Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript explores the comparative efficacy of AMC versus ERP in a rat model of ESBL-producing E. coli peritonitis, integrating microbiological, biochemical, proteomic, and histopathological readouts. The topic is clinically significant given the need to spare carbapenems when possible, in ESBL infections, and the multiparametric approach is a strength of the work. The overall dataset is coherent and primarily supports the idea that both antibiotics attenuate systemic and local inflammation compared with untreated infection. However, several conceptual, methodological, and reporting issues should be addressed before the manuscript is suitable for publication.

At present, the data show broadly similar trends between AMC and ERP, but the absence of a significant difference does not establish non-inferiority. I strongly recommend rephrasing throughout (title, abstract, introduction, results, and conclusions) to indicate that AMC showed “comparable” or “similar” effects to ERP within the limitations of a small exploratory animal study, rather than claiming non-inferiority.

The study’s design and reporting are missing several key ARRIVE 2.0 elements—including sample size justification, randomization, blinding, detailed inclusion/exclusion criteria, and animal welfare/monitoring information—and the authors are urged to systematically check their manuscript against the ARRIVE checklist and add these details, especially for randomization, blinding of histological and proteomic assessments, and criteria for excluding animals or data.

The Discussion states that the absence of a statistical difference between AMC and ERP in spleen cultures suggests AMC's non-inferiority; this should be toned down to emphasize similar trends, while acknowledging that the study is underpowered to rule out clinically relevant differences in bacteremia clearance.

It would help readers if you more clearly described in the Methods the exact quantification workflow (e.g., dot-blot–based densitometry normalized to GAPDH, then expressed as ratios p-protein/total protein or protein/GAPDH) and ensured that all axes in the bar plots specify the normalization used.

The lung involvement percentages (70% E. coli vs 50% AMC/ERP) would be more informative if presented as absolute numbers and, if possible, with a simple statistical comparison.

The study uses a single ESBL-producing E. coli strain, one infection model in healthy rats, and a relatively small sample size. A more cautious tone, explicitly framing the work as hypothesis-generating preclinical evidence that supports further clinical and pharmacokinetic studies, would be more appropriate.

Please ensure that all bacterial names are written in italics and that spacing and punctuation around parentheses are used correctly throughout the manuscript.

The study is interesting and potentially publishable, but its claims of non-inferiority, alignment with ARRIVE guidelines, statistical framework, and some aspects of language and reporting need substantial revision before it can be considered for acceptance.

Comments on the Quality of English Language

Numerous language and style issues could be improved with careful editing by a fluent English speaker or a professional service. A few examples (not exhaustive): “mural model” should be “murine model”; “anatomopathology” is better rendered as “histopathology” or “pathological examination”; “peritoneum tissue” should be “peritoneal tissue”; “GADPH” should be “GAPDH”; “ERP group…ER” in the figure legend for Figure 6 appears inconsistent; and several sentences in the Introduction and Discussion are long and difficult to follow. The abstract could also be streamlined, with a clearer description of the primary outcome(s) and a more cautious conclusion in line with the statistical limitations mentioned above.

Author Response

Response to Reviewer 1’s Comments

We would like to thank for the thorough and constructive evaluation of our manuscript.

All corrections and additions were written in red in the manuscript.

Please find below, point by point, answers to your comments.

 

 

Reviewer 1: Comments and Suggestions for Authors

This manuscript explores the comparative efficacy of AMC versus ERP in a rat model of ESBL-producing E. coli peritonitis, integrating microbiological, biochemical, proteomic, and histopathological readouts. The topic is clinically significant given the need to spare carbapenems when possible, in ESBL infections, and the multiparametric approach is a strength of the work. The overall dataset is coherent and primarily supports the idea that both antibiotics attenuate systemic and local inflammation compared with untreated infection. However, several conceptual, methodological, and reporting issues should be addressed before the manuscript is suitable for publication.

At present, the data show broadly similar trends between AMC and ERP, but the absence of a significant difference does not establish non-inferiority. I strongly recommend rephrasing throughout (title, abstract, introduction, results, and conclusions) to indicate that AMC showed “comparable” or “similar” effects to ERP within the limitations of a small exploratory animal study, rather than claiming non-inferiority.

 

Response:

 

• The title has been changed to: Comparative Evaluation of Amoxicillin–Clavulanate and Ertapenem in an Exploratory Rat Model of ESBL  coliPeritonitis

 

• Rephrasing was done in the abstract, introduction, results, and conclusion.

 

Reviewer 1

The study’s design and reporting are missing several key ARRIVE 2.0 elements—including sample size justification, randomization, blinding, detailed inclusion/exclusion criteria, and animal welfare/monitoring information—and the authors are urged to systematically check their manuscript against the ARRIVE checklist and add these details, especially for randomization, blinding of histological and proteomic assessments, and criteria for excluding animals or data.

 

Response:

A section was added in the Methods to address the ARRIVE 2.0 elements

 

 

Reviewer1

The Discussion states that the absence of a statistical difference between AMC and ERP in spleen cultures suggests AMC's non-inferiority; this should be toned down to emphasize similar trends, while acknowledging that the study is underpowered to rule out clinically relevant differences in bacteremia clearance.

 

Response:

We changed the text to: Although AMC and ERP showed similar downward trends in spleen culture positivity, the absence of a statistically significant difference between the two treatments does not imply equivalence or non‑inferiority. Instead, this finding likely reflects the limited statistical power of this small exploratory study, which is insufficient to rule out clinically relevant differences in bacteremia clearance

 

Reviewer 1

It would help readers if you more clearly described in the Methods the exact quantification workflow (e.g., dot-blot–based densitometry normalized to GAPDH, then expressed as ratios p-protein/total protein or protein/GAPDH) and ensured that all axes in the bar plots specify the normalization used.

 

Response:

The section was added to the methods.

 

Reviewer 1

The lung involvement percentages (70% E. coli vs 50% AMC/ERP) would be more informative if presented as absolute numbers and, if possible, with a simple statistical comparison.

 

Response:

This point was taken into consideration in the results section.

 

Reviewer 1

The study uses a single ESBL-producing E. coli strain, one infection model in healthy rats, and a relatively small sample size. A more cautious tone, explicitly framing the work as hypothesis-generating preclinical evidence that supports further clinical and pharmacokinetic studies, would be more appropriate.

 

Response:

This point was addressed in the Discussion section

 

Reviewer 1

Please ensure that all bacterial names are written in italics and that spacing and punctuation around parentheses are used correctly throughout the manuscript.

 

Response:

The fonts were checked.

Reviewer 1

The study is interesting and potentially publishable, but its claims of non-inferiority, alignment with ARRIVE guidelines, statistical framework, and some aspects of language and reporting need substantial revision before it can be considered for acceptance.

 

Response:

All was added in the manuscript

 

Reviewer 1: Comments on the Quality of English Language

Numerous language and style issues could be improved with careful editing by a fluent English speaker or a professional service. A few examples (not exhaustive): “mural model” should be “murine model”; “anatomopathology” is better rendered as “histopathology” or “pathological examination”; “peritoneum tissue” should be “peritoneal tissue”; “GADPH” should be “GAPDH”; “ERP group…ER” in the figure legend for Figure 6 appears inconsistent; and several sentences in the Introduction and Discussion are long and difficult to follow. The abstract could also be streamlined, with a clearer description of the primary outcome(s) and a more cautious conclusion in line with the statistical limitations mentioned above.

 

Response:

English was proofread.

Reviewer 2 Report

Comments and Suggestions for Authors

Find attached reviewer's comments

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript should be thoroughly proofread and edited to address several
grammatical inconsistencies and improve coherence.

Author Response

Response to Reviewer 2’s Comments

We thank the reviewer for the thoughtful and constructive comments, which have significantly improved the clarity and rigor of our manuscript. Below we provide a point‑by‑point response, indicating how each comment was addressed in the revised version.

Reviewer 2 comments

1. L40: Considering the model used in this study (animal), the small sample size, and the lack of resistance emergence analysis, I suggest that the authors rephrase the abstract’s conclusion to emphasize preclinical evidence and antibiotic-sparing potential, not treatment recommendations. Suggestion: “Findings from this experimental study suggests comparable efficacy of AMC and ertapenem in reducing inflammation and bacterial burden…”

Response: The Abstract conclusion has been revised to avoid therapeutic recommendations and now emphasizes preclinical exploratory findings and the comparable effects of AMC and ERP in this rat model: AMC and ERP produced broadly comparable effects on microbiological, biochemical, and inflammatory parameters in ESBL E. coli peritonitis.

2. In the Abstract and Introduction sections, the authors emphasized spontaneous bacterial peritonitis (SBP) in cirrhotic patients, yet the study uses a non-cirrhotic rat model without ascites. This disconnect should at least be acknowledged or justified.

Response: We added a full paragraph in both the Abstract and Introduction explicitly acknowledging that our model does not replicate cirrhosis‑associated SBP and explaining why extrapolation should be limited.

3. I suggest that the authors clearly outline the concept of “non-inferiority” and its relevance to this study in the Introduction or avoid the term and use more appropriate exploratory language.

Response: The term non‑inferiority has been removed throughout and replaced with “comparable effects” or “directionally similar outcomes.”

4. While antimicrobial resistance is introduced broadly in the Introduction section, the specific problem of carbapenem overuse and selection pressure is not clearly articulated. The authors should clearly frame the study within the context of antimicrobial stewardship, emphasizing the risks of carbapenem overuse (resistance amplification, ecological impact), and the clinical interest in narrower-spectrum alternatives for ESBL infections.

Response: We added clearer justification in the Introduction regarding carbapenem pressure and the rationale for narrower alternatives.

5. Line 65: This “IDSA 2024 Guidance on the Treatment of Antimicrobial Resistant Gram- Negative Infections(8)” appears as a standalone sentence fragment with no explanation.

Response: The sentence has been integrated properly into the paragraph and now reads as contextual justification for ESBL management.

6. Line 72: The discussion on animal models should be critically expanded to include the rationale for choosing direct inoculation over caecal ligation, suitability of animal models for antimicrobial efficacy studies, and other important concepts to improve the rigor of the section.

Response: The aim of our study is to assess the usage of AMC regarding ESBL not polymicrobial infections.

7. Line 77: “Mural model” or “murine model”?

Response: Corrected to murine model.

8. Line 102: Could the use of talc to prevent spontaneous healing not artificially amplify inflammation and bias treatment effects? The authors should provide a stronger justification for this.

Response: We significantly strengthened this point in the discussion.

9. Section “3.3” was labelled twice.

Response: Corrected.

10. The small sample sizes, particularly in the sham group and the proteomic subsamples,

 substantially limit the statistical power of the study. As a result, several key interpretations

 in the Discussion section such as superior peritoneal diffusion of amoxicillin–clavulanate,

 systemic efficacy, and control of bacteremia are based largely on non-significant trends

rather than robust statistical evidence. In addition, mortality outcomes are not informative, as only a single death occurred.

Response: We stated in the methods that “The selected group size was therefore appropriate for detecting directional trends across multiple biological endpoints and for informing future adequately powered hypothesis‑driven studies.” Our aim was to compare the outcome between AMC and ERP.

11. While the Discussion frequently attributes the absence of statistical significance to limited sample size, strong conclusions as highlighted above were still drawn from them, which represents a major interpretative inconsistency that should be addressed.

Response: We thoroughly revised the Discussion to temper conclusions.
Key modifications include:

• Removing claims of superiority or non-inferiority.
• Discussing this point and stating the rationale for conducting more robust work

12. The authors repeatedly extrapolate findings from the talc-induced rat peritonitis model to human ESBL spontaneous bacterial peritonitis, without sufficiently acknowledging the inherent biological and clinical limitations of this experimental system. Such overextension of conclusions from an artificial animal model to human disease is problematic and should be substantially tempered.

Response: We have tempered all extrapolations.

13. The manuscript should be thoroughly proofread and edited to address several grammatical inconsistencies and improve coherence.

Response: The entire manuscript has been thoroughly edited to improve style, consistency, clarity, and remove redundancies.

Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting and potentially clinically relevant experimental study comparing amoxicillin–clavulanate with ertapenem in a rat model of ESBL E. coli peritonitis. The work is timely in the context of antimicrobial stewardship and carbapenem-sparing strategies. However, several methodological, analytical, and reporting issues require clarification or revision before the manuscript can be considered for publication. My comments below aim to strengthen the scientific rigor, transparency, and translational interpretation of the study.

Major points

1)  Introduction (1st–2nd paragraphs) & Methods 2.2

The Introduction is largely framed around spontaneous bacterial peritonitis (SBP) in cirrhosis, whereas the experimental model represents acute induced ESBL E. coli peritonitis in healthy rats with intraperitoneal talc. This model does not replicate the pathophysiology of cirrhosis-associated SBP (portal hypertension, altered immunity, ascites, or bacterial translocation). The authors should explicitly clarify this distinction and temper translational claims.

2)  Methods 2.2

Intraperitoneal talc (125 mg) introduces a strong sterile inflammatory stimulus, which may independently influence cytokines (CRP, IL-6, TNF-α) and histology in peritoneum and lung. a) Provide justification for this exact talc dose with references, b) Discuss explicitly how talc may have contributed to inflammatory outcomes.

3)  Methods 2.2

Group sizes are markedly unbalanced (ERP n=10, AMC n=10, E. coli n=10, Sham n=3). The very small Sham group limits statistical robustness. Provide a rationale for n=3 in the Sham group, or acknowledge this as a major limitation and state whether an a priori sample size calculation was performed (if not, say so explicitly).

4)  Methods 2.2–2.6

The manuscript does not specify: whether animals were randomized to groups, or whether investigators were blinded during histopathological scoring and protein quantification. The authors must add: a) Randomization procedure (if used), b) Blinding status for histology and densitometry.

5) Methods 2.2

The doses (Ertapenem 150 mg/kg/day SC; AMC 100 mg/kg/day SC q12h) require clearer pharmacokinetic justification in rats (e.g., time above MIC or exposure equivalence). Need a brief rationale or reference supporting dose selection and comparability.

6)  Results 3.2 & Figure 1

The “E. coli growth score (0–5)” is not clearly defined in Methods. Required: a) Explicit definition of the scoring system, b) Clarify whether cultures were quantitative (CFU) or semi-quantitative.

7)  Results 3.3

Procalcitonin did not differ significantly among septic groups, yet the Discussion implies clear systemic benefit of treatment. Suggest a temper claims regarding systemic anti-inflammatory superiority of either antibiotic.

8)  Conclusion (lines 379–385)

The manuscript uses “non-inferiority” without a formal statistical non-inferiority framework. Either perform a formal non-inferiority analysis or replace with:

“comparable performance between AMC and ERP.”

12) Similarity score (32%)

Given the iThenticate result, authors should rephrase sections with substantial textual overlap and clarify originality.

Comments on the Quality of English Language

 The English is generally understandable but requires moderate revision for clarity, precision, and scientific style. Several sentences are lengthy or grammatically awkward, some terms are used inconsistently, and certain passages would benefit from clearer structure and more formal academic phrasing. Careful language editing is recommended prior to publication.

Author Response

Response to Reviewer 3’s Comments

We would like to thank for his positive feedback and valuable remarks.

All corrections and additions were written in red in the manuscript.

Please find below, point by point, answers to your comments.

 

 

 

Reviewer 3: Comments and Suggestions for Authors

This is an interesting and potentially clinically relevant experimental study comparing amoxicillin–clavulanate with ertapenem in a rat model of ESBL E. coli peritonitis. The work is timely in the context of antimicrobial stewardship and carbapenem-sparing strategies. However, several methodological, analytical, and reporting issues require clarification or revision before the manuscript can be considered for publication. My comments below aim to strengthen the scientific rigor, transparency, and translational interpretation of the study.

 

Major points

1)  Introduction (1st–2nd paragraphs) & Methods 2.2

The Introduction is largely framed around spontaneous bacterial peritonitis (SBP) in cirrhosis, whereas the experimental model represents acute induced ESBL E. coli peritonitis in healthy rats with intraperitoneal talc. This model does not replicate the pathophysiology of cirrhosis-associated SBP (portal hypertension, altered immunity, ascites, or bacterial translocation). The authors should explicitly clarify this distinction and temper translational claims.

 

Response:

This point has already been addressed in the revised version

 

2)  Methods 2.2

Intraperitoneal talc (125 mg) introduces a strong sterile inflammatory stimulus, which may independently influence cytokines (CRP, IL-6, TNF-α) and histology in peritoneum and lung. a) Provide justification for this exact talc dose with references, b) Discuss explicitly how talc may have contributed to inflammatory outcomes.

 

Response:

This point has already been addressed in the revised version

 

3)  Methods 2.2

Group sizes are markedly unbalanced (ERP n=10, AMC n=10, E. coli n=10, Sham n=3). The very small Sham group limits statistical robustness. Provide a rationale for n=3 in the Sham group, or acknowledge this as a major limitation and state whether an a priori sample size calculation was performed (if not, say so explicitly).

4)  Methods 2.2–2.6

The manuscript does not specify: whether animals were randomized to groups, or whether investigators were blinded during histopathological scoring and protein quantification. The authors must add: a) Randomization procedure (if used), b) Blinding status for histology and densitometry.

 

Response:

These points have already been addressed in the revised version

 

5) Methods 2.2

The doses (Ertapenem 150 mg/kg/day SC; AMC 100 mg/kg/day SC q12h) require clearer pharmacokinetic justification in rats (e.g., time above MIC or exposure equivalence). Need a brief rationale or reference supporting dose selection and comparability.

 

Response:

This point has already been addressed in the revised version

 

6)  Results 3.2 & Figure 1

The “E. coli growth score (0–5)” is not clearly defined in Methods. Required: a) Explicit definition of the scoring system, b) Clarify whether cultures were quantitative (CFU) or semi-quantitative.

 

Response:

This point has already been addressed in the revised version

 

7)  Results 3.3

Procalcitonin did not differ significantly among septic groups, yet the Discussion implies clear systemic benefit of treatment. Suggest a temper claims regarding systemic anti-inflammatory superiority of either antibiotic.

 

Response:

This point has already been addressed in the revised version

 

8)  Conclusion (lines 379–385)

The manuscript uses “non-inferiority” without a formal statistical non-inferiority framework. Either perform a formal non-inferiority analysis or replace with:

“comparable performance between AMC and ERP.”

 

Response:

This point has already been addressed in the revised version

 

 

 

 

 

12) Similarity score (32%)

Given the iThenticate result, authors should rephrase sections with substantial textual overlap and clarify originality.

 

Response:

This point has already been addressed in the revised version

 

 

Comments on the Quality of English Language

The English is generally understandable but requires moderate revision for clarity, precision, and scientific style. Several sentences are lengthy or grammatically awkward, some terms are used inconsistently, and certain passages would benefit from clearer structure and more formal academic phrasing. Careful language editing is recommended prior to publication.

 

Response:

English was proofread.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In my initial review, I raised significant concerns about the wording around non-inferiority, incomplete reporting of key ARRIVE 2.0 items, the clarity of the statistical framework, and several methodological and language issues. In the current version, the authors have carefully and satisfactorily addressed these points. The claims have been appropriately toned down to “comparable/similar effects” within the limits of an exploratory preclinical model, and the study is now clearly framed as hypothesis-generating. Methodological reporting has been substantially improved, with more precise descriptions of allocation, outcome assessment, and quantification procedures. The figures and legends are more accurate, bacterial nomenclature and technical terms have been corrected, and the overall English language and style have been significantly improved.

I do not have any remaining substantive scientific concerns. Any residual issues are minor editorial points that can be addressed during copy editing. I therefore consider the manuscript, in its present form, suitable for publication and recommend acceptance.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the necessary points in their revision. The changes made are appropriate and improve the manuscript. Therefore, the paper is suitable for acceptance.

Comments on the Quality of English Language

Improved and acceptable

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately addressed the major methodological and interpretative concerns raised in the previous review round. The manuscript has improved in clarity and transparency.