Anandamide Targets Membrane Integrity in Non-Albicans Candida: A Novel Antifungal Approach

Round 1

Reviewer 1 Report

The authors reported on anandamide targeting membrane integrity in non-albicans Candida. This is a continuation of their work on Streptococcus mutans.

This study lacks a decent working hypothesis and it is not apparent what the aim of this study was. The authors did a very similar study with Streptococcus mutans but this does not justify a similar study on non-albicans Candida strains. We have myriads of pathogenic bacteria and fungi and we have very effective antifungals – why this selection of species and why this compound?

The authors should offer some background information on anandamide to help the reader to put this study in a larger context. Anandamide is known for decades for its bioactivity and it is studied for its role as endogenous cannabinoid ligand for chronic pain management or anti-cancer activity. However, anandamide is not without side effects, e.g. neurotoxicity has been reported. How can this compound be regard as potential antifungal? This should be presented in the introduction.

Along these lines: The findings are all confirmation of known bioactivities of anandamide. Furthermore, this compound is a significantly less active compared to commercial antifungals. Both MICs and BMICs are lower for all strains for anandamide than for fluconazole. The authors should give a critical discussion of their findings and be much more realistic in their conclusions.

Check lines 79, 295, there are errors

Reference 13 is only a self-citation but does not contribute to the understanding of the text.

Figure 6 only fig. 6e, f and 6k, l are meaningful. The CLSM figures should go to the suppl. part.

Author Response

Reviewer 1:

 

The manuscript lacks sufficient scientific novelty and technical rigor to merit publication in this journal. The presented results are poorly illustrated, and the conclusions are not adequately supported by the provided data. Furthermore, the statistical analysis is absent or unclear, and the figures appear repetitive without demonstrating significant differences. Given that the manuscript significantly overstates the importance of the findings without providing satisfactory experimental evidence, I recommend rejection in its current form. 

 

Point 1: The results are overall poorly presented. The data do not support the results and discussion. The graphs look the same, and there is no significance ( I am not sure about the stats). The manuscript claims big but does not provide satisfactory data. 

 

Response 1: We thank the reviewer for his critical reading of the manuscript. We have modified the manuscript accordingly. Please find below our response. We believe that by addressing your concerns and comments the manuscript has been upgraded.

In accordance with all the reviewers' comments, the text of the manuscript, including the introduction, results and discussion have been edited to more critically evaluate the findings and the graphs rearranged with the significance more clearly stated to better explain the results found. 

Author Response File: Author Response.docx

Reviewer 2 Report

The results are overall poorly presented. The data do not support the results and discussion. The graphs look the same, and there is no significance ( I am not sure about the stats). The manuscript claims big but does not provide satisfactory data.

NA

Author Response

Point 1: The authors reported on anandamide targeting membrane integrity in non-albicans Candida. This is a continuation of their work on Streptococcus mutans. This study lacks a decent working hypothesis and it is not apparent what the aim of this study was. 

 

Response 1: We have edited the abstract and introduction to more clearly state the working hypothesis and aim of the manuscript:

 

Lines 14-20:  …causing more than half of all clinical cases with many strains gaining resistance to current treatments rapidly. Previously, N-arachidonoyl ethanolamine (anandamide, AEA) has been studied and shown to possess antibacterial and antifungal properties against various bacteria and Candida albicans. Given the previous findings, we aim here to expand the current preliminary research on AEA to investigate its antifungal activities against clinically relevant NAC species: Candida. glabrata, Candida. parapsilosis, and Candidaozyma. auris… 

 

Lines 136-141: We aim to study species-specific differences in their susceptibility following AEA exposure, analyze how the lipid membrane may play a role in AEA’s action on Candida as well as investigate why there are species-specific differences in their susceptibility to AEA. We hypothesized that AEA exhibits antifungal activity against NAC species by disrupting cellular membrane integrity and inducing downstream stress responses that impair fungal viability.

 

Point 2: The authors did a very similar study with Streptococcus mutans but this does not justify a similar study on non-albicans Candida strains. We have myriads of pathogenic bacteria and fungi and we have very effective antifungals – why this selection of species and why this compound?

 

Response 2:  We selected these non-albicans Candida (NAC) species because they are clinically important pathogens whose prevalence and antifungal resistance rates continue to rise. For example, Candida glabrata has become one of the leading causes of candidemia in the US (https://doi.org/10.1093/cid/ciag252), and several NAC species show increasing resistance trends (https://doi.org/10.5812/jjm.101767.) As the epidemiology of Candida shifts, it is reasonable to anticipate that NAC species will play an expanding role not only in invasive disease but also in colonization of mucosal sites, including the oral cavity.

AEA is an endogenous compound with documented antibacterial properties, and our group previously investigated its antifungal effects against Candida albicans. Given the growing clinical relevance of NAC species and the limited therapeutic options for some of them, we aimed to explore whether AEA could exert measurable anti-fungal activity or mechanistic effects on these microorganisms. Although AEA is not fungicidal, we believe that characterizing its mechanism of action is valuable, particularly as a foundation for future studies assessing potential synergistic combinations with established antifungals. 

 

 

Point 3: The authors should offer some background information on anandamide to help the reader to put this study in a larger context. Anandamide is known for decades for its bioactivity and it is studied for its role as endogenous cannabinoid ligand for chronic pain management or anti-cancer activity. However, anandamide is not without side effects, e.g. neurotoxicity has been reported. How can this compound be regard as potential antifungal? This should be presented in the introduction.

 

Response 3: Additional information on AEA has been added to the introduction (lines 107-131). We agree that AEA is a pleiotropic endogenous lipid mediator with diverse biological activities and that its therapeutic application requires careful evaluation. While some studies have reported adverse effects, including neurotoxicity under specific experimental conditions, others have demonstrated neuroprotective effects mediated through CB1 receptor activation, highlighting that the biological effects of anandamide are highly context-, receptor-, and tissue-dependent. The aim of the present study was not to propose AEA as an immediately translatable systemic antifungal therapy, but rather to investigate its antifungal activity and mechanism of action against multidrug-resistant NAC species in vitro. We believe that identifying the antifungal mechanisms of an endogenous lipid mediator may facilitate the future development of safer lipid-derived antifungal agents, structural analogues, or formulations with improved therapeutic indices. We have added a statement to the introduction (lines 129-131) and discussion (line 780) to clarify that further studies evaluating toxicity, pharmacokinetics, and efficacy in vivo are essential before considering clinical applications. 

Point 4: Along these lines: The findings are all confirmation of known bioactivities of anandamide. Furthermore, this compound is a significantly less active compared to commercial antifungals. Both MICs and BMICs are lower for all strains for anandamide than for fluconazole. The authors should give a critical discussion of their findings and be much more realistic in their conclusions.

Response 4: We have edited the results sections as well as the discussion to make it more critical of our findings (lines 780-785).

 

Point 5: Check lines 79, 295, there are errors

 

Response 5: Regarding Lines (Previously 79): The text has been rephrased accordingly: 

Lines 92-93: It is now the second most common cause of candidemia in many regions and is increasingly recognized as a cause of vaginal candidiasis.

 

Regarding Lines (Previously 295): The text has been slightly rephrased: 

Line 341-342: “At 125 µg/mL AEA, C. auris exhibited > 70 % reduction in ATP levels (p<0.001) and in metabolic activity (p<0.001), …

 

The calculation for these figures were calculated accordingly: 

(Value of Control - Value of Sample) / (Value of Control)) *100 = % reduction.

The calculations have been re-checked according to that equation and remain the same as they are written in the text. If there is a different error that we missed we apologize and would appreciate your clarification. The writing has been slightly edited to accommodate the request of all the reviewers comments. The above equation has been added to the methods section for greater clarity (lines 200-201).

 

Point 6: Reference 13 is only a self-citation but does not contribute to the understanding of the text.

 

Response 6: The citation of the manuscript is meant to give more information on the antibacterial effects of AEA tested on Streptococcus mutans previously. The study performed in that manuscript was a metabolomics assay with both untargeted and targeted pathways and reveals mechanistic insights into AEA’s mode of action against S. mutans. It is the first metabolomics study performed on S. mutans and is referenced here to help the reader understand more about AEA and how it works and provide a hypothesis for how it may also affect NAC species.

 

Point 7: Figure 6 only fig. 6e, f and 6k, l are meaningful. The CLSM figures should go to the suppl. Part.

 

Response 7: The CLSM images offer an important component of the main findings, as they provide direct visual support for the quantitative growth inhibition, viability, and dye retention presented in this study. Therefore, we have retained the CLSM images in the main manuscript and improved their resolution and quality (increased DPI) as suggested by the reviewers.

Author Response File: Author Response.docx

Reviewer 3 Report

 

1. The abstract only states AEA disrupts fungal membranes but fails to highlight the core innovation: this is the first direct comparison of AEA’s species-specific activity across three major MDR non-albicans Candida, with distinct differential membrane/oxidative stress responses between strains. Rewrite the final abstract sentence to explicitly emphasize this comparative novelty to distinguish from prior C. albicans-only AEA papers.
2. Add one brief clause noting that in vivo animal efficacy of AEA remains uncharacterized at the end of the conclusion sentence in the abstract.
3. Current keywords miss critical mechanistic terms; revise to Anandamide (AEA), non-albicans Candida, Candida auris, membrane fluidity, efflux pump inhibition, ROS stress; antibiofilm activity, and multidrug resistance. Remove overly broad, vague keywords.

4. The introduction lists separate background blocks (NAC threat, AEA bioactivity) but does not weave a clear unmet research gap. Add a dedicated standalone paragraph summarizing three critical gaps: (1) All prior AEA antifungal work only tested C. albicans, lacking cross-species NAC comparison; (2) No prior study linked AEA’s lipid bilayer disruption to ABC/MFS efflux pump dysfunction in drug-resistant Candida; (3) Species-dependent ROS/membrane susceptibility across clinical NAC clades remains unreported.
5. The intro cites outdated invasive candidiasis statistics; integrate recent 2023–2025 epidemiological references confirming NAC now accounts >60% of hospital candidemia, especially MDR C. auris outbreaks globally.
6. Delete repetitive descriptions of endocannabinoid biosynthesis twice in Section 1; condense AEA structural and biochemical properties into one tight subsection to reduce word bloat.

7. M&M Section 2.3 only briefly references CLSI M27-A3; fully detail standardized incubation temperature, medium pH, inoculum OD adjustment, and endpoint reading criteria to meet fungal susceptibility testing reproducibility standards.
8. Add critical negative/positive control groups missing in the methods: All membrane, ROS, and efflux assays lack positive control compounds (e.g., fluconazole, amphotericin B, and membrane-disrupting melittin). Supplement control treatment concentrations and incubation parameters for every fluorescence-based assay (Laurdan, DiS-C3(3), DCFH-DA, R6G).
9. Section 2.9 presents an incorrect, unlabeled Laurdan GP equation with misassigned wavelength numerators/denominators; replace with the standard Parasassi GP formula cited in membrane lipid literature and add a 1-sentence definition of GP value interpretation (high GP = rigid membrane, low GP = fluidized membrane).
10. The M&M statistical subsection lacks sample size rationale; add G*Power calculation notes for n=3 biological replicates, specify exact statistical software version, and clarify non-parametric vs parametric test selection criteria for Candida CFU and fluorescence data.
11. Different incubation times (12 h vs. 24 h) are randomly used for planktonic vs. biofilm cultures without explanation; standardize pre-culture growth stage (mid-log OD₆₀₀ = 0.6–0.8) across all phenotypic assays and add a cross-reference sentence in 2.2.

12. Every Results subsection only describes single-strain data without cross-comparison logic. Insert opening transition lines for each panel that explicitly contrast responses between C. auris, C. glabrata, and C. glabrata (e.g., “In contrast to C. parapsilosis, C. glabrata exhibited sustained ROS accumulation after AEA exposure”).
13. Currently ROS results (Fig. 5) appear before efflux pump Fig. 7: Reorder results sequence to match mechanistic cascade: growth/MIC → biofilm metabolism → membrane fluidity/potential → efflux inhibition → ROS generation → confocal live/dead imaging. Membrane damage precedes efflux failure and then oxidative stress, which aligns with your proposed mode of action.
14. Many text descriptions state a “marked difference” between strains without p-value markers; add exact statistical comparison notations (ns, *p<0.05, *p<0.01) for all interspecies comparisons in the main text, not only figure captions.
15. The manuscript repeatedly restates identical 64% biofilm reduction percentages across three paragraphs; condense duplicate numerical descriptions and only reference figure panels once per subsection.

16. The discussion currently treats three NAC strains as a single group without explaining why C. glabrata displays prolonged ROS stress while C. auris only transiently accumulates ROS. Create a standalone paragraph linking interspecies membrane lipid composition differences to differential AEFor susceptibility, cite Candida lipidome comparative literature.
17. Expand the efflux pump discussion to connect your R6G data to clinical MDR candidiasis treatment; elaborate on how AEA’s membrane-targeted action avoids the single-gene mutation resistance mechanisms common to azole drugs, and cite recent Cdr1 efflux inhibitor reviews.
18. Current limitations are overly brief, split into two clear subsections: (1) Limitations: lack of in vivo animal infection validation, only laboratory reference strains (no fresh clinical outbreak isolates tested), and single time-point membrane measurements; (2) Future directions: combinatorial AEA + azole antifungal testing, murine systemic candidiasis models, and topical formulation development for skin/hospital surface disinfection.

19. Crop blurry confocal microscopy raw panels and increase image resolution to 300 DPI minimum for J. Fungi requirements.
20. Lines 59-60: Please cite this reference (Khang TN, Binh HT, Dao VTT, Thao LTT and Duy TT, 2025. Antibacterial and antifungal abilities of Tacca (Tacca leontopetaloides) leaf extraction and its application in fresh mango preservation. International Journal of Agriculture and Biosciences 14(4): 556-564. https://doi.org/10.47278/journal.ijab/2025.034) as it aligns with your introduction’s core research gap and lack of natural lipid modulators tested on drug-resistant.
21. Lines 532-533: Please cite this reference (Elzaiat MA, Mandour AS, Youssef MAH, Wafa HA, Aljahdali SM, Shakak AO, Husnain LA, Alqahtani MM, Alghamdi MA, Abuzaid AO, Alqahtani TM, Al Gheffari HK, Bouqellah NA, Heakel RMY, 2024. Biochemical and molecular characterization of five basil cultivars extract for enhancing the antioxidant, antiviral, anticancer, antibacterial, and antifungal activities. Pak Vet J, 44(4): 1105-1119. http://dx.doi.org/10.29261/pakvetj/2024.279) because this article systematically compares multi-cultivar plant leaf extracts, quantifies total phenol/flavonoid phytochemical contents, and verifies broad-spectrum antibacterial + antifungal activity against pathogenic microbes; it also links phytochemical abundance to antimicrobial potency, fully matching the experimental logic of your Tacca leaf extract.
22. Lines 418-423: Please cite this reference (Rodjan P, Kaewnabon Y, Chimplee S, Pongpom M, Tedja I, Mitsuwan W and Jeenkeawpieam J 2025. Antifungal potential and safety evaluation of Thai Piper betle leaf extract and phenolics against animal pathogenic Candida species. Pak Vet J, 45(3): 1168-1178. http://dx.doi.org/10.29261/pakvetj/2025.232) because it establishes direct Southeast Asian tropical plant antifungal literature context, matching your Vietnam-based Tacca research.

Major Comments:

1. The abstract only states AEA disrupts fungal membranes but fails to highlight the core innovation: this is the first direct comparison of AEA’s species-specific activity across three major MDR non-albicans Candida, with distinct differential membrane/oxidative stress responses between strains. Rewrite the final abstract sentence to explicitly emphasize this comparative novelty to distinguish from prior C. albicans-only AEA papers.
2. Add one brief clause noting that in vivo animal efficacy of AEA remains uncharacterized at the end of the conclusion sentence in the abstract.
3. Current keywords miss critical mechanistic terms; revise to Anandamide (AEA), non-albicans Candida, Candida auris, membrane fluidity, efflux pump inhibition, ROS stress; antibiofilm activity, and multidrug resistance. Remove overly broad, vague keywords.

4. The introduction lists separate background blocks (NAC threat, AEA bioactivity) but does not weave a clear unmet research gap. Add a dedicated standalone paragraph summarizing three critical gaps: (1) All prior AEA antifungal work only tested C. albicans, lacking cross-species NAC comparison; (2) No prior study linked AEA’s lipid bilayer disruption to ABC/MFS efflux pump dysfunction in drug-resistant Candida; (3) Species-dependent ROS/membrane susceptibility across clinical NAC clades remains unreported.
5. The intro cites outdated invasive candidiasis statistics; integrate recent 2023–2025 epidemiological references confirming NAC now accounts >60% of hospital candidemia, especially MDR C. auris outbreaks globally.
6. Delete repetitive descriptions of endocannabinoid biosynthesis twice in Section 1; condense AEA structural and biochemical properties into one tight subsection to reduce word bloat.

7. M&M Section 2.3 only briefly references CLSI M27-A3; fully detail standardized incubation temperature, medium pH, inoculum OD adjustment, and endpoint reading criteria to meet fungal susceptibility testing reproducibility standards.
8. Add critical negative/positive control groups missing in the methods: All membrane, ROS, and efflux assays lack positive control compounds (e.g., fluconazole, amphotericin B, and membrane-disrupting melittin). Supplement control treatment concentrations and incubation parameters for every fluorescence-based assay (Laurdan, DiS-C3(3), DCFH-DA, R6G).
9. Section 2.9 presents an incorrect, unlabeled Laurdan GP equation with misassigned wavelength numerators/denominators; replace with the standard Parasassi GP formula cited in membrane lipid literature and add a 1-sentence definition of GP value interpretation (high GP = rigid membrane, low GP = fluidized membrane).
10. The M&M statistical subsection lacks sample size rationale; add G*Power calculation notes for n=3 biological replicates, specify exact statistical software version, and clarify non-parametric vs parametric test selection criteria for Candida CFU and fluorescence data.
11. Different incubation times (12 h vs. 24 h) are randomly used for planktonic vs. biofilm cultures without explanation; standardize pre-culture growth stage (mid-log OD₆₀₀ = 0.6–0.8) across all phenotypic assays and add a cross-reference sentence in 2.2.

12. Every Results subsection only describes single-strain data without cross-comparison logic. Insert opening transition lines for each panel that explicitly contrast responses between C. auris, C. glabrata, and C. glabrata (e.g., “In contrast to C. parapsilosis, C. glabrata exhibited sustained ROS accumulation after AEA exposure”).
13. Currently ROS results (Fig. 5) appear before efflux pump Fig. 7: Reorder results sequence to match mechanistic cascade: growth/MIC → biofilm metabolism → membrane fluidity/potential → efflux inhibition → ROS generation → confocal live/dead imaging. Membrane damage precedes efflux failure and then oxidative stress, which aligns with your proposed mode of action.
14. Many text descriptions state a “marked difference” between strains without p-value markers; add exact statistical comparison notations (ns, *p<0.05, *p<0.01) for all interspecies comparisons in the main text, not only figure captions.
15. The manuscript repeatedly restates identical 64% biofilm reduction percentages across three paragraphs; condense duplicate numerical descriptions and only reference figure panels once per subsection.

16. The discussion currently treats three NAC strains as a single group without explaining why C. glabrata displays prolonged ROS stress while C. auris only transiently accumulates ROS. Create a standalone paragraph linking interspecies membrane lipid composition differences to differential AEFor susceptibility, cite Candida lipidome comparative literature.
17. Expand the efflux pump discussion to connect your R6G data to clinical MDR candidiasis treatment; elaborate on how AEA’s membrane-targeted action avoids the single-gene mutation resistance mechanisms common to azole drugs, and cite recent Cdr1 efflux inhibitor reviews.
18. Current limitations are overly brief, split into two clear subsections: (1) Limitations: lack of in vivo animal infection validation, only laboratory reference strains (no fresh clinical outbreak isolates tested), and single time-point membrane measurements; (2) Future directions: combinatorial AEA + azole antifungal testing, murine systemic candidiasis models, and topical formulation development for skin/hospital surface disinfection.

19. Crop blurry confocal microscopy raw panels and increase image resolution to 300 DPI minimum for J. Fungi requirements.
20. Lines 59-60: Please cite this reference (Khang TN, Binh HT, Dao VTT, Thao LTT and Duy TT, 2025. Antibacterial and antifungal abilities of Tacca (Tacca leontopetaloides) leaf extraction and its application in fresh mango preservation. International Journal of Agriculture and Biosciences 14(4): 556-564. https://doi.org/10.47278/journal.ijab/2025.034) as it aligns with your introduction’s core research gap and lack of natural lipid modulators tested on drug-resistant.
21. Lines 532-533: Please cite this reference (Elzaiat MA, Mandour AS, Youssef MAH, Wafa HA, Aljahdali SM, Shakak AO, Husnain LA, Alqahtani MM, Alghamdi MA, Abuzaid AO, Alqahtani TM, Al Gheffari HK, Bouqellah NA, Heakel RMY, 2024. Biochemical and molecular characterization of five basil cultivars extract for enhancing the antioxidant, antiviral, anticancer, antibacterial, and antifungal activities. Pak Vet J, 44(4): 1105-1119. http://dx.doi.org/10.29261/pakvetj/2024.279) because this article systematically compares multi-cultivar plant leaf extracts, quantifies total phenol/flavonoid phytochemical contents, and verifies broad-spectrum antibacterial + antifungal activity against pathogenic microbes; it also links phytochemical abundance to antimicrobial potency, fully matching the experimental logic of your Tacca leaf extract.
22. Lines 418-423: Please cite this reference (Rodjan P, Kaewnabon Y, Chimplee S, Pongpom M, Tedja I, Mitsuwan W and Jeenkeawpieam J 2025. Antifungal potential and safety evaluation of Thai Piper betle leaf extract and phenolics against animal pathogenic Candida species. Pak Vet J, 45(3): 1168-1178. http://dx.doi.org/10.29261/pakvetj/2025.232) because it establishes direct Southeast Asian tropical plant antifungal literature context, matching your Vietnam-based Tacca research.

Comments for author File: Comments.pdf

Author Response

Point 1: The abstract only states AEA disrupts fungal membranes but fails to highlight the core innovation: this is the first direct comparison of AEA’s species-specific activity across three major MDR non-albicans Candida, with distinct differential membrane/oxidative stress responses between strains. Rewrite the final abstract sentence to explicitly emphasize this comparative novelty to distinguish from prior C. albicans-only AEA papers.

Response 1: The last lines of the abstract has been edited to reflect the reviewers suggestions: 

 

Lines 33-36: Together, these findings demonstrate that AEA exerts antifungal activity by disrupting membrane integrity and associated cellular functions and provide the first comparative characterization of species-specific membrane and oxidative stress responses to AEA across three major clinically relevant multidrug-resistant NAC species. 

 

Point 2: Add one brief clause noting that in vivo animal efficacy of AEA remains uncharacterized at the end of the conclusion sentence in the abstract.

Response 2: The following have been added to the abstract and discussion: 

Lines 21: … and Candidaozyma auris in vitro. 

Line 780: This study has several limitations, including the lack of in vivo testing…

Point 3: Current keywords miss critical mechanistic terms; revise to Anandamide (AEA), non-albicans Candida, Candida auris, membrane fluidity, efflux pump inhibition, ROS stress; antibiofilm activity, and multidrug resistance. Remove overly broad, vague keywords.

 

Response 3: The current keywords have been edited to reflect the reviewer’s suggestions:

Lines 40-43: Anandamide (AEA); non-albicans Candida (NAC) species; Candidozyma auris; Candida glabrata; Candida parapsilosis; membrane fluidity, efflux pump inhibition, ROS stress, antibiofilm activity, and multidrug resistance 

Point 4: The introduction lists separate background blocks (NAC threat, AEA bioactivity) but does not weave a clear unmet research gap. Add a dedicated standalone paragraph summarizing three critical gaps: (1) All prior AEA antifungal work only tested C. albicans, lacking cross-species NAC comparison; (2) No prior study linked AEA’s lipid bilayer disruption to ABC/MFS efflux pump dysfunction in drug-resistant Candida; (3) Species-dependent ROS/membrane susceptibility across clinical NAC clades remains unreported.

 

Response 4: We have added more of these points to the introduction as per the reviewer’s suggestions:

 

Lines 124-126: However, these findings are limited to a single C. albicans strain [20], leaving the activity of AEA against other clinically important NAC species largely unexplored.

 

Lines 135-139: this study investigates the anti-fungal activity of AEA against the three pathogenic NAC species described above. We aim to study species-specific differences in their susceptibility following AEA exposure, analyze how the lipid membrane may play a role in AEA’s action on Candida as well as investigate why there are species-specific differences in their susceptibility to AEA.

Point 5: The intro cites outdated invasive candidiasis statistics; integrate recent 2023–2025 epidemiological references confirming NAC now accounts >60% of hospital candidemia, especially MDR C. auris outbreaks globally.

 

Response 5: We have edited the introduction to reflect more recently published data and numbers regarding candidiasis.

 

Lines 54-55: …Candida glabrata, Candida parapsilosis and Candidozyma auris, which now account for more than 60% of all cases...

 

We selected these non-albicans Candida (NAC) species because they are clinically important pathogens whose prevalence and antifungal resistance rates continue to rise. For example, Candida glabrata has become one of the leading causes of candidemia in the US (https://doi.org/10.1093/cid/ciag252), and several NAC species show increasing resistance trends (https://doi.org/10.5812/jjm.101767.) As the epidemiology of Candida shifts, it is reasonable to anticipate that NAC species will play an expanding role not only in invasive disease but also in colonization of mucosal sites, including the oral cavity.

 

Regarding C. auris, the following has been added to the introduction as per the reviewer's suggestions:

 

Lines 74-89: 

Among the NAC species, C. auris has emerged as a major global concern since its first isolation from a bloodstream infection in South Korea in 1996[7]. It is now recognized as a healthcare-associated pathogen, causing hospital outbreaks and life-threatening infections worldwide[13]. Furthermore, it is difficult to identify, exhibits multidrug resistance, spreads rapidly in clinical settings, and persists on skin and environmental surfaces[7]. It is increasingly documented in bloodstream infections, with higher rates of reoccurrence, and treatment options unfortunately becoming more convoluted due to the growing number of multidrug resistant strains[13]. Six clades have been identified, with clade I, III and IV responsible for most of the outbreaks worldwide[7]. Mortality rates for C. auris candidemia are approximately 30%, though they vary by region, patient demographics and underlying conditions[7]. Resistance patterns differ among clades, but high levels of azole resistance are common[7], and individuals with prolonged hospitalizations, invasive procedures, or extensive antimicrobial exposure are at the greatest risk[7]. Despite targeted decolonization efforts, C. auris has proven exceptionally difficult to eradicate, leaving affected patients at an even higher risk for developing additional infections [13].

 

Point 6: Delete repetitive descriptions of endocannabinoid biosynthesis twice in Section 1; condense AEA structural and biochemical properties into one tight subsection to reduce word bloat.

Response 6: We have edited the paragraph on AEA to reflect the reviewer's suggestions:

 

Lines 106-120: Anandamide as a potential antifungal agent

Endocannabinoids are endogenous signaling molecules with diverse physiological functions including neuromodulatory, immunomodulatory, anti-inflammatory, and anti-oxidant activities [14]. Among them, N-arachidonoylethanolamine (anandamide; AEA),first identified by Mechoulam and colleagues in the 1990’s [15], has recently attracted attention for its antimicrobial properties against a range of bacterial pathogens[14,16 17]. Previously, our group demonstrated that AEA disrupts bacterial membrane integrity, resulting in membrane hyperpolarization, increased permeability, growth inhibition, altered membrane structure and rigidity, inhibition of drug efflux and impaired glucose uptake into the cell of Streptococcus mutans and Staphylococcus aureus [16,18,19].

Point 7: M&M Section 2.3 only briefly references CLSI M27-A3; fully detail standardized incubation temperature, medium pH, inoculum OD adjustment, and endpoint reading criteria to meet fungal susceptibility testing reproducibility standards.

Response 7: As per the reviewer's suggestions we have expanded the Methods section to include a detailed description of Section 2.3:

 

Lines 185-191: Briefly, yeast was cultured on SDA plates at 35 °C for 24-48 h. After which, colonies were taken from SDA plates and suspended in PBS medium and adjusted to McFarland standard 0.5. Then cells were resuspended in RPMI-1640 medium buffered with MOPS at pH 7.0 to 1:1000 dilution, seeded in sterile transparent U-bottom 96-well microplates (Sterilin^TM, Thermo Fisher Scientific, Loughborough, UK) and incubated in the presence or absence of AEA for 24 h at 35 °C. After incubation, growth was visualized in a microscope to determine the MIC and MBIC values. 

Point 8: Add critical negative/positive control groups missing in the methods: All membrane, ROS, and efflux assays lack positive control compounds (e.g., fluconazole, amphotericin B, and membrane-disrupting melittin). Supplement control treatment concentrations and incubation parameters for every fluorescence-based assay (Laurdan, DiS-C3(3), DCFH-DA, R6G).

Response 8: The following was added to the methods section for simplicity and to reduce redundancy: 

 

Lines 148-150: Ethanol (0.025%, v/v), fluconazole and untreated Candida cells were used as experimental controls in all experiments unless noted otherwise. 

 

All assays except for the efflux assay included ethanol and fluconazole as controls. The efflux assay was very time sensitive and the addition of another control risked missing critical measurement time points so we decided it should be left out to retain the accuracy of the experiment and the results.

With regards to the fluorescence-based assays, the treatment concentrations and incubation parameters were re-checked and should all be listed under their respective methods. The methods section for the membrane permeability assay, which was missing incubation parameters, has been edited to include this detail:

 

Line 250-253: Following incubation, all samples were adjusted to an OD600nm of 0.2, washed twice with PBS, and resuspended in 1mL of room temperature 10 µM DiS-C3(3) (diluted in PBS) where they were then read immediately. 

 

 

Point 9: Section 2.9 presents an incorrect, unlabeled Laurdan GP equation with misassigned wavelength numerators/denominators; replace with the standard Parasassi GP formula cited in membrane lipid literature and add a 1-sentence definition of GP value interpretation (high GP = rigid membrane, low GP = fluidized membrane).

Response 9: The following methods section has been edited to reflect the reviewer's suggestions and the results section updated to reflect these values:

 

Lines 276-279: 

 

GP = (I440- I490) / (I440- + I490),

(2)

where I440 and I490 are the fluorescence intensities at 440 and 490 nm, respectively. A higher GP value denotes a more rigid membrane while a lower GP value suggests a more fluid one (19).

Point 10: The M&M statistical subsection lacks sample size rationale; add G*Power calculation notes for n=3 biological replicates, specify exact statistical software version, and clarify non-parametric vs parametric test selection criteria for Candida CFU and fluorescence data.

 

Response 10: We did not perform any priori power analysis. Each experiment was performed using three independent biological replicates, each measured in technical triplicate where applicable, consistent with standard practice for in vitro microbiological studies. We have clarified the statistical software used in section 2.12. Differences between two groups were evaluated using a two-tailed unpaired Student's t-test. A P value < 0.05 was considered statistically significant. The manuscript does not include CFU assays. Statistical analysis was performed using an unpaired two-tailed Student's t-test for comparisons between two groups, and the Methods have been revised to specify the software used, the number of biological replicates, and the statistical test applied:

 

Lines 306-313: All experiments were performed using three independent biological replicates. Where applicable, measurements were performed in technical triplicates. Data are presented as a mean ± standard deviation. Statistical analyses were performed using the Student’s t-test in Microsoft Excel for Microsoft 365 (Version 2108, Microsoft Corp., Redmond, WA, USA). Comparisons between groups were performed using a two-tailed unpaired Student’s t-test, with a p-value of less than 0.05 considered significant when comparing treated versus control samples.

Point 11: Different incubation times (12 h vs. 24 h) are randomly used for planktonic vs. biofilm cultures without explanation; standardize pre-culture growth stage (mid-log OD₆₀₀ = 0.6–0.8) across all phenotypic assays and add a cross-reference sentence in 2.2.

Response 11: For all experiments, samples were incubated for 24 h with the exception of the ROS assay (Fig. 6) and efflux assay (Fig. 5) where the cells were incubated for 30 minutes and then assessed for the first hour(s) after treatment to investigate the immediate effects of AEA. 

 

Regarding standardizing growth stage - in our study, all phenotypic assays were performed using cells harvested after 24 h of growth and standardized to an OD₆₀₀ of 0.15–0.2 before each assay. We have clarified this standardized inoculum preparation in the Methods (lines 168-171). And we have added a cross-reference sentence in 2.2 lines (178-181) which can be seen reflected in the methods section.

 

Lines 168-171: All phenotypic assays were performed using cells harvested from the same standardized 24-h culture, washed twice with sterile PBS and resuspended in RPMI-1640 (L-glutamine, MOPS-buffered) and resuspended to OD₆₀₀ = 0.15 - 0.2 prior to experimentation. [15]. 

 

Lines 178-181: Cell preparation was performed as described in Section 2.1 before all phenotypic assays. 

 

Point 12: Every Results subsection only describes single-strain data without cross-comparison logic. Insert opening transition lines for each panel that explicitly contrast responses between C. auris, C. glabrata, and C. glabrata (e.g., “In contrast to C. parapsilosis, C. glabrata exhibited sustained ROS accumulation after AEA exposure”).

Response 12: The text has been modified to include better comparison and transition statements in the results section.

 

Point 13: Currently ROS results (Fig. 5) appear before efflux pump Fig. 7: Reorder results sequence to match mechanistic cascade: growth/MIC → biofilm metabolism → membrane fluidity/potential → efflux inhibition → ROS generation → confocal live/dead imaging. Membrane damage precedes efflux failure and then oxidative stress, which aligns with your proposed mode of action.

Response 13: The results have been reordered to reflect the reviewer's suggestion.

 

Point 14: Many text descriptions state a “marked difference” between strains without p-value markers; add exact statistical comparison notations (ns, *p<0.05, *p<0.01) for all interspecies comparisons in the main text, not only figure captions.

Response 14: We have added statistical notations to the results section as per the reviewer's request.

Point 15: The manuscript repeatedly restates identical 64% biofilm reduction percentages across three paragraphs; condense duplicate numerical descriptions and only reference figure panels once per subsection.

 

Response 15: We have condensed the results to reflect the reviewer's request. The reduction percentages have been decreased so as to not repeat identical percentages but we do find stating their values important for transparency of the manuscript. 

Point 16: The discussion currently treats three NAC strains as a single group without explaining why C. glabrata displays prolonged ROS stress while C. auris only transiently accumulates ROS. Create a standalone paragraph linking interspecies membrane lipid composition differences to differential AEA susceptibility, cite Candida lipidome comparative literature.

 

Response 16: The discussion has been edited so that there is now more explanation for the differences observed in ROS stress between the three NAC species (lines 748-769), as well as more explanation and discussion on membrane lipid differences and how this may affect their susceptibility to AEA (lines 601-613). However, as ROS is important feature in the life cycle of the yeast, much further investigations are needed to clear the mode of action and the reason for this observation.

 

Point 17: Expand the efflux pump discussion to connect your R6G data to clinical MDR candidiasis treatment; elaborate on how AEA’s membrane-targeted action avoids the single-gene mutation resistance mechanisms common to azole drugs,and cite recent Cdr1 efflux inhibitor reviews.

 

Response 16: The discussion has been edited to discuss in greater depth the various topics you have suggested with citations to recent studies about efflux inhibitors. These additions can be found from lines 669-679. Translation research remains very limited so the discussion on this remains more minimal.  

 

Lines 669-679: Targeting efflux pumps is becoming an increasingly popular area of research in NAC species. El-Ganiny et al. [54] demonstrated that Pantoprazole and haloperidole treated against various NAC species were able to chemosensitize fluconazole- resistant the isolates by counteracting overexpressed CDR1, MDR1, and ABC2 efflux genes, resulting in increased intracellular drug accumulation [54]. Despite many recent studies utilizing and repurposing drugs as efflux pump inhibitors [55–58], the clinical translation of this remains limited. Unlike azoles, which primarily target Erg11 and are susceptible to resistance arising from mutations or altered expression of a single gene [59,60], AEA’s disruption of the membrane integrity and associated membrane functions may be considered less vulnerable to resistance mediated by single-gene mutations because they affect multiple interconnected cellular processes simultaneously.

 

 

Point 18: Current limitations are overly brief, split into two clear subsections: (1) Limitations: lack of in vivo animal infection validation, only laboratory reference strains (no fresh clinical outbreak isolates tested), and single time-point membrane measurements; (2) Future directions: combinatorial AEA + azole antifungal testing, murine systemic candidiasis models, and topical formulation development for skin/hospital surface disinfection.

Response 18: The discussion has been revised to include a subsection discussing the limitations of the research according to the reviewers suggestions:

 

Lines 780-785: This study has several limitations, including the lack of in vivo testing, the use of laboratory reference strains rather than recent clinical outbreak isolates, and restriction to specific time points. Although AEA–fluconazole combination testing was performed and demonstrated an indifferent interaction (data not shown), future studies should further explore AEA combinations with additional antifungal agents and investigate their therapeutic potential in relevant infection models.

Point 19: Crop blurry confocal microscopy raw panels and increase image resolution to 300 DPI minimum for J. Fungi requirements.

 

Response 19: The images have been adjusted to an image resolution of 300 DPI. 

Point 20: Lines 59-60: Please cite this reference (Khang TN, Binh HT, Dao VTT, Thao LTT and Duy TT, 2025. Antibacterial and antifungal abilities of Tacca (Tacca leontopetaloides) leaf extraction and its application in fresh mango preservation. International Journal of Agriculture and Biosciences 14(4): 556-564. https://doi.org/10.47278/journal.ijab/2025.034) as it aligns with your introduction’s core research gap and lack of natural lipid modulators tested on drug-resistant.

 

Response 20: We have carefully evaluated the cited study and acknowledge its contribution to the broader field of natural antimicrobial agents. However, this study investigates solvent-derived Tacca leontopetaloides leaf extracts against Aspergillus niger and does not examine a defined lipid-based compound, membrane-targeting mechanism, or drug-resistant fungal pathogens. Therefore, we do not believe that this reference directly supports the specific research gap discussed in our Introduction regarding lipid-derived antifungal strategies against resistant Candida species. To maintain the focus and accuracy of the Introduction, we have not incorporated this reference. 

 

Point 21: Lines 532-533: Please cite this reference (Elzaiat MA, Mandour AS, Youssef MAH, Wafa HA, Aljahdali SM, Shakak AO, Husnain LA, Alqahtani MM, Alghamdi MA, Abuzaid AO, Alqahtani TM, Al Gheffari HK, Bouqellah NA, Heakel RMY, 2024. Biochemical and molecular characterization of five basil cultivars extract for enhancing the antioxidant, antiviral, anticancer, antibacterial, and antifungal activities. Pak Vet J, 44(4): 1105-1119. http://dx.doi.org/10.29261/pakvetj/2024.279) because this article systematically compares multi-cultivar plant leaf extracts, quantifies total phenol/flavonoid phytochemical contents, and verifies broad-spectrum antibacterial + antifungal activity against pathogenic microbes; it also links phytochemical abundance to antimicrobial potency, fully matching the experimental logic of your Tacca leaf extract.

Response 21: We thank the reviewer for this suggestion. We carefully evaluated the recommended reference. While the study provides valuable information on antioxidant, antiviral, anticancer, and antimicrobial activities of various basil cultivars, we believe it is not directly relevant to the focus of the present work, as it does not investigate Candida species, anandamide or related lipid-derived compounds, or mechanisms of antifungal activity. To maintain a focused and relevant Introduction, we have not included this citation.

 

Point 22: Lines 418-423: Please cite this reference (Rodjan P, Kaewnabon Y, Chimplee S, Pongpom M, Tedja I, Mitsuwan W and Jeenkeawpieam J 2025. Antifungal potential and safety evaluation of Thai Piper betle leaf extract and phenolics against animal pathogenic Candida species. Pak Vet J, 45(3): 1168-1178. http://dx.doi.org/10.29261/pakvetj/2025.232) because it establishes direct Southeast Asian tropical plant antifungal literature context, matching your Vietnam-based Tacca research.

Response 22:

We thank the reviewer for this suggestion. We carefully evaluated the recommended reference and have included a citation for it in our discussion.

 

Lines 574-579: The variability in responses between strains can be attributed, at least in part, to differences in plasma membrane structure and composition, in addition to other contributing factors. In fact, species-specific susceptibility among NAC species is not unusual, it has also been reported in response to other antifungal agents, including Thai Piper betle leaf extract, and has been attributed to differences in membrane composition, efflux pump activity, and metabolic adaptability [40].

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors made substantial changes and improved the overall manuscript, and now it is ready to be accepted for publication.

The authors made substantial changes and improved the overall manuscript, and now it is ready to be accepted for publication.