Characterization of a Vaginal Limosilactobacillus Strain Producing Anti-Virulence Postbiotics: A Potential Probiotic Candidate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript evaluated the ability of active postbiotics produced during the fermentation of Limosilactobaillus fermentum Lf 53 to inhibit QS-regulated characteristics of model pathogenic bacteria. Even though the work was systematically, there have many similar published papers. This study is lack of innovation, besides the strain Limosilactobaillus fermentum Lf 53 they found. In addition, the study is superficial and requires more experiments to determine its antibacterial components. It is not clear that the white star mentioned as “honeycomb” in FIG 4.

Author Response

Dear Editors Dr. Salva and Dr. Li,

please find the re-submition of Revised MS file entitled “Characterization of a Vaginal Limosilactobacillus Strain Producing Anti-Virulence Postbiotics: A Potential Probiotic Candidate”.

We have carefully considered all critical remarks, comments and suggestions of the Reviewers and have made all the necessary corrections/amendments accordingly, which are mentioned below. The corrections are outlined with track changes in the text. Our answers and explanations are as follows:

 

Reviewer 1

1. This manuscript evaluated the ability of active postbiotics produced during the fermentation of Limosilactobaillus fermentum Lf53 to inhibit QS-regulated characteristics of model pathogenic bacteria. Even though the work was systematically, there have many similar published papers. This study is lack of innovation, besides the strain Limosilactobaillus fermentum Lf 53 they found. In addition, the study is superficial and requires more experiments to determine its antibacterial components.

 

Reply: The present study encompasses a systematic approach that includes a comprehensive set of targeted methods aimed at evaluating the antivirulence properties of postbiotics derived from Limosilactobacillus fermentum. This work is part of the broader concept of contemporary antivirulence strategies, which seek to hinder bacterial pathogenesis. At the time of presenting the conducted research, an internet-based literature review was performed, revealing a limited number of publications focused on the antivirulence properties of strains belonging to the genus Limosilactobacillus. Approximately 600 articles were identified that address anti-biofilm activity; however, only 53 studies report on quorum sensing inhibition (e.g., pigment synthesis and related mechanisms). The innovative aspect of this research lies in the integration of probiotic characteristics with antivirulence data, which could, in turn, reduce the selective pressure on pathogens and their development of antimicrobial resistance. It is important to note that this study does not aim to elucidate the antibacterial properties of the strain.

2. It is not clear that the white star mentioned as “honeycomb” in FIG 4.

 

Reply: The data obtained from scanning electron microscopy provide a clear understanding of the spatial organization and architecture of bacterial cells within a given microbial community, as well as the presence of morphological aberrations (if any), which may result from specific treatments or, as in our case, from exposure to different pH conditions. In Figure 4, we present micrographs illustrating a dominant biofilm-like morphotypes in the arrangement of bacterial cells—resembling a “honeycomb” structure, indicated with a white star. The cells within these structures, similar to those in biofilm consortia, rise in height and form cavities at their base, within which individual cells and/or dead cells and extracellular DNA can be identified. In this context, we have placed clearer emphasis on the annotation of the figure (Section 3.3).

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents interesting findings on the anti-virulence properties of postbiotics from L. fermentum Lf53. However, significant revisions are required.

1. The phrase "A promising approach being explored is the potential of Lactobacillus postbiotics" could be more direct, e.g., "Exploration of Lactobacillus postbiotics represents a promising approach..."
2. "Biofilm inhibition of Pseudomonas aeruginosa revealed a notable suppressive effect by acidified and neutralized CFS and Lf53 lysates, each exceeding 50%." It's unclear if "each" refers to all three postbiotic types or if acidified and neutralized CFS are grouped. Clarify if lysates also exceeded 50% independently.
3. "methabiotics" - likely a typographical error for "metabiotics." Please verify. The introduction and consistent use of "methabiotics/metabiotics" and "parabiotics" require clear, early definition or justification for their necessity over "postbiotics" with descriptive qualifiers.
4. Detailed ultrasonication parameters (instrument, power/amplitude, duration, pulse, volume, temperature control) are essential for reproducibility and are missing. Verification method for cell lysis efficiency is also absent.
5. No mention of testing for ANOVA assumptions (normality and homogeneity of variances). Without this, the validity of ANOVA results is questionable. Alternative non-parametric tests should be considered if assumptions are not met.
6. "In the nCFS, the activity decreased but remained present, suggesting that the neutralization step is involved in modulating efficacy." This statement is vague. A more mechanistic interpretation of why neutralization alters efficacy (e.g., pH-dependent stability/activity of specific compounds) is needed, especially considering the variable effects on violacein vs. pyocyanin.
7. The differing activities of aCFS and nCFS against pyocyanin (nCFS much better) versus violacein (aCFS slightly better) warrant more in-depth speculation on the nature of the active compounds involved.
8. The novelty claim ("first reported data") needs to be very carefully framed and supported by an exhaustive literature search, as similar concepts of complex postbiotics or cell lysates having anti-virulence effects may exist for other strains or contexts.

Author Response

Dear Editors Dr. Salva and Dr. Li,

please find the re-submition of Revised MS file entitled “Characterization of a Vaginal Limosilactobacillus Strain Producing Anti-Virulence Postbiotics: A Potential Probiotic Candidate”.

We have carefully considered all critical remarks, comments and suggestions of the Reviewers and have made all the necessary corrections/amendments accordingly, which are mentioned below. The corrections are outlined with track changes in the text. Our answers and explanations are as follows:

Reviewer 2

1. The phrase "A promising approach being explored is the potential of Lactobacillus postbiotics" could be more direct, e.g., "Exploration of Lactobacillus postbiotics represents a promising approach..."

 

Reply: We thank the reviewer for the recommendation. The corrections made have been applied in the revised manuscript (Line 15-16).

2. "Biofilm inhibition of Pseudomonas aeruginosa revealed a notable suppressive effect by acidified and neutralized CFS and Lf53 lysates, each exceeding 50%." It's unclear if "each" refers to all three postbiotic types or if acidified and neutralized CFS are grouped. Clarify if lysates also exceeded 50% independently.

 

Reply: We thank the reviewer for the recommendation. The sentence has been corrected as follows: Biofilm inhibition of Pseudomonas aeruginosa revealed remarkable suppressive effects exerted by the three tested postbiotics, two of which (nCFS and aCFS) exhibited over 50% inhibition and more than 60% for lysates (Line 25-27).

3. "methabiotics" - likely a typographical error for "metabiotics." Please verify. The introduction and consistent use of "methabiotics/metabiotics" and "parabiotics" require clear, early definition or justification for their necessity over "postbiotics" with descriptive qualifiers.

Reply: We apologies this  typographical error and  thank you very much for reviewers’ attention. Additional a short definition for metabiotics was added in the introduction (Line 58-80).

4. The introduction and consistent use of "methabiotics/metabiotics" and "parabiotics" require clear, early definition or justification for their necessity over "postbiotics" with descriptive qualifiers.

Reply: Thank you very much for this valuable advice. The introduction was edited and the definitions of, parabiotics and metabiotics were added (Line 58-80).

5. Detailed ultrasonication parameters (instrument, power/amplitude, duration, pulse, volume, temperature control) are essential for reproducibility and are missing.

Reply: Thank you very much. The omitted details were added (Line 216-254).

6. Verification method for cell lysis efficiency is also absent.

Reply: Thank you very much, the missing details were added: Verification for cell lysis was made with microscopic control with a fresh covering microscopic preparation (800 x magnitude, Boeco) of the cell culture at the end of sonication procedure (5x10 sec. at 20% power pulse in ice).

7. No mention of testing for ANOVA assumptions (normality and homogeneity of variances). Without this, the validity of ANOVA results is questionable. Alternative non-parametric tests should be considered if assumptions are not met.

Reply: Thank you very much. We fully agree with the reviewer. However, the information for statistical details, carried out before ANOVA tests, were ommited and now we added (Line 332-337).

8. "In the nCFS, the activity decreased but remained present, suggesting that the neutralization step is involved in modulating efficacy." This statement is vague.

Reply: We fully agree with the respected reviewer. The neutralization step of cells – free supernatants (with 5N NaOH) was made to eliminate the inhibitory activity of produced lactic acid during the fermentation in MRS broth.  Several study showed a strong inhibition, due to the produced by LAB organic acid. The observed effect is probably due to another type of metabolites, that we visualize with weaker activity.

9. A more mechanistic interpretation of why neutralization alters efficacy (e.g., pH-dependent stability/activity of specific compounds) is needed, especially considering the variable effects on violacein vs. pyocyanin. The differing activities of aCFS and nCFS against pyocyanin (nCFS much better) versus violacein (aCFS slightly better) warrant more in-depth speculation on the nature of the active compounds involved.

Reply: Lactic acid bacteria and the metabolites synthesized represent a vital group capable of lowering pH, secreting natural antimicrobial compounds, and competing with pathogens for nutrients and/or adhesion sites. We hypothesize that the inhibitory effect on pigment production (violacein and pyocyanin) may result from one or more of the synthesized metabolites, such as lactic acid, acetic acid, or bacteriocins, produced during the analysis. We assume that the neutralization of the pH in the supernatants may lead to the inactivation or alteration of these metabolites, thereby diminishing their activity in pigment biosynthesis. Moreover, it has been demonstrated that the inactivation of such metabolites inhibits the production of autoinducer signaling molecules - AHLs and the virulence factors responsible for quorum sensing mechanisms that regulate pigment formation.

10. The novelty claim ("first reported data") needs to be very carefully framed and supported by an exhaustive literature search, as similar concepts of complex postbiotics or cell lysates having anti-virulence effects may exist for other strains or contexts.

Reply: We thank the reviewer, a correction has been made to the paragraph (Line 766-767).f

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Decipher the CFS in the abstract.
Lines 38-59 do not contain references, but sentences that require confirmation. For example, various studies have shown that ....... - no references.
The literature review is written very generalized and does not cover the original articles, the authors refer mainly to review articles.

There is a lot of self-citation in the article and review articles.

In the materials and methods, explain more clearly how the cells were treated. (incubation in sterile gastric juice for 2 hours) specifically in terms of cell fermentation.
In materials and methods there is no description of culturing in a Sartorius Biostat bioreactor, it is not clear from the article on which medium and carbon source the culturing was done. Also, the purpose of this experiment within the article is not clear.
Staphylococcus aureus strain 29213 is listed in the materials and methods, but it is not in the results? Explain the results obtained in the article with this strain.
The materials and methods do not describe what nCFS and aCFS are or how they were prepared. Only cell-free supernatants are listed in part 2.2 (first). Also describe how the lysates were prepared.
There are two parts 2.2 in the test.
Line 459 - Our results confirmed the study of L. plantarum strains from traditional dairy products [33]. - The reference is given without citing the journal. in general this statement is questionable.
The authors should more clearly state on which medium Lf53 was grown to produce lysate, nCFS and aCFS in the experiments described in Figures 5-8 and in the tables.
As for the discussion, it is too large and overloaded with references not relevant to this study. I think the authors should work on the structure of the discussion and make it more clear and precise.

Author Response

Dear Editors Dr. Salva and Dr. Li,

please find the re-submition of Revised MS file entitled “Characterization of a Vaginal Limosilactobacillus Strain Producing Anti-Virulence Postbiotics: A Potential Probiotic Candidate”.

We have carefully considered all critical remarks, comments and suggestions of the Reviewers and have made all the necessary corrections/amendments accordingly, which are mentioned below. The corrections are outlined with track changes in the text. Our answers and explanations are as follows:

Reviewer 3

1. Decipher the CFS in the abstract.

Reply: Thank you for the advice. The correction was made (Line 23).

2. Lines: 38-59 do not contain references, but sentences that require confirmation. For example, various studies have shown that ....... - no references.

Reply: Thank you very much. Our aim was to limit the number of references on these widely discussed aspects. In accordance with this useful recommendation of the reviewer, scientific publications on the different aspects have been added.

3. The literature review is written very generalized and does not cover the original articles, the authors refer mainly to review articles.

Reply: We thank the reviewer for the valuable comment. In response, we have included additional methodological references (Line 96-Ref. 15, 98-Ref.19, 114-Ref.20) and appropriate new data (Line 58-80).  

4. There is a lot of self-citation in the article and review articles.

Reply: Thank you. The self-citations have been reduced where possible.

5. In the materials and methods, explain more clearly how the cells were treated. (incubation in sterile gastric juice for 2 hours) specifically in terms of cell fermentation.

Reply: The details are presented in our manuscript (Line 216-254).

 

6. In materials and methods there is no description of culturing in a Sartorius Biostat bioreactor, it is not clear from the article on which medium and carbon source the culturing was done. Also, the purpose of this experiment within the article is not clear.

Reply: We apologize, for the omitted detail regarding the carbon source and fermentation for biomass collection. They were added at (Line 216-254).

7. Staphylococcus aureus strain 29213 is listed in the materials and methods, but it is not in the results? Explain the results obtained in the article with this strain.

Reply: The Gram-positive strain Staphylococcus aureus ATCC 29213 was included in the experiments related to biofilm formation. The effectiveness of the applied CFS on the strain’s biofilm development is presented in Figure 5 and described in the corresponding figure legend. The aim of the experiment was to compare two Gram-negative and two Gram-positive strains in a comparative framework, as structural and functional differences between these bacterial groups often result in variations in their ability to form biofilms, particularly in the presence of treatment agents.

8. The materials and methods do not describe what nCFS and aCFS are or how they were prepared. Only cell-free supernatants are listed in part 2.2 (first). Also describe how the lysates were prepared.

Reply: Thank you very much. The omitted details were added (Line 216-254).

 

9. There are two parts 2.2 in the test.

Reply: We thank the reviewer for the technical comment. The correction has been made.

10. Line 459 - Our results confirmed the study of L. plantarum strains from traditional dairy products [33]. - The reference is given without citing the journal. in general this statement is questionable.

Reply: We apologise for such technical mistake. It was corrected. Thank you.

11. The authors should more clearly state on which medium Lf53 was grown to produce lysate, nCFS and aCFS in the experiments described in Figures 5-8 and in the tables.

Reply: The conducted experiments, described in detail in the Materials and Methods section (see 2.2. and 2.3.), along with the corresponding results presented in the form of figures (Figures 5–8) and accompanying text, clearly define the use of a 10% v/v concentration of the three supernatants (lysate, nCFS, and aCFS). Each of the testing methodologies includes a description of the culture medium used (see 2.2. and 2.3.) as well as references to the relevant articles (2.3.1, 2.3.2, 2.3.3).

The cell –free supernatants (nCFS and aCFS) from L. fermentum LF53, were always collected after cultivation in modified MRS broth (meat extract and Tween 80 omitted), with glucose as carbon source (20 g/L), initial pH 6.5. at 37⁰C, Incubator Nuve EN400 (Ankara, Turkey) or in Bioreactor.

A microplate methods (using 96 wall plates) showed supplementation of the growth medium (for tested pathogens) with 10% v/v of tested postbiotics.

 

12. As for the discussion, it is too large and overloaded with references not relevant to this study. I think the authors should work on the structure of the discussion and make it more clear and precise.

Reply: The discussion (Section 4) has been adjusted according to the reviewer's requirements.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

It can be accept now.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed most of the comments.