Bacterial Extracellular Vesicles in Biotechnology: Current Challenges and Strategies for Production Enhancement

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article “Bacterial Extracellular Vesicles in Biotechnology: Challenges of Low Yield and Strategies for Production Enhancement” provides a solid overview of the advances in the study of extracellular vesicles, their functions, and their components. In addition, the authors propose strategies to standardize isolation methods in terms of both yield and composition.

The review article is well structured and guides the reader effectively; only a few minor comments are needed.

Table 1 is not cited within the main text.

Additionally, although several experimental strategies are suggested to increase vesicle yield, the authors should address whether they have any recommendations for managing the strain-dependent factors. It has been reported that different strains of the same species can secrete varying amounts and compositions of vesicles. Could the authors suggest any strategies to mitigate or control this issue? For example, would it be advisable to work exclusively with freshly obtained strains from certified cell banks such as ATCC? or what would the recommendation be?

Author Response

Comment 1: Table 1 is not cited within the main text.

Response: We are grateful to Reviewer for carefull reading. Table 1 was cited according to the suggestion.

 

Comment 2: Additionally, although several experimental strategies are suggested to increase vesicle yield, the authors should address whether they have any recommendations for managing the strain-dependent factors. It has been reported that different strains of the same species can secrete varying amounts and compositions of vesicles. Could the authors suggest any strategies to mitigate or control this issue? For example, would it be advisable to work exclusively with freshly obtained strains from certified cell banks such as ATCC? or what would the recommendation be?

Response: We sincerely thank the Reviewer for this insightful comment. This point regarding strain-dependent variability in vesicle production was indeed very helpful and prompted us to add a dedicated paragraph addressing this issue. In the new section (i.e. "2.2. Production of bacterial extracellular vesivles can vary among different strains of the same species") of the revised manuscript, we discuss strategies to mitigate such variability. We believe that incorporating this discussion enhances both the clarity and the practical relevance of our manuscript

 

Reviewer 2 Report

Comments and Suggestions for Authors

Review on manuscript

Bacterial Extracellular Vesicles in Biotechnology: Challenges of Low Yield and Strategies for Production Enhancement

 

The present manuscript is a well-structured and well-written review on bacterial extracellular vesicles (BEVs), which play critical roles in bacterial physiology, intercellular communication, and host–pathogen interactions. However, what I miss from the manuscript is the presentation of BEVs biogenesis. For example, there is any difference between outer membrane vesicles (OMVs) and BEVs?

The manuscript contains a wealth of information, and I suggest that this be better illustrated in the form of tables or sketches. I found Chapter 3 particularly interesting and useful, especially the section on the effects of antibiotics. Although it does not strictly belong to the topic of the article, I would be interested to know what happens, for example, in a living system. Are BEVs released, too?

Another issue what is not mentioned is the protein corona usually present on EVs. Do the authors some information on how different producing and storage circumstances affect the presence of protein corona. Is this question even relevant in the case of BEVs?

In summary, I guess that is a good review paper with well-chosen questions and answers, it can be published after minor revisions, especially OMVs vs BEVs and BEVs biogenesis in general.

Comments for author File: Comments.pdf

Author Response

Comments 1: However, what I miss from the manuscript is the presentation of BEVs biogenesis. For example, there is any difference between outer membrane vesicles (OMVs) and BEVs?

Response 1: We thank the Reviewer for carefull reading and for having pointed out this omission. We have addressed this comment by adding a clearer description of extracelluar biogenesis in Gram-positive and Gram-negative bacteria in the introduction of the manuscript.

 

Comments 2: The manuscript contains a wealth of information, and I suggest that this be better illustrated in the form of tables or sketches. I found Chapter 3 particularly interesting and useful, especially the section on the effects of antibiotics.

Response 2: We thank the Reviewer for the positive feedback. To improve the readability of the manuscript, we have summarized the key information from Chapter 3 in a comprehensive table (Table 2 in the revised version of the manuscript).

 

Comment 3: Although it does not strictly belong to the topic of the article, I would be interested to know what happens, for example, in a living system. Are BEVs released, too?

Response 3: We thank the Reviewer for this insightful comment. In response, we have added a dedicated paragraph (2.6 In vivo dynamics of BEVs: biodistribution and biological effects) summarizing the available evidences on the in vivo production of BEVs and on their fate following administration. Additionally, we have introduced a new section (2.5. Bacteriophage as modulators of bacterial extracellular vesicle production) discussing the influence of bacteriophages on BEV dynamics. We conclude that these aspects are crucial for better understanding, enhancing, and directing BEV production.

 

Comment 4: Another issue what is not mentioned is the protein corona usually present on EVs. Do the authors some information on how different producing and storage circumstances affect the presence of protein corona. Is this question even relevant in the case of BEVs?

Response 4: We thank the Reviewer for raising this important point. At present, no direct evidence is available regarding the formation or modulation of a protein corona on BEVs, as the existing literature mainly focuses on extracellular vesicles of eukaryotic origin, particularly those produced by mammalian cells. Nevertheless, we have added a specific paragraph (4.6 Protein corona of BEVs: an overlooked aspect) in the revised manuscript in which we speculate that a similar phenomenon could potentially occur in BEVs, considering their structural and biochemical features.

Reviewer 3 Report

Comments and Suggestions for Authors

Bacterial extracellular vesicles (BEV) have shifted from being studied mainly as virulent factors in pathogens to emerging as versatile biotechnological tools. This review synthesizes recent advances in genetic analysis, physiological modulation, physicochemical stimuli, and bioprocess optimization aimed at enhancing BEV production and stabilizing cargo profiles. The manuscript is reasonable and has originality. However, the trends in prior research related to the title “Challenges of Low Yield and Strategies for Production Enhancement” are limited, and the manuscript structure is visually difficult to comprehend. Therefore, some revisions seem to be necessary in this journal.

 

The author needs to create a section summarizing the in vivo dynamics of BEV. Understanding BEV behavior when applied to living organisms is crucial for biotechnological applications in fields such as biomedicine.

 

What are the key challenges moving forward with commercialization in mind? Demonstrating the importance of standardization experiments for BEV is essential. The author should carefully discuss with the current situation: that BEV has a shorter history than mammalian-derived EVs, and that consensus on the ISEV guidelines remains insufficient (DOI: 10.1002/jev2.12404).

 

The information regarding production enhancement methods, microbial strains, and the extent of enhancement (based on protein content or particle count) is presented in a highly technical manner, making it difficult to comprehend as a review. Would it be possible to create a table to summarize this information?

 

The citation style is inconsistent in the References section. (Publication years are sometimes bold or plain. Additionally, some papers lack DOIs.)

Author Response

Comments 1: The author needs to create a section summarizing the in vivo dynamics of BEV. Understanding BEV behavior when applied to living organisms is crucial for biotechnological applications in fields such as biomedicine.

Response 1: We thank the Reviewer for careful reading and for this valuable suggestion. In response, we have added a dedicated section (2.6 In vivo dynamics of BEVs: biodistribution and biological effects) summarizing the current evidence on the in vivo dynamics of BEVs, with particular attention to their biodistribution and biological effects in living organisms. We agree that understanding BEV behavior in vivo is crucial for biotechnological and biomedical applications, and we believe that this new section strengthens the scientific relevance and clarity of the manuscript.

 

Comments 2: What are the key challenges moving forward with commercialization in mind? Demonstrating the importance of standardization experiments for BEV is essential. The author should carefully discuss with the current situation: that BEV has a shorter history than mammalian-derived EVs, and that consensus on the ISEV guidelines remains insufficient (DOI: 10.1002/jev2.12404).

Response 2: We thank the Reviewer for raising this important point. As suggested, we have expanded the discussion in the section “4. Fermentation and process optimization strategies for extracellular vesicle production”, emphasizing the urgent need for standardization in BEV research. In this context, we note that BEVs have a much shorter research history compared to mammalian-derived EVs and that consensus on the application of ISEV-based guidelines is still limited. We have also addressed the issue of reproducibility in the paragraph “2.2. Production of bacterial extracellular vesicles can vary among different strains of the same species” of the revised manuscript. Additionally, following your comment and in order to better capture the broader challenges beyond yield alone, we have modified the title of the revised manuscript to reflect a wider scope accordingly.

 

Comments 3: The information regarding production enhancement methods, microbial strains, and the extent of enhancement (based on protein content or particle count) is presented in a highly technical manner, making it difficult to comprehend as a review. Would it be possible to create a table to summarize this information?

Response 3: We thank the Reviewer for this useful suggestion. To improve clarity and readability, we have summarized the information regarding production‑enhancement in a comprehensive table (Table 2 of the revised manuscript) within Chapter 3. We believe this addition greatly facilitates the understanding of the technical aspects presented in the review.

 

Comments 4: The citation style is inconsistent in the References section. (Publication years are sometimes bold or plain. Additionally, some papers lack DOIs.)

Response 4: We thank the Reviewer for pointing out this issue. We have carefully revised the entire References section to ensure a consistent citation style, standardizing the formatting of publication years and adding the missing DOIs where available.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Accept in present form