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funded by the Deanship of Research and Graduate Studies at King Khalid University through small group research under grant number RGP 1/4/46

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funded by the Deanship of Research and Graduate Studies at King Khalid University through small group research under grant number RGP 1/4/46

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Additional Editor Comments :

Two reviewers assessed your manuscript and recommended substantial revisions

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Partly

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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-->5. Review Comments to the Author

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Reviewer #1: The manuscript is well written and organized. Figures are well demonstrated with good reflection. Statistical results are mentioned properly. Pharmacokinetic parameters are not given. You can add PK study in this paper.

Reviewer #2: The study "Cyclodextrin Inclusion Complexes Enhance the Solubility and Anti-Virulence Activity of Metronidazole Against Uropathogenic Proteus mirabilisis" is methodologically sound and internally consistent, and the experimental dataset is substantial. However, over‑interpretation, conceptual ambiguity, and insufficient critical depth currently limit its suitability for publication without major revision. follwing are the major concerns and recommendations for authors.

Major Scientific Concerns

1. Anti‑virulence vs. Antibacterial Activity

The manuscript repeatedly alternates between antibacterial efficacy and anti‑virulence activity without clearly delineating the two concepts.

- MIC reduction is used as evidence of improved “anti‑virulence” activity, yet MIC is a growth inhibition metric, not a virulence‑specific endpoint.

- True anti‑virulence strategies are typically defined by effects at sub‑MIC levels without affecting growth. While sub‑MIC experiments are mentioned later (motility, urease, biofilm), the manuscript does not consistently frame or interpret results within this paradigm.

2. Over‑Interpretation of Molecular Docking Results

- Docking scores of −4 to −5 kcal/mol for CD inclusion are modest and fall within a range where enthalpy–entropy compensation and solvent effects dominate. Claiming “perfect fit” or “strongest interactions” is overstated.

- The assertion that metronidazole’s nitro group chelates both Ni²⁺ ions in urease similarly to acetohydroxamic acid is highly speculative. Nitroimidazoles are not established urease chelators in vivo.

- No experimental urease kinetics (IC₅₀, mode of inhibition) are provided to validate docking predictions.

3. Misinterpretation of FTIR and DSC as Proof of Inclusion

FTIR peak “weakening/disappearance” and DSC melting point suppression are repeatedly described as proof of complete inclusion complexation.

- FTIR spectral overlap with cyclodextrins frequently masks drug peaks even in simple physical mixtures.

- DSC melting peak disappearance can result from amorphization or dilution, not necessarily inclusion.

Authors can support these results with complementary techniques (PXRD, phase solubility diagrams, Job’s plot, NMR ROESY).

4. Dissolution Methodology and Interpretation

The “in vitro dissolution” experiment uses a Franz diffusion cell with a dialysis membrane, which measures diffusion + dissolution, not classical dissolution.

This setup is more representative of permeation release than intrinsic solubility.

- Interpretation as dissolution enhancement alone is therefore misleading.

Rename this experiment to “in vitro release/diffusion study” and clarify its limitations. Consider acknowledging that CDs may alter membrane transport rather than dissolution alone.

5. MIC Values Remain Extremely High

Even after formulation, MIC values remain in the mg/mL range (5–20 mg/mL), which is:

- Orders of magnitude higher than clinically relevant antibiotic MICs

- Not discussed critically in terms of translational relevance

Claiming “significant enhancement” without contextualizing these absolute values risks overstatement.

Discuss clinical relevance candidly. Emphasize anti‑virulence potential at sub‑MIC levels rather than antimicrobial potency.

6. Urease Assay Interpretation

The Christensen’s urea agar assay measures phenotypic urease activity, not direct enzyme inhibition.

- Reduced color change may reflect growth suppression, altered metabolism, or diffusion limitations, not enzyme inhibition.

- Yet results are discussed as “urease enzyme inhibition.”

Rephrase throughout to “suppression of urease activity/expression” rather than enzyme inhibition, unless biochemical assays are added.

Missing ares, author need to add out come of these

1. Phase solubility theory (AL/AN/AP types)– fundamental to CD inclusion science.

2. Comparative discussion of CD derivatives beyond HP‑β‑CD (e.g., SBE‑β‑CD, RM‑β‑CD).

3. Limitations of cyclodextrins (cholesterol extraction, membrane toxicity at high concentrations).

4. Recent anti‑virulence frameworks (QS inhibition vs metabolic interference).

5. In vivo relevance– no discussion of urinary dilution, urine composition, or catheter flow dynamics.

- Results are restated rather than critically analyzed.

- Contradictions are not explored (e.g., α‑CD showing activity despite poor inclusion).

- No comparison is made with alternative solubility enhancers or non‑CD systems.

- Why HP‑β‑CD outperforms β‑CD beyond solubility (steric shielding, hydration shell).

- Whether observed anti‑virulence effects stem from CDs themselves.

What are the limitations of docking‑driven mechanistic ?.

Data Presentation and Interpretation

- Tables lack statistical annotations.

- MIC table lacks confidence intervals or replicate variance.

- Molecular docking content is duplicated across sections.

Condense docking description into a single cohesive subsection and tighten the Discussion.

Professional language editing is strongly advised.

Limitations

- Conceptual novelty is incremental, not transformative.

- No new formulation principle or mechanistic model is proposed.

Specific Recommendations for Improvement

1. Rewrite Introduction to clearly define anti‑virulence vs antibacterial activity.

2. Soften mechanistic claims from docking and FTIR/DSC.

3. Clarify dissolution vs diffusion methodology.

4. Reframe urease results as phenotypic suppression.

5. Add a limitations subsection in Discussion.

6. Tighten language and reduce redundancy.

7. Consider removing or shortening repetitive docking descriptions.

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Reviewer #1: No

Reviewer #2: No

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Cyclodextrin Inclusion Complexes Enhance the Solubility and Anti-Virulence Activity of Metronidazole Against Uropathogenic Proteus mirabilis

PONE-D-25-60159R1

Dear Dr. Abd El-Baky,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Reham Mokhtar ELTarabili

Academic Editor

PLOS One

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