Review Reports - Bacteriophages 6phi8, 6phi10, and 6phi13 Isolated from the Therapeutic Cocktail “Sextaphag<sup>®</sup>”

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents and interesting premise, connecting AMR, E. coli clinical relevance, and the need to characterize therapeutic phage-cocktail isolates as a precursor to clinical or formulation use. The hypothesis is more descriptive than mechanistic, focusing more on characterization of phages rather than delineating and testing a focused biological hypothesis.

The results generally demonstrate isolation, plaque morphology, killing activity, pH/thermal stability, genome annotation, and comparative taxonomy for the three phages. The genomic and bioinformatic pipelines (e.g., Illumina sequencing, SPAdes for assembly, Pharokka for annotation, and ViPTree for phylogenetics) are well chosen for supporting accurate classification, but the technical impact and implications are narrow. Specifically, the study is less effective in providing a strong basis to argue therapeutic relevance, though, since testing is focused on E. coli MG1655 rather than broader clinical E. coli/Enterobacteriaceae panels. MG1655 is a highly domesticated, non-pathogenic laboratory strain that lacks many of the defense mechanisms found in both conventional wild-type and multidrug-resistant clinical isolates. Such deficiencies present major limitations on the practical impact of the study, as well as tangible relevance to safety assessments.

Beyond this, the writeup relies largely on qualitative observations trends, without clearly reported statistical tests, confidence intervals, effect sizes, or formal comparisons linking experimental results to the conclusions. Rigor is limited by narrow host-range testing, short killing assays, no electron microscopy or adsorption/latent-period/burst-size data, and limited evidence that the isolates represent clinically useful cocktail components. These shed some doubt on the safety discussions; they do not contravene the more modest claims and promises in the manuscript, but do further limit the real-world relevance of the study.

Fundamentally, there is academic merit in the study, and it may thus warrant publication. It is generally well written, and mostly avoids undue claims. It is not a blockbuster, however. The authors may improve the positioning of the work by stating clearly how this study might be used to design more impactful follow-ups.

Author Response

Dear Reviewer 1,

We are grateful for the constructive comments and for recognizing the scientific merit of our study.

"The manuscript presents and interesting premise, connecting AMR, E. coli clinical relevance, and the need to characterize therapeutic phage-cocktail isolates as a precursor to clinical or formulation use. The hypothesis is more descriptive than mechanistic, focusing more on characterization of phages rather than delineating and testing a focused biological hypothesis.

We agree that evaluation of the phages against a broader panel of clinical and multidrug-resistant E. coli isolates would provide valuable information regarding their therapeutic potential. Since our laboratory is primarily focused on fundamental bacteriophage biology and does not have direct access to collections of clinical isolates, evaluation of phage activity against clinical isolates is currently not feasible.

"Specifically, the study is less effective in providing a strong basis to argue therapeutic relevance, though, since testing is focused on E. coli MG1655 rather than broader clinical E. coli/Enterobacteriaceae panels. MG1655 is a highly domesticated, non-pathogenic laboratory strain that lacks many of the defense mechanisms found in both conventional wild-type and multidrug-resistant clinical isolates. Such deficiencies present major limitations on the practical impact of the study, as well as tangible relevance to safety assessments."

The bacteriophages investigated in this study are not newly proposed therapeutic candidates. All three phages are already included in the widely used therapeutic phage cocktail "Sextaphag" (Microgen), which can be purchased at a pharmacy (https://microgen.ru/en/products/bakteriofagi/sekstafag-piobakteriofag-polivalentnyy/). Microgen has been producing therapeutic phage cocktails for over two decades. Since the genetic nature of the phages in these cocktails has not been established, the primary objective of the present work was to provide a comprehensive characterization of their genomic features and basic physicochemical properties. We believe that detailed genomic characterization, together with assessment of fundamental biological properties, represents an important component of the characterization and quality assessment of bacteriophages used in therapeutic phage cocktails. To better reflect this scope, we have made a revision of the manuscript to clarify the positioning of the study and to avoid overemphasizing its clinical implications.

"Beyond this, the writeup relies largely on qualitative observations trends, without clearly reported statistical tests, confidence intervals, effect sizes, or formal comparisons linking experimental results to the conclusions. Rigor is limited by narrow host-range testing, short killing assays, no electron microscopy or adsorption/latent-period/burst-size data, and limited evidence that the isolates represent clinically useful cocktail components. These shed some doubt on the safety discussions; they do not contravene the more modest claims and promises in the manuscript, but do further limit the real-world relevance of the study."

In all figures with the experimental results data shown with error bars (Figures 2-4). We decided not to analyze phage virions using transmission electron microscopy because such analysis had already been done for phages of the same genus, as the virions look similar (Т4-like and T7-phage like). We also replaced the adsorption/latent-period/burst-size characterization with a killing assay due to its clarity and speed. Killing assays most clearly demonstrate lytic characteristics and allow us to distinguish virulent phages from temperate ones. Detailed characteristics such as adsorption/latent period/burst size are typically specific to the phage-host pair and also depend on external factors such as temperature, nutrient medium composition, and agitation/mixing. Given that we used a model E. coli strain, we decided to limit bacteriophage characterization to the most effective methods (i.e. killing assay, and pH- and thermostability).

"Fundamentally, there is academic merit in the study, and it may thus warrant publication. It is generally well written, and mostly avoids undue claims. It is not a blockbuster, however. The authors may improve the positioning of the work by stating clearly how this study might be used to design more impactful follow-ups."

Our next study – to use these bacteriophages in in vitro assembly system, based in coupled in vitro transcription – in vitro translation. We believe that use of therapeutic phages will increase the impact of our publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive biological and genomic characterization of three Escherichia bacteriophages isolated from the commercial therapeutic cocktail Sextaphag®. The study combines phenotypic characterization, whole-genome sequencing, comparative genomics, and taxonomic analyses, providing valuable information on bacteriophages with potential therapeutic application. The genomic analyses are comprehensive, the manuscript is generally well organized, and the figures are of good graphical quality. The identification of bacteriophage 6phi10 as a putative novel species within the genus Berlinvirus further increases the scientific interest of the study. Overall, this work has merit and addresses a relevant topic in the field of phage biology and antimicrobial resistance.
Nevertheless, I believe that several aspects should be improved before the manuscript is suitable for publication.

Major comments

The manuscript would benefit from a more comprehensive description of the commercial product Sextaphag®. Since the study is entirely based on bacteriophages isolated from this therapeutic cocktail, readers need a clearer understanding of the product itself. The manuscript currently provides only a brief description of the bacterial genera targeted by Sextaphag®, without presenting references describing its development, formulation, validation, regulatory status, or clinical use. Moreover, it remains unclear whether the three bacteriophages characterized in this study represent the complete anti-E. coli fraction of the product or only a subset of a more complex phage mixture. This lack of contextual information limits the interpretation of the biological relevance of the results and affects the Discussion and Conclusions. The Introduction should therefore include a more detailed description of Sextaphag®, supported by appropriate references.
The biological characterization remains relatively limited considering the therapeutic perspective proposed throughout the manuscript. The study evaluates plaque morphology, killing assays, physicochemical stability, and genomic characteristics using only the laboratory strain E. coli MG1655. Although these analyses are technically sound, additional information regarding host range, efficiency of plating, adsorption kinetics, latent period, burst size, or, at minimum, a discussion acknowledging these limitations, would considerably strengthen the biological significance of the work. Consequently, statements regarding the therapeutic potential of these phages should be presented more cautiously.
The manuscript suggests that the studied bacteriophages may infect bacterial species beyond Escherichia coli, based primarily on their phylogenetic relationships with bacteriophages isolated from Shigella, Salmonella, Yersinia, Enterobacter, and other members of the family Enterobacteriaceae. However, this conclusion is not supported by experimental evidence presented in the manuscript. If host range experiments demonstrating cross-species infectivity have been performed, these results should be included or, if previously published, appropriately cited. Otherwise, the discussion should clearly state that these observations represent a hypothesis based on genomic relatedness rather than experimentally demonstrated infectivity. Furthermore, considering the therapeutic context of Sextaphag®, the authors are encouraged to discuss both the potential advantages (e.g., broader bacterial coverage) and disadvantages (e.g., reduced specificity, possible effects on the microbiota, and regulatory implications) of incorporating bacteriophages with a broader host range into commercial phage formulations.
Some experimental results require better integration into the manuscript. In particular, Figure 6 presents the restriction profile of phage 6phi10 DNA obtained in vitro and in silico, but this experiment is only briefly mentioned in the text and is not effectively discussed. The manuscript does not explain how the experimental restriction profile validates the genomic assembly or the identification of direct terminal repeats, nor does it interpret the observed banding pattern. If the authors consider this experiment relevant, additional explanation should be incorporated into the Results and Discussion. Otherwise, removal of this figure should be considered, as it currently adds little to the scientific narrative.
The Discussion is comprehensive but could be more concise and analytical. Several sections, particularly those describing sequence conservation of structural proteins and detailed comparisons with closely related phages, become highly descriptive. Condensing these sections would improve readability and allow greater emphasis on the biological implications, novelty, and significance of the findings.

Author Response

Dear Reviewer 2,

We are grateful for the careful evaluation of our manuscript and for the constructive comments and suggestions.

"The manuscript presents a comprehensive biological and genomic characterization of three Escherichia bacteriophages isolated from the commercial therapeutic cocktail Sextaphag®. The study combines phenotypic characterization, whole-genome sequencing, comparative genomics, and taxonomic analyses, providing valuable information on bacteriophages with potential therapeutic application. The genomic analyses are comprehensive, the manuscript is generally well organized, and the figures are of good graphical quality. The identification of bacteriophage 6phi10 as a putative novel species within the genus Berlinvirus further increases the scientific interest of the study. Overall, this work has merit and addresses a relevant topic in the field of phage biology and antimicrobial resistance."

We appreciate the positive assessment of the scientific merit of our work, the genomic analyses, and the overall organization of the manuscript. We have carefully considered all comments and revised the manuscript where appropriate. Our point-by-point responses are provided below.

"Nevertheless, I believe that several aspects should be improved before the manuscript is suitable for publication.

Major comments

Comment 1:

The manuscript would benefit from a more comprehensive description of the commercial product Sextaphag®. Since the study is entirely based on bacteriophages isolated from this therapeutic cocktail, readers need a clearer understanding of the product itself. The manuscript currently provides only a brief description of the bacterial genera targeted by Sextaphag®, without presenting references describing its development, formulation, validation, regulatory status, or clinical use.

Moreover, it remains unclear whether the three bacteriophages characterized in this study represent the complete anti-E. coli fraction of the product or only a subset of a more complex phage mixture. This lack of contextual information limits the interpretation of the biological relevance of the results and affects the Discussion and Conclusions. The Introduction should therefore include a more detailed description of Sextaphag®, supported by appropriate references."

The bacteriophages characterized in this study were isolated from the commercial therapeutic phage cocktail Sextaphag®, manufactured by Microgen (Russia). We added more description of Sextaphag® in the introduction section and added a reference with available description from producer: https://microgen.ru/en/products/bakteriofagi/sekstafag-piobakteriofag-polivalentnyy/”. To the best of our knowledge, no publications describing the development, formulation, validation, or complete composition of Sextaphag® are currently available. Existing studies are limited to individual bacteriophages isolated from this therapeutic cocktail rather than the formulation itself. Furthermore, detailed information regarding the composition and manufacturing of Sextaphag® is proprietary and has not been publicly disclosed by the manufacturer. Consequently, it is not possible to determine whether the three bacteriophages characterized in the present study represent the complete Escherichia-infecting fraction of Sextaphag® or only a subset of a more complex phage mixture. It is likely that the three bacteriophages characterized in this study represent only a subset of the Escherichia-targeting phages present in the therapeutic cocktail.

"Comment 2:

The biological characterization remains relatively limited considering the therapeutic perspective proposed throughout the manuscript. The study evaluates plaque morphology, killing assays, physicochemical stability, and genomic characteristics using only the laboratory strain E. coli MG1655. Although these analyses are technically sound, additional information regarding host range, efficiency of plating, adsorption kinetics, latent period, burst size, or, at minimum, a discussion acknowledging these limitations, would considerably strengthen the biological significance of the work. Consequently, statements regarding the therapeutic potential of these phages should be presented more cautiously."

Response 2:

We agree that additional experiments, such as host range determination, efficiency of plating, adsorption kinetics, latent period, and burst size analyses, would provide a more comprehensive biological characterization of the bacteriophages. However, (i) these analyses were beyond the scope of the present study and would substantially increase its scope and length; (ii) we believed that significant part of such assays were done when the therapeutic cocktail were designed; (iii) for the treatment of a individual clinical case of bacterial infection, a biomaterial suitability to Sextaphag® cocktail currently available in large diagnostic laboratories networks such as Gemotest (https://gemotest.ru/moskva/catalog/infektsii/bakterioskopiya-i-posevy/chuvstvitelnost-k-antibiotikam-antimikotikam-i-bakteriofagam/opredelenie-chuvstvitelnosti-vozbuditelya-k-bakteriofagam/) or Citylab (https://citilab.ru/moskva/catalog/dopolnitelnye_bakteriologicheskie_issledovaniya_106/chuvstvitelnost_k_bakteriofagam/). The primary aim of this work was to characterize the genomes and fundamental physicochemical properties of three isolated bacteriophages. Following the Reviewer's recommendation, we have revised the Discussion to explicitly acknowledge these limitations and to outline these analyses as important directions for future research.

"Comment 3:

The manuscript suggests that the studied bacteriophages may infect bacterial species beyond Escherichia coli, based primarily on their phylogenetic relationships with bacteriophages isolated from Shigella, Salmonella, Yersinia, Enterobacter, and other members of the family Enterobacteriaceae. However, this conclusion is not supported by experimental evidence presented in the manuscript. If host range experiments demonstrating cross-species infectivity have been performed, these results should be included or, if previously published, appropriately cited. Otherwise, the discussion should clearly state that these observations represent a hypothesis based on genomic relatedness rather than experimentally demonstrated infectivity. Furthermore, considering the therapeutic context of Sextaphag®, the authors are encouraged to discuss both the potential advantages (e.g., broader bacterial coverage) and disadvantages (e.g., reduced specificity, possible effects on the microbiota, and regulatory implications) of incorporating bacteriophages with a broader host range into commercial phage formulations."

Response 3:

Our observations may only suggest the possibility of a broader host range based on phylogenetic and genomic relatedness and do not provide experimental evidence of cross-species infectivity. To avoid overinterpretation, we have revised the corresponding sections of the manuscript to clearly emphasize the hypothetical nature of this statement.

"Comment 4:

Some experimental results require better integration into the manuscript. In particular, Figure 6 presents the restriction profile of phage 6phi10 DNA obtained in vitro and in silico, but this experiment is only briefly mentioned in the text and is not effectively discussed. The manuscript does not explain how the experimental restriction profile validates the genomic assembly or the identification of direct terminal repeats, nor does it interpret the observed banding pattern. If the authors consider this experiment relevant, additional explanation should be incorporated into the Results and Discussion. Otherwise, removal of this figure should be considered, as it currently adds little to the scientific narrative."

Response 4:

We appreciate the Reviewer's observation regarding Figure 6 and agree that the purpose of this experiment was not sufficiently explained in the original version of the manuscript. The determination of genome termini is an important step in bacteriophage genome characterization, as it provides information on the DNA packaging strategy. In the case of bacteriophage 6phi10, which is closely related to T7-like phages, prediction of direct terminal repeats (DTRs) was facilitated by comparison with closely related bacteriophages possessing experimentally validated DTRs, together with analysis of raw sequencing read coverage, where elevated coverage at the genome termini is consistent with the presence of terminal repeats. Nevertheless, to further validate the predicted DTRs, we performed restriction analysis of the phage genomic DNA. The experimentally obtained restriction profile allows visualization of the terminal genome fragments, and agreement between the observed fragment sizes and the in silico restriction pattern supports the correctness of the selected genome termini and DTR boundaries. To improve clarity, we have expanded the Results and Discussion sections to better explain the purpose of this experiment.

"Comment 5:

The Discussion is comprehensive but could be more concise and analytical. Several sections, particularly those describing sequence conservation of structural proteins and detailed comparisons with closely related phages, become highly descriptive. Condensing these sections would improve readability and allow greater emphasis on the biological implications, novelty, and significance of the findings."

Response 5:

Thank you for this helpful suggestion. We agree that maintaining a balance between comprehensiveness and conciseness is important. However, we believe that the current Discussion adequately covers all major findings presented in the Results and discusses their biological significance in sufficient detail. For this reason, we have retained the overall content of the Discussion, while making minor editorial revisions to improve its readability.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript includes several useful clarifications and improvements. However, after carefully comparing the authors’ responses with the revised text, I consider that a number of the original concerns have been only partially addressed. In several instances, the response letter provides relevant explanations that are not fully incorporated into the manuscript, and the absence of page and line references makes it difficult to verify the extent of the revisions. My remaining comments are presented below.

Comment 1 – Description and composition of Sextaphag®

This comment has been only partially addressed. The revised Introduction now identifies the manufacturer, describes the bacterial genera targeted by Sextaphag®, and refers to the product as a sterile filtrate of bacteriophage lysates. However, the additional information remains insufficient to provide the requested context regarding the product’s development, formulation, validation, regulatory status, and clinical use.

Reference 6 consists only of a manufacturer’s webpage and does not provide adequate scientific or regulatory support for all statements made about the commercial preparation. Moreover, the manuscript does not establish whether the current product information corresponds to the specific batch used in this study, namely batch P813 from December 2016. The authors should clarify the relevance of this source to the tested product and, where available, include appropriate scientific, regulatory, or product-specific documentation.

The response letter also states that previous studies have characterized individual bacteriophages isolated from Sextaphag®. However, these studies have not been identified or cited in the revised Introduction. If such publications are available, they should be cited and briefly discussed to place the present isolates in the context of previous work involving the same commercial cocktail.

In addition, there is an important inconsistency between the response letter and the revised manuscript. In the response, the authors correctly acknowledge that it is not possible to determine whether the three bacteriophages represent the complete Escherichia-infecting fraction of Sextaphag® or only a subset. Nevertheless, the revised Introduction states that these bacteriophages “therefore represent only a subset” rather than the complete anti-E. coli component. This conclusion is not supported by the available information. The fact that Sextaphag® targets several bacterial genera demonstrates only that the three isolates represent a subset of the complete multicomponent cocktail; it does not establish whether they constitute all or only part of its Escherichia-targeting fraction.

The text should therefore be revised to state explicitly that the composition of the Escherichia-targeting fraction remains unknown because the complete formulation has not been publicly disclosed.

Comment 2 – Limited biological characterization

This comment has not been adequately addressed, and substantial concerns remain. The authors acknowledge that host-range determination, efficiency-of-plating assays, adsorption kinetics, latent-period determination, and burst-size analysis would provide a more comprehensive biological characterization. However, the response does not clearly identify where the corresponding limitations and revisions were incorporated into the manuscript.

The statement that a substantial proportion of these assays was presumably performed during the development of the therapeutic cocktail is not scientifically acceptable without supporting evidence. It remains unknown whether such experiments were conducted, which methods and bacterial strains were used, whether the data refer specifically to phages 6phi8, 6phi10, and 6phi13, and whether the results are publicly available. If published studies or technical documentation exist, they should be cited. Otherwise, this assumption should be removed from the response.

Similarly, the availability of commercial diagnostic services that evaluate susceptibility to the complete Sextaphag® formulation does not substitute for the biological characterization of the individual bacteriophages examined in this study. Such services do not provide host-range, efficiency-of-plating, adsorption, latent-period, or burst-size data for phages 6phi8, 6phi10, and 6phi13.

The use of only the laboratory strain Escherichia coli MG1655 remains a major limitation of the biological component of the study. The current experiments demonstrate activity against this single strain but do not establish the host range, infective efficiency, or therapeutic relevance of the three phages against diverse E. coli isolates.

The authors should therefore either:

provide additional biological data, particularly host-range and efficiency-of-plating results using a representative panel of E. coli strains; or
substantially restrict the scope and wording of the manuscript, clearly defining the work as a genomic and preliminary physicochemical characterization and moderating all statements regarding therapeutic potential.

At minimum, the Discussion and Conclusions should explicitly state that the therapeutic relevance of these phages cannot be established from experiments performed exclusively with E. coli MG1655 and requires validation using clinical and antimicrobial-resistant isolates.

Comment 3 – Interpretation of the potential host range

This comment has been only partially addressed. The authors appropriately acknowledge in the response letter that phylogenetic and genomic relatedness can only suggest a possible broader host range and does not constitute experimental evidence of cross-species infectivity. This clarification is scientifically appropriate and helps prevent overinterpretation.

However, the response does not identify which sections of the manuscript were revised, nor does it provide page and line numbers or reproduce the modified text. The statement that the “corresponding sections” were revised is too vague to allow verification that all unsupported claims regarding infection of Shigella, Salmonella, Yersinia, Enterobacter, or other Enterobacteriaceae have been consistently removed or qualified throughout the manuscript.

The authors should clearly indicate where the revisions were introduced and ensure that the Abstract, Results, Discussion, and Conclusions consistently distinguish genomic relatedness from experimentally demonstrated host range. Any suggestion of cross-species infectivity should be explicitly presented as a hypothesis requiring experimental validation.

In addition, the second part of the original comment has not been explicitly addressed in the response. The authors were encouraged to discuss both the potential advantages and disadvantages of including broader-host-range bacteriophages in commercial therapeutic formulations. The response does not indicate whether the revised Discussion addresses potential benefits, such as increased bacterial coverage, or potential limitations, including reduced specificity, possible effects on the microbiota, and regulatory implications.

The authors should therefore provide the precise location of the revised passages and explicitly address these biological and therapeutic implications in the Discussion. In the absence of host-range experiments, the manuscript should not imply that the studied bacteriophages infect bacterial species beyond E. coli.

Comment 4 – Integration and methodological description of the restriction analysis

This comment has been only partially addressed, and a relevant methodological omission remains. The authors provide a technically appropriate explanation in the response letter regarding the relevance of genome-terminus determination, sequencing-read coverage, direct terminal repeat prediction, and experimental restriction analysis. The expanded description in the Results improves the presentation of the experiment and clarifies its intended role in supporting the proposed genome termini and DTR boundaries.

However, although the authors state that both the Results and Discussion were expanded, the additional explanation appears to have been incorporated only into the Results section.

The original comment specifically requested that this experiment be integrated into both the Results and Discussion. The Results should clearly identify the expected and observed restriction fragments, indicate which bands correspond to terminal fragments, and explain the degree of agreement between the experimental and in silico profiles. The Discussion should interpret how this agreement supports the proposed genome assembly, genome termini, and DTR boundaries and explain its implications for the DNA-packaging strategy of phage 6phi10.

In addition, the restriction analysis shown in Figure 6 is not adequately described in the Materials and Methods section. The manuscript should provide sufficient methodological information to permit evaluation and reproducibility of the experiment, including:

the restriction enzyme or enzymes used;
the amount of DNA and digestion conditions;
the electrophoresis parameters;
the molecular size marker;
the DNA visualization procedure; and
the software or computational procedure used to generate the in silico restriction profile.

The authors should also explain how the experimentally observed bands were matched to the predicted fragments and how the terminal fragments were identified.

The absence of this methodological description is particularly relevant because the restriction profile is used as experimental support for the proposed genome termini and DTR boundaries. A result presented in the manuscript and used to support a genomic conclusion must be accompanied by a corresponding and reproducible experimental protocol.

The authors should also use cautious wording. The agreement between the experimental and predicted restriction profiles provides support for the proposed termini and DTR boundaries, but it should not be presented as definitive validation unless alternative genome-end arrangements have been adequately excluded.

The authors should therefore:

include a complete description of the experimental and in silico restriction analyses in Materials and Methods;
provide a more explicit comparison between predicted fragment sizes and observed bands in the Results;
incorporate the genomic and biological interpretation of this experiment into the Discussion; and
identify the precise page and line numbers of all corresponding revisions.

Comment 5 – Discussion

This comment has been only partially addressed. The authors indicate that they chose to retain the overall structure and content of the Discussion while making only minor editorial revisions to improve readability. However, the response does not specify which sections were revised, nor does it provide page and line numbers or reproduce the modified text. Consequently, it is not possible to assess the extent of the editorial changes.

The original comment did not question the scientific quality of the Discussion but suggested improving its balance between descriptive content and biological interpretation. Several sections continue to provide detailed descriptions of structural protein conservation and comparisons with closely related bacteriophages, much of which reiterates information already presented in the Results. Condensing these descriptive passages would improve readability and create more space for discussion of the biological implications, novelty, limitations, and broader significance of the findings.

Although the authors are not required to remove all comparative genomic detail, the Discussion would benefit from a clearer emphasis on interpretation rather than description. In particular, greater attention could be given to the significance of the proposed novel Berlinvirus, the limitations of the current biological characterization, and the implications of these findings for bacteriophage biology and future therapeutic applications.

If the authors choose to retain the current structure, they should provide a more explicit scientific justification for doing so and clearly identify the editorial revisions made in response to the review.

General comment on the revision process

I would also like to make a general observation regarding the revision process. Throughout the response letter, the authors repeatedly state that the manuscript has been revised, expanded, clarified, or edited; however, none of the responses identifies the corresponding page and line numbers or reproduces the modified text. This lack of traceability makes it unnecessarily difficult to verify whether the original comments have been fully addressed.

The authors should provide the precise location of every modification and, whenever appropriate, quote the revised passages directly in the response letter. A point-by-point response should allow the reviewer to readily identify both what was changed and where the change was introduced.

In addition, several scientific explanations presented in the response letter are more complete and informative than those incorporated into the revised manuscript. Although the responses sometimes clarify the authors’ rationale, important methodological details, interpretations, limitations, and discussion points remain absent or insufficiently developed in the manuscript itself.

Since the published article, rather than the response letter, constitutes the permanent scientific record, all information necessary to understand, evaluate, and reproduce the study must be incorporated directly into the manuscript.

The authors should therefore submit a revised point-by-point response in which each modification is clearly identified by page and line number and ensure that all relevant explanations currently restricted to the response letter are appropriately incorporated into the manuscript.

Author Response

We thank the Reviewer for the careful assessment of our manuscript and for the constructive comments and suggestions. We have carefully considered all of the points raised and have revised the manuscript accordingly. Below, we provide a point-by-point response to the Reviewer’s comments and describe the changes introduced in the revised manuscript. We added a file comparing previous version of the manuscript with current version (v2_vs_v3).

Comment 1 (Part 1)

«This comment has been only partially addressed. The revised Introduction now identifies the manufacturer, describes the bacterial genera targeted by Sextaphag®, and refers to the product as a sterile filtrate of bacteriophage lysates. However, the additional information remains insufficient to provide the requested context regarding the product’s development, formulation, validation, regulatory status, and clinical use.»

Response 1 (Part 1)

We thank the Reviewer for this comment. We incorporated all relevant information available from publicly sources into the manuscript. Detailed information on the product’s development, formulation, validation, regulatory status, and clinical use is not publicly available. We therefore did not add unsupported information. We also added the explicitly state that the exact composition and number of bacteriophages in the commercial preparation are unknown (page 2, lines 68-71).

Comment 1 (Part 2)

« Reference 6 consists only of a manufacturer’s webpage and does not provide adequate scientific or regulatory support for all statements made about the commercial preparation. Moreover, the manuscript does not establish whether the current product information corresponds to the specific batch used in this study, namely batch P813 from December 2016. The authors should clarify the relevance of this source to the tested product and, where available, include appropriate scientific, regulatory, or product-specific documentation»

Response 1 (Part 2)

Reference 6 was cited only to document the publicly available information provided by the manufacturer regarding the commercial preparation and its declared antibacterial spectrum. The batch number and production date (P813, December 2016) refer to the specific Sextaphag® vial used in this study and were recorded directly from the product packaging. Thus, Reference 6 was not intended to substantiate the batch information. We also added national drug registration number ЛП-№(003826)-(РГ-RU) into the text (page 2, line 65).

Comment 1 (Part 3)

«The response letter also states that previous studies have characterized individual bacteriophages isolated from Sextaphag®. However, these studies have not been identified or cited in the revised Introduction. If such publications are available, they should be cited and briefly discussed to place the present isolates in the context of previous work involving the same commercial cocktail. »

Response 1 (Part 3)

We have added the previously characterized phages iF6 and Sxt1, both isolated from Sextaphag®, to the Introduction and cited the corresponding publications [7, 8] (phage 2, lines 71-74).

Comment 1 (Part 4)

«In addition, there is an important inconsistency between the response letter and the revised manuscript. In the response, the authors correctly acknowledge that it is not possible to determine whether the three bacteriophages represent the complete Escherichia-infecting fraction of Sextaphag® or only a subset. Nevertheless, the revised Introduction states that these bacteriophages “therefore represent only a subset” rather than the complete anti-E. coli component. This conclusion is not supported by the available information. The fact that Sextaphag® targets several bacterial genera demonstrates only that the three isolates represent a subset of the complete multicomponent cocktail; it does not establish whether they constitute all or only part of its Escherichia-targeting fraction.»

Response 1 (Part 4)

We agree that the available information does not allow us to determine whether the three bacteriophages represent the complete Escherichia-targeting fraction of Sextaphag® or only a subset. We have therefore removed the statement that these phages “represent only a subset” of the anti-E. coli component and revised the Introduction accordingly (page 2, line 75-78).

Comment 2 (Part 1)

«This comment has not been adequately addressed, and substantial concerns remain. The authors acknowledge that host-range determination, efficiency-of-plating assays, adsorption kinetics, latent-period determination, and burst-size analysis would provide a more comprehensive biological characterization. However, the response does not clearly identify where the corresponding limitations and revisions were incorporated into the manuscript.

Response 2 (Part 1)

We acknowledge that these additional characteristics could provide useful information for a more comprehensive biological characterization;

During use, bacteriophage-based drugs included in the phage therapeutic cocktail may encounter previously unknown bacterial strains. It is important to ensure that the bacteriophages used are capable of combating the bacterial strains that cause infections. It is also important to conduct experiments to determine the lytic activity of phages against bacterial strains while requiring reasonable resources. In our opinion, a killing assay (Figure 4) is such an experiment.

Our laboratory does not have the capability to work with clinical isolates. Therefore, we excluded the lytic spectrum and EOP from the bacteriophage characterization and relied on the manufacturer's data.

The latent-period and burst-size data were excluded because these parameters depend on the propagation strain and many other factors, such as temperature, growth medium composition, and the growth stage of the bacterial culture. For example, bacteriophage T7 (subfamily Studiervirinae, which also includes phage 6phi10) shows considerable variability in burst size. The average burst size reported for T7 varies from ~200 to 300 virions per infected bacterium. The latent period also varies from ~10 min at 37 °C to ~30 min at 30 °C. For phage T4, a representative of the subfamily Tevenvirinae (as are 6phi8 and 6phi13), the latent period increased from 21 min to 35 min when glucose was replaced with acetate as the carbon source, whereas the burst size decreased from 150 to 12 virions per infected bacterium, respectively. Because these parameters have already been investigated for representatives of these subfamilies, and because the phages examined in this study were isolated from therapeutic cocktails, we restricted the investigation of their lytic activity to the killing assay.

We changed the statement in the Abstract from “The results expand current knowledge of the biological and molecular properties of phages included in therapeutic cocktails…” to “The results expand current knowledge of the genetic properties of phages included in therapeutic cocktails…”, because the paper is primarily focused on genetic characterization (page 1. Lines 22-25)

Comment 2 (Part 2)

«The use of only the laboratory strain Escherichia coli MG1655 remains a major limitation of the biological component of the study. The current experiments demonstrate activity against this single strain but do not establish the host range, infective efficiency, or therapeutic relevance of the three phages against diverse E. coli isolates.

The authors should therefore either:

provide additional biological data, particularly host-range and efficiency-of-plating results using a representative panel of E. coli strains; or
substantially restrict the scope and wording of the manuscript, clearly defining the work as a genomic and preliminary physicochemical characterization and moderating all statements regarding therapeutic potential.

Response 2 (Part 2)

The manuscript clearly states that the biological activity of the three bacteriophages was assessed using the laboratory strain E. coli MG1655 (page 3 lines 90, 123). We therefore agree that these experiments alone do not establish the host range or infective efficiency of the phages against diverse E. coli isolates. However, we do not make claims regarding the therapeutic potential of these phages. The three phages characterized in this study were isolated from Sextaphag®, a commercially available therapeutic phage cocktail, and are already components of this formulation.

The limitations of the present biological characterization and the need for further studies are explicitly addressed in the Discussion. In particular, we state that “These findings provide a basis for follow-up studies assessing host range and killing efficiency against clinical and multidrug-resistant Enterobacteriaceae isolates, which will be required to more fully establish the therapeutic and formulation potential of these phages” (page 20 lines 564-567).

The scope of the study is already clearly defined in the manuscript as a genomic and preliminary physicochemical characterization of the three bacteriophages. This is explicitly stated in the Introduction (page 2, lines 75-78) and reiterated in the Discussion (page 20, line 560).

Comment 3 (Part 1)

«This comment has been only partially addressed. The authors appropriately acknowledge in the response letter that phylogenetic and genomic relatedness can only suggest a possible broader host range and does not constitute experimental evidence of cross-species infectivity. This clarification is scientifically appropriate and helps prevent overinterpretation.

Response 3 (Part 1)

We thank the Reviewer for this comment. No host-range analysis was performed in the present study, and all statements regarding a potentially broader host range based on genomic data are explicitly presented as hypothetical. In the Discussion (page 20, lines 549-550), we state: “However, based solely on genomic evidence, the studied phages can only be hypothesized to possess a broad host range, which requires direct experimental confirmation.” Thus, any broader host range inferred from genomic relatedness is presented explicitly as a hypothesis requiring experimental validation, rather than as evidence of cross-species infectivity.

Comment 3 (Part 2)

«In addition, the second part of the original comment has not been explicitly addressed in the response. The authors were encouraged to discuss both the potential advantages and disadvantages of including broader-host-range bacteriophages in commercial therapeutic formulations. The response does not indicate whether the revised Discussion addresses potential benefits, such as increased bacterial coverage, or potential limitations, including reduced specificity, possible effects on the microbiota, and regulatory implications.

Response 3 (Part 2)

The potential advantages and limitations of a broader host range are already discussed in the Discussion (page 20, lines 556-559). Specifically, we note that a broader host range could extend cocktail coverage beyond E. coli, while potentially reducing target specificity, increasing the possibility of off-target effects on commensal microbiota, and introducing additional regulatory considerations for formulation approval.

Comment 4 (Part 1)

«This comment has been only partially addressed, and a relevant methodological omission remains. The authors provide a technically appropriate explanation in the response letter regarding the relevance of genome-terminus determination, sequencing-read coverage, direct terminal repeat prediction, and experimental restriction analysis. The expanded description in the Results improves the presentation of the experiment and clarifies its intended role in supporting the proposed genome termini and DTR boundaries.

However, although the authors state that both the Results and Discussion were expanded, the additional explanation appears to have been incorporated only into the Results section.

Response 4 (Part 1)

We thank the Reviewer for the detailed comment and careful attention to the determination of the direct terminal repeats. The expected and observed restriction fragments are already indicated in Figure 6. The fragments containing the terminal repeats are marked with red triangles and correspond to 1605 bp and 705 bp fragments, respectively. The Results section further states that the in silico restriction profile agrees with the experimentally observed profile (page 10, lines 277-285), with Figure 6 provided as the corresponding experimental evidence. Therefore, the requested information regarding the observed fragments, terminal fragments, and agreement between the experimental and in silico profiles is already provided in the manuscript.

The Discussion has been revised (page 20, lines 535-542) to include a comparison of the predicted DTRs of phage 6phi10 with those of T3, T7, and its closest phylogenetic relatives, as well as a discussion of their implications for the genome maturation and DNA-packaging strategy characteristic of T7-like bacteriophages.

Comment 4 (Part 2)

«In addition, the restriction analysis shown in Figure 6 is not adequately described in the Materials and Methods section. The manuscript should provide sufficient methodological information to permit evaluation and reproducibility of the experiment, including:

the restriction enzyme or enzymes used;
the amount of DNA and digestion conditions;
the electrophoresis parameters;
the molecular size marker;
the DNA visualization procedure; and
the software or computational procedure used to generate the in silico restriction profile.»

Response 4 (Part 2)

To provide a more detailed and transparent description of the restriction analysis procedure, a dedicated paragraph (Section 2.7) has been added to the Materials and Methods, including all of the methodological information requested by the Reviewer (page 5, lines 188-201).

Comment 4 (Part 3)

«The authors should also explain how the experimentally observed bands were matched to the predicted fragments and how the terminal fragments were identified.

Response 4 (Part 3)

To address this point, we have clarified how the experimentally observed restriction bands were matched to the predicted restriction fragments and how the terminal fragments were identified. Specifically, we explain that the observed restriction profile was compared with the in silico restriction pattern predicted from the assembled genome sequence, and that the terminal fragments were identified as those corresponding to the predicted genome termini and DTR regions (page 5, lines 198-201).

Comment 4 (Part 4)

«The authors should also use cautious wording. The agreement between the experimental and predicted restriction profiles provides support for the proposed termini and DTR boundaries, but it should not be presented as definitive validation unless alternative genome-end arrangements have been adequately excluded.»

Response 4 (Part 4)

We agree with the Reviewer that the interpretation of the restriction analysis should be presented more cautiously. Accordingly, we have revised the corresponding statement (page 10, line 280-282) to use more conservative wording, indicating that the agreement between the experimental and in silico restriction profiles provides independent support for the proposed genome termini and DTR boundaries, rather than presenting them as definitively validated.

Comment 4 (Part 5)

«The authors should therefore:

include a complete description of the experimental and in silico restriction analyses in Materials and Methods;
provide a more explicit comparison between predicted fragment sizes and observed bands in the Results;
incorporate the genomic and biological interpretation of this experiment into the Discussion; and
identify the precise page and line numbers of all corresponding revisions.»

Response 4 (Part 5)

The manuscript has been revised accordingly. A complete description of the experimental and in silico restriction analyses has been added to the Materials and Methods (Section 2.7, page 5, lines 188-201). The Results section has been revised to clarify how the experimentally observed restriction bands were compared with the predicted in silico restriction fragments and how the terminal fragments were identified (page 5, lines 198-201). The Discussion has also been supplemented with an interpretation of the restriction analysis in the context of the proposed genome assembly, genome termini, DTR boundaries, and the DNA-packaging strategy characteristic of T7-like bacteriophages (page 20, lines 535-542). The locations of all corresponding revisions are indicated throughout the present response.

Comment 5

«This comment has been only partially addressed. The authors indicate that they chose to retain the overall structure and content of the Discussion while making only minor editorial revisions to improve readability. However, the response does not specify which sections were revised, nor does it provide page and line numbers or reproduce the modified text. Consequently, it is not possible to assess the extent of the editorial changes.

Response 5

We thank the Reviewer for this constructive and detailed comment and for acknowledging that the scientific quality of the Discussion was not in question. The Discussion section was significantly modified; some blocks of Discussion section were removed. Please follow the additional file “v2_vs_v3” to see the exact changes.

General comment on the revision process

«I would also like to make a general observation regarding the revision process. Throughout the response letter, the authors repeatedly state that the manuscript has been revised, expanded, clarified, or edited; however, none of the responses identifies the corresponding page and line numbers or reproduces the modified text. This lack of traceability makes it unnecessarily difficult to verify whether the original comments have been fully addressed.

We thank the Reviewer for this important general observation. We agree that our previous response letter did not provide sufficient traceability between the Reviewer’s comments, our responses, and the corresponding revisions in the manuscript. Statements that the text had been “revised,” “expanded,” or “clarified” without providing page and line numbers or reproducing the modified passages made it unnecessarily difficult to evaluate whether each comment had been fully addressed.

To resolve this issue, we have comprehensively revised the point-by-point response letter. For each modification, we now provide (i) the precise page and line numbers in the revised manuscript; (ii) a concise description of the change made.

The revised manuscript now includes additional or expanded information The corresponding locations and revised passages are specified individually in our responses to each Reviewer comment. We have additionally checked that each explanation required to understand the study is included in the manuscript itself rather than being restricted to the response letter.

We appreciate the Reviewer’s emphasis on transparency and traceability during the revision process. We believe that the revised point-by-point response now allows every modification to be readily identified and verified, while the revised manuscript contains the methodological and interpretative information necessary to evaluate the study independently of the response letter.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have substantially improved the manuscript and the point-by-point response. Most of the concerns raised during the previous review have now been adequately addressed. In particular, the additional contextual information on Sextaphag®, the clarification of the hypothetical nature of the broader host-range inference, the methodological and interpretative improvements concerning the restriction analysis, and the revision of the Discussion have strengthened the manuscript. The newly provided page and line references, together with the version-comparison document, also greatly improve the traceability of the revisions.

One issue, however, still requires correction before publication.

The authors state in their response that the manuscript is primarily focused on genomic characterization and have appropriately revised several sections to define the study as a physicochemical and genomic characterization. The concluding paragraph of the Discussion now follows this approach, and the Abstract has also been partially revised by replacing the reference to “biological and molecular properties” with “genomic properties.”

Nevertheless, this revision has not been applied consistently throughout the manuscript. The Abstract still begins by stating that the study provides a “biological and genomic characterization” of the three bacteriophages, while the Key Contribution states that the phages were “comprehensively characterized at the biological and genomic levels.”

These statements are not fully supported by the experimental design, since the biological activity of all three phages was evaluated exclusively using the laboratory strain Escherichia coli MG1655. The present data demonstrate lytic activity against this strain and provide useful physicochemical information, but they do not constitute a comprehensive biological characterization in the absence of host-range, efficiency-of-plating, or related analyses.

I therefore recommend that the terminology in the Abstract and Key Contribution be made consistent with the revised scope of the manuscript. “Biological and genomic characterization” could be replaced by “physicochemical and genomic characterization,” or by another appropriately cautious formulation. Similarly, the statement that the phages were “comprehensively characterized at the biological and genomic levels” should be revised to avoid overstating the extent of the biological characterization.

Apart from this remaining issue, I consider that the authors have satisfactorily addressed the major concerns raised during the previous review.

Author Response

«The authors have substantially improved the manuscript and the point-by-point response. Most of the concerns raised during the previous review have now been adequately addressed. In particular, the additional contextual information on Sextaphag®, the clarification of the hypothetical nature of the broader host-range inference, the methodological and interpretative improvements concerning the restriction analysis, and the revision of the Discussion have strengthened the manuscript. The newly provided page and line references, together with the version-comparison document, also greatly improve the traceability of the revisions.

One issue, however, still requires correction before publication.

Apart from this remaining issue, I consider that the authors have satisfactorily addressed the major concerns raised during the previous review.»

Thank you for this comment. We have revised the terminology throughout the relevant sections to ensure consistency with the scope of the study. Specifically, “biological and genomic characterization” was replaced with “physicochemical and genomic characterization” in the Abstract (Page 1, line 11). In addition, the Key Contribution was revised to state that the bacteriophages were “characterized at the physicochemical and genomic levels,” and the term “comprehensively” was removed (Page 1, lines 28-29).