Evaluating the Antagonistic Activity of Lactic Acid Bacteria in Cadaverine Production by Vibrio Strains during Co-Culture

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

The summary is pleasant to read, it is understandable, it shows methodology, results and conclusions, however, it should be improved by showing concrete results in numbers.   Furthermore, I suggest that the summary be better articulated with the title, this is because the word co-culture is mentioned in the title, however, the summary does not mention this word.

Keywords:

I suggest changing the words cadverin, co-culture, lactic acid bacteria, because these words are already in the title

Introduction:

The introduction is carefully written. It shows the justification, the problem, and the way in which some authors have approached the problem. End with a clear objective.

Materials and Methods

Strains and Growth Conditions: I am struck by the fact that this section indicates that all strains were incubated at 37°C for 24 hours. Please justify this, because each strain has different optimal growth conditions.

Screening of LAB against CAD-producing Vibrio Strains: What method was used to seed the lactic acid bacteria? What size plate was used? How was the circular zone of inhibition measured?

Co-culture of Vibrio spp. and LAB 126: This section should be rewritten in detail. It is not understood what the investigators did. Please remember the importance of detailing the methodology, since all research must be reproducible.

Quantitative Analysis of CAD Using HPLC: This section must be described in detail, it is not reproducible. For example, what was wine used for? What was grape must used for?

Results and Discussion

Screening of LAB Against CAD-producing Vibrio Strains: I am struck by the fact that a response variable that was indicated in the methodology as a quantitative measurement (measurement of halo diameter), is presented in the results as a qualitative variable. I consider that this should be corrected and left quantitative, in order to be able to do the corresponding statistical analysis.

In vitro CAD Production and Bacteria Cell Growth in Lysine Decarboxylase Broth: I suggest that (in the text), the reduction of cadaverine due to the co-culture of VC and VP is given in percentage terms, this allows the effect to be better visualized.

How are the results of in vitro tests explained?

In vivo CAD Production and Bacterial Cell Growth in Shrimp: Similarly, for the In vivo test I suggest presenting the cadaverine reduction in percentage. How are the results of In Vivo tests explained?

Furthermore, when the growth results of VC and VP are presented due to the effect of coculture with lactic acid bacteria, I suggest expressing these results in terms of reduction in logarithmic units and not in number of microorganisms. 

Regarding the following authors cited: Also, Sorée (2023), Mahmoud, (2014), and Shirazinejad (2010), I consider that they are not relevant to compare results, since in these studies they directly use lactic acid. In the present investigation, the lactic acid produced by fermentation was not quantified, so it cannot be compared, nor can it be stated that it serves for disinfection. 

Conclusion

The conclusion could be improved. I suggest removing the talk about using lactic acid to kill Vibrio cells, since this was not what they investigated.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

<Summary> Comments 1:  The summary is pleasant to read, it is understandable, it shows methodology, results and conclusions, however, it should be improved by showing concrete results in numbers.  Furthermore, I suggest that the summary be better articulated with the title, this is because the word co-culture is mentioned in the title, however, the summary does not mention this word. Response 1: We revised the abstract as you suggested. Please review.

<Keywords> Comments 2: I suggest changing the words cadverin, co-culture, lactic acid bacteria, because these words are already in the title Response 2: As you suggested, I changed the 'keywords' by excluding the words mentioned in the title.

<Introduction> Comments 3: The introduction is carefully written. It shows the justification, the problem, and the way in which some authors have approached the problem. End with a clear objective. Response 3: We made some changes to the introduction to clearly state the goals of the study.   <Materials and Methods> Comments 4: Strains and Growth Conditions: I am struck by the fact that this section indicates that all strains were incubated at 37°C for 24 hours. Please justify this, because each strain has different optimal growth conditions. Response 4: The strains and their optimal growth conditions used in the experiment are as follows. The optimal culture temperature for all strains matches the temperature and time conditions we set (source: the website of the standard strain supplier). Therefore, we cultured the strains at 37°C for 24 hours and used Lysine Decarboxylase Broth (LDB) to observe the biogenic amine production reactions (decarboxylase reactions) of these strains. Strain Temperature Time Medium Lactiplantibacillus plantarum NCIMB 6105 30 - 40℃ 24 hours MRS Le. mesenteroides ATCC 10830 37℃ 24 hours MRS V. cholerae NCCP 13589 37℃ 24 hours LB V. parahaemolyticus ATCC 27969 37℃ 24 hours LB   Comments 5: Screening of LAB against CAD-producing Vibrio Strains: What method was used to seed the lactic acid bacteria? What size plate was used? How was the circular zone of inhibition measured? Response 5: Additional details are provided in Section 2.3.   Comments 6: Co-culture of Vibrio spp. and LAB 126: This section should be rewritten in detail. It is not understood what the investigators did. Please remember the importance of detailing the methodology, since all research must be reproducible. Response 6: Additional details are provided in Section 2.4.   Comments 7: Quantitative Analysis of CAD Using HPLC: This section must be described in detail, it is not reproducible. For example, what was wine used for? What was grape must used for? Response 7: There was a typo (wine, grape), so I edited it again on line 307.

<Results and Discussion>

Comments 8: Screening of LAB Against CAD-producing Vibrio Strains: I am struck by the fact that a response variable that was indicated in the methodology as a quantitative measurement (measurement of halo diameter), is presented in the results as a qualitative variable. I consider that this should be corrected and left quantitative, in order to be able to do the corresponding statistical analysis.

Response 8: As a result of Lactic acid bacteria screening for Cadaverine-producing Vibrio strains, two strains (Lactiplantibacillus plantarum NCIMB 6105 and Leuconostoc mesenteroides ATCC 10830) with large inhibition zones were qualitatively selected. In the subsequent study (section 2.5.), the amount of cadaverine produced during mono-culture and co-culture was quantitatively analyzed focusing on these two strains.

 

Comments 9: In vitro CAD Production and Bacteria Cell Growth in Lysine Decarboxylase Broth: I suggest that (in the text), the reduction of cadaverine due to the co-culture of VC and VP is given in percentage terms, this allows the effect to be better visualized.

Response 9: Percentage values ​​are displayed on lines 425-433..

 

Comments 10: How are the results of in vitro tests explained?

Response 10: As an in vitro experiment, we cultured Vibrio spp and lactic acid bacteria mono-cultured or co-cultured in lysine decarboxylase broth as in Section 2.4. The results are described in Section 3.2.

 

Comments 11: In vivo CAD Production and Bacterial Cell Growth in Shrimp: Similarly, for the In vivo test I suggest presenting the cadaverine reduction in percentage. How are the results of In Vivo tests explained?

Response 11: In vivo test results are described in Section 3.3, and as suggested, percentages are included so that the reduction rate can be easily checked (lines 476-480).

 

Comments 12: Furthermore, when the growth results of VC and VP are presented due to the effect of coculture with lactic acid bacteria, I suggest expressing these results in terms of reduction in logarithmic units and not in number of microorganisms. 

Response 12: I agree with the reviewer's comment. Our result data already indicate the reduction rate on a logarithmic scale (in figure 2). If this is not the point you were referring to, could you please specify the particular aspect so that we can make the necessary corrections?

 

Comments 13: Regarding the following authors cited: Also, Sorée (2023), Mahmoud, (2014), and Shirazinejad (2010), I consider that they are not relevant to compare results, since in these studies they directly use lactic acid. In the present investigation, the lactic acid produced by fermentation was not quantified, so it cannot be compared, nor can it be stated that it serves for disinfection.

Response 13: We have further reviewed the pointed-out section and have removed the citation because it is not closely related to the content of the manuscript.

 

<Conclusion>

Comments 14: The conclusion could be improved. I suggest removing the talk about using lactic acid to kill Vibrio cells, since this was not what they investigated.

Response 14 : The mention of 'lactic acid' in line 512 was a typographical error; it should have been 'lactic acid bacteria.' This has been corrected.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Please see below my comments.

 

In general, the document contains too many abbreviations.
Why do you use abbreviations for putrescine (PUT) and cadaverine (CAD) but not for other compounds like Histamine? Anyway, if the abbreviation addresses only a word, in my point of view it is pointless.

 

ABSTRACT

It is suggested to skip abbreviations in the abstract.  (LAB) (FBPs) (CAD) (LDB) (BAs) can be reported for the first time in the introduction.

 

 

INTRODUCTION

Lines 55-56

Microorganism in microorganisms

Delete bacteria after Vibrio sp. In the whole document. Any microbiologist knows Vibrio is a bacteria.  

Maladiesthat in maladies that (moreover It is suggested to change “maladies” in “diseases”)

 

Line 58: Vibrio in italics

 

From line 58 to line 64: it is a mixture of concepts disconnected and of little interest.
I would delete all.  For example: the additional information about “Fresh fish predom-61 inantly harbored the genera Bacillus, Vibrio and Flavobacterium.” Does add value to the introduction?

 

Line 70. “Some of bacteria”. Please be more specific. In the same sentence, the secondary compounds are usually defined as “antimicrobial compounds” (AMPs). If you refer to lactic and acetic acid, the sentence should be rephrased as well.

 

Line 74: are you assuming the LAB are “probiotics” without introducing their pathogens counteraction capabilities? It is suggested to tell their main potentials (lines 76-81) and then declare them as probiotics/beneficial (line 74).

 

Lactobacillus plantarum isolated in shrimps? Really? Do you have a citation for this? Because this bacterium is usually associated with plants…

 

Moreover please upgrade the scientific nomenclature. For example Lactobacillus plantarum is now Lactiplantibacillus plantarum. It was renamed in 2021.

 

Moreover, the bibliography in the whole introduction is scarce.

 

M&M

The paragraph a 2.1 is extremely confusing.
It is suggested to make a table with all the strains used (pathogenic and LAB), with their source of isolation /purchase, and their aim in the work (Eg: Bacillus cereus à negative control)

 

Paragraph 2.2.

It is suggested to rename it “cultivation of Vibrio strains”.

 

Table 1: please also add the melting temperature for every amplicon. It is very important information for the specificity of the reaction and reproducibility of the experiment in other laboratories worldwide.

 

The section describing the statistics used and data analysis software/packages  to generate figures is completely missing.
please provide details on the applied statistic.

 

RESULTS and D
 Figure 1: please increase quality and mark significant differences with a “ * “.

The same for Figure 2.

 

 

The reduction of pathogenic bacteria by LAB is a good result. But would you define it as biological relevance? Or the CFU of Vibrio after the inoculation of LAB is anyway a concern for shrimps and human health? In the same way, cadaverine and putrescine are reduced below the threshold level of danger? Please better discuss this in the manuscript.

Comments on the Quality of English Language

Please see below my comments.

 

In general, the document contains too many abbreviations.
Why do you use abbreviations for putrescine (PUT) and cadaverine (CAD) but not for other compounds like Histamine? Anyway, if the abbreviation addresses only a word, in my point of view it is pointless.

 

ABSTRACT

It is suggested to skip abbreviations in the abstract.  (LAB) (FBPs) (CAD) (LDB) (BAs) can be reported for the first time in the introduction.

 

 

INTRODUCTION

Lines 55-56

Microorganism in microorganisms

Delete bacteria after Vibrio sp. In the whole document. Any microbiologist knows Vibrio is a bacteria.  

Maladiesthat in maladies that (moreover It is suggested to change “maladies” in “diseases”)

 

Line 58: Vibrio in italics

 

From line 58 to line 64: it is a mixture of concepts disconnected and of little interest.
I would delete all.  For example: the additional information about “Fresh fish predom-61 inantly harbored the genera Bacillus, Vibrio and Flavobacterium.” Does add value to the introduction?

 

Line 70. “Some of bacteria”. Please be more specific. In the same sentence, the secondary compounds are usually defined as “antimicrobial compounds” (AMPs). If you refer to lactic and acetic acid, the sentence should be rephrased as well.

 

Line 74: are you assuming the LAB are “probiotics” without introducing their pathogens counteraction capabilities? It is suggested to tell their main potentials (lines 76-81) and then declare them as probiotics/beneficial (line 74).

 

Lactobacillus plantarum isolated in shrimps? Do you have a citation for this? Because this bacterium is usually associated with plants…

 

Moreover please upgrade the scientific nomenclature. For example Lactobacillus plantarum is now Lactiplantibacillus plantarum. It was renamed in 2021.

 

Moreover, the bibliography in the whole introduction is scarce.

 

M&M

The paragraph a 2.1 is extremely confused.
It is suggested to make a table with all the strains used (pathogenic and LAB), with their source of isolation /purchase, and their aim in the work (Eg: Bacillus cereus à negative control)

 

Paragraph 2.2.

It is suggested to rename it “cultivation of Vibrio strains”.

 

Table 1: please also add the melting temperature for every amplicon. It is very important information for the specificity of the reaction and reproducibility of the experiment in other laboratories worldwide.

 

The section describing the statistics used and data analysis software/packages  to generate figures is completely missing.
please provide details on the applied statistic.

 

RESULTS and D
 Figure 1: please increase quality and mark significant differences with a “ * “.

The same for Figure 2.

 

 

The reduction of pathogenic bacteria by LAB is a good result. But would you define it as biological relevance? Or the CFU of Vibrio after the inoculation of LAB is anyway a concern for shrimps and human health? In the same way, cadaverine and putrescine are reduced below the threshold level of danger? Please better discuss this in the manuscript.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

 

<Summary> Comments 1: In general, the document contains too many abbreviations. Why do you use abbreviations for putrescine (PUT) and cadaverine (CAD) but not for other compounds like Histamine? Anyway, if the abbreviation addresses only a word, in my point of view it is pointless. Response 1: We completely agree with reviewer's comment. While it is generally appropriate to use abbreviations when listing various biogenic amines, in this manuscript, there are not many references to other biogenic amines besides cadaverine. Therefore, we have removed and revised the abbreviations.   <Abstract> Comments 2: It is suggested to skip abbreviations in the abstract.  (LAB) (FBPs) (CAD) (LDB) (BAs) can be reported for the first time in the introduction. Response 2: We have removed all abbreviations from the abstract.   <Introduction> Comments 3: Lines 55-56. Microorganism in microorganisms Delete bacteria after Vibrio sp. In the whole document. Any microbiologist knows Vibrio is a bacteria. Maladies that in maladies that (moreover It is suggested to change “maladies” in “diseases”) Response 3: We have deleted the word 'bacteria' in line 91 (lines 55-56 of original manuscript) and changed 'maladies' to 'disease' as per your suggestion.   Comments 4: Line 58: Vibrio in italics Response 4: Vibrio has been changed to italics (in lines 58 of original manuscript).   Comments 5: From line 58 to line 64: it is a mixture of concepts disconnected and of little interest. I would delete all.  For example: the additional information about “Fresh fish predom-61 inantly harbored the genera Bacillus, Vibrio and Flavobacterium.” Does add value to the introduction? Response 5: As per your suggestions, we have improved the sentence structure and removed unnecessary content in the manuscript.   Comments 6: Line 70. “Some of bacteria”. Please be more specific. In the same sentence, the secondary compounds are usually defined as “antimicrobial compounds” (AMPs). If you refer to lactic and acetic acid, the sentence should be rephrased as well. Response 6: We have adjusted the arrangement of the paragraph and refined some sentences in accordance with your comments (line 104-112).   Comments 7: Line 74: are you assuming the LAB are “probiotics” without introducing their pathogens counteraction capabilities? It is suggested to tell their main potentials (lines 76-81) and then declare them as probiotics/beneficial (line 74). Response 7: We have adjusted the arrangement of the paragraph and refined some sentences in accordance with your comments (line 113-119)   Comments 8: Lactobacillus plantarum isolated in shrimps? Really? Do you have a citation for this? Because this bacterium is usually associated with plants… Response 8: I wrote this after reading the attached reference paper No. 20. Lactobacillus plantarum used in the experiment was a strain isolated from whiteleg shrimp (Litopenaeus vannamei) (Vieira et al., 2007).   Comments 9: Moreover, please upgrade the scientific nomenclature. For example, Lactobacillus plantarum is now Lactiplantibacillus plantarum. It was renamed in 2021. Response 9: According to the revised nomenclature, I have changed Lactobacillus plantarum to Lactiplantibacillus plantarum.   Comments 10: Moreover, the bibliography in the whole introduction is scarce. Response 10: Our manuscript has been prepared to meet the word limit for a short communication, totaling less than 5,000 characters. Due to the restriction on length, we did not add more references to the introduction. However, to aid the reader's understanding, we have made some revisions, including modifying certain content and restructuring the paragraphs in the introduction.   Comments 11: The paragraph a 2.1 is extremely confusing. It is suggested to make a table with all the strains used (pathogenic and LAB), with their source of isolation /purchase, and their aim in the work (Eg: Bacillus cereus à negative control) Response 11: Our manuscript is a short communication, not an article, and therefore there is a limitation on the number of tables allowed. We apologize for not being able to present the list of strains used in a table format. However, to enhance readability, we have made revisions to section 2.1.   Comments 12: Paragraph 2.2. It is suggested to rename it “cultivation of Vibrio strains”. Response 12: In accordance with the reviewer's advice, we have made the necessary revisions.   Comments 13: Table 1: please also add the melting temperature for every amplicon. It is very important information for the specificity of the reaction and reproducibility of the experiment in other laboratories worldwide Response 13: Added melting points to all amplicons in Table 1.   Comments 14: The section describing the statistics used and data analysis software/packages to generate figures is completely missing. please provide details on the applied statistic. Response 14: We appreciate the reviewer's attention to detail regarding statistical analysis. However, we did not perform any statistical analysis on our data. Our study was primarily focused on observing qualitative changes in cadaverine production by Vibrio strains in the presence of LAB. As such, the results presented are descriptive and observational.   <Results and Discussion> Comments 15: Figure 1: please increase quality and mark significant differences with a “ * “. The same for Figure 2. Response 15: We acknowledge the reviewer's suggestion to enhance the quality of Figures 1 and 2 and to mark significant differences. However, given that no statistical analysis was performed, we cannot mark statistically significant differences.   Comments 16: The reduction of pathogenic bacteria by LAB is a good result. But would you define it as biological relevance? Or the CFU of Vibrio after the inoculation of LAB is anyway a concern for shrimps and human health? In the same way, cadaverine and putrescine are reduced below the threshold level of danger? Please better discuss this in the manuscript. Response 16: In this study, we confirmed that the colony count of cadaverine-producing Vibrio strains decreased due to the action of LAB, and that the production of cadaverine also decreased under various culture conditions (mono-culture or co-culture). Specifically, as shown in Fig 1, the Vibrio strain produced up to approximately 570 μg/mL of cadaverine during cultivation. However, with the inoculation of LAB, the cadaverine production decreased by more than 90%. However, since there is currently no allowable intake or safety standard for cadaverine (including putrescine) in food, we did not include any conclusions related to human health. Presently, the only biogenic amine standard pertains to histamine, which generally should be detected at levels below 200 mg/kg in food, with consumption over 500 mg/kg considered toxic to humans.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

See comments to Editor

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and in the tracked changes in the re-submitted files. Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and in the tracked changes in the re-submitted files. 

Comments 1: I note that some of the suggestions were corrected. However, one of the major corrections that should have been made to the manuscript corresponded to the description of co-culture. In the original version of the article this part of the methodology was not understood at all. In this second corrected version I observe that the methodology does not correspond to a Co-culture. The term co-culture implies that the two microbial species are being grown together on the same medium, which is not what is described in the corrected paragraph. The experiment appears to focus on tests of microbial antagonism, evaluating the effect of lactic acid bacteria on Vibrio species when grown in separate media. This serious inconsistency left me baffled, since co-cultivation has a great degree of complexity, which made the article very interesting, and now I note that this is not the case.

Response 1 (co-culture): Thank you for your valuable feedback. We apologize for the misunderstanding regarding the methodology described in our manuscript. The following text is a corrected explanation of the co-culture method used in our experiment:

Each strain was individually activated and grown on their respective optimal media to ensure their survival by eliminating contaminants before co-cultivation in lysine decarboxylase broth or shrimp extract broth. Subsequently, the activated lactic acid bacteria and Vibrio strains were co-cultured together on the same medium (section 2.4).

We understand that the term "co-culture" generally implies that two microbial species are grown together on the same medium, as you rightly pointed out. Our experiment indeed followed this methodology, where both the lactic acid bacteria and Vibrio strains were co-cultured on the same medium to directly observe their interactions. Therefore, we believe our use of the term "co-culture" is accurate in describing our experimental approach. Thank you for bringing this to our attention.

 

Comments 2: Likewise, the title and abstract indicate that in vivo tests were done. The term in vivo is traditionally used to refer to experiments carried out inside a living organism, such as an animal or a plant, and the researchers used a biological extract. That is to say, the article creates expectations that are not met.

Response 2 (in vivo test): Thank you for your insightful feedback. We apologize for the confusion regarding the use of the term "in vivo" in our title and abstract. Here is our corrected explanation:

The term "in vivo" is generally understood to refer to experiments conducted inside a living organism or under conditions that closely mimic a natural living environment. In our study, we used shrimp extract to create a medium for observing the antagonistic interactions between bacteria. We initially considered this approach to constitute an "in vivo" test because the medium was derived from a biological organism (shrimp). However, we acknowledge that most readers might interpret "in vivo" as experiments conducted within a living organism (e.g., within the gut).

To address this potential misunderstanding, we have reclassified all experiments described in our manuscript as "in vitro" tests. We have made the necessary corrections to the terminology throughout the text to accurately reflect the nature of our experiments.

 

Comments 3: Furthermore, although as you can see I am not an authority on English language corrections, I found several typographical and grammatical errors in small paragraphs, which indicates a lack of care when correcting the manuscript.

Response 3 (English language corrections): After incorporating all the aforementioned revisions, we completed a thorough check for typos and an English language review through a professional editing service. We have attached the proofreading certificate. Thank you.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I have re-read the manuscript and find that the authors have made several important changes and have changed the title, so that the current title better fits the content of the manuscript. However, the description of the coculture is still poor. As described, the coculture is not a reproducible experiment. I believe that once this part of the manuscript is detailed, the article can be published. Performing a co-culture is a very complex task, as the nutritional needs and physical conditions of pH, agitation and temperature of each microorganism are different, and no conditions are specified in the manuscript. It is only stated: "Each resuspended culture was inoculated in 20 mL of lysine decarboxylase broth medium,

individually and in co-culture, and incubated at 37°C under anaerobic conditions for 48 h". Also, the results do not address co-culture, which is a very important part of this manuscript.

I ask the authors to describe the step-by-step of the co-culture, and justify the growth conditions chosen, and also include a paragraph in the discussion of what might occur when the pathogen growth conditions are favored, and what might occur when the antagonistic microorganism growth conditions are favored. Finally, many typographical errors continue to appear in the new version of the manuscript.

Author Response

Dear Reviewer,
Thank you for your thorough review of our manuscript and for highlighting areas that require further clarification. We appreciate your constructive feedback and have made several revisions to address your concerns.

1.    Detailed Description of Co-culture Experiments:
o    We have provided a step-by-step description of the co-culture procedure in the "Materials and Methods" section. This includes specific details about the pH, agitation speed, and temperature conditions used during the experiments. Additionally, we have justified the choice of these growth conditions based on the optimal requirements for both Vibrio strains and lactic acid bacteria.

2.    Discussion of Co-culture Results:
o    We have added a paragraph in the "Discussion" section to elaborate on the potential outcomes when pathogen growth conditions are favored versus when conditions favor the growth of antagonistic microorganisms. This discussion provides insights into the dynamics of co-culture environments and their impact on cadaverine production and bacterial growth.

3. Correction of typo: 
We re-checked the entire manuscript and corrected typos.

We believe these revisions address your concerns and enhance the clarity and reproducibility of our experiments. Thank you for your valuable suggestions, which have significantly improved our manuscript.
We look forward to your favorable response.