Antifungal Activity of Four Medium-Chain Fatty Acids and γ-Undecalactone Against Candida albicans
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the Current Issues in Molecular Biology, theoretical, scientific and instrumental foundations are required to obtain a reasonable explanation of the phenomena that occur in complex systems, to obtain a conclusive response to the research work.
Minor revisions:
- Change the title, it's very simple.
- Add important numerical values of your work in the abstract.
- Highlights the limitations of the work.
- The results and discussion require further comparison and justification of the scientific contribution.
- The conclusion they are the statements derived from the results and the discussion; they should not exceed the scope of the results and discussion, nor be a summary thereof. Care must be taken to ensure that the conclusions are fully consistent with the objectives set out at the beginning of the study. This section should only include facts proven in the study, so they should not contain hypotheses or speculation, nor should recommendations be made to guide future work.
- Add Pearson correlation coefficients or orthogonal contrasts with respect to the answer with the greatest weight in the research work.
The English could be improved to more clearly express the research.
Author Response
Reviewer 1
Thank you for your useful comment. According your comments, we revised in red letters described below.
Change the title, it's very simple.
We changed the title to “Antifungal activity of undecanoic acid and undecalactone against Candida albcans.”
Add important numerical values of your work in the abstract.
Thank you for your useful comment. According your comments, we added the sentence described below in Abstract.
Page 1, Line 29-31
The equation derived by multiple regression analysis revealed that the logS value was significantly related to the MIC of fatty acids containing 8 to 11 carbon atoms (R=0.95, p<0.01).
The results and discussion require further comparison and justification of the scientific contribution.
The conclusion they are the statements derived from the results and the discussion; they should not exceed the scope of the results and discussion, nor be a summary thereof. Care must be taken to ensure that the conclusions are fully consistent with the objectives set out at the beginning of the study. This section should only include facts proven in the study, so they should not contain hypotheses or speculation, nor should recommendations be made to guide future work.
Thank you for your useful comment. According your comments, we revised the sentences in Result, Disccusion and Conclusion section. The revised parts are written in red in the manuscript.
Add Pearson correlation coefficients or orthogonal contrasts with respect to the answer with the greatest weight in the research work.
Thank you for your comment. According your comment, we added the sentences in Discussion section described below.
Page 9, Line 316-323
A strong regression is considered to exist when the partial regression coefficient R of the multiple regression analysis is 0.7 or higher. The obtained multiple regression equa-tion (partial correlation coefficient was 0.95) suggested that it was possible to predict the MICs of Compound (a) to (f) using the log S value. A strong correlation is considered to exist when the Pearson correlation coefficient is between 0.7 and 1.0. The Pearson correla-tion coefficient between theobserved and predicted MICs was 0.95, suggesting that the multiple regression equation for predicting MICs derived by multiple regression analysis can predict the observed MICs of Compound (a) to (f).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
The manuscript addresses an interesting and potentially relevant topic: the evaluation of the anticandidal activity of. The idea of testing medium chain lactones as antifungals is attractive. However, the manuscript requires revisions before it can be considered for publication
Abstract needs clarification in several parts and language revision (re-check the construction of sentences)
For example, written part:
“Our results demonstrate that γ-undecalactone has potent antifungal activity against C. albicans in the tested com-pounds, induced perforations on the fungal surface, and killed and deactivated C. albicans via the outflow of cell content without hemodialysis as cytotoxicity”
Should be corrected to
“Our results show that γ-undecalactone has a potent antifungal effect against C. albicans in the tested compounds, as it induces perforations on the fungal surface, and kills and inactivates C. albicans through efflux of cellular contents without hemodialysis as cytotoxicity.”
Lines 22-23 change the presentation style. Was written
(Octanoic acid; 1.73±0.00 mM, Nonanoic acid; 1.58±0.00 mM
Change to
(octanoic acid, 1.73±0.00 mM; nonanoic acid, 1.58±0.00 mM;
Keyword list: why don’t you reverse the order for the two firstly mentioned, and edit like medium chain fatty acids and medium chain fatty acid lactones?
Line 83-84 “Unfortunately, macrolide antibiotics that contain a lactone ring are not suitable for antifungal use” revise the sentence considering info that mostly used antifungals such as nystatin and amphotericin B are polyene macrolide antibiotics (look for the literature source, eg Brautaset T, Sletta H, Degnes KF, Sekurova ON, Bakke I, Volokhan O, Andreassen T, Ellingsen TE, Zotchev SB. New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei. Appl Environ Microbiol. 2011 Sep;77(18):6636-43. doi: 10.1128/AEM.05780-11).
From the abstract, keywords and introduction, the reader gets the impression that the number of lactones/fluorinated fatty acids examined is larger, but when one gets to the Materials and Methods section, one realizes that there are only one/two... a bit of disappointing.. Please be specific, and correct this ambiguity by mentioning these compounds by name.
From section 2.1 exclude word chemical, refer to the corresponding compound by its letter designation, for example (a) or (d)..
Apart from necessity of language polishing (unclear) you have a severe methodology fault. It is a well known fact is that DMSO in concentrations above 10% affects microbial growth, there is no need to confirm this in tests again! My suggestion is to leave out the parts where DMSO concentrations were higher than 10% (15 and 30%). If you had any particular difficulty with solubility, you should have mentioned that, and also explained what you used to overcome the situation.
The method used for testing MIC was not stated. What methodology did you use to determine the MIC?
line 107 italicize C. albicans
Lines 138, 226 Marvinsketch convert to MarvinSketch
Author Response
Reviewer 2
Thank you for your useful comment. According your comments, we revised in red letters described below.
“Our results demonstrate that γ-undecalactone has potent antifungal activity against C. albicans in the tested com-pounds, induced perforations on the fungal surface, and killed and deactivated C. albicans via the outflow of cell content without hemodialysis as cytotoxicity”
Should be corrected to
“Our results show that γ-undecalactone has a potent antifungal effect against C. albicans in the tested compounds, as it induces perforations on the fungal surface, and kills and deactivates C. albicans through efflux of cellular contents without hemodialysis as cytotoxicity.”
Thank you for your comment. According your comment, we changed described below.
Page 1, Line 34-37
Our results show that γ-undecalactone has a potent antifungal effect against C. albicans in the tested compounds, as it induces perforations on the fungal surface, and kills and deactivates C. albicans through efflux of cellular contents without hemodialysis as cytotoxicity.
Lines 22-23 change the presentation style. Was written
(Octanoic acid; 1.73±0.00 mM, Nonanoic acid; 1.58±0.00 mM
Change to
(octanoic acid, 1.73±0.00 mM; nonanoic acid, 1.58±0.00 mM;
Thank you for your comment. According your comment, we changed described below.
Page 1, Line 24-28
(octanoic acid, 1.73±0.00 mM; nonanoic acid, 1.58±0.00 mM; decanoic acid, 0.73±0.00mM; undecanoic acid, 0.45±0.19 mM), whereas the antifungal activity of medium-chain fatty acid lactones, in which the carboxyl group of the medium-chain fatty acid was converted to a lactone ring, was greatly enhanced(γ-undecalactone, 0.15±0.00mM; δ-undecalactone, 0.30±0.00 mM).
Keyword list: why don’t you reverse the order for the two firstly mentioned, and edit like medium chain fatty acids and medium chain fatty acid lactones?
Thank you for your comment. According your comment, we changed described below.
Page 1, Line 38, 39
Keywords: undecanoic acid; undecalactone; antifungal activity; Candida albicans; minimum inhibitory concentration.
Line 83-84 “Unfortunately, macrolide antibiotics that contain a lactone ring are not suitable for antifungal use” revise the sentence considering info that mostly used antifungals such as nystatin and amphotericin B are polyene macrolide antibiotics (look for the literature source, eg Brautaset T, Sletta H, Degnes KF, Sekurova ON, Bakke I, Volokhan O, Andreassen T, Ellingsen TE, Zotchev SB. New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei. Appl Environ Microbiol. 2011 Sep;77(18):6636-43. doi: 10.1128/AEM.05780-11).
Thank you for your comment. According your comment, we changed described below and added two references.
Page 2, Line 85-87
The antifungal agents commonly used are polyene macrolide antibiotics such as nystatin and amphotericin B [25, 26].
- Brautaset, T.; Sletta, H.; Degnes, K.F.; Sekurova, O.N.; Bakke, I.; Volokhan, O.; Andreassen, T.; Ellingsen, T.E.; Zotchev, S.B.; New nystatin-related antifungal polyene macrolides with modified polyol domains generated by biosynthetic engineering of Streptomyces noursei. Appl Environ Microbiol. 2011, 77, 6636-6643. https://doi.org/10.1128/AEM.05780-11
- Akinosoglou, K.; Rigopoulos, E.A.; Papageorgiou, D.; Schinas, G.; Polyzou, E.; Dimopoulou, E.; Gogos, C.; Dimopoulos, G. Amphotericin B in the Era of New Antifungals: Where Will It Stand? 2024, 10, 278. https://doi.org/10.3390/jof10040278.
From the abstract, keywords and introduction, the reader gets the impression that the number of lactones/fluorinated fatty acids examined is larger, but when one gets to the Materials and Methods section, one realizes that there are only one/two... a bit of disappointing.. Please be specific, and correct this ambiguity by mentioning these compounds by name.
Thank you for your comment. According your comment, we revised described below.
Page 1, Line 20, 21, Page 3, Line 14, Page 11, Line 420
medium chain fatty acid with 8 to 11 carbon atoms
From section 2.1 exclude word chemical, refer to the corresponding compound by its letter designation, for example (a) or (d)..
Thank you for your comment. According your comment, we excluded word chemical described below. For SEM and TEM results, we left "Chemical" as it should not be confused with the Fig number.
Page 3, Line 115-119
Medium chain fatty acids (octanoic acid, nonanoic acid, decanoic acid, and undecanoic acid)((a) to (d)), γ-undecalactone (e), and δ-undecalactone (f) were purchased from Tokyo chemical industry Co., Ltd. (Tokyo, Japan). 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoroundecanoic acid (g) was purchased from Merck KGaA (Darmstadt, Germany).
Apart from necessity of language polishing (unclear) you have a severe methodology fault. It is a well known fact is that DMSO in concentrations above 10% affects microbial growth, there is no need to confirm this in tests again! My suggestion is to leave out the parts where DMSO concentrations were higher than 10% (15 and 30%). If you had any particular difficulty with solubility, you should have mentioned that, and also explained what you used to overcome the situation.
Thank you for your comment. According your comment, we left out the parts where DMSO concentrations were 15 and 30% described below.
Page 3, Line 130
The final concentration of DMSO were 0.02, 0.03, 0.05, 0.1, 0.2, 0.5, 0.9, 1.9, 3.8 and 7.5 %.
The method used for testing MIC was not stated. What methodology did you use to determine the MIC?
Thank you for your comment. According your comment, we changed described below.
Page 3, Line 123-138
2.2. Determination of Minimum Inhibitory Concentrations (MICs)
The antibacterial activity of (a) to (g) was characterised by determining its MICs which standardized by Clinical Laboratory Standard Institute, CLSI. MIC was determined as described previously [36-39] with minor modifications. The C. albicans strain was di-luted to 4 × 104 CFU/mL in yeast and mold broth at pH 6.2. The plates were incubated at 27°C for 48 h after dispersing 150 µL of the culture into a 96-well microtiter plate well (6.0 × 103 CFU/well). Sample solutions diluted by DMSO were prepared and yeast and mold broth contained yeast and maltose. The final concentration of DMSO were 0.02, 0.03, 0.05, 0.1, 0.2, 0.5, 0.9, 1.9, 3.8 and 7.5 %. Although not shown in the data, it was confirmed that a DMSO content of 10% or less had no effect on yeast growth. The final concentrations of (a) to (d) in solution were 0.97, 1.95, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 μg/mL. The final concentrations of Compound(e) and (f) in solution were 1.75, 3.51, 7.03, 14.1, 28.1, 56.3, 112.5, 225, 450, and 900 μg/mL. The final concentrations of (g) in solution were 2.93, 5.86, 11.7, 23.4, 46.9, 93.8, 187.5, 375, 750 and 1500 μg/mL. Each analysis was performed in triplicate. The MIC was determined as the minimum compound concentration at which the well was clear, and indicated that no bacterial growth had occurred.
line 107 italicize C. albicans
Thank you for your comment. According your comment, we changed described below.
Page 3, Line 35
- albicans in the tested compounds
Lines 138, 226 Marvinsketch convert to MarvinSketch
Thank you for your comment. According your comment, we changed described below.
Page 1, Line 141
The physicochemical parameters of medium chain fatty acids and medium chain fatty acid lactones were derived from MarvinSketch (ChemAxon Ltd.).
Thank you for your comment. According your comment, we changed described below.
Page 4, Line 223
The following physicochemical properties of the seven compounds (four medium chain fatty acids, two lactones, and partially fluorinated fatty acid), except for the molecular weight (MW), were obtained by MarvinSketch:
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript is devoted to the study of antifungal activity of medium chain fatty acid, their lactones and partially fluorinated derivative. It was shown that the antifungal activity of fatty acid with chain length C8-C11 is increased with the increasing chain length. The lactones C11 are the most active while the partially fluorinated derivative is the less efficient.
However, the manuscript requires language correction. It contains many repeats and unnecessary explanations. This spoils the impression of the presented research. For example, paragraph of Results (lines 205-220) contains many repeats. The sentence “The MIC became small, exhibiting an increase in antifungal activity.” (line 210) can be deleted. Instead of “medium chain fatty acids”, studied fatty acids or indicated fatty acids can be used. In Discussion, the repeat is “In this study, we used racemic undecalactone. Lactones tested in this study was a racemic mixture.” (lines 317,318). Besides, Lactones …were racemic mixtures.
The nonaic acid is incorrect title (the legend for Fig. 1). The nonanoic acid should be used.
Lines 346,347: “Although not listed in Table 1, heptadecafluorononanoic acid also had no antifungal activity (MIC; 450μg/mL).” Heptadecafluorononanoic acid is listed in Table 1 (Chemical g), and its antifungal activity (MIC in mM), apparently, is indicated incorrect (0.91±0.00). In Abstract its MIC = 3.05±0.00 mM.
What is the correlation coefficient between the predicted values obtained by multiple regression analysis and the actual observed values? R = 0.95 (lines 234, 238) or 0.96 (line 245).
Has the method for MICs determination been described anywhere? It will be desirable to give reference.
Author Response
Reviewer 3
Thank you for your useful comment. According your comments, we revised in red letters described below.
It contains many repeats and unnecessary explanations. This spoils the impression of the presented research. For example, paragraph of Results (lines 205-220) contains many repeats. The sentence “The MIC became small, exhibiting an increase in antifungal activity.” (line 210) can be deleted. Instead of “medium chain fatty acids”, studied fatty acids or indicated fatty acids can be used. In Discussion, the repeat is “In this study, we used racemic undecalactone. Lactones tested in this study was a racemic mixture.” (lines 317,318). Besides, Lactones …were racemic mixtures.
Thank you for your comment. According your comment, we deleted “The MIC became small, exhibiting an increase in antifungal activity” and changed described below.
Page 9, Line 313,314
Besides, lactones which used as food additives and quasi-drugs were racemic mixtures.
“medium chain fatty acids”, studied fatty acids or indicated fatty acids can be used.
The nonaic acid is incorrect title (the legend for Fig. 1). The nonanoic acid should be used.
Thank you for your comment. According your comment, we changed described below.
Page 5, Line 199, 202
Figure 1. Chemical structures of medium chain fatty acids (a) Octanoic acid, (b) nonanoic acid, (c) decanoic acid, (d) undecanoic acid, lactones (e) γ-undecalactone, (f) σ-undecalactone, and partially fluorinated fatty acids (g) 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoroundecanoic acid.
Chemical structures of medium chain fatty acids (a) Octanoic acid, (b) nonanoic acid,
Lines 346,347: “Although not listed in Table 1, heptadecafluorononanoic acid also had no antifungal activity (MIC; 450μg/mL).” Heptadecafluorononanoic acid is listed in Table 1 (Chemical g), and its antifungal activity (MIC in mM), apparently, is indicated incorrect (0.91±0.00). In Abstract its MIC = 3.05±0.00 mM.
Thank you for your comment. As you pointed out, there were errors in the description. We sincerely apologize for the error. We have corrected it as follows:
Page 5
Table 1
|
Chemical |
(a) |
(b) |
(c) |
(d) |
(e) |
(f) |
(g) |
miconazole |
|
MIC (µg/mL) |
250.0±0.0 |
250.0±0.0 |
125.0±0.0 |
83.3±36.1 |
28.1±0.0 |
56.3±0.0 |
1500.0±0.0 |
11.25±0.00 |
|
MIC (mM) |
1.73±0.00 |
1.58±0.00 |
0.73±0.00 |
0.45±0.19 |
0.15±0.00 |
0.31±0.00 |
3.05±0.00 |
0.03±0.00 |
Page 3, Line 135, 136
The final concentrations of (g) in solution were 2.93, 5.86, 11.7, 23.4, 46.9, 93.8, 187.5, 375, 750 and 1500 μg/mL.
Page 10, Line351, 352
Although not listed in Table 1, 2H,2H,3H,3H-Perfluorononanoic acid also had no antifungal activity (MIC; 1500 µg/mL).
What is the correlation coefficient between the predicted values obtained by multiple regression analysis and the actual observed values? R = 0.95 (lines 234, 238) or 0.96 (line 245).
Thank you for your comment. According your comment, we added the sentences in Discussion section described below.
Page 9, Line 316-323
A strong regression is considered to exist when the partial regression coefficient R of the multiple regression analysis is 0.7 or higher. The obtained multiple regression equa-tion (partial correlation coefficient was 0.95) suggested that it was possible to predict the MICs of Compound (a) to (f) using the log S value. A strong correlation is considered to exist when the Pearson correlation coefficient is between 0.7 and 1.0. The Pearson correla-tion coefficient between theobserved and predicted MICs was 0.95, suggesting that the multiple regression equation for predicting MICs derived by multiple regression analysis can predict the observed MICs of Compound (a) to (f).
Has the method for MICs determination been described anywhere? It will be desirable to give reference.
Thank you for your comment. According your comment, we revised the sentences in Materials and Methods section described below.
Page 3 Line 123-138
The antibacterial activity of (a) to (g) was characterised by determining its MICs which standardized by Clinical Laboratory Standard Institute, CLSI. MIC was determined as described previously [36-39] with minor modifications. The C. albicans strain was di-luted to 4 × 104 CFU/mL in yeast and mold broth at pH 6.2. The plates were incubated at 27°C for 48 h after dispersing 150 µL of the culture into a 96-well microtiter plate well (6.0 × 103 CFU/well). Sample solutions diluted by DMSO were prepared and yeast and mold broth contained yeast and maltose. The final concentration of DMSO were 0.02, 0.03, 0.05, 0.1, 0.2, 0.5, 0.9, 1.9, 3.8 and 7.5 %. Although not shown in the data, it was confirmed that a DMSO content of 10% or less had no effect on yeast growth. The final concentrations of (a) to (d) in solution were 0.97, 1.95, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 μg/mL. The final concentrations of Compound(e) and (f) in solution were 1.75, 3.51, 7.03, 14.1, 28.1, 56.3, 112.5, 225, 450, and 900 μg/mL. The final concentrations of (g) in solution were 2.93, 5.86, 11.7, 23.4, 46.9, 93.8, 187.5, 375, 750 and 1500 μg/mL. Each analysis was performed in triplicate. The MIC was determined as the minimum compound concentration at which the well was clear, and indicated that no bacterial growth had occurred.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe article presents research on the antifungal activity of γ-undecalactone against Candida albicans, where the authors compare its effectiveness with other fatty acids and their derivatives. The study makes an interesting contribution to understanding the potential of modified FAs as alternative antifungal agents. However, despite the nice topic, the publication suffers from a few drawbacks.
The text contains many linguistic, grammatical, and stylistic errors, which make it difficult to read. The paper appears not to have undergone good language editing. Many sections are repetitive or lack logical flow, namely in the introduction and discussion.
As for the methodology, comparing the activity of the modified fatty acid with that of other medium-chain fatty acids was a very good methodological choice.
However, in 2.2, the authors state: “The C. albicans strain was diluted to 4 × 10⁴ CFU/mL in yeast and mold broth at pH 6.2.” (lines 131–132), it is not clearly specified what exact medium formulation was used ? Moreover, performing the MIC assays at 27 °C raises concerns, as the study’s main focus is on the medical relevance of the antifungal activity. C.albicans exhibits notable temperature-dependent physiological changes stress tolerance can vastly differ between 30 °C and 37 °C. Additionally, why the authors did not determine the MFC, which is a simple, straightforward follow–up after MIC assay?
Lines 171–2: does really the cost of sheep blood is a good argument here?
The conclusion that the fatty acids “form pores” is too far-reaching — in the SEM images, these are not actual holes but ‘inward indentations’, which are commonly observed after treatment with stressors that alter the lipid organization of the plasma membrane or cause localized chitin accumulation. Both possibilities would, however, require further confirmation using additional assay (such as propidium iodide or calcofluor white staining).
lines 402–408: the authors suggest that the compound might be active against azole-resistant strains. This is a too far-reaching conclusion, as no experimental data on resistant strains was present in the study.
Lines 409: the discussion of “sustainable development” (SDG) feels misplaced and purely declarative. It doesn’t add any real scientific value to the paper.
In conclusion, the topic is relevant, and the paper does have some strong points (comparing γ-undecalactone with other medium-chain fatty acids). However, other parts are weak, both methodologically and in presentation. To sum up, the study requires major revision before it can be considered for publication.
Author Response
Reviewer 4
Thank you for your useful comment. According your comments, we revised in red letters described below.
Many sections are repetitive or lack logical flow, namely in the introduction and discussion.
Thank you very much for your valuable comments. I have made the necessary revisions based on the reviewer's suggestions.
However, in 2.2, the authors state: “The C. albicans strain was diluted to 4 × 10⁴ CFU/mL in yeast and mold broth at pH 6.2.” (lines 131–132), it is not clearly specified what exact medium formulation was used ? Moreover, performing the MIC assays at 27 °C raises concerns, as the study’s main focus is on the medical relevance of the antifungal activity. C.albicans exhibits notable temperature-dependent physiological changes stress tolerance can vastly differ between 30 °C and 37 °C. Additionally, why the authors did not determine the MFC, which is a simple, straightforward follow–up after MIC assay?
Thank you for your comments. Yeast and mold broth contained yeast and maltose. We conducted the experiment at 27°C, where Candida albicans has the highest proliferation and infectivity. It has been suggested that C. albicans grown at room temperature, such as 27°C, grows faster and is more virulent in mice than those grown at 37 oC [41]. MIC measurements were performed at 27 oC, where C. albicans grows easily and have highly infectious in this study. The MIC, an index of bacteriostatic activity, is used based on the idea that if the growth of pathogenic microorganisms can be prevented in the case of infection in patients with normal immune function, they will be killed by the immune system and expelled from the body. Also, the measurement method of MIC has been standardized world-wide to ensure measurement accuracy and reproducibility. Consequently, MIC as fungi-static activity index was determined in this study.We added the sentences described below in Discussion section.
Page 3, Line 130
yeast and mold broth contained yeast and maltose
Page 10, Line 363-371
It has been suggested that C. albicans grown at room temperature, such as 27oC, grows faster and is more virulent in mice than those grown at 37 oC [41]. MIC measurements were performed at 27 oC, where C. albicans grows easily and have highly infectious in this study. The MIC, an index of bacteriostatic activity, is used based on the idea that if the growth of pathogenic microorganisms can be prevented in the case of infection in patients with normal immune function, they will be killed by the immune system and expelled from the body. Also, the measurement method of MIC has been standardized worldwide to ensure measurement accuracy and reproducibility. Consequently, MIC as fungistatic activity index was determined in this study.
Lines 171–2: does really the cost of sheep blood is a good argument here?
Thank you for your comment. The misleading statement has been revised and reference added.
Page 4, Line 174, 175
The hemolytic activity was evaluated using sheep red blood cells by a modified previously reported method [40]
- Kannan, T.R.; Baseman J.B. Hemolytic and Hemoxidative Activities in Mycoplasma penetrans. Infect Immun, 2000, 68, 6419-6422 doi: 10.1128/iai.68.11.6419-6422.2000
The conclusion that the fatty acids “form pores” is too far-reaching — in the SEM images, these are not actual holes but ‘inward indentations’, which are commonly observed after treatment with stressors that alter the lipid organization of the plasma membrane or cause localized chitin accumulation. Both possibilities would, however, require further confirmation using additional assay (such as propidium iodide or calcofluor white staining).
Thank you for your useful comment. “perforation” was replaced by “‘inward indentations” in the manuscript. In our next further study, we will conduct assays to clarify the mechanism. We added the sentences in Disscusion section.
Page 11, Line 391-396
Addition of undecalactone to C. albicans caused inward indentations on the surface of the fungus. The inward indentations, which are commonly observed after treatment with stressors that alter the lipid organization of the plasma membrane or cause localized chitin accumulation. Both possibilities would in this study. To confirm the mechanism, It is required another assay (such as propidium iodide or calcofluor white staining).
lines 402–408: the authors suggest that the compound might be active against azole-resistant strains. This is a too far-reaching conclusion, as no experimental data on resistant strains was present in the study.
Thank you for your useful comment. We deleted the far-reaching sentences described below.
Azole antifungal drugs inhibit the synthesis of ergosterol in the endoplasmic reticulum. Fungi that have lost ergosterol, the main component of the cell membrane, are inhibited from growing. It was suggested that γ-decanolactone may be adsorbed to the surface of C. albicans and destroy the cell surface due to hydrophobic interactions between the alkyl group, which is a hydrophobic functional group in the structure, and the hydrophobic substances on the surface of C. albicans. The medium chain fatty acid lactone, γ-undecalactone was suggested to have an antifungal effect mechanism unlike azole antifungal drugs, and thus may have an antifungal effect on C. albicans that retain the above-mentioned drug-resistant C. albicans. JCM1542T is type strain of C. albicans. In our previous paper [38], the susceptibility of JCM1542T to the azole antifungal agent miconazole has been confirmed. Further investigation whether medium-chain fatty acid lactone would have antifungal activity to drug-resistant C. albicans would be required.
Lines 409: the discussion of “sustainable development” (SDG) feels misplaced and purely declarative. It doesn’t add any real scientific value to the paper.
Thank you for your comment. We revised the sentences described below.
Page 11, Line 414-417
Our results in this paper showed the possibility that γ-undecalactone deadened C. albicans causing the opportunistic infection. The undecalactone is used as food additive. Ensure healthy lives and promote well-being for all at all ages
In conclusion, the topic is relevant, and the paper does have some strong points (comparing γ-undecalactone with other medium-chain fatty acids). However, other parts are weak, both methodologically and in presentation. To sum up, the study requires major revision before it can be considered for publication.
Thank you very much for your valuable comments. I have made the necessary revisions based on the reviewer's suggestions. I would like to ask you to review it again.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsIn the revised version of the paper cimb-3945645-peer-review-v2, the Authors responded to the most comments I had. However, before reaching the final version few additional details should be corrected
I do not understand the use of certain words written with a capital letter within sentences (such as the names of compounds “Octanoic” (line 201) or Chemical (line 249) and, in my opinion, completely random words such as “Sterile” (line 164), “Predicted” (line 241), “Observed” (line 241) and others. The entire manuscript should be corrected to eliminate such shortcomings.
Also not all are capital letters within the subtitle
2.3. Physical Property-Antifungal Activity Correlation in Medium Chain Fatty Acids and
Medium Chain Fatty Acid Lactones by Multiple Regression Analysis
The authors are inconsistent in the use of certain compound names like on page 5, in Figure 1 caption, and line 202 not s-undecalactone, but d-undecalactone. Please, correct this.
and on page 9 (line 350) not 2H,2H,3H,3H-Perfluorononanoic acid but 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoic acid. Please correct this
page 2 “gram-positive and -negative bacteria” (line 55) should be as on page 2 “Gram-positive and Gram-negative bacteria” (line 85)
C. albicans (line 391) or Candida albicans are not always written in italic
Two paragraphs related to Candida albicans membrane composition are missing references: page 9 (lines 328-340) and page 11 (lines 396-404)
Once the above updates are submitted, I will be more than happy to assist in evaluating the revised contribution for the CIMB journal
Author Response
Reviewer 2
I do not understand the use of certain words written with a capital letter within sentences (such as the names of compounds “Octanoic” (line 201) or Chemical (line 249) and, in my opinion, completely random words such as “Sterile” (line 164), “Predicted” (line 241), “Observed” (line 241) and others. The entire manuscript should be corrected to eliminate such shortcomings.
Also not all are capital letters within the subtitle
2.3. Physical Property-Antifungal Activity Correlation in Medium Chain Fatty Acids and
Medium Chain Fatty Acid Lactones by Multiple Regression Analysis
Thank you for your comments. We revised capital letter within sentences to lowercase letter.
The authors are inconsistent in the use of certain compound names like on page 5, in Figure 1 caption, and line 202 not s-undecalactone, but d-undecalactone. Please, correct this.
and on page 9 (line 350) not 2H,2H,3H,3H-Perfluorononanoic acid but 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoic acid. Please correct this
We revised 2H,2H,3H,3H-Perfluorononanoic acid to 4,4,5,5,6,6,7,7,8,8,9,9,9-Tridecafluorononanoic acid
page 2 “gram-positive and -negative bacteria” (line 55) should be as on page 2 “Gram-positive and Gram-negative bacteria” (line 85)
We revised according your comment.
- albicans (line 391) or Candida albicans are not always written in italic
Thank you for your comment. We revised it.
Two paragraphs related to Candida albicans membrane composition are missing references: page 9 (lines 328-340) and page 11 (lines 396-404)
We added two references.
- Derkacz, D.; Krasowska, A.; Alterations in the Level of Ergosterol in Candida albicans' Plasma Membrane Correspond with Changes in Virulence and Result in Triggering Diversed Inflammatory Response. Int J Mol Sci 2023 Feb 16;24(4):3966. doi: 10.3390/ijms24043966.
- Sanglard, D.; Ischer F.; Tania Parkinson, T.; Falconer, D.; Bille, J.; Candida albicans Mutations in the Ergosterol Biosynthetic Pathway and Resistance to Several Antifungal Agents. Antimicrob Agents Chemother. 2003 47(8):2404–2412.
Once the above updates are submitted, I will be more than happy to assist in evaluating the revised contribution for the CIMB journal
Thank you for reviewing of our revised manuscript. We would like to thank you for reviewing our manuscript to see if it was worthy of publication and for your comments on how to improve it.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have made the necessary corrections. The article can be published. However, it seems to me that English requires correction.
Author Response
Reviewer 3
The authors have made the necessary corrections. The article can be published. However, it seems to me that English requires correction.
Thank you for your comment. According to the suggestion of reviewer 4, we re-examined all seven compounds using the temperature conditions of 37oC instead of 27oC, and performed MIC measurements, MBC measurements, PI staining, SEM, and hemolytic activity evaluation. Regarding TEM, due to a mechanical problem, it was not possible to perform re-measurement by the deadline. Following the comment of reviewer 4, to clarify the mechanism of antifungal activity, we performed a PI staining experiment and examined the fungal killing effect in MFC.We entirely revised the manuscript due to the re-experiment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsI get the impression (reading the authors’ responses) that they are not entirely confident in their approach and seem to be navigating the issues somewhat blindly.
First: the authors refer to CLSI methodology for MIC determination and describe their results as “antibacterial,” yet they only tested fungi... In addition, although they cite the CLSI, the description they provide does not correspond to the referenced procedure... Furthermore, they state that “were prepared and yeast and mold broth contained yeast and maltose”, however, they do not clearly specify which broth was used? nor how it relates to the broth recommended in the CLSI guidelines? Finally, they write “The MIC was determined as the minimum compound concentration at which the well was clear, and indicated that no bacterial growth had occurred.” C. albicans is not a bacterium, and the wording suggests a misunderstanding of the organism being tested.
The authors performed MIC testing at 27 °C. In response to my question regarding the incubation temperature, they cited a publication in which the virulence of C. albicans inocula grown at 27 °C vs. 37 °C was compared in a mouse infection model. This appears to be a misunderstanding of the cited work. In their revised manuscript, the authors state: “MIC measurements were performed at 27 °C, where C. albicans grows easily and have highly infectious in this study”. In their reply, they add: “It has been suggested that C. albicans grown at room temperature, such as 27 °C, grows faster and is more virulent in mice than those grown at 37 °C.”
However, C. albicans cannot be more virulent in mice at 27 °C, since the body temperature of mice is ~37.5 °C. The cited study compared inocula grown at different temperatures before infection; it did not suggest that C. albicans is more virulent within a host at 27 °C. Rather, blastoconidial cells grown at lower temperatures may evade immune recognition longer, whereas hyphal forms grown at 37 °C are more rapidly detected by the immune system, hence differences in killing kinetics. Importantly, this rationale is not relevant to in-vitro MIC testing, where immune pressure is absent and the culture plate remains at a single temperature.
Additionally, the authors reference CLSI methodology, which clearly specifies incubation at 37 °C for MIC determination against Candida species. Performing MIC assays at 27 °C is therefore inconsistent with the cited standard, and the justification provided does not support deviating from established guidelines and logic.
Author Response
Reviewer 4
I get the impression (reading the authors’ responses) that they are not entirely confident in their approach and seem to be navigating the issues somewhat blindly.
First: the authors refer to CLSI methodology for MIC determination and describe their results as “antibacterial,” yet they only tested fungi... In addition, although they cite the CLSI, the description they provide does not correspond to the referenced procedure... Furthermore, they state that “were prepared and yeast and mold broth contained yeast and maltose”, however, they do not clearly specify which broth was used? nor how it relates to the broth recommended in the CLSI guidelines? Finally, they write “The MIC was determined as the minimum compound concentration at which the well was clear, and indicated that no bacterial growth had occurred.” C. albicans is not a bacterium, and the wording suggests a misunderstanding of the organism being tested.
The authors performed MIC testing at 27 °C. In response to my question regarding the incubation temperature, they cited a publication in which the virulence of C. albicans inocula grown at 27 °C vs. 37 °C was compared in a mouse infection model. This appears to be a misunderstanding of the cited work. In their revised manuscript, the authors state: “MIC measurements were performed at 27 °C, where C. albicans grows easily and have highly infectious in this study”. In their reply, they add: “It has been suggested that C. albicans grown at room temperature, such as 27 °C, grows faster and is more virulent in mice than those grown at 37 °C.”
However, C. albicans cannot be more virulent in mice at 27 °C, since the body temperature of mice is ~37.5 °C. The cited study compared inocula grown at different temperatures before infection; it did not suggest that C. albicans is more virulent within a host at 27 °C. Rather, blastoconidial cells grown at lower temperatures may evade immune recognition longer, whereas hyphal forms grown at 37 °C are more rapidly detected by the immune system, hence differences in killing kinetics. Importantly, this rationale is not relevant to in-vitro MIC testing, where immune pressure is absent and the culture plate remains at a single temperature.
Additionally, the authors reference CLSI methodology, which clearly specifies incubation at 37 °C for MIC determination against Candida species. Performing MIC assays at 27 °C is therefore inconsistent with the cited standard, and the justification provided does not support deviating from established guidelines and logic.
Thank you for your valuable comments. We used Yeast Extract-Malt Extract broth (YM broth). According to your suggestion, we re-examined all seven compounds using the temperature conditions of 37oC instead of 27oC, and performed MIC measurements, MBC measurements, PI staining, SEM, and hemolytic activity evaluation. Regarding TEM, due to a mechanical problem, it was not possible to perform re-measurement by the deadline. Following the comment of you, to clarify the mechanism of antifungal activity, we performed a PI staining experiment and examined the fungal killing effect in MFC.
Thank you for your suggestions on how to evaluate antimicrobial activity against fungi. we learned many important points.
Author Response File: Author Response.pdf
