Inhibitory Effects and Mechanism of Action of Elsinochrome A on Candida albicans and Its Biofilm
Round 1
Reviewer 1 Report
It is an interesting and novel study showing the activity of effect of Elsinochrome A (EA) against different stage of C. albicans biofilms. However, there are multiple flaws, mainly in the presentation of results.
There are many grammar errors, and the use of acronyms is not followed.
Line 39-40 check the sentence, it is an alternative method for fungal infection?.
line 59, it is not clear whether the results obtained are from multiple strains. In the methodology, it is mentioned *Strains were*
(if multiple strains, please provide results for each strain)
Line 63: check the sentence:
Fluconazole was purchased from Solarbio as a positive control
Line 69: explain why 10e6 cells were used if you followed CLSI guidelines (this is extremely important).
Line 73 fluconazol
Line 75: how long?
Line 80 (all manuscript): please organise the concentrations in ascending order, e.g. 2-128
Line 84: meaning of the abbreviation
Line 95: fluconazol
Line 113: You are not quoting a reference method, you are quoting the method of the reference 20. Please adjust the sentence.
Line 195 meaning of the abbreviation
line 206, table 1, add a column of references (provide the references).
table 2: specify which one is biofilm
line 225: drug
Line 227 why didn't you use the abbreviation before? FLZ
Lines 238-240 Reached - over use
Figure 2: remove min, it is duplicated
line 247, why 90 min? (see line 240)
Lines 261-263 we, CV, And.
figure 3: if there is the same statistical difference, why use the lines (b)? Why don't you use them in the other graph (a)?
Figure 4 color conventions
a, b, c, d ?
Figure 5: What are the small dots in (8-32 ug/mL photos)?
Figure 8: check asterisks arrangement
B, y-axis title
line 343: check the sentence
Line 369 PACT
Line 370 ROS
Line 383: Why did you use fluconazole as a control?, it has only moderate antibiofilm activity and resistance is occurring. please discuss it.
Line 388: add a specific reference for fungal biofilms
Line 435 ROS
Discussion, Add a paragraph about difficulties and weaknesses of this study
Line 448, discuss how it could be used in this scenario
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Title: suggest to rephrase- suggestion: inhibitory effects and mechanism of action of elsinochrome A .......
Abstract - grammar and language changes are required. Suggest to improve the clarity of the abstract presentation
Introduction - suggest to provide more information on the source and properties of elsinochrome A
Materials and methods – need some information on
i. C. albicans strain, on its fluconazole sensitivity, biofilm producing ability
ii. the method for preparation of elsinochrome A. What was the method to prepare purified elsinochrome A for testing and the concentration required?
iii. Basic properties of elsinochrome A- solubility, solvent used?
iv. Light source (for example, instrument, LED, Energy Density (Fluence) (J/cm2) Spot Size/Fiber Surface Area (cm2) etc..
v. Did the light source affect Candida growth? Why 470 nm was selected?
Not too clear with the method described in section 2.9..
If instrument or kit were used, please include the manufacturers information in the text, refer to section 2.11.1, 2.12
Results
Lane 214 – yeast instead of bacteria
Figure 4 – no indication of A, B, C, D in the figure
Figure 7 – top panel, the images are not clear. Is it possible to use higher magnification
Discussion
Is elsinochrome A safe for application – what will be the route of administration for therapy?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Some issues to consider
Line 41: `Photodynamic antimicrobial chemotherapy (PACT) is one of the alternative methods for traditional fungal infection treatment`. However, the reference used is about PACT in cancer. Why you said *traditional* method for fungal infection treatment? e.g., References (6-9) 2020-2022.
(innovator method?).
Line 70 meaning of the acronym YPD
Line 72 16 days?
Line 73 adequate water? Please specify
Line 73-77. Reference?
Line 84 replace the word measured by determined
155 please remove the word designed
Line 287 showed
Line 304 remove crystal violet
Figure 4. Significant differences?
figure 10 b is not clear, please replace or improve the image quality.
Line 419. Consider add a reference about candida albicans mucosal infections
e.g DOI: 10.4103/jgid.jgid_74_19
line 438. Add in a FLC-sensitive strain (MIC 0.5, SMIC80 >1024). To confirm those results in a resistant strain is recommended (please consider mentioning this).
Line 440 Full stop at the end.
Line 448 showed
Add that a weakness is that EA was only evaluated in one strain, more strains are needed mainly resistant to antifungals with different mechanisms, e.g. mutations in genes associated with efflux pumps.
add information about the strain CAMS-CCPM-D-52061 (source, date, clinical specimen?)
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Line 17: is it RT-qPCR (give full name)
Line 32: is it immune defence rather than homeostasis
Line 70: what is EA(98%)? Percentage shows EA quality? How the authors determine the purity of EA (based on which lab method? ). I noted that the source of EA (Shiraia bambusicola) was different from the initial report by Chen et al. This is not explained.
Line 81: Solarbio (source, country)
Line 91: FLC (concentration range?)
Line 93: which instrument was used for light irradiation experiment? Explain why the irradiation parameters were chosen? Any pilot study to show that the parameters chosen were the best?
Line 135: give citation for reference 24
Line 188: conductivity meter (brand, manufacturer)
Line 236: t-test? P value <0.05?
Line 105, 248: log instead of lg
Table 2: Please clarify which one is planktonic MIC? And which one is biofilm MIC? Did fluconazole-treated C. albicans cultures demonstrated any increased antifungal effects upon light irradiation?
Line 254: SMIC stands for sessile MIC? Not stated. or it should be MBIC?
Line 259: Figure 1, why the starting inocula (y axis, log CFU/ml) for all EA with various concentration at 0 h were different? I thought the inocula should be standardized at 0 hour.
Line 274: EA upon light irradiation?
Figure 2: What caused C. albicans growth inhibition at 0 irradiation hour. This was not explained.
Figure 4: Was there any significant difference in the metabolic activity of C. albicans upon treatment with different concentrations of EA?
Line 302: scavenging – means biofilm eradication?
Figure 7. Are we expected to see some fluorescence for Fig 7A1, B1, C1, and D1. Also would like to suggest the authors to give more explanation for their observation for Fig 7 A2-D2 in the legend. The small dots represent whole cells of C. albicans, but why there were lower number of untreated cells (A2) and B2? any positive control was used?
Figure 8. What is DCF, explain Figure 8A in the legend, any positive control was used?
Figure 9: Add some explanation to Figure 9. any positive control was used?
Figure 10. How to interpret Figure 10 result on nuclear fragmentation, any positive control was used?
Line 513: any supporting evidence for nano-targeted drug delivery system
Line 520: The treatment with 1024μg/mL EA and irradiation (470nm; 90mW/cm2; 324J/cm2) had significant effects on pre-formed mature biofilms, with a clearance rate of 35.16%. The significance effects were not indicated in Figure 4
Author Response
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Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
The authors have answered my queries.
qRT-PCR- Real-Time Quantitative Reverse Transcription PCR
The light emitting diode (LED) lamp (90 mW cm2; Cidly Optoelectronic Technology Co., Ltd, China), although mentioned in the authors response, should be also mentioned in the manuscript
Was it not necessary to have positive controls for some of the experiments? May be this can be discussed in the limitation and future experiments
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
