Review Reports - Bacteriophages as Potential Sustainable Alternatives to Antibiotics for Controlling <i>Salmonella</i> in the Poultry Value Chain

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review manuscript provided a concise summary of the application of bacteriophages in the poultry industries, with a particular focus on their utilisation in combating Salmonella spp., including Salmonella antibiotic-resistant serovars, throughout the poultry value chain. The review article will provide a comprehensive overview of the application of phages in the poultry industry, offering valuable insights and perspectives that will contribute to the advancement of research in this field.

Here is some minor revision:

Line79/81: “As of 2024, the WHO has classified Salmonella as a high-priority research pathogen due to its resistance to antibiotics, particularly to fluoroquinolones”. The appropriate should be cited here.

Line 82: Salmonella spp. “.” Is missing!

Line 101: Enterobacteriaceae is italic.

Line 110: Salmonella spp. “.” Is missing!

Line 218: Replacre the reference “(Mehdi et al., 2018)” with number in the citation.

Line 369: correct, S. Enteritidis. Check and correct in whole manuscript.

Line 372: correct “S. Typhimurium”

Comment 1: It is imperative that the abbreviation 'PFU' (phage forming unit) is written in full for the first instance, or alternatively, it may be written under the table as well as “SPF”

Line 449: correct, antimicrobial resistance (AMR) genes.

Comment 2: Prior to the implementation of phage application in the poultry industry, it is imperative that the author provides detailed information regarding the phage tapes that have been attached to S. Enteritidis or S. Typhimurium, along with their characterisation. For instance, Salmonella Typhimurium (DT Types): DT104, DT204c, DT12, DT66, DT110, DT193 and DT135. Salmonella Enteritidis (PT Types): The following letters are to be noted: PT19, PT47, PT29, PT6, PT8 and PT4.

Author Response

Here is some minor revision:

Response: Thank you so much for your valuable comment. The statement has now been supported with a citation.

Line 82: Salmonella spp. “.” Is missing!

Response: Thank you so much for your valuable comment. The error has been corrected.

Line 101: Enterobacteriaceae is italic.

Response: Thank you so much for your valuable comment. The word has been corrected.

Line 110: Salmonella spp. “.” Is missing!

Response: Thank you so much for your valuable comment. The error has been corrected.

Line 218: Replacre the reference “(Mehdi et al., 2018)” with number in the citation.

Response: Thank you very much for your valuable comment. The error has been corrected.

Line 369: correct, S. Enteritidis. Check and correct in whole manuscript.

Response: Thank you very much for your valuable comment. This section has been revised, and the error has been corrected.

Line 372: correct “S. Typhimurium”

Response: Thank you very much for your valuable comment. The error has been corrected.

Comment 1: It is imperative that the abbreviation 'PFU' (phage forming unit) is written in full for the first instance, or alternatively, it may be written under the table as well as “SPF”

Response: Thank you very much for your valuable comment. The error has been corrected.

Line 449: correct, antimicrobial resistance (AMR) genes.

Response: Thank you very much for your valuable comment. The error has been corrected.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the abstract accordingly (Line 284-293).

Reviewer 2 Report

Comments and Suggestions for Authors

The abstract states the global burden of salmonellosis exceeds 20 million cases annually with ~150,000 deaths (lines 60–62), but line 127 gives figures of 93 million infections and 155,000 deaths. These are dramatically different estimates and are attributed to different sources without explanation.
Line 121 reads "According to Sonalika et al. [39] (2020)…" but reference [39] in the reference list is a CDC source, while Sonalika et al. (2020) is reference [38]. Please correct this.

3. Section 6 mentions CRISPR-Cas as a phage resistance mechanism (line 494) but has a typo: “CRIPR-Cas9”. Further, bacteria employ multiple CRISPR-Cas types beyond Cas9, plus entirely different resistance mechanisms (receptor modification, restriction-modification systems, superinfection exclusion, abortive infection). The review should at minimum enumerate these, particularly since resistance is presented as a key limitation.

4. Line 218 contains a parenthetical in-text citation "(Mehdi et al., 2018)" that is inconsistent with the numbered citation style used throughout.

5. The Conclusion section largely restates the Abstract. It should instead summarize what is specifically known, what remains unknown, and what research is most urgently needed.

Author Response

1. The abstract states the global burden of salmonellosis exceeds 20 million cases annually with ~150,000 deaths (lines 60–62), but line 127 gives figures of 93 million infections and 155,000 deaths. These are dramatically different estimates and are attributed to different sources without explanation.

Response: Thank you very much for your valuable comment. The first statement (lines 60–62) refers to disease burden of salmonellosis in general, while the second statement (line 127) refers to Salmonella gastroenteritis only and its associated mortality. Therefore, the statement in line 127 has been revised to clarify and distinguish the two statements (Line 163-164).

2. Line 121 reads "According to Sonalika et al. [39] (2020)…" but reference [39] in the reference list is a CDC source, while Sonalika et al. (2020) is reference [38]. Please correct this.

Response: Thank you very much for your valuable comment. This error has been correct accordingly.

Response: Thank you very much for your valuable comment. This error has been correct accordingly and additional mechanisms enumerated as suggested.

4. Line 218 contains a parenthetical in-text citation "(Mehdi et al., 2018)" that is inconsistent with the numbered citation style used throughout.

Response: Thank you very much for your valuable comment. This has been revised and error has been correct accordingly.

5. The Conclusion section largely restates the Abstract. It should instead summarize what is specifically known, what remains unknown, and what research is most urgently needed.

Response: Thank you very much for your valuable comment. This has been revised accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

Bacteriophages as Sustainable Alternatives to Antibiotics for Controlling Salmonella in the Poultry Value Chain

In this review, the authors examine the antibacterial activity of bacteriophages against Salmonella, an important bacterial pathogen in the poultry industry. While the topic is relevant and potentially valuable, several limitations reduce the overall quality of the review. These include an unclear research aim, insufficient discussion of the limitations of phage therapy, and a lack of information regarding the current stage of research and whether any phage-based treatments have been approved for use. In addition, the review does not adequately explore strategies or alternative approaches to enhance the efficiency of phage therapy. Overall, the manuscript remains narrowly focused on summarizing selected studies on the antibacterial activity of certain bacteriophages against Salmonella in poultry, without providing a comprehensive or critical analysis of the field.

Following requests are necessary to be addressed to improve quality of the study.

Title:

In my opinion, given the various limitations associated with phage application and the potential role of bacteriophages in facilitating the transfer of resistance genes between bacterial genera, it is premature to consider phages as a definitive alternative to antibiotics. Therefore, it is recommended to revise the title and remove any phrasing that suggests bacteriophages are a direct substitute for antibiotics.

Abstract:

Line 25: A clear and well-defined aim should be provided to better delineate the objectives and scope of the review
Line 28-33: The abstract lacks specific findings from the reviewed studies that support the authors’ hypothesis or their claim that bacteriophages could serve as an alternative to antibiotics for controlling Salmonella in the poultry industry. The authors should highlight impactful and well-supported research in this field to strengthen the validity of their conclusions.
Line 38: The authors should include a clear statement outlining the significance of this review, specifically what new insights it provides and how it contributes to the current body of knowledge in the field.
Line 40: A clear statement outlining the limitations of the approach, along with how advances in molecular biology could help address these challenges, should be included at the end of this section, together with specific suggestions for future research directions.

Introduction

A detailed paragraph should be included describing the available methods for controlling Salmonella in poultry, along with the limitations of each approach. This will help support the aim of the review by providing a balanced context and demonstrating why bacteriophage therapy may represent a promising strategy for controlling Salmonella in the poultry industry.
At the end of this section, the authors should clearly present the main components of the review, along with its aim, and briefly describe the methodology used to collect and select the relevant articles on the use of bacteriophages for controlling Salmonella in poultry. This will improve clarity, help readers better understand the structure of the review, and provide a smooth transition to the subsequent sections.

Line 100: The title should be revised to first reflect the broader group of bacteria relevant to food safety, and then specifically highlight the importance of Salmonella among them.
Line 145: This figure is also applicable to Campylobacter. Therefore, the authors should clearly justify why Salmonella was selected as the focus of the study, and why Campylobacter was not included or discussed in this context.

Line 249: Before describing the application of bacteriophages against Salmonella, the mechanism of phage action should be clearly explained. This should include key aspects such as their spectrum of activity, species-related effects, and other relevant characteristics of their antibacterial properties.
Table 1 line 381: Most of the effects of bacteriophages presented in the table focus primarily on the reduction in Salmonella counts. However, the authors should also include other significant findings from the reviewed studies, such as the broad-spectrum activity or high specificity of bacteriophages, to provide a more comprehensive overview of their antibacterial characteristics.
Line 382: The authors are requested to include, if available, a table summarizing the types of bacteriophages approved for use against Salmonella, along with the corresponding countries where such approvals have been granted.
Line 473: The section on challenges should be expanded in detail to comprehensively address the limitations of bacteriophage use in both clinical and industrial settings. This should include potential impacts on animal and human health, as well as the possible role of bacteriophages in the dissemination of virulence and antibiotic resistance genes among different bacterial genera, and across animal, human, and environmental bacterial populations.

Conclusion

In need to explain if the result gotten from some reviewed article is logical or some of them is unexpected
Clearly explain the limitations of the study and recommend further research
Include a section that highlights and explains the clear contributions and implications of this study.

Author Response

Bacteriophages as Sustainable Alternatives to Antibiotics for Controlling Salmonella in the Poultry Value Chain

Following requests are necessary to be addressed to improve quality of the study.

Title:

In my opinion, given the various limitations associated with phage application and the potential role of bacteriophages in facilitating the transfer of resistance genes between bacterial genera, it is premature to consider phages as a definitive alternative to antibiotics. Therefore, it is recommended to revise the title and remove any phrasing that suggests bacteriophages are a direct substitute for antibiotics.

Response: Thank you very much for your valuable comment. The topic has been revised accordingly.

Abstract:

Line 25: A clear and well-defined aim should be provided to better delineate the objectives and scope of the review

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the abstract accordingly (Line 25-28).

Line 28-33: The abstract lacks specific findings from the reviewed studies that support the authors’ hypothesis or their claim that bacteriophages could serve as an alternative to antibiotics for controlling Salmonella in the poultry industry. The authors should highlight impactful and well-supported research in this field to strengthen the validity of their conclusions.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the abstract accordingly (Line 31-34).

Line 38: The authors should include a clear statement outlining the significance of this review, specifically what new insights it provides and how it contributes to the current body of knowledge in the field.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the abstract accordingly (Line 43-47).

Line 40: A clear statement outlining the limitations of the approach, along with how advances in molecular biology could help address these challenges, should be included at the end of this section, together with specific suggestions for future research directions.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the abstract accordingly (Line 49-54).

Introduction

A detailed paragraph should be included describing the available methods for controlling Salmonella in poultry, along with the limitations of each approach. This will help support the aim of the review by providing a balanced context and demonstrating why bacteriophage therapy may represent a promising strategy for controlling Salmonella in the poultry industry.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly (Line 77-86).

At the end of this section, the authors should clearly present the main components of the review, along with its aim, and briefly describe the methodology used to collect and select the relevant articles on the use of bacteriophages for controlling Salmonella in poultry. This will improve clarity, help readers better understand the structure of the review, and provide a smooth transition to the subsequent sections.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly (Line 115-128).

Line 100: The title should be revised to first reflect the broader group of bacteria relevant to food safety, and then specifically highlight the importance of Salmonella among them.

Response: Thank you very much for your valuable comment. This section has been revised and incorporated into the manuscript accordingly (Line 131-137).

Line 145: This figure is also applicable to Campylobacter. Therefore, the authors should clearly justify why Salmonella was selected as the focus of the study, and why Campylobacter was not included or discussed in this context.

Response: Thank you very much for your valuable comment. The reason for focusing on Salmonella has been justified in the revised text (Line 131-137).

Line 249: Before describing the application of bacteriophages against Salmonella, the mechanism of phage action should be clearly explained. This should include key aspects such as their spectrum of activity, species-related effects, and other relevant characteristics of their antibacterial properties.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly (Line 297-312).

Table 1 line 381: Most of the effects of bacteriophages presented in the table focus primarily on the reduction in Salmonella counts. However, the authors should also include other significant findings from the reviewed studies, such as the broad-spectrum activity or high specificity of bacteriophages, to provide a more comprehensive overview of their antibacterial characteristics.

Response: Thank you very much for your valuable comment. The table has been revised, and the suggestions have been incorporated accordingly.

Line 382: The authors are requested to include, if available, a table summarizing the types of bacteriophages approved for use against Salmonella, along with the corresponding countries where such approvals have been granted.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly (Line 479-482).

Line 473: The section on challenges should be expanded in detail to comprehensively address the limitations of bacteriophage use in both clinical and industrial settings. This should include potential impacts on animal and human health, as well as the possible role of bacteriophages in the dissemination of virulence and antibiotic resistance genes among different bacterial genera, and across animal, human, and environmental bacterial populations.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly.

Conclusion

In need to explain if the result gotten from some reviewed article is logical or some of them is unexpected

Clearly explain the limitations of the study and recommend further research

Include a section that highlights and explains the clear contributions and implications of this study.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly (Line 663-681).

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript presents a review of bacteriophage applications as alternatives to antibiotics for controlling Salmonella across the poultry value chain. This subject is pertinent, especially regarding the rising antimicrobial resistance and the necessity for enduring food safety measures. The manuscript offers a comprehensive overview and aggregates a substantial body of material; yet, it predominantly stays descriptive. The scientific contribution would be enhanced by more thorough critical analysis, better organization, and clearer expression of originality.

The manuscript primarily recapitulates previously published studies without offering adequate critical analysis. Sections 4.1–4.3 reference multiple research indicating reductions in Salmonella after bacteriophage application; nevertheless, the heterogeneity in results among these studies is little analyzed. The study design lacks thorough examination of potential limitations, like variations in experimental settings, phage strains, or sample numbers, which could affect the reported outcomes. Moreover, the manuscript inadequately tackles the issues of converting these insights into practical poultry production systems, encompassing scalability, cost, and environmental unpredictability.
Although the manuscript presents numerous quantitative findings, such as log reductions in Salmonella counts and multiplicity of infection values, these data are not effectively synthesized or interpreted. The results are largely reported in isolation, without establishing clear benchmarks for what constitutes effective control or comparing outcomes across studies.
This section on regulatory and industry viewpoints succinctly addresses regional disparities in regulatory methodologies. Nonetheless, it lacks to deliver a systematic comparison of frameworks across critical regions, like the United States, European Union, and Asia. The topic is also ambiguous regarding regulatory procedures, including the criteria for safety assessment and the commercialization of phage-based goods.
Certain conclusions in the manuscript seem more robust than what the existing evidence adequately substantiates. While bacteriophages provide a viable alternative to antibiotics, the text intermittently portrays them as a well-established and generally applicable remedy. The concluding parts inadequately address significant limitations, including regulatory restraints, the potential for phage resistance, and obstacles associated with large-scale adoption.
The manuscript contains grammatical errors, inconsistent punctuation, and repetitive phrasing. Terminology is not always used consistently, and some sentences lack clarity.

Author Response

1. The manuscript primarily recapitulates previously published studies without offering adequate critical analysis. Sections 4.1–4.3 reference multiple research indicating reductions in Salmonella after bacteriophage application; nevertheless, the heterogeneity in results among these studies is little analyzed. The study design lacks thorough examination of potential limitations, like variations in experimental settings, phage strains, or sample numbers, which could affect the reported outcomes. Moreover, the manuscript inadequately tackles the issues of converting these insights into practical poultry production systems, encompassing scalability, cost, and environmental unpredictability.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly.

2. Although the manuscript presents numerous quantitative findings, such as log reductions in Salmonella counts and multiplicity of infection values, these data are not effectively synthesized or interpreted. The results are largely reported in isolation, without establishing clear benchmarks for what constitutes effective control or comparing outcomes across studies.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly.

3. This section on regulatory and industry viewpoints succinctly addresses regional disparities in regulatory methodologies. Nonetheless, it lacks to deliver a systematic comparison of frameworks across critical regions, like the United States, European Union, and Asia. The topic is also ambiguous regarding regulatory procedures, including the criteria for safety assessment and the commercialization of phage-based goods.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly (Line 546-550, 597-600).

4. Certain conclusions in the manuscript seem more robust than what the existing evidence adequately substantiates. While bacteriophages provide a viable alternative to antibiotics, the text intermittently portrays them as a well-established and generally applicable remedy. The concluding parts inadequately address significant limitations, including regulatory restraints, the potential for phage resistance, and obstacles associated with large-scale adoption.

Response: Thank you very much for your valuable comment. This section has been revised, and the suggestions have been incorporated into the manuscript accordingly.

5. The manuscript contains grammatical errors, inconsistent punctuation, and repetitive phrasing. Terminology is not always used consistently, and some sentences lack clarity.

Response: Thank you very much for your valuable comment. These errors have been revised and incorporated into the manuscript accordingly.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Most of the requested corrections have been completed. Thank you.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors has addressed all the comments and revised the manuscript as per suggestions.