Antibacterial Efficacy of Feline-Derived Lactic Acid Bacteria against Enteropathogenic Escherichia coli: A Comprehensive In Vitro Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Several studies report that lactic ferments can rebalance the intestinal bacterial population by restoring its physiological functions. They are able to resist the action of gastric juice and bile secretion, therefore, they are able to settle in the intestine: by adhering to the epithelial cells, they prevent the proliferation of pathogenic bacterial strains, giving rise to a healthier and more balanced intestinal bacterial flora. Furthermore, through the production of lactic acid, acetaldehyde, diacetyl and other substances, they are also able to inhibit the growth of unwanted microorganisms. Starting from this knowledge, this study was based on the isolation and screening of strains of lactic acid bacteria (LAB) from feces of healthy cats to find probiotics that effectively fight enteropathogenic Escherichia coli (EPEC), as a possible alternative to conventional antibiotics.

The topic covered falls within the aims and scope of the Journal.

The Introduction is clearly and correctly written and ends with the purpose of the research.

The Materials and Methods section reports the procedures in detail.

The Results and Discussion are well presented with an adequate number of Tables and Figures.

The References are appropriate and related to previously published works.

Overall, the structure of the manuscript is well organized. However, some changes are required to improve the presentation of the work.

It is recommended to add other keywords other than those already present in the title.

Check throughout the text that one of the selected and used strains is always ZY25 and not ZA25 (for example: line 371).

Add a list of the abbreviations used in the manuscript.

Comments on the Quality of English Language

The English used in the text is readable.

Author Response

Reviewer 1

Comments and Suggestions for Authors

Point 1: It is recommended to add other keywords other than those already present in the title.

Thank you for your thorough review and valuable suggestions. We understand your recommendation to include additional keywords beyond those already present in the title. To more comprehensively cover the content of the manuscript, we have added the following two keywords to complement the existing ones:

Host-specific probiotics: In our study, we emphasize the advantages of feline-derived lactic acid bacteria strains compared to non-host-specific strains in terms of gut colonization and pathogen inhibition. This keyword better reflects our focus on the research and potential applications of host-specific probiotics.

Organic acid production: Our results demonstrate that the antibacterial activity of feline-derived lactic acid bacteria is largely attributed to the production of organic acids such as lactic acid and acetic acid. Including this keyword highlights our exploration of the antibacterial mechanisms of these strains and their role in inhibiting pathogenic bacteria.

Please see P1 L26-27

Keywords: Enteropathogenic Escherichia coli; Feline; Probiotics; Antibacterial activity; Host-specific probiotics; Organic acid production

Point 2: Check throughout the text that one of the selected and used strains is always ZY25 and not ZA25 (for example: line 371).

Thank you for your careful observation and for pointing out the inconsistency in the strain designation. We have reviewed the manuscript thoroughly and confirmed that the correct strain designation is ZY25. We have corrected any instances where the strain was mistakenly referred to as ZA25, including in line 371 and throughout the entire text.

We appreciate your attention to detail, which has helped us improve the clarity and accuracy of our manuscript.

Point 3: Add a list of the abbreviations used in the manuscript.

Thank you for your suggestion. We have compiled a list of abbreviations used in the manuscript to improve clarity and accessibility for readers. The list has been added to the beginning of the manuscript, following the abstract section.

Please see P1 L28-31

Abbreviations: EPEC: Enteropathogenic Escherichia coli, LAB: Lactic Acid Bacteria, MRS: de Man, Rogosa, and Sharpe (medium), PCR: Polymerase Chain Reaction, SGF: Simulated Gastric Fluid, SIF: Simulated Intestinal Fluid, CFU: Colony-Forming Unit, HPLC: High-Performance Liquid Chromatography, OD: Optical Density, SEM: Standard Error of the Mean

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors evaluated antibacterial efficacy of lactic acid bacteria. My comments are as follows:

 

1. Page 1, lines 7-15: Can authors provide formal university issued e-mail address instead of commercial one?

 

2. Page 4, line 186: ZY33, ZY35, and ZY25. Why not follow numeral order?

 

3. Page 5, line 232: the authors already mentioned ‘measured at 2-hour intervals up to 24 hours’. Making ‘(specifically at 2, 4….24 hours)’ redundant.

 

4. Page 6, line 241: Milli-Q system (city name, country name).

 

5. Page 6, line 253: Xcalibur 4.1 and TracerFinder^TM 4.1 Clinical. Please provide www link, references, and so on.

 

6. Page 6, line 256: SPSS 22.0 (company name, city name, country name)

 

7. Page 6, Figure 1: Try to place figure and legend on the same page.

 

8. Page 10, lines 316-317: ZY21,….and ZY33. Why not follow numeral order?

 

9. Page 11, Figure 3. Please increase resolution, and also enlarge font size.

 

10. Page 12, Figure 4. The title of x-axis. Are treatments better than times?

 

11. Page 12, Figure 5. Try to place figure and legend on the same page.

 

12. Page 13, line 371: ZA25, typo? ZY25?

 

13. Page 13, line 374: Telracycline, typo? Tetracyclin?

 

14. Page 13, lines 390 and 391: ZA25, typo? Also page 14, line 400; page 15, lines 430, 439 and 441.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Point 1: Page 1, lines 7-15: Can authors provide formal university issued e-mail address instead of commercial one?

Thank you for your suggestion. We have reviewed all authors' email addresses. However, some of the authors, including PhD and Master's students, have already graduated, and their university email accounts will no longer be active. Therefore, we have provided the curricula vitae of these authors as supplementary documents to validate their affiliations and contributions to the study.

We hope this solution meets the requirements and ensures transparency and credibility.

CVs (curricula vitae) of the authors

Weiwei Wang, Ph.D. Assistant Researcher. Research interests include the selection of functional lactic acid bacteria and the development of functional pet food products.

Hao Dong, M.Sc. Specializes in the research and development of functional pet food products, with a particular focus on juvenile pet nutrition.

Xiaohan Chang, M.Sc. Researches and develops functional pet food products, concentrating on herbal-based formulations for pets.

Longjiao Wang, Ph.D. Postdoctoral Fellow at China Agricultural University. His research focuses on the mechanisms of anemia in patients with chronic kidney disease, the effects of abnormal bile acid metabolism on bodily functions, and the development and efficacy evaluation of functional pet food products.

Chengyi Miao, M.Sc. Veterinarian. His primary research interest is in alleviating stress in pets.

Shuxing Chen, Ph.D. Professor. Engaged in the long-term research and development of functional dairy products and cheese processing.

Lishui Chen, Ph.D. Senior Engineer and Deputy Director of the Zhongyuan Food Laboratory. In the past five years, he has been involved in various research and development projects focused on the nutritional and health aspects of snack foods.

Shaoyang Ge, Ph.D. Professor. His primary research focuses on the development and industrialization of probiotic strains.

Wei Xiong, Ph.D. Head of the Pet Food Team at Zhongyuan Food Laboratory. His research is focused on pet nutrition and the development of functional pet food products.

Point 2: Page 4, line 186: ZY33, ZY35, and ZY25. Why not follow numeral order?

Thank you for your observation. We agreed that following a numerical order (ZY25, ZY33, and ZY35) would enhance clarity and consistency in the manuscript. We have updated the text accordingly to present the strains in the correct numerical order throughout the manuscript.

We appreciate your attention to this detail.

Please see P 4, L190.

Fresh bacterial strains ZY25, ZY33, and ZY35 were streaked on Columbia blood agar plates.

Point 3: Page 5, line 232: the authors already mentioned ‘measured at 2-hour intervals up to 24 hours’. Making ‘(specifically at 2, 4….24 hours)’ redundant.

Thank you for your suggestion. We agreed that the detailed specification of time intervals was redundant in this context. We have removed the phrase “(specifically at 2, 4….24 hours)” to improve the clarity and conciseness of the manuscript.

Point 4: Page 6, line 241: Milli-Q system (city name, country name).

Thank you for your suggestion. We have revised the manuscript to include the city and country of the Milli-Q system's manufacturer. The updated information is as follows:

"Milli-Q system (Merck Millipore, Burlington, MA, USA)." Please see P6, L243.

We appreciate your attention to detail and have ensured that the necessary updates have been made.

Point 5: Page 6, line 253: Xcalibur 4.1 and TracerFinderTM 4.1 Clinical. Please provide www link, references, and so on.

Thank you for raising this excellent point. Xcalibur 4.1 and TraceFinder™ 4.1 Clinical are indeed two separate software programs that do not require an online connection during operation. I have included the relevant information and provided references for both software packages in the manuscript. Please see P6, L255-257.

The data were processed with Xcalibur 4.1 (Thermo Scientific) and TraceFinder™ 4.1 Clinical (Thermo Scientific), and the results were outputted in Excel format [21].

21. Khan, N.; Ali, S.; Zandi, P.; Mehmood, A.; Babar, A. Role of sugars, amino acids and organic acids in improving plant abiotic stress tolerance. Pak. J. Bot. 2020, 52, 2. https://doi.org/10.30848/PJB2020-2(24).

Point 6: Page 6, line 256: SPSS 22.0 (company name, city name, country name).

Thank you. I have already revised the manuscript to include the company name, city, and country for SPSS. The updated information is as follows:

“SPSS 22.0 (IBM Corp., Armonk, NY, USA). " Please see P6, L260.

Point 7: Page 6, Figure 1: Try to place figure and legend on the same page.

Thank you for your suggestion. I have adjusted the formatting to ensure that Figure 1 and its legend are now placed together on the same page for better readability and presentation.

Point 8: Page 10, lines 316-317: ZY21,….and ZY33. Why not follow numeral order?

Thank you for your observation. We agreed that presenting the strains in numerical order would improve clarity and consistency. We have revised the sentence as follows:

"As shown in the phylogenetic tree (Figure 3), strains ZY21, ZY23, ZY25, ZY33, ZY35, and ZY39 were distinctly classified among various species of lactic acid bacteria."

Please see P10, L321-322.

Point 9: Page 11, Figure 3. Please increase resolution, and also enlarge font size.

Thank you for your suggestion. I have increased the resolution and enlarged the font size of Figure 3 to improve its readability and clarity. The updated figure has been included in the revised manuscript to ensure that all details are clear and legible.

Point 10: Page 12, Figure 4. The title of x-axis. Are treatments better than times?

Thank you. I have made the necessary changes in the figure, replacing the x-axis title with "Treatments" instead of "Times" as recommended.

Point 11: Page 12, Figure 5. Try to place figure and legend on the same page.

Thank you for your suggestion. I have adjusted the layout to ensure that Figure 5 and its legend are now placed together on the same page for improved readability and presentation.

Point 12: Page 13, line 371: ZA25, typo? ZY25?

Thank you for your careful observation and for pointing out the inconsistency in the strain designation. We have reviewed the manuscript thoroughly and confirmed that the correct strain designation is ZY25. We have corrected any instances where the strain was mistakenly referred to as ZA25, including in line 371 and throughout the entire text.

We appreciate your attention to detail, which has helped us improve the clarity and accuracy of our manuscript.

Point 13: Page 13, line 374: Telracycline, typo? Tetracyclin?

Thank you for pointing out the typo. You are correct; the correct term is “Tetracycline.” I have corrected this error in the manuscript to ensure accuracy. Please see P13, L379.

Point 14: Page 13, lines 390 and 391: ZA25, typo? Also page 14, line 400; page 15, lines 430, 439 and 441.

Thank you for your careful observation and for pointing out the inconsistency in the strain designation. We have reviewed the manuscript thoroughly and confirmed that the correct strain designation is ZY25. We have corrected any instances where the strain was mistakenly referred to as ZA25, including in line 400, 430, 439, 411 and throughout the entire text.

Author Response File: Author Response.pdf