 3a. For example, majority of cells in Mock 2 in Figure 3a shows red color in the top middle figure, which means dead cells. If you only comment on the combined images on the very right, you should also adjust exposure and incubation times so that you don’t get that much red signal with the red filter. Propidium iodide only stains the dead cells under optimal conditions. If you incubate longer than required (or longer exposure during image capture) even the live cells would give red signal under microscopy.

Comparing Mock samples of P. aeruginosa and HBD-3, it seems that you have equal density of dead cells. But you said “greater number of dead cells” in line 221.

There might be another way of commenting these results. If you only keep green channel images, it will totally overlap with your graphs in Figure 2. However, including red channel causing confusion. Live and dead staining requires a precise and fine handling. You might want to repeat the experiment if you want to include red channel.

You can also try to use same exposure time of green and red channel. Avoiding “auto exposure or saturation functions of the software” might help.

15. Did you include a housekeeping gene in your Q-PCR experiment? If so, please add them.
16. Figure 4. Giving the gene groups as separate figures will help to understand the differences. For example, the difference level of LasI transcription can be easily compared between treatments. So that, you can use a smaller “Relative Gene Expression” axis for the other genes.
17. Graphs could use pattern rather than colors, this will help readers when they print it in black and white.

 Thanks

Author Response

REVIEWER 3:

 

Dear authors, I found your study quite well built and enjoyed reading it. However, there are some parts needed more attention.

 

Dear Reviewer, thank you very much for the positive opinion on our manuscript and for the valuable suggestions you have provided.

 

Here are my comments to help publishing your manuscript.

 

• Line 67-70; This sentence could use a reference.

 

Done

 

• Line 94-96; When you read this sentence, you got a meaning that PIA alone could build the whole biofilm structure. Biofilm formation is a complex phenomenon including many metabolic pathways and physiological activities. Instead, you can say she. like “PIA is one of the most important biofilm compenent ….”

 

Done

 

• Line 104; “Typhimurium” should be decapitalized.

 

Done

 

• Line 125-127; You mentioned that you have tested the Cell-free supernatants for their HBD-2 and 3 content with ELISA. Showing your results with a graph will help your audience. This will also prove that the HBD-2 expressed in transfected Caco-2 cell lines can be secreted out of the cells. Or these antimicrobial peptides come from the dead cells? You might want to add an experiment by bursting the cells and check the HBD levels with supernatant samples, if applicable. This is not required for your manuscript to be published but it will be a leader study for the other researchers.

 

This is a very correct observation. The data has not been shown as it was previously published in our first work on transfected Caco-2 cells, in which we performed the experimental system, evaluating the production levels of antimicrobial peptides. In this regard, we have added the reference of our previous work [ref. 10]. However, if you think it would be helpful, we can add the chart to supplemental materials. 

 

• Line 143-148; Too long to follow. Please break this sentence into smaller one. The meaning is not clear. Please rephrase.

 

According with your suggestions and Reviewer 2's, we have divided in two separate sentences.

 

• Line 148; Please define how you created the anaerobic conditions. Did you use a jar and a pouch system, or did you burn the oxygen in a desiccator? Or any other method.

 

We are sorry for the mistake. Bacterial cultures were conducted under aerobic conditions.

 

• Line 158; “thrice” did you mean “twice”.

 

Done

 

• Line 157-161; You could use a reference for the method.

 

Done

 

• Line 161; Please put a space before the sentence.

 

Done

 

• Line 162; “Fluorescens” should be corrected as “Fluorescence”

 

Done

 

• Line 183; “Primers sequences” should be corrected as “Primer sequences”. Same thing can be applied to headline of the table. Inside the table, please remove the spaces before “˚” signs. Please be consistent. Also consider typing those gene names in italic characters.

 

Done

• Line 193; Species names should be italicized. Same thing goes for the line 200, 206, and 209 too. Please also check the rest of the paper.

 

Done

 

• In figure 3. “P. aeruginosa” and “S. aureus” first letters should be italicized.

 

Done

 

• In figure 3, it seems that the differentiation of live and dead cells are not distinctive enough (or most the cells are already dead). In other words, the color differences can not be easily identified. To be frank, these results are confusing. Live and dead staining should not overlap that much red and green signals in the mock samples assuming most of the bacterial cells are still alive. In the red filter of the mock samples of aeruginosa cells, there are many dead cells. This is a biofilm only treated with Caco-2 cell line supernatant. This means the supernatant of Caco-2 cells (with no HBD2 or 3 expression) can kill almost all bacterial cells in these biofilms. The case is more like a biofilm removal in HBD-2 and HBD-3 samples. You might want to try to optimize the staining or treatment time or even the concentration of the dyes within the limits of manufacturer suggestion. You can also check the exposure time when you are capturing the florescent images.

 

We are sorry that the images in Fig. 3 are unclear to you. Unfortunately, as regards the possibility of repeating the experiment, this would not change the result as the treatment time and the concentration of the coloring solution are optimized according to the manufacturer's instructions, and the exposure times to green and red light are the same. themselves. It could be a limitation of our microscope.

In fact, what we think makes the difference and makes the result more understandable is the image resulting from the fusion of the two colors, in which the type of cells (living or dead) visibly takes over.

The fact that in the supernatants of non-transfected cells the percentage of red bacteria is also higher could be due to a higher bacterial density, caused by the absence of antimicrobial peptides. We did however change the sentence in the Results section.

However, if you really believe that this explanation is not satisfactory and that it would be better to show only the images in green, we will make this change in the final version of the paper.

 

• Fluorescence analysis of live and dead cells from your treatments should include more details in material and methods section. There is no details about incubation time and conditions of the biofilms with cell culture supernatants for this experiment. It is clear to follow biofilm experiment with crystal violet experiment but not with fluorescent one. Please define the discs and other experimental details. For example, how old were the supernatant of cell culture, did you just use culture media to kill the already grown biofilms or did you grow the biofilms with the supernatants. How old were the biofilms on the discs? Were the discs glass or steel?

 

A correct description of the method has been added to the Materials and Methods section

 

• Line 218-219; Is said that “clear majority of live cells in the supernatants of untransfected Caco-2 cells.” Assuming these samples were shown under the name of “Mock” in Figure 3a. For example, majority of cells in Mock 2 in Figure 3a shows red color in the top middle figure, which means dead cells. If you only comment on the combined images on the very right, you should also adjust exposure and incubation times so that you don’t get that much red signal with the red filter. Propidium iodide only stains the dead cells under optimal conditions. If you incubate longer than required (or longer exposure during image capture) even the live cells would give red signal under microscopy. Comparing Mock samples of P. aeruginosa and HBD-3, it seems that you have equal density of dead cells. But you said “greater number of dead cells” in line 221.

There might be another way of commenting these results. If you only keep green channel images, it will totally overlap with your graphs in Figure 2. However, including red channel causing confusion. Live and dead staining requires a precise and fine handling. You might want to repeat the experiment if you want to include red channel.

You can also try to use same exposure time of green and red channel. Avoiding “auto exposure or saturation functions of the software” might help.

 

Please, for this question refer to the previous answer

 

• Did you include a housekeeping gene in your Q-PCR experiment? If so, please add them.

 

In the Real-Time PCR assays we did not include housekeeping genes, while the 16S was used following the extraction of the bacterial mRNA, before proceeding with the Real-Time, to verify the success of the extraction. The procedure has been added to the material and methods section.  

 

• Figure 4. Giving the gene groups as separate figures will help to understand the differences. For example, the difference level of Las transcription can be easily compared between treatments. So that, you can use a smaller “Relative Gene Expression” axis for the other genes. Graphs could use pattern rather than colors; this will help readers when they print it in black and white.

 

Indeed, the graph of the Pseudomonas aeruginosa genes does not allow to better appreciate the significance of the relative expression of all the genes analyzed. However, we think that making 4 different graphs can make the figure confusing, and this problem does not occur in the graph of S. aureus. We therefore decided to follow your suggestion by modifying the structure of the y axis, dividing it into 3 sections which makes the results obtained for all genes more appreciable, and we also applied the pattern for all the figures as requested by you.

Author Response File: Author Response.pdf