Review Reports - Targeting Dermatophyte Biofilms: Effects of <i>Lavandula stoechas</i> subsp. <i>luisieri</i> Essential Oil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well written and addresses a very important topic. The compilation of data is good and the representation of data is clear. However, some additions and improvements are needed before publication.

1. Please update the references in the Introduction with recent studies from 2023 to 2025.

2. Add a section on the limitations for using essential oils at commercial and industrial scale.

3. Explain why industries are still not widely adopting essential oils for biofilm control.

4. Include toxicity or cytotoxicity data for these essential oils on human cells.

5. Mention which specific compounds in essential oils are most responsible for anti-biofilm activity.

6. Provide details of positive controls used in the antimicrobial and anti-biofilm assays and compare results with standard antibiotics.

7. Clarify the incubation time, temperature, and number of replicates used for biofilm formation in the Methods section.

8. The MIC values and biofilm inhibition percentages in Table 2 do not fully correlate. Please explain this observation.

9. Add SEM or confocal microscopy images to support the claim that essential oils disrupt the EPS matrix of biofilms.

10. Include a future perspective on combinations of essential oils with nanoparticles or antibiotics to improve industrial application.

Comments on the Quality of English Language

Thought i am not a native speaker, but it seems the paper iw well written.

Author Response

Reviewer 1:

Please update the references in the Introduction with recent studies from 2023 to 2025.

Response: We sincerely thank the reviewer for emphasizing the importance of updating the references in the Introduction section. In response to this valuable suggestion, we thoroughly revised the bibliography and updated the reference list wherever appropriate to better reflect the most recent advances in the field. As a result, we incorporated several studies published between 2023 and 2025, increasing the proportion of recent references from 45.1% to 68.5% of the total citations included in the manuscript. These revisions ensure that the manuscript is supported by up-to-date literature while maintaining the scientific rigor, contextual relevance, and completeness of the information presented.

Add a section on the limitations for using essential oils at commercial and industrial scale.

Response: We sincerely thank the reviewer for this valuable suggestion. We agree that discussing the limitations associated with the commercial and industrial application of essential oils strengthens the manuscript and provides a more balanced perspective on their potential translation into practical use. Accordingly, we have added a new paragraph to the Discussion addressing the main challenges associated with large-scale implementation.

The following paragraph has been added to the Discussion:

“Nevertheless, despite these promising biological properties and their potential therapeutic applications, the translation of essential oils from laboratory research to large-scale industrial use still faces important technological and economic challenges. Their chemical composition is strongly influenced by factors such as geographical origin, climatic conditions, harvesting season, and extraction methods, which may compromise batch-to-batch reproducibility and biological consistency [103]. Further-more, essential oils are highly volatile and susceptible to oxidation and degradation by light, oxygen, and temperature, reducing their stability during storage and formulation [104–106]. An additional limitation is the relatively low essential oil yield, which may increase production costs and hinder large-scale manufacturing [107,108]. Therefore, standardized cultivation and extraction procedures, as well as quality control proto-cols, together with comprehensive toxicological and stability studies, remain essential before these natural products can be widely implemented in industrial settings.

Explain why industries are still not widely adopting essential oils for biofilm control.

Response: We sincerely thank the reviewer for this valuable comment. We agree that the reasons limiting the industrial adoption of essential oils for biofilm control deserve further discussion. Accordingly, we expanded the Discussion section by adding a paragraph that addresses the main barriers to their large-scale commercial and industrial implementation. In addition to challenges related to chemical variability, limited stability, low extraction yields, and the need for standardized cultivation, extraction methods, and quality control, the new text also discusses regulatory requirements, the lack of standardized formulations, and the limited evidence demonstrating efficacy under real conditions. These factors currently hinder the widespread industrial application of essential oils for biofilm control.

The following paragraph has been added to the Discussion:

“Furthermore, the industrial implementation of essential oils for biofilm control remains limited by additional challenges, including the lack of standardized formulations, regulatory constraints, and the scarcity of studies evaluating their efficacy under real conditions. Factors such as scalability, long-term stability, compatibility with manufacturing processes, and the reproducibility of antibiofilm activity must still be thoroughly investigated. Consequently, although essential oils represent promising alternatives for controlling microbial biofilms, further technological development, regulatory validation, and industrial-scale studies are required before they can be widely adopted in commercial applications.”

Include toxicity or cytotoxicity data for these essential oils on human cells.

Thank you for this valuable comment. We agree that evaluating the safety of essential oils in human cells is essential for assessing their potential topical application. In the present study, cytotoxicity was investigated using 3T3 fibroblasts, which constitute a well-established mammalian model for the preliminary assessment of biocompatibility. The results demonstrated low cytotoxicity at the concentrations associated with antifungal activity. Nevertheless, we acknowledge that additional studies using human skin-derived cell models, such as human keratinocytes and dermal fibroblasts, would provide further evidence regarding the safety profile of the essential oil for topical applications. To address this limitation, we have included the following statement in the section Discussion of the manuscript:

“Although 3T3 fibroblasts represent a relevant mammalian model for cytotoxicity evaluation, further studies using human skin-derived cell models would be valuable to confirm its safety for topical applications.”

Mention which specific compounds in essential oils are most responsible for anti-biofilm activity.

Response: We sincerely thank the reviewer for this valuable comment. We agree that discussing the potential contribution of specific essential oil constituents to the observed anti-biofilm activity is important. However, the contribution of individual compounds cannot be confirmed based on the current experimental design, as no assays were performed using isolated constituents. Essential oils are complex mixtures, and their biological activities are generally influenced by synergistic or additive interactions among multiple components. Therefore, while some major constituents may contribute to the observed activity, the involvement of other compounds and possible interactions between oil components cannot be excluded. The revised text has been carefully worded to include this comment., as follow:

“Taken together, these findings suggest that elevated levels of necrodyl derivatives, particularly trans-α-necrodyl acetate and trans-α-necrodol, may contribute not only to the antidermatophytic activity but also to the inhibition of biofilm formation exhibited by the essential oil. However, this hypothesis should be interpreted cautiously, as the contribution of other constituents, as well as their possible interactions, cannot be excluded.”

Provide details of positive controls used in the antimicrobial and anti-biofilm assays and compare results with standard antibiotics.

Response: Thank you for this important comment. We would like to clarify that standard antifungal agents were not included as positive controls in the antimicrobial and antibiofilm assays performed in the present study. Instead, fluconazole was selected as a reference antifungal for comparative purposes, as previously published susceptibility data obtained for the same dermatophyte strains were available, allowing contextual interpretation of the activity observed for L. stoechas subsp. luisieri essential oil. Accordingly, we have clarified this aspect in the revised manuscript and included the following statement in the Discussion:

“Although fluconazole was used as the reference antifungal to enable comparison with previously published susceptibility data for the same dermatophyte strains, future studies should include first-line antifungal agents, particularly terbinafine and itraconazole, to provide a more clinically relevant assessment of the therapeutic potential of L. stoechas subsp. luisieri essential oil.”

Clarify the incubation time, temperature, and number of replicates used for biofilm formation in the Methods section.

Thank you for this comment. We have revised the Materials and Methods section to clarify the biofilm experimental conditions, including the incubation time, temperature, and the number of replicates, as follows:

“For both assays, fungal conidia were initially allowed to adhere to the wells for 3 h at 37 °C. Following removal of non-adherent cells by washing with PBS, biofilms were allowed to develop for 72 h at 37 °C. In the biofilm formation inhibition assay, the es-sential oil was added immediately after the adhesion phase and maintained through-out the biofilm development period. In the mature biofilm disruption assay, estab-lished 72 h-old biofilms were exposed to the same concentration range of the essential oil for an additional 24 h at 37 °C to evaluate its ability to disrupt pre-formed biofilm structures. In both experimental conditions, three parameters were assessed: biofilm biomass, ECM deposition, and fungal cell viability. All assays were performed in triplicate and repeated in three independent experiments.”

The MIC values and biofilm inhibition percentages in Table 2 do not fully correlate. Please explain this observation.

Response: We sincerely thank the reviewer for this important observation. We agree that the MIC values and the biofilm inhibition percentages presented in Table 2 do not show a direct correlation. This can be explained by the fact that MIC assays and biofilm assays evaluate distinct biological processes. While MIC determination reflects the ability of the essential oil to inhibit planktonic fungal growth, biofilm assays assess more complex processes, including cell adhesion, biofilm maturation, extracellular matrix production, and the metabolic activity of biofilm-associated cells.

In the present study, biofilm formation inhibition required concentrations slightly higher than those affecting planktonic growth, which is consistent with the increased tolerance generally exhibited by biofilm-associated fungal cells. Notably, the concentration of 25 µg/mL, although effective in some growth-related parameters, did not significantly inhibit biofilm formation, whereas higher concentrations within the tested range showed a more pronounced inhibitory effect. This finding highlights that the establishment of a biofilm structure requires different or higher exposure levels compared with the inhibition of free-living fungal cells.

Therefore, the absence of a direct relationship between MIC values and biofilm inhibition percentages is not unexpected, as these assays reflect different stages of fungal development and different biological responses to the essential oil. The revised manuscript has been modified to clarify this point and to avoid implying a direct equivalence between antifungal and anti-biofilm activity.

The fllowing text was included:

“However, the antibiofilm effects did not directly correlate with the MIC values, as MIC assays evaluate inhibition of planktonic fungal growth, whereas biofilm assays assess more complex processes, including biomass accumulation, extracellular matrix production, and biofilm-associated cell viability. In the present study, inhibition of biofilm formation required slightly higher concentrations than those affecting planktonic growth, which is consistent with the increased tolerance of biofilm-associated cells and the structural complexity of fungal biofilms. Therefore, the antifungal activity observed in planktonic conditions cannot be directly extrapolated to predict antibiofilm efficacy.”

Add SEM or confocal microscopy images to support the claim that essential oils disrupt the EPS matrix of biofilms.

Response: We sincerely thank the reviewer for this valuable suggestion. As SEM images were already included in the manuscript, we improved their interpretation by adding arrows highlighting fungal hyphae and extracellular matrix in untreated biofilms and their reduction/disorganization following essential oil treatment. These morphological observations are consistent with the quantitative reduction in ECM production observed in our assays and further support the effect of L. stoechas subsp. luisieri essential oil on biofilm structural organization.

Include a future perspective on combinations of essential oils with nanoparticles or antibiotics to improve industrial application.

Response: We sincerely thank the reviewer for this valuable suggestion. We have revised the manuscript by adding a future perspective on the potential integration of L. stoechas subsp. luisieri essential oil with conventional antifungal agents and nanoparticle-based delivery systems. This addition highlights how such strategies may enhance therapeutic efficacy, improve formulation stability, and support future pharmaceutical and industrial applications. The following statement has been added to the conclusion section in the revised manuscript:

"In addition, the combination of essential oils with conventional antifungal drugs or nanoparticle-based delivery systems represents a promising strategy to enhance therapeutic efficacy, improve bioavailability and stability, and facilitate their future pharmaceutical and industrial application."

Reviewer 2 Report

Comments and Suggestions for Authors

Could you more clearly delineate the principal scientific contribution of the present study in relation to your previous publications? Specifically, which findings are genuinely novel and have not been reported or demonstrated in your earlier work?
In the Discussion, the authors state that the essential oil exhibits higher activity than fluconazole. What was the rationale for selecting fluconazole as the reference antifungal agent, given that terbinafine and itraconazole are more commonly used as first-line therapies for dermatophytoses in clinical practice? Would it not be more appropriate to compare the antifungal activity of the essential oil with these current therapeutic standards?
The antibiofilm activity was evaluated exclusively against Epidermophyton floccosum. On what basis do the authors consider these findings to be generalizable to the other dermatophyte species included in the MIC/MFC analysis? Please justify the rationale for selecting floccosum as the sole experimental model.
The conclusions regarding the effects of the essential oil in the ex vivo model appear to rely predominantly on qualitative observations from SEM images. Could the authors provide quantitative measurements to statistically support the observed inhibition of fungal colonization and spread? For example, image-based morphometric analysis, fungal biomass quantification, hyphal coverage, or other objective metrics would substantially strengthen the evidence supporting the reported ex vivo antifungal efficacy.
The authors propose that the high content of trans-α-necrodyl acetate is the principal determinant of the observed antifungal activity. Are there any experimental data that directly support this conclusion, or does this interpretation remain speculative? If the latter, the Discussion should clearly acknowledge that the proposed role of trans-α-necrodyl acetate is hypothetical and that the observed antifungal activity may also result from synergistic interactions among multiple constituents of the essential oil.

Author Response

Reviewer 2:

Could you more clearly delineate the principal scientific contribution of the present study in relation to your previous publications? Specifically, which findings are genuinely novel and have not been reported or demonstrated in your earlier work?

We thank the reviewer for this important comment. The main scientific contribution of the present study has been clarified in the revised manuscript. Previous studies on L. stoechas subsp. luisieri essential oil focused primarily on planktonic antifungal activity, including MIC/MFC determination and effects on fungal germination. In contrast, the present study provides new insights by investigating, for the first time, the activity of this essential oil against fungal biofilm development, specifically evaluating its effects on Epidermophyton floccosum biofilms, as well as its performance in an ex vivo model of Trichophyton rubrum-infected skin. These approaches extend the knowledge beyond conventional susceptibility assays by addressing clinically relevant stages of dermatophyte infection, including biofilm establishment and fungal colonization in a tissue-like environment. Therefore, the novelty of this work does not rely solely on the antifungal activity of the essential oil itself, but on the characterization of its antibiofilm properties and its potential activity in an ex vivo infection model, which have not been previously demonstrated for this essential oil.

To clarify this point, we included the following statement:

“Although L. stoechas subsp. luisieri essential oil is well recognized for its biological properties, our previous studies have primarily focused on its activity against planktonic fungal cells. These investigations have evaluated antifungal susceptibility through minimum inhibitory and minimum fungicidal concentrations, as well as its effects on germ tube germination. However, whether this essential oil can interfere with dermatophyte biofilm formation and fungal colonization under conditions that more closely mimic the infected tissue environment remains unknown. Addressing these knowledge gaps is particularly important, as biofilm-associated growth and tissue colonization are key determinants of persistent infection and therapeutic failure that are not adequately captured by conventional planktonic susceptibility assays.”

In the Discussion, the authors state that the essential oil exhibits higher activity than fluconazole. What was the rationale for selecting fluconazole as the reference antifungal agent, given that terbinafine and itraconazole are more commonly used as first-line therapies for dermatophytoses in clinical practice? Would it not be more appropriate to compare the antifungal activity of the essential oil with these current therapeutic standards?

Thank you for this important comment. We agree that terbinafine and itraconazole are currently the preferred first-line systemic antifungal agents for the treatment of most dermatophytoses. However, the aim of our study was not to compare the efficacy of L. stoechas subsp. luisieri essential oil with current standard-of-care antifungal drugs, but rather to characterize its antifungal activity against dermatophytes.

Fluconazole was selected as the reference antifungal because susceptibility data for the same dermatophyte strains had been previously reported under comparable experimental conditions. This enabled a direct comparison with our results and provided a consistent benchmark for interpreting the antifungal activity of the essential oil. In contrast, equivalent susceptibility data for terbinafine and itraconazole were not available for the complete strain panel used in this study, precluding a meaningful side-by-side comparison.

We acknowledge that comparison with the antifungal agents currently recommended for the treatment of dermatophytosis would further strengthen the translational relevance of our findings. Accordingly, we have revised the Discussion to clarify the rationale for using fluconazole as the reference antifungal and to state that future studies should directly compare the activity of L. stoechas subsp. luisieri essential oil with first-line agents, particularly terbinafine and itraconazole, to better define its therapeutic potential, as follows:

The antibiofilm activity was evaluated exclusively against Epidermophyton floccosum. On what basis do the authors consider these findings to be generalizable to the other dermatophyte species included in the MIC/MFC analysis? Please justify the rationale for selecting floccosum as the sole experimental model.

Thank you for this important comment. Epidermophyton floccosum was selected because it is a well-established dermatophyte model that reliably forms homogeneous and reproducible biofilms under in vitro conditions, making it particularly suitable for standardized evaluation of antibiofilm activity. Moreover, as a clinically relevant etiological agent of dermatophytosis, it provides an appropriate proof-of-concept model for investigating the antibiofilm potential of L. stoechas subsp. luisieri essential oil. We agree, however, that the results should not be extrapolated to all dermatophyte species, as biofilm architecture and susceptibility may vary among species. Accordingly, we have revised the Discussion as follows:

“The ability of dermatophytes to form biofilms has been widely recognized as an important factor contributing to infection persistence and reduced susceptibility to an-tifungal therapy [10,14–16]. Epidermophyton floccosum was selected as the experimental model because it is a clinically relevant dermatophyte with a well-documented ability to form homogeneous and reproducible biofilms in vitro, making it particularly suita-ble for standardized antibiofilm assays [78,79]. As this was a proof-of-concept study, the antibiofilm activity of L. stoechas subsp. luisieri essential oil was evaluated in a sin-gle, well-characterized dermatophyte model. Therefore, these findings should be in-terpreted specifically for E. floccosum, and future studies should determine whether similar effects are observed in other clinically relevant dermatophyte species.”

The conclusions regarding the effects of the essential oil in the ex vivo model appear to rely predominantly on qualitative observations from SEM images. Could the authors provide quantitative measurements to statistically support the observed inhibition of fungal colonization and spread? For example, image-based morphometric analysis, fungal biomass quantification, hyphal coverage, or other objective metrics would substantially strengthen the evidence supporting the reported ex vivo antifungal efficacy.

Thank you for this valuable comment. We agree that a more detailed visualization of fungal colonization and morphological alterations is important for the interpretation of the ex vivo model. To strengthen the SEM analysis, we have revised the corresponding figures by including higher magnification images and annotations highlighting the main fungal structures and morphological changes observed following treatment with L. stoechas subsp. luisieri essential oil. These additions provide a clearer representation of the differences between untreated and treated samples, particularly regarding hyphal development, organization, and colonization patterns, and improve the interpretation of the observed antifungal effects in the ex vivo model.

The authors propose that the high content of trans-α-necrodyl acetate is the principal determinant of the observed antifungal activity. Are there any experimental data that directly support this conclusion, or does this interpretation remain speculative? If the latter, the Discussion should clearly acknowledge that the proposed role of trans-α-necrodyl acetate is hypothetical and that the observed antifungal activity may also result from synergistic interactions among multiple constituents of the essential oil.

Thank you for this important comment. We agree that the specific contribution of trans-α-necrodyl acetate to the antifungal activity observed in the present study cannot be directly demonstrated, as assays with isolated compounds were not performed. Accordingly, we have revised the Discussion as follows:

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled „Targeting dermatophyte biofilms: effects of Lavandula stoechas subsp. luisieri essential oil” investigates the potential of lavender essential oil to inhibit dermatophyte biofilm formation and to disrupt established (mature) biofilms.

The manuscript is relevant, interesting, and well-structured. However, several sections require clarification to improve the overall quality and readability of the work. Therefore, I recommend minor revision before the manuscript can be considered for publication.

I did not fully understand the rationale behind the selection of fungal strains throughout the study. Initially seven fungal strains were investigated; subsequently, E. floccosum was selected, but the final experiments were performed with T. rubrum. Please clarify the reason for changing the model organism during the research. Since the manuscript emphasize E. floccosum as a model organism for dermatophyte research, it would be helpful to explain why the subsequent experiments were not continued using this species.

Please carefully revise the manuscript, as several typographical errors remain throughout the text. In addition, I recommend checking and improving the quality, labelling and consistency of all figures and graphical presentations. Addressing these issues will significantly improve the clarity and overall quality of the manuscript.

Detailed comments:

Materials and methods

Lines 113-120

The study includes seven fungal strains. Have all of these strains been previously verified to form biofilms? If so, please, provide the appropriate references or supporting evidence in the manuscript

Lines 155-158

I believe that the explanation, regarding E. floccosum does not belong in the materials and methods section. It would be more appropriate to move this information to the Results section, for example, around lines 360-362.

Results

Lines 289-291

I did not fully understand the format and presentation of Table 1. Some of the reported MIC and MFC values show very wide ranges. It may be more appropriate to present the mean values obtained from the experimental replicates together with the corresponding measures of variability (e.g. standard deviation or standard error). This would provide a clearer representation of the experimental results.

Lines 313-356

I would like to ask whether images of the control samples without DMSO were obtained and whether these results were compared with the DMSO-containing results. Were there any observable differences between the control samples with and without DMSO?

Additionally, please note that the formatting of fungal genus names is not consistent throughout the manuscript. The font style used for these names should be standardized. are not uniform.

Lines 382-392

Please note that the labels presented next to the graph in Figure 2 are not in English (controle instead of control). Please correct this terminology. Additionally, I would appreciate clarification regarding the concentration ranges indicated in the figure. Do the reported ranges (800–200 μg/mL) indicate that the observed pattern was consistent across all tested concentrations within this range? The same clarification is needed for the concentration range of 6.25–1.56 μg/mL. Please specify whether the results represented a range tested concentrations or whether to a specific concentration-dependent effect.

Lines 418-433

Please note that the statistical significance indicators (asterisks) are missing from figure 3 B. Please include the appropriate statistical annotations to clearly indicate significant differences between groups. Additionally, the term controle is used again in the figure label. This should be corrected to control to ensure accurate English in the manuscript.

Lines 454-488

Please revise Figure 4. The letters (A, I, E, M, B, etc.) mentioned in the figure caption are not displayed in the figure itself, making it difficult to understand. Please, ensure that all labels referred to in the caption are clearly indicated in the figure.

Lines 494-501

Could you please clarify the meanings of the zero viability values shown in Figure 5 for the doses of 3,12 and 1,56? Additionally, the meaning of the asterisk indicating statistical significance is not provided in the figure legend. Please include an explanation of the statistical annotations used in figure 5. forgot to write down the meaning of asterixis in Figure 5.

Author Response

Reviewer 3:

I did not fully understand the rationale behind the selection of fungal strains throughout the study. Initially seven fungal strains were investigated; subsequently, E. floccosum was selected, but the final experiments were performed with T. rubrum. Please clarify the reason for changing the model organism during the research. Since the manuscript emphasize E. floccosum as a model organism for dermatophyte research, it would be helpful to explain why the subsequent experiments were not continued using this species.

Thank you for this important comment. The different dermatophyte models used in this work were selected according to the specific biological question addressed in each experimental approach. The initial screening of seven dermatophyte strains was performed to establish the antifungal spectrum of L. stoechas subsp. luisieri essential oil against a diverse panel of clinically relevant dermatophytes. Following this screening, Epidermophyton floccosum was selected for the in vitro antibiofilm assays because this species has been extensively used as a reproducible biofilm-forming model, allowing consistent evaluation of biofilm development and inhibition under standardized laboratory conditions. For the ex vivo model, Trichophyton rubrum was selected because the objective was not to evaluate biofilm formation, but rather to investigate fungal colonization in a host-derived environment. T. rubrum is the predominant causative agent of human dermatophytoses and onychomycosis and is therefore a clinically relevant model for studying tissue colonization and infection-associated growth. Its use in the ex vivo model provides greater clinical relevance for assessing the potential effect of the essential oil under conditions that better resemble the infected tissue environment. We have revised the manuscript to clarify this rationale and avoid any perception of inconsistency in strain selection.

Please carefully revise the manuscript, as several typographical errors remain throughout the text. In addition, I recommend checking and improving the quality, labelling and consistency of all figures and graphical presentations. Addressing these issues will significantly improve the clarity and overall quality of the manuscript.

Thank you for this helpful comment. The manuscript has been thoroughly checked and carefully revised to identify and correct typographical errors, inconsistencies, and issues related to formatting throughout the text. In addition, all figures and graphical presentations were reviewed.

Detailed comments:

Materials and methods

Lines 113-120 - The study includes seven fungal strains. Have all of these strains been previously verified to form biofilms? If so, please, provide the appropriate references or supporting evidence in the manuscript

Thank you for this important comment. The seven dermatophyte strains included in the initial screening were selected to evaluate the antifungal activity spectrum of L. stoechas subsp. luisieri essential oil against clinically relevant dermatophytes. Although biofilm formation has been described for several dermatophyte species, the ability to form biofilms and the characteristics of these biofilms may vary considerably among species and strains. For the antibiofilm experiments, Epidermophyton floccosum was specifically selected because it is a well-established and reproducible biofilm-forming model among dermatophytes, allowing standardized evaluation of biofilm development and inhibition. Therefore, the selection of E. floccosum was based on its suitability for a controlled in vitro biofilm model rather than on the assumption that all strains included in the initial screening display equivalent biofilm-forming capacity. This information was included in the manuscript.

Lines 155-158 - I believe that the explanation, regarding floccosum does not belong in the materials and methods section. It would be more appropriate to move this information to the Results section, for example, around lines 360-362.

Thank you for this helpful suggestion. We agree that the explanation regarding the selection of Epidermophyton floccosum as the biofilm model was not ideally placed in the Materials and Methods section. Accordingly, this information has been removed from the Materials and Methods and relocated to the appropriate sections of the manuscript. Following the reviewer’s suggestions, the rationale for the selection of E. floccosum and its relevance as a biofilm model has now been further clarified in the Discussion section, where it is better integrated with the interpretation of the findings and the overall study rationale.

Lines 289-291 - I did not fully understand the format and presentation of Table 1. Some of the reported MIC and MFC values show very wide ranges. It may be more appropriate to present the mean values obtained from the experimental replicates together with the corresponding measures of variability (e.g. standard deviation or standard error). This would provide a clearer representation of the experimental results.

Thank you for this valuable comment. We would like to clarify that reporting mean MIC and MFC values is not considered appropriate for the present dataset because these parameters are determined as discrete endpoints obtained from predefined serial dilution concentrations (800, 400, 200, 100, 50, 25, 12.5, 6.25 µg/mL, etc.). Therefore, calculating an average value could generate an artificial concentration that was not directly tested and does not correspond to an experimentally determined inhibitory or fungicidal endpoint. In this study, MIC and MFC assays were initially performed in triplicate. When concordant results were obtained, a single MIC or MFC value was assigned. When discrepancies were observed among replicates and no unique endpoint value could be established after additional confirmation assays, the range of concentrations consistently observed was reported. Thus, the values presented in Table 1 reflect the experimentally determined antifungal activity intervals rather than statistical variation around a continuous measurement. To improve clarity, the Table 1 legend has been revised to explicitly describe the serial dilution approach and the rationale for reporting values as ranges.

“Data are expressed in µg/mL. MIC, Minimum Inhibitory Concentration; MFC, Minimum Fungi-cidal Concentration. Values represent the range obtained from independent experiments (n ≥ 3). When concordant MIC/MFC values were obtained among replicates, a single value was assigned; when no unique endpoint could be established after confirmation assays, the observed concentration range is reported.”

Lines 313-356 - I would like to ask whether images of the control samples without DMSO were obtained and whether these results were compared with the DMSO-containing results. Were there any observable differences between the control samples with and without DMSO?

Thank you for this important comment. In the present study, DMSO was used as the solvent for the preparation and dilution of the essential oil samples; therefore, a DMSO control was included to evaluate any potential effects associated with the solvent itself. A control without DMSO was also considered, as it represents the untreated condition. No observable differences were detected between the controls with and without DMSO in the evaluated parameters. Since the DMSO-containing control is the most appropriate comparison for the essential oil treatments, as it accounts for the solvent exposure present in the experimental conditions, this control was selected for the statistical analysis and interpretation of the results.

Additionally, please note that the formatting of fungal genus names is not consistent throughout the manuscript. The font style used for these names should be standardized. are not uniform.

Thank you for this helpful comment. A thorough revision of the manuscript was performed to ensure consistency in the formatting of fungal genus and species names throughout the text, tables, and figure legends.

Lines 382-392 - Please note that the labels presented next to the graph in Figure 2 are not in English (controle instead of control). Please correct this terminology. Additionally, I would appreciate clarification regarding the concentration ranges indicated in the figure. Do the reported ranges (800–200 μg/mL) indicate that the observed pattern was consistent across all tested concentrations within this range? The same clarification is needed for the concentration range of 6.25–1.56 μg/mL. Please specify whether the results represented a range tested concentrations or whether to a specific concentration-dependent effect.

Thank you for this valuable comment. The terminology errors identified in Figure 2 have been corrected. Regarding the concentration ranges indicated in the figure, all concentrations within the reported ranges were experimentally tested. These ranges were used to summarize concentrations that produced a comparable effect, and therefore only one representative image was selected for visualization in the figure. The graphical representation includes the complete range of concentrations tested, while the representative images illustrate observed effect patterns.

Lines 418-433 - Please note that the statistical significance indicators (asterisks) are missing from figure 3 B. Please include the appropriate statistical annotations to clearly indicate significant differences between groups. Additionally, the term controle is used again in the figure label. This should be corrected to control to ensure accurate English in the manuscript.

Thank you for this valuable comment. We apologize for this oversight. No statistical differences were found between the experimental groups and therefore this information was removed from figure legend. In addition, the terminology error in the figure label has been corrected to “control” to ensure consistency and proper English usage throughout the manuscript.

Lines 454-488

Thank you for this helpful comment. The figure has been revised accordingly, and all labels referred to in the caption have now been clearly included in the figure itself.

Lines 494-501

Thank you for this valuable comment. We sincerely apologize for this oversight. The concentrations of 3.12 and 1.56 were not included in the experimental design and were inadvertently represented in Figure 5. These values have now been removed from the graph to ensure that only the tested concentrations are presented. In addition, the statistical annotations in Figure 5 have been revised, and the meaning of the asterisks indicating statistical significance has now been included in the figure legend. All related errors have been corrected to improve the accuracy and clarity of the figure presentation.