Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an interesting study; however, there are significant considerations that should be addressed before it can be considered for publication.

Title
• Does the title accurately reflect the work conducted in the manuscript? As currently written, it appears limited in relation to the breadth of the study.

Abstract and Keywords
• It is recommended to avoid repeating the same terms used in the title, as this will help broaden the manuscript’s visibility in database searches.

Introduction
• The section lacks sufficient background to properly contextualize the study and support its scope. It is necessary to clearly establish what has been previously done, what remains unexplored, and how this research addresses that gap.
• The narrative flow requires improvement, as the ideas appear fragmented. The connection between sections is not clearly articulated, which hinders the reader’s understanding of the progression and rationale of the study.

Materials and Methods
• A dedicated Materials section is missing. Detailed information should be provided regarding reagents, media, and materials, including product codes, as well as the city, state, and country of origin.
• What microbial strains are present in the SCOBY? What is the structure and order of diversity within the population? Is the microbial population uniform?
• It is unclear whether appropriate controls were included to prevent false positives or false negatives.
• How is it ensured that the observed optical density (OD) is attributable to microbial death rather than the formation of biofilms or biopolymers that may influence OD measurements?
• The statistical analysis should be consolidated into a single section, as some of this information appears to be repeated.
• For the presentation of treatments, the use of tables is recommended to improve clarity.
• What is the relevance and necessity of Figure 2 within the methodology section?

Results
• How was the microbial load in the kombucha standardized?
• The use of significant figures in tables should be carefully reviewed.
• Data should be reported as mean ± standard deviation.
• Statistical analysis is not reported.
• Subheadings should be included to improve organization.
• There are inconsistencies in the reported particle size across figures, results, and discussion. What is the correct value? It is recommended to categorize sizes into ranges and to report size distribution, including mean, median, and mode, or percentage-based distributions.
• The purpose of Figure 5 is unclear. Consider presenting the full spectrum of the analyzed elements.
• In the FTIR analysis, the reported changes appear subjective. It is recommended to clearly indicate bands of interest below 2000 cm⁻¹ and, if relevant, also evaluate regions above 2000 cm⁻¹.
• The presentation of results in Figure 7 does not appear optimal; consider using tables or formats similar to Figure 10.
• Based on the results shown in Figure 8, the selected graph type does not effectively illustrate the observed changes, unlike Figure 9.
• Information presented in tables or figures should not be duplicated in the text. Instead, the text should emphasize trends and key findings rather than restating numerical values.
• All tables and figures should be explicitly referenced in the text.
• Table 2 could be more effectively presented as a figure similar to Figure 10 to enhance clarity.
• The Results section should be limited to the description of findings, without interpretation.

Discussion
• The discussion does not fulfill its purpose, as it primarily reiterates results accompanied by references.
• This section reads as a condensed continuation of the Results section.
• The objective of the first paragraph is unclear.
• The role of references within the discussion is not clearly defined.

Conclusions
• The section summarizes results rather than providing clear and concise conclusions.

General Comments
• Avoid the use of abbreviations in the abstract, objectives, and conclusions.
• Improve the quality of the figures.
• Not all ideas or paragraphs are supported by references.
• The citation style is inconsistent.
• Several references are cited only once.
• The reference formatting is not uniform.

Author Response

Response to Reviewer 1 Comments

 

We thank Reviewer 1 for the careful reading of the manuscript and for the constructive comments. Below we provide a point-by-point response and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. Manuscript modifications are indicated where applicable.

Comment 1: Does the title accurately reflect the work conducted in the manuscript? As currently written, it appears limited in relation to the breadth of the study.

Response 1: The current title, “Kombucha-Mediated Silver Nanoparticles with Broad-Spectrum Fungicidal Activity against WHO Priority Candida Pathogens: In Vitro and Galleria mellonella Evaluation”, captures the synthesis vehicle (kombucha), the material (silver nanoparticles), the activity profile (broad-spectrum fungicidal), the target organisms (WHO priority Candida pathogens), and both validation arms (in vitro and G. mellonella). We believe this encompasses the full scope of the study. However, we are open to specific suggestions from the reviewer regarding aspects they feel are underrepresented. No change has been made at this stage.

 

Comment 2: It is recommended to avoid repeating the same terms used in the title, as this will help broaden the manuscript’s visibility in database searches.

Response 2: We appreciate this suggestion. However, for biomedical literature indexed in PubMed and Scopus, keyword overlap with the title is standard practice and is explicitly recommended by most indexing guidelines to strengthen discoverability under the primary search terms of the study. Removing core terms such as “silver nanoparticles” or “Candida” from the keywords would reduce rather than improve database retrieval. We have reviewed the keyword list and believe the current selection (silver nanoparticles, kombucha, Candida, antifungal, Galleria mellonella) provides appropriate coverage. No change has been made.

 

Comment 3: The section lacks sufficient background to properly contextualize the study and support its scope. It is necessary to clearly establish what has been previously done, what remains unexplored, and how this research addresses that gap.

Response 3: We structured the Introduction around four thematic blocks, the first tackling the global burden and species epidemiology of invasive candidiasis, including the WHO Fungal Priority Pathogens List and the clinical challenges posed by biofilm-forming and multidrug-resistant species (paragraph 1, references 1–7). The second, the rationale for silver nanoparticles as antifungal candidates, including multi-target mechanisms and the advantages of green synthesis, with specific focus on kombucha tea as a synthesis substrate (paragraph 2, references 8–18), followed by the justification for the G. mellonella model as a translational readout (paragraph 3, references 19–22), and finally, a clear statement of the study aims and the gap this work addresses (paragraph 4). We believe this structure does establish what has been done (paragraphs 1–3), what remains unexplored (the final sentences of paragraphs 2 and 3), and how this study addresses the gap (paragraph 4). Nevertheless, we have added a brief transitional sentence at the beginning of paragraph 4 to make the novelty statement more explicit (Lines 96-99).

 

Comment 4: The narrative flow requires improvement, as the ideas appear fragmented. The connection between sections is not clearly articulated, which hinders the reader’s understanding of the progression and rationale of the study.

Response 4: We have reviewed the Introduction and added transitional phrasing between the thematic blocks to improve narrative continuity. Specifically, we have strengthened the bridge between the epidemiological framing and the nanotechnology rationale (Line 61-63) and between the kombucha synthesis section and the G. mellonella model justification (Lines 83-86).

Comment 5: A dedicated Materials section is missing. Detailed information should be provided regarding reagents, media, and materials, including product codes, as well as the city, state, and country of origin.

Response 5: Reagent details, supplier information, and locations were initially provided within each methodological subsection where the materials are used. However, upon your suggestion we added in a new subsection (2.1. Materials – Lines 110-154) that provides all information. We have also taken this chance to review all materials added supplier locations and/or equipment manufacturer information where they were incomplete. As a consequence, all subsections numbering of the Materials and Methods section have been modified.

 

Comment 6: What microbial strains are present in the SCOBY? What is the structure and order of diversity within the population? Is the microbial population uniform?

Response 7: The SCOBY was obtained as a commercial starter culture from Kombucha Now, LLC (Madison, USA) [ref. 23]. Detailed metagenomic or culture-based characterization of the SCOBY microbial community (16S rRNA and ITS profiling) was beyond the scope of the present study, which focuses on the phytochemical profile of the fermented tea extract as the nanoparticle synthesis substrate rather than on the microbiology of the SCOBY itself. The chemical composition of the fermentate, which is the relevant parameter for nanoparticle synthesis, was characterized longitudinally by LC-MS/MS across four fermentation time points (Table 1), providing a functional chemical fingerprint that is more directly relevant to synthesis reproducibility than taxonomic composition. We have added a brief statement in Section 2.2 noting that detailed SCOBY community profiling was not performed (Lines 114-117).

 

Comment 7: It is unclear whether appropriate controls were included to prevent false positives or false negatives.

Response 7: Controls are explicitly described in the manuscript. For the MIC/MFC assay (Section 2.5.1), positive control wells contained fungal suspensions in RPMI without antimicrobial agent (to confirm viability), negative control wells contained RPMI only (to confirm sterility), all assays were run in quadruplicate on seven separate days, and inoculum density was verified by a separate plating step. For the G. mellonella experiments (Sections 2.6.2 and 2.6.3), DPBS-injected negative controls, K-AgNP-only groups (toxicity controls), and untreated infected controls were included in every model. We have added a clarifying sentence in the Methods overview to draw attention to these control groups more prominently (Lines 205-208).

 

Comment 8: How is it ensured that the observed optical density (OD) is attributable to microbial death rather than the formation of biofilms or biopolymers that may influence OD measurements?

Response 8: The OD-based turbidity reading was used for MIC determination (Section 2.5.1), while MFC was confirmed by a separate MTT metabolic viability assay (Section 2.5.2), which specifically measures the ability of viable cells to reduce tetrazolium salt to formazan. The absence of purple formazan production in wells at and above the MFC concentration confirms that no metabolically active cells remain, regardless of any optical interference from biofilm or biopolymer material. The combination of spectrophotometric MIC and MTT-based MFC provided confirmation that the observed endpoints reflect genuine antifungal activity. We have added a sentence to the Results (Section 3.2.1) to make this validation more explicit (Lines 525-528).

 

Comment 9: The statistical analysis should be consolidated into a single section, as some of this information appears to be repeated.

Response 9: Thank you for your suggestion. We have added a new “Statistical analysis” subsection (Lines 420-440) to the Material and Methods section that encompasses all analysis performed, while removing the text from the original sections.

 

Comment 10: For the presentation of treatments, the use of tables is recommended to improve clarity.

Response 10: We appreciate this suggestion. The experimental groups for all G. mellonella experiments are defined in text (Sections 2.6.2 and 2.6.3), and MIC/MFC values are reported in text (Section 3.2.1). We have now added a summary table of the G. mellonella experimental groups (species, inoculum, treatment, n per group) as a new supplementary table to improve at-a-glance clarity and mentioned it in text where appropriate (Subsubsection 2.6.3 and 3.3.1).

 

Comment 11: What is the relevance and necessity of Figure 2 within the methodology section?

Response 11: Figure 2 presents representative microscopy images of viable and non-viable G. mellonella hemocytes as assessed by methylene blue dye exclusion. This figure serves two purposes, first to distinguish viable from non-viable cells, which is essential for methodological reproducibility, and second, it provides visual validation that the staining protocol reliably differentiates membrane-intact from membrane-compromised cells across different hemocyte subtypes. We believe this figure adds methodological value and have retained it.

Comment 12: How was the microbial load in the kombucha standardized?

Response 12: No microbial load was present in the synthesis substrate. As described in subsection 2.4, the kombucha tea extract was filter-sterilized by sequential filtration through 0.45 µm and 0.22 µm membranes prior to use as a reducing agent for nanoparticle synthesis. The synthesis substrate is therefore a cell-free, sterile filtrate whose relevant properties are its phytochemical composition (characterized by LC-MS/MS, Table 1) and pH, not its microbial load.

 

Comment 13: The use of significant figures in tables should be carefully reviewed.

Response 13: We have reviewed all tables and standardized the number of significant figures to be consistent within each column and appropriate for the precision of each measurement.

 

Comment 14: Data should be reported as mean ± standard deviation.

Response 14: Data are reported as mean ± standard deviation throughout the manuscript. MIC/MFC values are reported as modal values from independent runs, which is the standard reporting convention for EUCAST microdilution [ref. 29].

 

Comment 15: Statistical analysis is not reported.

Response 15: In the revised manuscript, a dedicated Statistical Analysis subsection (Section 2.7) has been added to the Materials and Methods to consolidate all analytical details in one place and we have added a Supplementary Table 2 for the extended statistical analysis for hemocyte viability. Statistical analyses are reported extensively throughout the manuscript. Specifically: Kaplan–Meier survival analysis with log-rank tests and Holm-corrected pairwise comparisons (Sections 2.7 and 3.4, Figures 8–9 with significance annotations), two-way ANOVA with Tukey's multiple comparisons test for total hemocyte counts (Section 2.7 and Figure 10 with significance annotations), and two-way ANOVA with partial eta-squared effect sizes, Group × Time interaction testing, Shapiro–Wilk and Levene's diagnostics, and Tukey's pairwise comparisons for hemocyte viability (Sections 2.7 and 3.5, Figure 11, and Supplementary Table 2). All p-values, effect sizes, and post-hoc groupings are reported in the Results text and figures. Please let us know if there are any specific statistical analysis that we have not covered since the first revision.

 

Comment 16: Subheadings should be included to improve organization.

Response 16: We have changed the Results section to include more subsubheadings for a better organizational structure. Subsection 3.3. (In vivo experiments using Galleria mellonella larvae now) now has 2 new Subsubsections: 3.3.1. (Nanoparticle toxicity in G. mellonella larvae and) and 3.3.2. (K-AgNPs treatment of fungal infection model in G. molenella larvae). We’ve also changed the name of subsection 3.2. to “Antifungal effects” as to better represent the content of the subsection. The other Subsections, namely 3.1. (Kombucha tea and biosynthesized K-AgNPs characteristics), 3.4. (Total Hemocyte counts), and 3.5. (Hemocyte Viability) have remained unchanged.

 

Comment 17: There are inconsistencies in the reported particle size across figures, results, and discussion. What is the correct value? It is recommended to categorize sizes into ranges and to report size distribution, including mean, median, and mode, or percentage-based distributions.

Response 17: Two size measurements are reported, and both are correct as they measure different physical parameters. The TEM-derived core particle diameter is 19.4 ± 7.9 nm (mean ± SD, n = 234 particles, Figure 4), which reflects the metallic core size measured under vacuum. The NTA-derived hydrodynamic diameter is 88 nm (Section 3.1), which reflects the core particle plus its adsorbed polyphenol-glycoside corona and the solvation layer in aqueous suspension. This discrepancy is expected and commonly reported for surface-functionalized nanoparticles. We have added a clarifying sentence in Section 3.1 explicitly noting that the difference reflects the hydrodynamic contribution of the organic capping layer (Lines 483-486), and we have ensured consistent terminology throughout ("TEM-derived core diameter" versus "NTA-derived hydrodynamic diameter"). The TEM size distribution histogram (Figure 4) already presents the full particle size distribution.

 

Comment 18: The purpose of Figure 5 is unclear. Consider presenting the full spectrum of the analyzed elements.

Response 18: Figure 5 presents the UV-Vis absorption spectrum across the 300–800 nm range, which is the standard spectral window for confirming silver nanoparticle formation via the surface plasmon resonance band. The spectrum shows the characteristic SPR peak at 415 nm. This is not an elemental analysis (e.g. EDX or XPS) but a spectroscopic confirmation of nanoparticle formation, which is a standard characterization step in every biogenic AgNP study. We have improved the figure legend to make this purpose more explicit (Lines 499-501).

 

Comment 19: In the FTIR analysis, the reported changes appear subjective. It is recommended to clearly indicate bands of interest below 2000 cm⁻¹ and, if relevant, also evaluate regions above 2000 cm⁻¹.

Response 19: The FTIR analysis (Section 3.1, final paragraph) reports specific band positions for both the kombucha reference and the K-AgNPs spectrum, with the direction and magnitude of each shift described quantitatively (e.g. O–H stretching shifted from ~3422 to ~3385 cm⁻¹, C=O/C=C at ~1621 shifting to ~1566 cm⁻¹, COO⁻ symmetric stretching at 1407 and 1386 cm⁻¹, and C–O–C glycosidic bands at ~1054–1135 cm⁻¹). These assignments are correlated with LC-MS/MS-identified functional groups and referenced to published FTIR data for green tea-mediated AgNPs [refs. 17, 35]. The region above 2000 cm⁻¹ (O–H at ~3385–3422 cm⁻¹ and C–H at ~2927 cm⁻¹) is already discussed. No changes have been made, as the existing analysis provides quantitative band assignments throughout the full spectral range.

 

Comment 20: The presentation of results in Figure 7 does not appear optimal; consider using tables or formats similar to Figure 10.

Response: Figure 7 presents photographic images of the MTT assay plates, which serve as direct visual evidence of the presence or absence of metabolic activity (purple formazan formation versus colourless wells). This type of visual documentation is standard in antimicrobial susceptibility publications and provides qualitative confirmation that complements the quantitative MIC/MFC data in the text. We believe replacing plate photographs with numerical tables would remove this visual evidence. No change has been made.

 

Comment 21: Based on the results shown in Figure 8, the selected graph type does not effectively illustrate the observed changes, unlike Figure 9.

Response 21: Both Figure 8 (toxicity) and Figure 9 (infection models) use Kaplan-Meier survival curves, which is the standard and methodologically appropriate format for time-to-event data. The visual difference between the two reflects the biological effects followed. In the toxicity experiment (Figure 8), survival remains high across all groups with minimal separation, whereas in the infection models (Figure 9), treatment groups diverge substantially from infected controls. The near-flat survival curves in Figure 8 are the expected and desired result, as they demonstrate the absence of K-AgNP toxicity. We have improved the text in this results Section 3.3.1. to make this interpretation more explicit (Lines 560-569).

 

Comment 22: Information presented in tables or figures should not be duplicated in the text. Instead, the text should emphasize trends and key findings rather than restating numerical values.

Response 22: We acknowledge this point. We have removed from Section 3.5 an interpretive paragraph that restated hemocyte viability findings already presented in the Discussion (Section 4), retaining only data description and statistical outcomes in the Results.

 

Comment 23: All tables and figures should be explicitly referenced in the text.

Response 23: We have verified that all tables and figures are referenced in the text.

 

Comment 24: Table 2 could be more effectively presented as a figure similar to Figure 10 to enhance clarity.

Response 24: We agree. Table 2 has been converted to bar chart figures with the same visual format as Figure 10 (grouped bar charts with significance annotations), which improves visual interpretability and consistency across the hemocyte data.

 

Comment 25: The Results section should be limited to the description of findings, without interpretation.

Response 25: We have reviewed the Results section. An interpretive paragraph in Section 3.5 that discussed the mechanistic implications of hemocyte viability patterns has been removed, as this content is already presented in the Discussion (Section 4). The Results section now contains only data description and statistical outcomes.

 

Comment 26: The discussion does not fulfill its purpose, as it primarily reiterates results accompanied by references. This section reads as a condensed continuation of the Results section. The objective of the first paragraph is unclear. The role of references within the discussion is not clearly defined.

Response26: We respectfully disagree with the characterization that the Discussion primarily reiterates results. We structured the Discussion section around the following the study’s main result points. First, the phytochemical rationale for selecting the 21-day fermentate (linking LC-MS/MS data to documented biotransformation pathways and capping chemistry). Second, mechanistic correlation of FTIR band shifts with specific surface-coordinating groups identified by LC-MS/MS. Third, contextualisation of K-AgNP MIC/MFC values against published data for biogenic AgNPs and EUCAST breakpoints for conventional antifungals. Fourth, interpretation of species-specific susceptibility differences in terms of resistance determinants, fifth, comparison of G. mellonella survival outcomes with published nanomaterial efficacy studies and discussion of translational relevance, sixth, mechanistic interpretation of THC escalation as evidence of immunostimulatory activity, and lastly, analysis of hemocyte viability trajectories as reflections of species-specific virulence strategies.

References in the Discussion serve to benchmark our findings against the published literature, identify mechanistic parallels, and highlight where our results converge with or diverge from prior work, which is the standard function of references in a scientific discussion. The first paragraph provides a high-level synthesis of the main findings and the integrative rationale of the study.

Nevertheless, we have reviewed the Discussion and confirmed that each paragraph opens with an interpretive claim rather than a data summary, and that cited references serve to benchmark, contextualize, or mechanistically interpret findings rather than merely accompany them. No structural changes were required.

 

Comment 27: The section summarizes results rather than providing clear and concise conclusions.

Response 27: The Conclusions section is structured in three paragraphs that move from the synthesis rationale and substrate characterization, through the antifungal potency profile and its mechanistic implications, to the in vivo safety and efficacy evidence and future directions. Each paragraph already opens with a conclusive statement (“demonstrates,” “achieved,” “confirmed”) and draws a synthetic conclusion rather than merely restating individual data points. No changes were made at this time.

Comment 28: Avoid the use of abbreviations in the abstract, objectives, and conclusions.

Response 28: We have reviewed the Abstract and Conclusions and ensured that all abbreviations are defined at first use. K-AgNPs is defined at first mention in the Abstract ("kombucha tea extract (K-AgNPs)"), and standard abbreviations (MIC, MFC, FTIR, UV-Vis, LC-MS/MS) are widely understood by the readership of this journal. All abbreviations are defined at first use in the Abstract. We corrected and minimized the use in of abbreviations in the Conclusions section, leaving only K-AgNPs as the only used abbreviation.

 

Comment 29: Improve the quality of the figures.

Response 29: We have reviewed all figures and improved resolution where possible. TEM images, FTIR spectra, and Kaplan-Meier survival curves have been re-exported at higher resolution (minimum 300 dpi). The MTT plate photographs (Figure 7) are inherently limited by the photographic capture conditions but have been optimized for contrast and clarity.

 

Comment 30: Not all ideas or paragraphs are supported by references.

Response 30: We have reviewed the manuscript and confirmed that all claims are supported by references where appropriate. Statements describing our own experimental results or observations do not require external references. The reviewer does not identify specific unsupported claims, and we were unable to identify any upon review.

 

Comment 31: The citation style is inconsistent. Several references are cited only once. The reference formatting is not uniform.

Response 31: Thank you for bringing this to our attention. We have reviewed and standardized the reference formatting throughout the manuscript to ensure consistency with Vancouver style, we have modified 3 references (14, 15, and 38). Regarding references cited only once, the frequency of citation does not determine the relevance of a reference. Each citation serves a specific purpose (methodological source, comparative data, or contextual support), and single-citation references are appropriate when they provide unique or non-redundant information. No references have been removed on the basis of citation frequency alone.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Kombucha-Mediated Silver Nanoparticles with Broad-Spectrum Fungicidal Activity against WHO Priority Candida Pathogens: In Vitro and Galleria mellonella Evaluation" presents an important interface between in vivo and nano-mycology, featuring intriguing tests and timely findings. However, the authors fail to include positive controls necessary for publication.

K-AgNPs Synthesis
The green synthesis via kombucha (21-day fermented extract, 10 mM AgNO₃, pH 5) is robust, with LC-MS/MS detailing the polyphenolic profile (EGC dominant, EGCG/gallic for capping). Yet, it could quantify yield (% Ag reduced) and test stability in RPMI (MIC medium). UV-Vis alone confirms formation (SPR peak at 415 nm indicates spherical Ag⁰ <50 nm) but does not provide size/morphology data.

MIC Assessment
Concordant MIC/MFC values (0.8-1.6 μg/mL) against 8 Candida spp. (including ATCC standards and clinical C. auris) are potent via EUCAST (RPMI microdilution, MTT), confirming fungicidal activity. Using standard ATCC strains (e.g., C. albicans 64548) is correct, but conventional antifungals are missing as controls (fluconazole MIC₉₀ ~1-4 μg/mL for susceptible strains; anidulafungin 0.03-0.25 μg/mL). The absence of standard treatments is inadequate for claiming "broad-spectrum."

Suggest the authors include comparisons with azoles/echinocandins in MIC/survival assays to contextualize potency. Characterization of AgNPs needs expansion (XRD, long-term DLS for stability) and anti-biofilm testing. The authors discuss biofilm challenges but perform no anti-biofilm treatment.

G. mellonella Survival Curve
Kaplan-Meier/log-rank (n=30, triplicated, seasonally controlled batches) shows significant benefit (p<0.001 for C. albicans/glabrata), with no toxicity (98% K-AgNPs survival). Methodologically correct (0.5 McFarland inoculum, 37°C, 7 days), but incomplete without conventional antifungal arms (e.g., fluconazole 32 μM as used in similar studies). THC/hemocyte viability supports immunomodulation, but comparisons are lacking.

Hemocyte Viability Analyses
The analyses are adequate but have important methodological limitations that may weaken claims of "negligible toxicity." The method uses methylene blue exclusion (viable cells unstained; dead stained) with Thoma chamber counting. Hemolymph collection by decapitation is well-explained. However, where is the two-way ANOVA table? Interactions? P-values? Were normality tests performed (Shapiro-Wilk/Levene)?

Another point: lack of quantitative hemolysis (LDH release) or apoptosis (annexin V/PI), as methylene blue underestimates late necrosis. Polyphenolic-capped AgNPs (FTIR: OH/COO shifts) reduce expected toxicity (ROS/membrane damage in erythrocytes), but comparisons with non-capped AgNPs or antifungals are missing. Human erythrocyte testing (hemolysis <5% per ASTM F756) is recommended for translation; hemocyte viability supports biocompatibility but does not replace it. Suggestion: add flow cytometry for subpopulations and ROS (DCFH-DA).

The manuscript is well-written and, with these corrections, could be eligible for publication.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

 

We thank Reviewer 1 for the careful reading of the manuscript and for the constructive comments. Below we provide a point-by-point response and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. Manuscript modifications are indicated where applicable.

Comment 1: UV-Vis alone confirms formation (SPR peak at 415 nm indicates spherical Ag⁰ <50 nm) but does not provide size/morphology data.

Response 1: We respectfully note that UV-Vis spectroscopy was not the sole characterization technique employed. As detailed in Section 2.3 and Section 3.1 of the manuscript, nanoparticle characterization comprised a multi-technique suite including transmission electron microscopy (TEM, Hitachi H-7650, 120 kV) with manual measurement of 234 individual particles from five micrographs, confirming spherical morphology and an average diameter of 19.4 ± 7.9 nm (Figure 3 and Figure 4), nanoparticle tracking analysis (NTA, Nanosight NS300) providing hydrodynamic diameter (88 nm) and zeta potential (−14.52 ± 0.04 mV),  UV-Vis spectroscopy confirming a surface plasmon resonance peak at 415 nm, and FTIR spectroscopy confirming polyphenol-glycoside surface capping. This characterization suite, encompassing both core particle properties and surface chemistry, is consistent with current standards in the biogenic AgNP literature [Widatalla et al. 2022 (ref. 17), Afandy et al. 2023 (ref. 35), Nkosi et al. 2024 (ref. 10)]. All characterization data are presented in the original submission.

Comment 2: Yet, it could quantify yield (% Ag reduced) and test stability in RPMI (MIC medium).

Response 2: We acknowledge that reporting the percentage of Ag⁺ reduced to Ag⁰ would provide a useful metric of synthesis efficiency. In the present study, the silver concentration of the final colloidal stock was determined gravimetrically (400 µg/mL), but inductively coupled plasma spectroscopy (ICP-OES or ICP-MS), which would enable precise determination of residual unreduced Ag⁺, was not available for this project. We have added a sentence to the limitations paragraph of the revised Discussion acknowledging this gap and noting that ICP-based yield quantification is planned for the next phase of formulation optimization (Lines 929-933).

Regarding stability in RPMI 1640, K-AgNPs remained colloidally stable throughout the 24 h incubation period of the microdilution assay, with no visible aggregation, colour change, or sedimentation observed in any of the seven independent assay runs. A sentence confirming this observation has been added to Section 3.2. of the revised manuscript (Lines 545-547).

 

Comment 3: Characterization of AgNPs needs expansion (XRD, long-term DLS for stability).

Response 3: We appreciate the suggestion. XRD would provide confirmatory crystallographic data for the face-centred cubic (fcc) structure of metallic silver. In the present study, the narrow, symmetric SPR peak at 415 nm and the spherical morphology confirmed by TEM are both consistent with crystalline Ag⁰ nanoparticles, and FTIR provided detailed surface chemistry data. The combined TEM, NTA, UV-Vis, and FTIR characterization suite is well aligned with the characterization depth presented in recent biogenic AgNP antifungal studies, including those published in high-ranking journals [Widatalla et al. 2022 (ref. 17), Malik et al. 2022 (ref. 36), Alnahdi et al. 2025 (ref. 40)]. XRD analysis is planned for ongoing formulation optimization work and will be included in subsequent publications.

Regarding long-term DLS stability monitoring, NTA (which provides particle-by-particle size resolution superior to ensemble DLS) was performed, and the zeta potential of −14.52 ± 0.04 mV was reported. While formal time-course colloidal stability studies (e.g. 30, 60, 90 day DLS monitoring) are appropriate for formulation development, they exceed the scope of the present biological activity study. We have added this as a future direction in the revised limitations section (Lines 945-949).

Comment 4: Using standard ATCC strains (e.g., C. albicans 64548) is correct, but conventional antifungals are missing as controls (fluconazole MIC₉₀ ~1–4 µg/mL for susceptible strains; anidulafungin 0.03–0.25 µg/mL). The absence of standard treatments is inadequate for claiming "broad-spectrum."

Response 4: We understand the reviewer’s desire to see K-AgNP potency benchmarked against conventional antifungals. However, we respectfully note that experimental inclusion of fluconazole and echinocandin MIC arms was not part of the present study’s design, which aimed to characterize K-AgNP antifungal activity and safety rather than conduct a head-to-head comparative pharmacological trial. The ATCC reference strains used in this study (C. albicans ATCC 64548, C. glabrata ATCC 90030, C. krusei ATCC 6258, C. parapsilosis ATCC 22019, C. tropicalis ATCC 750, C. dubliniensis ATCC MYA-646, C. kefyr ATCC 38296) are EUCAST-recommended quality control and reference organisms for which extensive published MIC data against standard antifungals exist.

To address this point, we have added a new paragraph to the revised Discussion (Section 4) that contextualizes the K-AgNP MIC/MFC values (0.80–1.60 µg/mL) against published EUCAST MIC data for fluconazole, anidulafungin, and micafungin for the same species (Lines 799-818). For context, EUCAST clinical breakpoints for C. albicans are S ≤2 / R >4 mg/L for fluconazole and S ≤0.03 / R >0.03 mg/L for anidulafungin, with wild-type MIC distributions of 0.12–0.5 mg/L for fluconazole and 0.008–0.03 mg/L for anidulafungin [Arendrup et al. 2020, Pfaller et al. 2010, Meletiadis et al. 2022]. This comparison demonstrates that K-AgNP MIC values fall within the same order of magnitude as conventional antifungal agents against susceptible strains, and we make this explicit in the revised text.

Importantly, the term "broad-spectrum" in our manuscript refers to the range of species inhibited (eight Candida species including the WHO critical-priority pathogen C. auris), not to a claim of superior potency over conventional antifungals. Direct comparison with azoles and echinocandins would require a separate pharmacological study with formal isobolographic or checkerboard design, which is beyond the scope of this work but is noted as planned future research.

Comment 5: The authors discuss biofilm challenges but perform no anti-biofilm treatment.

Response 5: The reviewer is correct that antibiofilm assays were not included in the present study. The manuscript already acknowledges this as a limitation (Discussion, paragraph 7): "the study did not assess antibiofilm activity, which represents a key clinical advantage of AgNPs over conventional antifungals, and quantitative biofilm inhibition and eradication assays will be addressed in subsequent work."

 

Comment 6: Methodologically correct (0.5 McFarland inoculum, 37°C, 7 days), but incomplete without conventional antifungal arms (e.g., fluconazole 32 µM as used in similar studies).

Response 6: We acknowledge that inclusion of a conventional antifungal comparator arm (e.g. fluconazole) in the G. mellonella survival experiments would enable direct head-to-head in vivo comparison. However, the primary objective of the present in vivo study was to establish efficacy and safety of K-AgNPs in the G. mellonella model, not to conduct a comparative therapeutic trial. This design is consistent with the majority of published nanoparticle G. mellonella efficacy studies, which characterize the nanoparticle treatment effect relative to untreated infected and vehicle controls rather than conventional drug arms [Thomaz et al. 2020 (ref. 42), Gottardo et al. 2023 (ref. 43), Saberi Moqaddam et al. 2025 (ref. 41)].

Extensive published data on fluconazole dose-response in G. mellonella against C. albicans and C. glabrata are available from the studies cited in our manuscript [Jemel et al. 2020 (ref. 22), Borman 2018 (ref. 21), Marena et al. 2025 (ref. 20)], and the K-AgNP MIC values are contextualized against published EUCAST breakpoints in the revised Discussion (see response to Point 4 above). Direct in vivo comparison with standard antifungals in the larval model is planned for subsequent work and has been noted as a future direction in the revised manuscript (Line 952).

 

Comment 7: However, where is the two-way ANOVA table? Interactions? P-values? Were normality tests performed (Shapiro-Wilk/Levene)?

Response 7: We thank the reviewer for this important methodological point. In the revised manuscript, the statistical analysis subsection (Section 2.7) now explicitly states that normality of residuals was assessed with the Shapiro–Wilk test and homogeneity of variances with Levene's test for the hemocyte viability data. Effect sizes are reported as partial eta-squared (η²p), and all significant main effects and interactions were followed by Tukey's multiple comparisons test at each time point.

The Results section (Section 3.5) reports Group main effect p-values (all p < 0.001), partial η²p values for each species (ranging from 0.480 for C. albicans to 0.839 for C. krusei), and Group × Time interaction significance for each species (significant in five of six models, p < 0.001 to p = 0.013), with C. tropicalis as the only model where the interaction was non-significant (p = 0.229). Pairwise comparisons are presented directly on the bar charts in Figure 11 using standard significance brackets.

To address the reviewer's request for full ANOVA output, we have prepared a new Supplementary Table 2 containing the complete two-way ANOVA summary for all six species, including sums of squares, degrees of freedom, mean squares, F-statistics, p-values, partial η²p, and the results of the Shapiro–Wilk and Levene's diagnostic tests. Furthermore, the hemocyte viability data previously presented in tabular form (Table 2) have been reformatted as grouped bar charts with significance brackets (Figure 11), improving visual interpretability while the full statistical output remains available in the supplementary material.

Comment 8: Lack of quantitative hemolysis (LDH release) or apoptosis (annexin V/PI), as methylene blue underestimates late necrosis. [...] Suggestion: add flow cytometry for subpopulations and ROS (DCFH-DA).

Response 8: We appreciate the reviewer’s suggestion of additional cytotoxicity endpoints. The methylene blue dye exclusion assay is the established and widely used standard method for hemocyte viability assessment in G. mellonella studies. Trypan blue and methylene blue dye exclusion remain the reference approach for insect hemocyte viability in published protocols [Browne et al. 2013 (ref. 46), Senior et al. 2020 (F1000Research 9:1372)]. While LDH release assays, annexin V/PI apoptosis staining, and flow cytometric subpopulation analysis (including ROS measurement via DCFH-DA) would provide deeper mechanistic resolution, adding these analysis is not possible for the present study, but future studies would greatly benefit from them and have acknowledged this in the revised Discussion (Lines 949-954).

Comment 9: Polyphenolic-capped AgNPs (FTIR: OH/COO shifts) reduce expected toxicity (ROS/membrane damage in erythrocytes), but comparisons with non-capped AgNPs or antifungals are missing.

Response 9: Including a chemically synthesized (non-capped) AgNP control arm in the hemocyte assays would isolate the contribution of the polyphenolic corona to biocompatibility, and we agree this is a mechanistically informative comparison. However, this experiment requires synthesis, characterization, and biological testing of an additional nanoparticle type under matched conditions, which constitutes a separate study. Our manuscript already discusses the mechanistic basis for reduced toxicity of polyphenol-capped versus citrate- or uncoated AgNPs by referencing the FTIR-confirmed multi-component surface corona and citing relevant literature on capping-dependent toxicity modulation [Mussin and Giusiano 2022 (ref. 9), Opris et al. 2021 (ref. 28)]. We have added a sentence to the revised limitations section explicitly noting that comparative evaluation with non-capped AgNPs is warranted and planned in addition to antifungal head-to-head (Line 948).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

- Line 55. N. glabrata.

- Line 88. From here on, include G. mellonella.

- Line 92. Candidozyma auris.

- Line 93. Nakaseomyces glabrata.

- Line 173. Name of the species with changes.

- Lines 180 and 182. 10⁵.

- Line 170. Remove "minimum fungicidal concentration" from the title; only the MIC is determined in this section.

- line 185. Does the medium contain MOPS?

- Using MTT (metabolic viability) could determine the MIC value; however, only by plating on agar plates (each well) can the CMF be determined.

- Studies recommend the use of menadione or similar agents when using MTT, as its absence can result in lower values.

- 235. Italics in vivo.

- 352. G. mellonella.

- The authors should improve the quality and image of Figure 4.

- Line 495. Are the MIC results 80% (line 200)? The authors measured the absorbance at 530 nm, which corresponds to the MIC value at 100%, and calculated the 80% MIC from this. Therefore, you should indicate in the results section of the manuscript that the data correspond to the MIC80. Why don't the authors give the 100% MIC value, that is, the value they obtained when measuring at 530 nm?

- Line 499. Nakaseomyces glabrata.

- Line 530. C. albicans.

- Line 535. C. auris and C. krusei.

- Lines 537-358. C. parapsilosis and C. tropicalis. Review the rest to standardize the manuscript.

Author Response

We thank Reviewer 3 for the careful reading of the manuscript and for the constructive comments. Below we provide a point-by-point response and the corresponding revisions highlighted in track changes in the resubmitted files. Manuscript modifications are indicated where applicable.

 

Comment 1: Line 55. N. glabrata.

Response 1: Corrected. Candida glabrata has been updated to Nakaseomyces glabrata (abbreviated N. glabrata after first use).

Comment 2: Line 88. From here on, include G. mellonella.

Response 2: Corrected. Galleria mellonella is now abbreviated as G. mellonella thereafter.

Comment 3: Line 92. Candidozyma auris.

Response 3: This nomenclature was already used in the original submission. We have verified that Candidozyma auris appears consistently throughout the manuscript.

Comment 4: Line 93. Nakaseomyces glabrata.

Response 4: Corrected, as described in Response 1. The updated nomenclature now appears at Line 93 and at all subsequent mentions.

Comment 5: Line 173. Name of the species with changes.

Response 5: Corrected. All species names at Line 173 have been updated to reflect current nomenclature, including Nakaseomyces glabrata and Candidozyma auris. The full strain designations with ATCC numbers have been retained.

Comment 6: Lines 180 and 182. 10⁵.

Response 6: Corrected. The inoculum density has been formatted with proper superscript notation (10⁵ CFU/mL) at Lines 180 and 182.

Comment 7: Line 170. Remove “minimum fungicidal concentration” from the title; only the MIC is determined in this section.

Response 7: The reviewer is correct that the MIC determination by broth microdilution and the MFC confirmation by MTT viability assay are described in separate subsections (Sections 2.5.1 and 2.5.2, respectively). The section title has been revised to reflect only the MIC determination, while the MFC methodology retains its own subsection heading.

Comment 8: Line 185. Does the medium contain MOPS?

Response 8: Yes. The RPMI 1640 medium used throughout this study was supplemented with 0.165 M MOPS, in accordance with the EUCAST E.Def 7.4 reference method for broth microdilution of yeasts. This detail is now present in the new Materials section (Section 2.1, Line 120). We also have added the MOPS specification to the MIC methods subsection for clarity (Line 185).

Comment 9: Using MTT (metabolic viability) could determine the MIC value; however, only by plating on agar plates (each well) can the MFC be determined.

Response 9: We appreciate this important methodological distinction. The reviewer is correct that the reference standard for minimum fungicidal concentration (MFC) determination, as defined by the CLSI M27-A3 document and by Cantón et al. (2009), involves subculturing aliquots from MIC wells onto solid agar medium and defining the MFC as the lowest concentration producing a 99.9% (3-log) reduction in colony count relative to the starting inoculum. We acknowledge that our MTT-based approach does not constitute a classical MFC determination under this definition.

In our study, the MTT assay (Section 2.5.2) was used as a metabolic viability endpoint that complements the turbidity-based MIC. The complete absence of formazan production (purple colour) in wells at and above a given concentration confirms that no metabolically active cells remain, which is functionally consistent with a fungicidal outcome. This approach has been widely adopted in the biogenic nanoparticle literature precisely because nanoparticle suspensions can interfere with the optical density readings required for standard agar-subculture colony counting due to their inherent absorbance and light-scattering properties. Numerous published studies on silver nanoparticle antifungal activity have employed MTT or XTT viability assays as the primary or confirmatory fungicidal endpoint without agar subculture (Mussin and Giusiano 2022 [ref. 9 of manuscript], Nqakala et al. 2021 [ref. 11 of manuscript]).

References:

Cantón E, Pemán J, Viudes A, Quindós G, Gobernado M, Espinel-Ingroff A. Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species. Diagn Microbiol Infect Dis. 2003;45(3):203–206. doi:10.1016/S0732-8893(02)00525-3

Comment 10: Studies recommend the use of menadione or similar agents when using MTT, as its absence can result in lower values.

Response 10: We thank the reviewer for raising this point. The requirement for exogenous electron-coupling agents (menadione or phenazine methosulfate) is well established for the XTT reduction assay in Candida studies, where menadione is added to facilitate extracellular reduction of the tetrazolium salt (Kuhn et al. 2003, J Clin Microbiol 41:506–508). However, the MTT assay operates by a distinct mechanism. MTT is taken up by viable cells and reduced intracellularly by mitochondrial and cytosolic dehydrogenases, producing insoluble formazan crystals that are subsequently solubilized for spectrophotometric quantification. Because the reduction occurs intracellularly, the need for exogenous electron carriers is substantially lower than for XTT, which relies on extracellular reduction. Published MTT protocols for Candida planktonic susceptibility testing routinely omit menadione (Mosmann 1983, Levitz and Diamond 1985), and our protocol follows this established methodology.

The potential for underestimation of viable cell counts in the absence of menadione applies primarily to biofilm-associated cells with reduced metabolic accessibility, not to the planktonic suspensions used in our MIC/MFC assay.

References:

Kuhn DM, Balkis M, Chandra J, Mukherjee PK, Ghannoum MA. Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J Clin Microbiol. 2003;41(1):506–508. doi:10.1128/JCM.41.1.506-508.2003

Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. doi:10.1016/0022-1759(83)90303-4

Comment 11: Line 235. Italics in vivo.

Response 11: Corrected. In vivo has been formatted in italics at Line 235 and verified throughout the manuscript.

Comment 12: Line 352. G. mellonella.

Response 12: Corrected. G. mellonella is now abbreviated.

Comment 13: The authors should improve the quality and image of Figure 4.

Response 13: Figure 4 (TEM-derived particle size distribution histogram) has been regenerated at higher resolution (minimum 300 dpi) with improved axis formatting. The histogram was generated from manual measurement of 234 individual nanoparticles across five representative TEM micrographs using ImageJ, and the revised figure now includes clearer bin edges, axis labels, and annotation of the mean ± SD.

Comment 14: Line 495. Are the MIC results 80% (line 200)? The authors measured the absorbance at 530 nm, which corresponds to the MIC value at 100%, and calculated the 80% MIC from this. Therefore, you should indicate in the results section of the manuscript that the data correspond to the MIC₈₀. Why don’t the authors give the 100% MIC value, that is, the value they obtained when measuring at 530 nm?

Response 14: We thank the reviewer for this important clarification request. The MIC endpoint used in this study was defined as the lowest K-AgNP concentration producing ≥80% reduction in optical density at 530 nm relative to the drug-free growth control. We have now stated this definition explicitly in Section 2.5.1 of the revised manuscript and explained the reasoning behind its use (Lines 263-272).

The 80% inhibition threshold was selected because silver nanoparticle suspensions contribute to background optical density through surface plasmon resonance absorbance and light scattering, which cannot be fully subtracted in wells containing both nanoparticles and biological material. This renders lower inhibition thresholds less reliable for colloidal agents. Notably, this endpoint is more stringent than the ≥50% inhibition specified by the current EUCAST E.Def 7.4 and CLSI M27-A4 reference methods for azoles, echinocandins, and flucytosine, meaning that our MIC values represent a conservative estimate of antifungal potency. The 80% spectrophotometric endpoint has precedent in the antifungal susceptibility literature (Espinel-Ingroff et al. 1995, J Clin Microbiol 33:3154–3158) and has been adopted in biogenic AgNP studies (Kota et al. 2017, BioNanoScience 7:628–637).

Regarding the 100% value, the MTT metabolic viability assay (Section 2.5.2) provides the complementary zero-viability fungicidal endpoint. The complete absence of formazan production at and above the MFC concentration confirms that no metabolically active cells remain, which functionally corresponds to the 100% inhibition endpoint the reviewer refers to. The distinction between the turbidity-based MIC (≥80% growth inhibition) and the MTT-based MFC (zero metabolic viability) is now made explicit in both the Methods (Lines 298-301) and Results (Section 3.2, Lines 548-554) sections.

References:

EUCAST. EUCAST definitive document E.Def 7.4: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. 2023. Available from: https://www.eucast.org

CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts. 4th ed. CLSI standard M27. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.

Espinel-Ingroff A, Rodriguez-Tudela JL, Martinez-Suarez JV. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J Clin Microbiol. 1995;33(12):3154–3158. doi:10.1128/jcm.33.12.3154-3158.1995

Kota S, Dumpala P, Anantha RK, Verma MK, Kandepu S. Green synthesis of silver nanoparticles and their antifungal properties. BioNanoScience. 2017;7(4):628–637. doi:10.1007/s12668-017-0481-4

Comment 15: Line 499. Nakaseomyces glabrata.

Response 15: Corrected,.

Comment 16: Line 530. C. albicans.

Response 16: Corrected.

Comment 17: Line 535. C. auris and C. krusei.

Response 17: Corrected.

Comment 18: Lines 537–538. C. parapsilosis and C. tropicalis. Review the rest to standardize the manuscript.

Response 18: Corrected. We have performed a full review of the manuscript to standardise all species name formatting, ensuring that genus names are spelled out in full at first mention in each section and abbreviated thereafter, with consistent use of the updated nomenclature (Candidozyma auris and Nakaseomyces glabrata) throughout.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

First, thank you for your responses to the questions raised; they are crucial for the manuscript to reach its highest potential quality. Furthermore, providing a better description of the methods, results, discussion, and conclusion will help future studies investigate and reproduce the authors' findings.

Points that still raise concerns:

1) Demonstrating the reduction/stability within the RPMI 1640 culture medium is fundamental. RPMI 1640 medium contains a relatively high amount of chloride, approximately 103.5 mmol/L of NaCl and 5.3 mmol/L of KCl, which, given the low solubility of AgCl, would allow for some precipitation. The authors state that the nanoparticles "remained colloidally stable throughout the 24 h incubation period... with no visible aggregation, colour change, or sedimentation observed". For nanotechnology, visual inspection is insufficient. The correct approach would have been to monitor the UV-Vis spectrum (checking for a shift or flattening of the SPR band at 415 nm) or particle size via DLS/NTA directly in the RPMI over time. In my view, there could be a background absorbance contribution during the photometric reading of the antifungal activity, which compromises the reliability of the MIC.

 

2) The Minimum Inhibitory Concentration (MIC) obtained for the K-AgNPs is valid on its own as an intrinsic property of this new molecule/particle against the standardized ATCC strains. However, without a parallel positive control (commercial drugs), the internal validation of the assay on the day of testing is lost. A control with Fluconazole/Anidulafungina serves to guarantee that the methodology performed according to EUCAST guidelines. The authors present a "historical comparison" in the discussion; while this might be accepted by some journals, it weakens the study's argumentative strength.

 

3) The term "broad-spectrum" is incorrectly applied. The authors are engaging in scientific hyperbole; inhibiting eight species of Candida spp. does not constitute a broad spectrum. Instead, this represents a restricted antifungal activity or, at most, a relevant action against opportunistic yeasts. It could only be considered broad-spectrum if the K-AgNPs demonstrated concomitant action against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and yeasts. Therefore, the correct term should be "expanded anti-Candida activity" or "broad activity against the genus Candida"; otherwise, it is a misuse of the term or an exaggeration.

4) The authors have confused certain concepts. The Checkerboard assay and the Isobologram are used to evaluate combinative interactions (Synergism, Antagonism, or Additivity) between two drugs administered simultaneously, rather than to compare isolated potencies. To compare potencies, the pipeline should involve the simultaneous determination of the MICs for K-AgNPs, Fluconazole, and Anidulafungina against all strains. Additionally, Time-Kill curves could be performed to assess the rate of cell death, and the selectivity index (SI) should be calculated as the ratio between the cytotoxic dose in human cells/hemocytes and the MIC against the fungus. The higher the SI, the more potent and safer the agent.

5) Photometric readings were performed at 530 nm. Why did the authors not use 620 nm? It is well known that Surface Plasmon Resonance (SPR) is a broad band with a Rayleigh scattering tail that can easily extend up to 550 nm, depending on the concentration and aggregation state. This could be bypassed if the authors used a background control (medium + nanoparticles without fungi) electronically subtracted from each well, though it remains unclear whether this was done. Switching to longer wavelengths (such as 620 nm or 630 nm) drastically minimizes interference from the silver plasmon.

 

6) Thank you for providing Supplementary Table 2 and showing the results of the normality (Shapiro-Wilk) and homoscedasticity (Levene's) tests. However, in the footnotes of the tables, all Shapiro-Wilk and Levene's tests returned p < 0.05, indicating deviations from normality and homoscedasticity. The authors justify that "Given the balanced design (n = 12 per cell) and the robustness of ANOVA to such violations under balanced conditions, the parametric analysis was retained.". While it is true in theoretical statistical literature that ANOVA is robust to deviations from normality when the design is balanced (n equal across all groups), there are strict limits. For example:

• For Candida krusei and Candida parapsilosis: The interaction term (Group x Time) presents massive F-values (F = 69.06 and F = 24.74, respectively) with p < 0.001. In these cases, the effect size (n²p = 0.758 and 0.529) is so overwhelming that even the violation of homoscedasticity by Levene's test is unlikely to alter the conclusion that a statistically significant difference exists.

• In the case of Candidozyma auris (C. auris): The Group x Time interaction yielded a p = 0.013 with a low effect size (n²p = 0.113). Because both Levene's and Shapiro-Wilk tests were severely violated (p < 0.001), the Type I error rate (false positive) inflates considerably. Asserting that a significant interaction exists at p = 0.013 while violating both basic assumptions is statistically imprudent.

Suggestions:

• a) Apply a data transformation (Logarithmic, square root, or Box-Cox transformation) to attempt to stabilize the variance and bring the residuals closer to normality.

• b) If data transformation fails, the analysis should migrate to non-parametric models (such as the Scheirer-Ray-Hare test, which is the non-parametric extension of the Two-Way ANOVA) or utilize Generalized Linear Models (GLM) adjusted for distributions that accommodate heteroscedasticity.

Author Response

Response to Reviewer 2 — Second Round

Manuscript ID: cimb-4306174

Title: Kombucha-Mediated Silver Nanoparticles with Broad Anti-Candida Fungicidal Activity against WHO Priority Candida Pathogens: In Vitro and Galleria mellonella Evaluation

We thank Reviewer 2 for the careful re-reading of the manuscript and for the additional comments. Each of the six points has been addressed below, with corresponding revisions made to the manuscript where feasible (highlighted as tracked changes in the resubmitted file). Where we have not been able to add new experimental data within the revision window, we have stated this transparently, defended the existing evidence on its methodological merits, and added explicit acknowledgments to the limitations section so that the reader is informed of the scope of the present study and of the experiments planned for follow-up work.

Comment 1. Demonstrating the reduction/stability within the RPMI 1640 culture medium is fundamental. RPMI 1640 medium contains a relatively high amount of chloride, approximately 103.5 mmol/L of NaCl and 5.3 mmol/L of KCl, which, given the low solubility of AgCl, would allow for some precipitation. The authors state that the nanoparticles “remained colloidally stable throughout the 24 h incubation period... with no visible aggregation, colour change, or sedimentation observed”. For nanotechnology, visual inspection is insufficient. The correct approach would have been to monitor the UV-Vis spectrum (checking for a shift or flattening of the SPR band at 415 nm) or particle size via DLS/NTA directly in the RPMI over time. In my view, there could be a background absorbance contribution during the photometric reading of the antifungal activity, which compromises the reliability of the MIC.

Response 1: We accept the reviewer’s point that macroscopic inspection alone is insufficient to claim true colloidal stability of K-AgNPs in RPMI 1640, particularly in light of the chloride content of the medium and the low aqueous solubility of AgCl. We have therefore softened the corresponding sentence in the Results (Section 3.2.1) so that it no longer asserts colloidal stability in chemical terms, and we have added an explicit limitation in the Discussion (Section 4, Sixth limitation) acknowledging that in situ monitoring of the SPR band by UV-Vis or of hydrodynamic size and zeta potential by DLS/NTA in RPMI over the 24 h assay window was not performed and that Ag+/AgCl speciation cannot be excluded.

At the same time, we respectfully note that several design features of the present study reduce the likelihood that silver speciation in RPMI materially affected interpretation of the biological endpoints. First, antifungal activity was confirmed using an independent MTT-based MFC assay, in which formazan production depends on enzymatic NAD(P)H-driven reduction by metabolically viable fungal cells rather than on turbidity measurements, light scattering, or nanoparticle surface plasmon absorbance. The complete absence of formazan production at and above the MFC concentration for every species tested (Figure 7) indicates loss of fungal metabolic activity regardless of whether the silver was present as colloidal Ag0, dissolved Ag+, or partially precipitated AgCl. Second, per-concentration nanoparticle background-correction wells containing K-AgNPs in RPMI without fungal inoculum were included and subtracted from the corresponding test wells during MIC determination. In addition, MIC values were defined using a stringent ≥80% inhibition threshold rather than the EUCAST ≥50% endpoint, providing a more conservative criterion and reducing the likelihood that minor residual optical contributions influence MIC assignment. While these measures cannot substitute for direct physicochemical monitoring of nanoparticle behaviour in RPMI, they provide safeguards against optical artefacts affecting the biological interpretation of the assay. Finally, MIC, MFC, hemocyte viability, and Galleria mellonella survival data showed internally consistent biological responses across the tested Candida species. Although Ag+/AgCl speciation in RPMI cannot be excluded, the collective findings indicate that biologically active silver species remained present throughout the assay period and support the validity of the reported antifungal activity.

 

Comment 2. The Minimum Inhibitory Concentration (MIC) obtained for the K-AgNPs is valid on its own as an intrinsic property of this new molecule/particle against the standardized ATCC strains. However, without a parallel positive control (commercial drugs), the internal validation of the assay on the day of testing is lost. A control with Fluconazole/Anidulafungina serves to guarantee that the methodology performed according to EUCAST guidelines. The authors present a “historical comparison” in the discussion; while this might be accepted by some journals, it weakens the study’s argumentative strength.

Response 2: The reviewer is correct that contemporaneous parallel MIC determination of a conventional comparator antifungal on the day of testing is the strongest form of internal QC for EUCAST E.Def 7.4 microdilution. We accept that benchmarking against published EUCAST wild-type distributions and clinical breakpoints, while informative for situating K-AgNP potency in the licensed-drug landscape, does not substitute for assay-day validation, and we have explicitly added this as a limitation in the revised Discussion (Section 4, Seventh limitation).

In defense of the existing data, we would respectfully highlight the following methodological safeguards that were in place. The panel included two ATCC strains that are themselves EUCAST quality-control organisms for antifungal susceptibility testing, namely Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019, whose growth characteristics in RPMI 1640 at the inoculum used in this study are well-defined in the EUCAST QC literature. Each plate included growth controls (drug-free fungal suspension), sterility controls (medium only), nanoparticle background controls (K-AgNP dilutions in medium without fungus), and an independent inoculum CFU verification step (Section 2.5.1). The assay was repeated in quadruplicate on seven separate days, with modal MIC and MFC values reported, and concordant MIC and MFC endpoints were obtained across all eight species. While these features do not replace a fluconazole or anidulafungin comparator arm, they do support the technical reproducibility of the assay across seven independent runs.

We have not added new comparator MIC data within this revision because that would require de novo dilutions of fluconazole and anidulafungin against all eight species under the same conditions, which is a separate experimental campaign rather than a same-batch addition. We have, however, identified contemporaneous head-to-head MIC determination of K-AgNPs versus licensed antifungals against all eight species (including expanded C. auris clinical isolates) as a priority for the next phase of this work, and this is now stated explicitly in the limitations.

 

Comment 3. The term “broad-spectrum” is incorrectly applied. The authors are engaging in scientific hyperbole; inhibiting eight species of Candida spp. does not constitute a broad spectrum. Instead, this represents a restricted antifungal activity or, at most, a relevant action against opportunistic yeasts. It could only be considered broad-spectrum if the K-AgNPs demonstrated concomitant action against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and yeasts. Therefore, the correct term should be “expanded anti-Candida activity” or “broad activity against the genus Candida”; otherwise, it is a misuse of the term or an exaggeration.

Response 3: We accept this terminological correction in full. The term “broad-spectrum” has been removed from the title, the abstract, the introduction, and the conclusion of the manuscript. The phrase “broad antifungal activity through multiple targets” in the Introduction has been retained because it refers to the literature-supported mechanistic plurality of silver nanoparticles (membrane and cell wall damage, ROS generation, interference with adhesion and morphogenesis, biofilm disruption) and is sourced to general AgNP reviews [refs 8, 9, 10], not to a spectrum claim about K-AgNPs themselves.

Comment 4. The authors have confused certain concepts. The Checkerboard assay and the Isobologram are used to evaluate combinative interactions (Synergism, Antagonism, or Additivity) between two drugs administered simultaneously, rather than to compare isolated potencies. To compare potencies, the pipeline should involve the simultaneous determination of the MICs for K-AgNPs, Fluconazole, and Anidulafungina against all strains. Additionally, Time-Kill curves could be performed to assess the rate of cell death, and the selectivity index (SI) should be calculated as the ratio between the cytotoxic dose in human cells/hemocytes and the MIC against the fungus. The higher the SI, the more potent and safer the agent.

Response 4: We thank the reviewer for this clarification and agree that checkerboard and isobologram analyses are designed to evaluate interactions between two agents administered simultaneously and are not appropriate methods for direct potency comparison between independent antifungal compounds. The reference to these approaches appeared only in our previous response letter and not in the manuscript itself. We apologize for the imprecise wording.

We also agree that additional studies such as head-to-head MIC determination against licensed antifungals, time-kill analyses, and calculation of a selectivity index based on mammalian-cell cytotoxicity data would provide valuable complementary information regarding comparative potency and therapeutic potential. However, these experiments address questions beyond the primary objective of the present study, which was to determine the antifungal activity of the newly synthesized K-AgNPs against WHO-priority Candida species and to establish an initial in vivo safety profile in Galleria mellonella.

The current work demonstrates reproducible MIC and MFC activity across eight Candida species together with low toxicity in the invertebrate model. We agree that comparative pharmacodynamic studies and mammalian-cell selectivity assessments represent important next steps and have identified these as priorities for future investigation in the revised Discussion.

Comment 5. Photometric readings were performed at 530 nm. Why did the authors not use 620 nm? It is well known that Surface Plasmon Resonance (SPR) is a broad band with a Rayleigh scattering tail that can easily extend up to 550 nm, depending on the concentration and aggregation state. This could be bypassed if the authors used a background control (medium + nanoparticles without fungi) electronically subtracted from each well, though it remains unclear whether this was done. Switching to longer wavelengths (such as 620 nm or 630 nm) drastically minimizes interference from the silver plasmon.

Response 5: We are grateful to the reviewer for this question and acknowledge that the original methods text was not sufficiently explicit about how nanoparticle background was handled. We can confirm, and have edited the methods accordingly, that per-concentration nanoparticle background-correction wells were used. The methods section (Section 2.5.1) has been updated to eliminate expression ambiguity of “microorganism-free wells” as rightly highlighted.

On the choice of 530 nm, we agree with the reviewer that the Ag0 surface plasmon resonance band centered at 415 nm has a long-wavelength tail that can extend into the 530–570 nm region, particularly under conditions of aggregation, and that readings at 620–630 nm would further minimise this potential contribution. The 530 nm endpoint was selected to align with the visible-light range conventionally used for fungal turbidity measurements and with the EUCAST E.Def 7.4 protocol. To minimise optical interference, per-concentration nanoparticle background-correction wells were included for every K-AgNP dilution and their absorbance values were subtracted from the corresponding test wells. In addition, MIC values were defined using a stringent ≥80% inhibition threshold rather than the EUCAST ≥50% endpoint, providing a more conservative criterion that reduces the likelihood that minor residual absorbance or scattering effects influence MIC assignment. We have, however, added an explicit limitation point (Section 4, Eighth limitation) recommending that future MIC determinations of colloidal silver agents be performed at 620–630 nm with parallel comparator drug arms, as the reviewer suggests.

Comment 6. Thank you for providing Supplementary Table 2 and showing the results of the normality (Shapiro-Wilk) and homoscedasticity (Levene’s) tests. However, in the footnotes of the tables, all Shapiro-Wilk and Levene’s tests returned p < 0.05, indicating deviations from normality and homoscedasticity. The authors justify that “Given the balanced design (n = 12 per cell) and the robustness of ANOVA to such violations under balanced conditions, the parametric analysis was retained.” While it is true in theoretical statistical literature that ANOVA is robust to deviations from normality when the design is balanced (n equal across all groups), there are strict limits. For example, for Candida krusei and Candida parapsilosis: the interaction term presents massive F-values (F = 69.06 and F = 24.74, respectively) with p < 0.001 and effect sizes (η²p = 0.758 and 0.529) so overwhelming that even violations are unlikely to alter the conclusion. In the case of Candidozyma auris: the Group × Time interaction yielded p = 0.013 with a low effect size (η²p = 0.113), and because both Levene’s and Shapiro–Wilk were severely violated (p < 0.001), the Type I error rate inflates considerably; asserting a significant interaction at p = 0.013 while violating both basic assumptions is statistically imprudent. Suggestions: (a) apply a data transformation (logarithmic, square root, or Box-Cox transformation); (b) if data transformation fails, the analysis should migrate to non-parametric models (such as the Scheirer-Ray-Hare test) or use Generalized Linear Models (GLM) adjusted for distributions that accommodate heteroscedasticity.

Response 6: We are grateful to the reviewer for this rigorous methodological critique, which we accept in full. The argument that Type I error inflates when ANOVA assumptions are severely violated and effect sizes are modest is exactly correct, and the C. auris interaction term is the case where this concern bites hardest in the present dataset.

Following the reviewer’s recommended pipeline, we first attempted to rescue the parametric framework via variance-stabilising transformations of the percentage viability data. Four transformations appropriate to bounded proportion data were evaluated (arcsin-square-root, smoothed logit, reflected square-root, and reflected log). For C. tropicalis, these transformations restored both normality and homoscedasticity, but for the other five species no transformation jointly satisfied both assumptions. The hemocyte viability analysis has therefore been migrated to a non-parametric framework, as the reviewer recommended. The Scheirer–Ray–Hare test is reported as the primary two-factor analysis, with effect sizes expressed as epsilon-squared (ε²). Because the Scheirer–Ray–Hare test is known to underpower interaction tests in factorial designs owing to the confounding of main-effect rankings with the interaction signal, the Aligned Rank Transform ANOVA is also reported as a sensitivity analysis. Pairwise contrasts within each time point are now tested by Dunn’s test with Holm–Bonferroni correction following an omnibus Kruskal–Wallis test, replacing the Tukey HSD post-hoc used for the parametric pipeline. The total hemocyte count analysis, where the data are unbounded and the assumption violations were milder, has been retained under the two-way ANOVA + Tukey HSD framework. The complete re-analysis is presented in the revised Supplementary Table 2 (omnibus analyses with raw and arcsin-transformed assumption diagnostics) and the new Supplementary Table 3 (all 108 pairwise Dunn’s contrasts). Section 2.7 (Statistical analysis), the opening paragraph of Section 3.5 (Hemocyte Viability), and the per-species results narrative in Section 3.5 have been re-written accordingly. Figure 11 has been re-rendered as Tukey-style box-and-whisker plots, with significance brackets reflecting Dunn’s pairwise results, and its caption has been updated to describe the new framework.

 

We are grateful to the reviewer for the depth and precision of these comments, which have improved methodological transparency and the calibration of the manuscript’s claims. We hope the revisions and the added limitations adequately address the concerns raised.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is suitable for publication.

Author Response

Thank you for your work and careful review of our manuscript. 

 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed nearly all of the reviewer’s comments and suggestions. However, a few critical points regarding conceptual consistency, methodology, and the tone of the conclusions still require clarification before the manuscript can be considered for publication.

1. Conceptual Consistency and Scope (Invasive Candidiasis & Biofilms)

There appears to be an ongoing inconsistency throughout the manuscript regarding the distinction between in vitro activity against specific pathogens and actual therapeutic/translational applicability:

Invasive Candidiasis: The authors demonstrated antifungal activity against species known to cause invasive candidiasis; therefore, it is appropriate to state that the nanoparticles exhibit activity against these species. However, no data on the route of administration, pharmacokinetics, biodistribution, plasma concentrations, metabolism, excretion, or systemic toxicity were provided. Any direct association with the treatment of invasive candidiasis represents an extrapolation beyond the presented pharmacological data.

Biofilms: The manuscript relies heavily on a biofilm-related biological rationale in the Introduction (discussing MBIC, MBEC, formation, and eradication). While silver nanoparticles could theoretically be applied to medical devices, no biofilm-related experiments were performed. Unless biofilm inhibition or eradication is evaluated, there is no strong rationale for emphasizing this aspect, and it remains unclear how these topics connect to the actual scope of the study.

2. Methodological Contextualization (Absence of Comparator)

Overall, the manuscript successfully demonstrates antifungal activity against clinically relevant Candida isolates using a standardized and validated framework (EUCAST-based methodology). However, the absence of a contemporaneous comparator antifungal agent (such as a licensed drug) precludes meaningful pharmacological contextualization of the reported MIC values. Without a head-to-head comparison, claims regarding "potency" cannot be supported, which significantly limits the translational significance that can be inferred from the findings.

3. Nanoparticle Stability in RPMI.

The authors acknowledge in their rebuttal that Ag+/AgCl speciation cannot be excluded because nanoparticle stability in RPMI was not measured. Yet, the Results section still states: "No macroscopic aggregation, colour change, or sedimentation was observed..." * While not a direct contradiction, this statement risks being interpreted as a claim of colloidal stability. As the rebuttal correctly notes, visual inspection alone is insufficient.

Recommendation: The manuscript should either (a) soften or remove the macroscopic‑inspection phrasing and explicitly state that in situ physicochemical monitoring (e.g., UV‑Vis, DLS/NTA) was not performed, or (b) present such measurements if available.

 

4. Abstract, Conclusions, and Overall Tone

Given the acknowledged absence of a contemporaneous antifungal comparator and the lack of physicochemical validation in RPMI, the tone of the conclusions must be more cautious and better aligned with the data:

Abstract Wording: The phrase "potent and biocompatible antifungal agent with broad activity across the WHO Fungal Priority Pathogens Candida tier" is ambiguous and should be qualified or removed since no direct comparison against licensed agents was performed.

Concluding Remarks: Sweeping claims of a "compelling platform for further development toward antifungal applications in clinical and infection‑control contexts" should be replaced with more measured language.

Suggested Alternative: "The present findings demonstrate antifungal activity of K‑AgNPs and provide justification for further investigation, including head‑to‑head comparisons with licensed antifungals and physicochemical validation of nanoparticle stability under assay conditions."

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 — Third Round

Manuscript ID: cimb-4306174

Title: Kombucha-Mediated Silver Nanoparticles with Fungicidal Activity against WHO Priority Candida Pathogens: In Vitro and Galleria mellonella Evaluation

We thank Reviewer 2 for the careful third-round assessment and for the precision of the four remaining points. Each comment has been addressed below, with corresponding revisions made to the manuscript. The scope of the experimental dataset has not changed in this revision; only the language used to frame and describe that dataset has been adjusted, so that the manuscript states only what the data directly supports and explicitly identifies the additional work required before any translational reading can be advanced. Where the reviewer offered a specific alternative phrasing, we have incorporated it. In addition, we have applied internal-consistency edits throughout the Discussion and Conclusions so that the calibrated language requested by the reviewer is carried through the manuscript rather than confined to the most prominent sentences.

Comment 1. Conceptual Consistency and Scope (Invasive Candidiasis & Biofilms). There appears to be an ongoing inconsistency throughout the manuscript regarding the distinction between in vitro activity against specific pathogens and actual therapeutic/translational applicability: Invasive Candidiasis: The authors demonstrated antifungal activity against species known to cause invasive candidiasis; therefore, it is appropriate to state that the nanoparticles exhibit activity against these species. However, no data on the route of administration, pharmacokinetics, biodistribution, plasma concentrations, metabolism, excretion, or systemic toxicity were provided. Any direct association with the treatment of invasive candidiasis represents an extrapolation beyond the presented pharmacological data. Biofilms: The manuscript relies heavily on a biofilm-related biological rationale in the Introduction (discussing MBIC, MBEC, formation, and eradication). While silver nanoparticles could theoretically be applied to medical devices, no biofilm-related experiments were performed. Unless biofilm inhibition or eradication is evaluated, there is no strong rationale for emphasizing this aspect, and it remains unclear how these topics connect to the actual scope of the study.

Response 1: We accept both sub-points in full. The reviewer is correct that the present study, while demonstrating activity against species that cause invasive candidiasis, does not include the pharmacokinetic, biodistribution, route-of-administration, or systemic toxicity data required to support any direct claim regarding the treatment of invasive candidiasis as a clinical condition. We have therefore rephrased the Abstract, the closing paragraph of the Discussion (Section 4), and the second paragraph of the Conclusions (Section 5) so that the manuscript states only what is shown, namely activity against the relevant species in standardised in vitro and in vivo settings, and explicitly identifies the missing pharmacological work as a prerequisite for any translational claim.

On the biofilm sub-point, we agree that the biofilm-related rationale previously present in the Introduction is not supported by the present dataset, which evaluates planktonic susceptibility and in vivo larval survival but does not include MBIC or MBEC assays. The biofilm framing in the Introduction (Section 1) has therefore been substantially trimmed. We respectfully note for the record that the current Introduction does not contain the acronyms MBIC or MBEC, which may correspond to a recollection from an earlier draft, but we have nevertheless reduced the remaining biofilm-tolerance phrasing in the spirit of the comment. The acknowledgement that antibiofilm evaluation is a key gap is retained in the Discussion limitations (Section 4, Fourth limitation) and is now explicitly listed among the prerequisites for any translational reading in the revised Conclusions.

Comment 2. Methodological Contextualization (Absence of Comparator). Overall, the manuscript successfully demonstrates antifungal activity against clinically relevant Candida isolates using a standardized and validated framework (EUCAST-based methodology). However, the absence of a contemporaneous comparator antifungal agent (such as a licensed drug) precludes meaningful pharmacological contextualization of the reported MIC values. Without a head-to-head comparison, claims regarding “potency” cannot be supported, which significantly limits the translational significance that can be inferred from the findings.

Response 2: Accepted. Evaluative "potency" language has been removed throughout the Abstract, Discussion, and Conclusions, and replaced with descriptive MIC-based phrasing. The EUCAST contextualisation paragraph in Section 4 has been amended so that the comparison is now framed only as contextualisation against published wild-type and breakpoint distributions, with no potency claim relative to licensed agents retained. The corresponding Discussion limitation (Section 4, Seventh limitation) has been preserved, since it already identifies the absence of comparator arms and flags head-to-head MIC determination as a priority for follow-up work.

Comment 3. Nanoparticle Stability in RPMI. The authors acknowledge in their rebuttal that Ag⁺/AgCl speciation cannot be excluded because nanoparticle stability in RPMI was not measured. Yet, the Results section still states: “No macroscopic aggregation, colour change, or sedimentation was observed...” While not a direct contradiction, this statement risks being interpreted as a claim of colloidal stability. As the rebuttal correctly notes, visual inspection alone is insufficient. Recommendation: The manuscript should either (a) soften or remove the macroscopic-inspection phrasing and explicitly state that in situ physicochemical monitoring (e.g., UV-Vis, DLS/NTA) was not performed, or (b) present such measurements if available.

Response 3: Accepted. We have taken the cleaner of the two options the reviewer offered and removed the macroscopic-inspection sentence from the Results section entirely. The in-medium stability paragraph of the Discussion limitations (Section 4, Sixth limitation) has been refined to explicitly name UV-Vis tracking of the surface plasmon resonance band, dynamic light scattering, and nanoparticle tracking analysis as the in situ measurements that were not performed under assay conditions, and the "assessed only macroscopically" framing has been removed. The chloride-driven Ag⁺/AgCl speciation rationale and the MIC/MFC consistency observation across all eight species are preserved verbatim.

Comment 4. Abstract, Conclusions, and Overall Tone. Given the acknowledged absence of a contemporaneous antifungal comparator and the lack of physicochemical validation in RPMI, the tone of the conclusions must be more cautious and better aligned with the data. Abstract Wording: The phrase “potent and biocompatible antifungal agent with broad activity across the WHO Fungal Priority Pathogens Candida tier” is ambiguous and should be qualified or removed since no direct comparison against licensed agents was performed. Concluding Remarks: Sweeping claims of a “compelling platform for further development toward antifungal applications in clinical and infection-control contexts” should be replaced with more measured language. Suggested Alternative: “The present findings demonstrate antifungal activity of K-AgNPs and provide justification for further investigation, including head-to-head comparisons with licensed antifungals and physicochemical validation of nanoparticle stability under assay conditions.”

Response 4: Accepted. The reviewer's suggested alternative phrasing has been incorporated essentially verbatim at the end of the Discussion (Section 4), and the same calibrated tone has been applied to the Abstract and the Conclusions (Section 5). The terms "potent", "compelling platform", "multidimensional preclinical foundation", "clinical and infection-control contexts", and "safety window at therapeutic concentrations" have been removed, and the Conclusions now state explicitly the work required before any translational claim can be supported, comprising head-to-head comparison with licensed antifungals, physicochemical validation of nanoparticle stability under assay conditions, antibiofilm evaluation, expanded clinical isolate panels, and mammalian safety assessment including pharmacokinetic and biodistribution data.

We are grateful to the reviewer for the precision of these comments. The revised manuscript no longer makes claims that extend beyond the experimental dataset, the calibrated language requested by the reviewer is carried through the Abstract, Discussion, and Conclusions rather than confined to isolated sentences, and the prerequisites for any future translational reading are stated openly. We hope the manuscript is now acceptable for publication and remain available to address any further point that may arise.

Author Response File: Author Response.pdf

Round 4

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have comprehensively addressed all points and systematically improved the manuscript. Consequently, I believe the manuscript is now suitable for publication.