Review Reports - Phenotypic and Genomic Characterization of Novel Straboviridae Bacteriophages Targeting Multidrug-Resistant <i>Salmonella enterica</i> subspecies <i>enterica</i> Serovar Enteritidis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for a large and useful study. At the same time, there are several comments on the manuscript that could improve the results and make them clear.

Title:

Phenotypic and Genomic Characterization of Novel Straboviridae Bacteriophages Targeting Multidrug-Resistant Salmonella enterica Serovar Enteritidis

I think the name of bacteria under study is Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis)

Abstract: Please clearly state the research objective at the beginning of the abstract.

In my opinion, the Introduction could be expanded to include information on the way of controlling Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) studies. The goal and problems of the study should be clearly outlined.

The Materials and Methods section require significant expansion. Some of the methods used are not described.

Were the water samples collected in different locations? The location of samples is not indicated.

Is it necessary to indicate the chemical composition of the swage water used in this study. Does this data affect the experimental results in any way?

The rule is that Latin names of genera and species are written in italics. This applies to the entire manuscript:

Page 17, lines 646, 652

Page 18 Line 675, 690, 696: x g should be written the letter x according to scientific as italic

Page 18: line 679

Pages 18 and 19 LB, PFU, CFU, MOI and DLA: Write the term in full without abbreviation when it is mentioned for the first time, and then use the abbreviation afterward.

Page 18: line 690: Write the name of equipment and the originality of this machine (shaken at 150 rpm)

Sometimes use 5 minutes and other time you use 5 min. Please use the standard units in all the manuscript.

2- Lines 655 and 656: For preservation, glycerol stocks (50%, v/v; 655 Fisher Chemical, Waltham, MA, USA) were prepared and stored at −20 °C.

This data is wrong with the concentration of glycerol between 15-20% not 50% and stored at -80 not -20 C.

3- Line 673: 50 mL of each sample: this volume is little; the sample should be one liter for each sample.

The Results section could be shortened by removing unnecessary duplication of information. Some tables and figures are missing links in the text.

Discussion: Please remove unnecessary duplication and focus on isolated pathogenic bacteria and phages as an effective means of combating multidrug-resistant bacteria and reducing antibiotic use as a new approach, and what the future prospects are in this field.

Author Response

Reviewer 1: Comments

Thank you very much for your constructive and insightful comments. We sincerely appreciate the reviewer’s careful evaluation of our manuscript. All comments were carefully considered, and the manuscript has been revised accordingly. Below, we provide a detailed point-by-point response.

Comment 1

Title: “Phenotypic and Genomic Characterization of Novel Straboviridae Bacteriophages Targeting Multidrug-Resistant Salmonella enterica Serovar Enteritidis”

I think the name of bacteria under study is Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis).

Response:

Thank you for this important observation. The title has been revised accordingly to reflect the complete and taxonomically accurate bacterial nomenclature. The revised title now reads:

“Phenotypic and Genomic Characterization of Novel Straboviridae Bacteriophages Targeting Multidrug-Resistant Salmonella enterica subspecies enterica Serovar Enteritidis”

Comment 2

Abstract: Please clearly state the research objective at the beginning of the abstract.

Response:

Thank you for this valuable suggestion. The abstract was revised to clearly introduce the study objective at the beginning. Specifically, the following statement was added:

“This study aimed to isolate and comprehensively characterize novel lytic bacteriophages targeting multidrug-resistant Salmonella enterica subspecies enterica serovar Enteritidis isolates from Lebanon.”

This revision improves the clarity and structure of the abstract.

Comment 3

In my opinion, the Introduction could be expanded to include information on the way of controlling Salmonella enterica subspecies enterica serovar Enteritidis studies. The goal and problems of the study should be clearly outlined.

Response:

We thank the reviewer for this helpful recommendation. The Introduction was substantially expanded to include additional discussion regarding current Salmonella Enteritidis control strategies, including biosecurity measures, sanitation programs, vaccination, surveillance systems, and antibiotic-based interventions. Their limitations, particularly in the context of multidrug resistance, were also discussed.

In addition, the study objectives and research questions were clarified and explicitly stated near the end of the Introduction. The revised Introduction now clearly outlines:

the clinical and public health importance of MDR Enteritidis,
the rationale for investigating bacteriophages as alternative biocontrol agents,
the major knowledge gaps addressed by the study,
and the specific aims of the phenotypic and genomic characterization performed.

Comment 4

The Materials and Methods section require significant expansion. Some of the methods used are not described.

Response:

Thank you for this important comment. The Materials and Methods section was extensively revised and expanded to improve methodological clarity, reproducibility, and transparency.

Additional methodological details were incorporated throughout the section, including:

bacterial strain preparation and antimicrobial susceptibility interpretation,
wastewater sample collection strategy,
enrichment and purification procedures,
adsorption and one-step growth methodologies,
MOI calculation procedures,
bacteriolytic assays,
pH and thermal stability protocols,
phage DNA extraction methodology,
sequencing and bioinformatic workflows,
genome assembly and annotation pipelines,
and statistical/data presentation considerations.

These revisions substantially improve the reproducibility and completeness of the methodological description.

Comment 5

Were the water samples collected in different locations? The location of samples is not indicated.

Response:

Thank you for pointing this out. The manuscript was revised to clearly indicate that untreated sewage samples were collected from geographically distinct locations across Lebanon, including:

Beirut,
Mount Lebanon,
Bekaa,
and South Lebanon.

We additionally specified that the samples originated from municipal wastewater discharge points and poultry farm effluents. These details have now been included in Section 4.2 (“Sewage Sample Collection and Processing”).

Comment 6

Is it necessary to indicate the chemical composition of the sewage water used in this study? Does this data affect the experimental results in any way?

Response:

We appreciate this thoughtful question. A clarification was added in the Materials and Methods section stating that the physicochemical composition of the wastewater samples was not analyzed because the primary objective of the study was bacteriophage isolation and characterization rather than environmental wastewater profiling.

We also clarified that the enrichment-based isolation strategy employed in this study was designed to selectively recover bacteriophages infecting the targeted MDR Salmonella hosts, independently of detailed wastewater chemistry characterization. Accordingly, the absence of physicochemical wastewater profiling does not affect the interpretation of the phage characterization results presented in this study.

Comment 7

The rule is that Latin names of genera and species are written in italics. This applies to the entire manuscript.

Response:

Thank you for highlighting this formatting issue. The entire manuscript was carefully revised to ensure that all Latin genus and species names are consistently written in italics according to scientific nomenclature standards.

Comment 8

Page 17, lines 646, 652 / Page 18 Line 675, 690, 696: x g should be written the letter x according to scientific as italic.

Response:

Thank you for noting this formatting inconsistency. All centrifugation units throughout the manuscript were corrected and standardized using the proper scientific notation format “× g”.

Comment 9

Pages 18 and 19: LB, PFU, CFU, MOI and DLA: Write the term in full without abbreviation when it is mentioned for the first time, and then use the abbreviation afterward.

Response:

We thank the reviewer for this important observation. The manuscript was revised to ensure that all abbreviations are introduced in full upon first mention before subsequent abbreviation use. This includes:

Luria–Bertani (LB),
plaque-forming unit (PFU),
colony-forming unit (CFU),
multiplicity of infection (MOI),
and double-layer agar (DLA).

This revision improves readability and consistency throughout the manuscript.

Comment 10

Page 18: line 690: Write the name of equipment and the originality of this machine (shaken at 150 rpm).

Response:

Thank you for this comment. The corresponding section was revised to include the equipment details and manufacturer information. Specifically, the orbital shaking incubator used during enrichment was identified as:

“orbital shaking incubator (New Brunswick Scientific, Edison, NJ, USA)”

This information has now been incorporated into the Materials and Methods section.

Comment 11

Sometimes use 5 minutes and other time you use 5 min. Please use the standard units in all the manuscript.

Response:

We appreciate the reviewer’s attention to consistency. The manuscript was thoroughly revised to standardize all scientific units and abbreviations according to journal conventions. Time measurements are now consistently presented using SI-style abbreviations (e.g., “min”, “h”).

Comment 12

Lines 655 and 656: For preservation, glycerol stocks (50%, v/v; Fisher Chemical, Waltham, MA, USA) were prepared and stored at −20 °C. This data is wrong with the concentration of glycerol between 15–20% not 50% and stored at −80 °C.

Response:

We thank the reviewer for identifying this error. The manuscript was corrected accordingly. The revised statement now reads:

“For preservation, glycerol stocks (20%, v/v; Fisher Chemical, Waltham, MA, USA) were prepared and stored at −80 °C.”

We appreciate the reviewer’s careful attention to this methodological detail.

Comment 13

Line 673: 50 mL of each sample: this volume is little; the sample should be one liter for each sample.

Response:

We thank the reviewer for this valuable observation. We clarified in the revised manuscript that although larger sample volumes are frequently used in environmental virome studies, the present study employed an enrichment-based isolation strategy that enabled successful bacteriophage recovery from smaller sample volumes.

We additionally clarified that the study objective was targeted phage isolation rather than comprehensive environmental virome characterization. Despite the smaller processed volume, the enrichment strategy successfully yielded four distinct lytic bacteriophages suitable for downstream phenotypic and genomic characterization.

Comment 14

The Results section could be shortened by removing unnecessary duplication of information. Some tables and figures are missing links in the text.

Response:

Thank you for this important recommendation. The Results section was carefully revised to reduce unnecessary repetition and improve readability. Redundant descriptions between the text, figures, and tables were minimized while preserving essential scientific interpretation.

In addition, all figures, supplementary figures, and tables were carefully cross-checked and explicitly linked within the text to improve manuscript organization and continuity.

Comment 15

Discussion: Please remove unnecessary duplication and focus on isolated pathogenic bacteria and phages as an effective means of combating multidrug-resistant bacteria and reducing antibiotic use as a new approach, and what the future prospects are in this field.

Response:

We sincerely appreciate this valuable suggestion. The Discussion section was substantially revised to reduce redundancy and improve focus on the translational significance of bacteriophage-based biocontrol against multidrug-resistant Salmonella.

The revised Discussion now places stronger emphasis on:

the potential role of lytic bacteriophages as targeted alternatives to conventional antibiotics,
their relevance in reducing antimicrobial usage and mitigating antimicrobial resistance,
the importance of phage cocktails in limiting bacterial resistance emergence,
applications in food safety and poultry-associated biocontrol,
and future prospects including in vivo validation, food matrix studies, and phage–antibiotic synergistic strategies.

Several repetitive statements were removed or consolidated to improve clarity and overall manuscript flow.

We again sincerely thank the reviewer for the constructive and insightful comments, which significantly improved the quality, clarity, and scientific rigor of the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Overall Comments: This study by Abou Daoud et al. presents the isolation and comprehensive characterization of four novel bacteriophages against multidrug-resistant Salmonella Enteritidis ST11 from Lebanon, a region with limited prior data on such phages. The work is methodologically sound, combining thorough phenotypic assays (host range, one-step growth, pH/thermal stability, bacteriolytic activity) with whole-genome sequencing and phylogenetic analysis to confirm the lytic, safe nature of the isolates. The identification of phages with complementary profiles (e.g., broad host range of EDA03/06 vs. high burst size of EDA05) provides a rational basis for future cocktail development, and the genomic safety screening (absence of lysogeny, virulence, or AMR genes) supports their biocontrol potential. Overall, this is a valuable contribution to the fields of phage biology and antimicrobial resistance mitigation, and it addresses an important regional knowledge gap.

1.Since the data has been replicated three times, it is fully eligible for statistical analysis. We strongly urge the author to supplement the statistical analysis data, such as temperature and pH data，and figure 5, to make it more scientific and instructive. Additionally, the statistical analysis method should be supplemented.

2.Line501, "(29–32)."The referenced literature is in bold font, while other parts are not. Please standardize the referencing format of the text.

3.The caption of Figure 5 is not clear. There are a total of four figures, A, B, and D. However, it is not indicated or explained which bacteriophage each figure corresponds to. Please write it clearly in the figure and caption.

4.In Figure 5, the results indicate that the bacteria have recovered after 5 hours, and the titer of bacteriophages in Table A3 is also very high, such as EDA05, which can reach 10 ¹¹ PFU/mL. There are contradictions between these results, please focus on discussing them in the discussion and find references with similar situations for discussion.

5.The inhibition of Salmonella by different MOI of different bacteriophages in Figure 5 did not show a significant dose-dependent effect. Please modify this expression in the text and indicate the differences in concentration inhibition of each bacteriophage in the result description of Figure 5.

6.Please standardize the format of the references, especially the[31] reference. Do not look for online links, please cite the official source of the reference.

Author Response

Reviewer 2: Comments:

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We highly appreciate the reviewer’s insightful feedback, which has helped us improve the clarity, scientific rigor, and overall quality of the manuscript. All comments have been carefully considered, and the manuscript has been revised accordingly. Our detailed responses are provided below.

Comment 1:

Since the data has been replicated three times, it is fully eligible for statistical analysis. We strongly urge the author to supplement the statistical analysis data, such as temperature and pH data, and Figure 5, to make it more scientific and instructive. Additionally, the statistical analysis method should be supplemented.

Response:
We thank the reviewer for this valuable comment regarding the inclusion of statistical analyses. In response, we have expanded the “Data Presentation and Statistical Considerations” section to clearly describe the statistical approach applied in this study. Specifically, phage stability under different pH and temperature conditions was analyzed using two-way ANOVA, with phage identity and treatment condition as fixed factors. When significant main effects or interactions were detected, Dunnett’s multiple-comparison post-hoc test was performed using pH 7 and 37 °C as the respective reference conditions. Statistical significance was defined as p < 0.05, and all data are presented as mean ± SD from three independent biological replicates.

Comment 2:

Line 501, “(29–32).” The referenced literature is in bold font, while other parts are not. Please standardize the referencing format of the text.

Response:
We thank the reviewer for identifying this formatting inconsistency. The reference formatting throughout the manuscript has been carefully revised and standardized to ensure uniform citation style and text formatting across all sections of the manuscript.

Comment 3:

The caption of Figure 5 is not clear. There are a total of four figures, A, B, and D. However, it is not indicated or explained which bacteriophage each figure corresponds to. Please write it clearly in the figure and caption.

Response:
We appreciate the reviewer’s observation. The caption of Figure 5 has been revised to explicitly indicate the corresponding bacteriophage represented in each panel. The revised caption now clearly specifies: (A) EDA02, (B) EDA03, (C) EDA05, and (D) EDA06, thereby improving figure clarity and interpretability.

Comment 4:

In Figure 5, the results indicate that the bacteria have recovered after 5 hours, and the titer of bacteriophages in Table A3 is also very high, such as EDA05, which can reach 10¹¹ PFU/mL. There are contradictions between these results, please focus on discussing them in the discussion and find references with similar situations for discussion.

Response:
We thank the reviewer for this insightful comment. We agree that the coexistence of high phage titers and partial bacterial regrowth requires additional clarification. Accordingly, the Discussion section has been substantially expanded to address this phenomenon in greater detail. Specifically, we clarified that sustained or increasing extracellular phage titers do not necessarily indicate complete bacterial eradication, as bacterial regrowth may occur through the emergence of resistant or phenotypically tolerant subpopulations following the initial lytic phase. We further discussed potential mechanisms underlying this regrowth, including receptor modification, reduced adsorption efficiency, phase variation, and activation of intracellular bacterial defense pathways. In addition, we incorporated several references reporting similar phage–host dynamics in Salmonella and other enterobacterial systems. These additions improve the biological interpretation of the bacteriolytic assays and further support the rationale for future multi-phage cocktail development.

Comment 5:

The inhibition of Salmonella by different MOI of different bacteriophages in Figure 5 did not show a significant dose-dependent effect. Please modify this expression in the text and indicate the differences in concentration inhibition of each bacteriophage in the result description of Figure 5.

Response:

We thank the reviewer for this important observation. We agree that the inhibition profiles observed across different MOIs did not demonstrate a strong or strictly dose-dependent pattern. Accordingly, the text describing the bacteriolytic assays has been revised to avoid overinterpretation of MOI-dependent effects. The Results section now more accurately states that bacterial growth suppression varied modestly across MOIs and depended primarily on phage identity and infection dynamics. In addition, the revised description now explicitly distinguishes the inhibition profiles of EDA02, EDA03, EDA05, and EDA06 at different MOIs, while acknowledging that bacterial regrowth occurred under several conditions despite initial suppression.

Comment 6:

Please standardize the format of the references, especially the [31] reference. Do not look for online links, please cite the official source of the reference.

Response:
We appreciate the reviewer’s careful assessment of the reference formatting. The reference list has been thoroughly revised and standardized according to the journal formatting guidelines. In particular, Reference [31] has been corrected to remove non-standard online citation formatting and has been replaced with the official journal citation source where available. Additional formatting inconsistencies throughout the reference section were also corrected to ensure consistency and compliance with journal requirements.