Single-Cell Optical Nanomotion of Candida albicans in Microwells for Rapid Antifungal Susceptibility Testing

Round 1

Reviewer 1 Report

The authors used a novel technique for antifungal susceptibility testing of one of the most important human fungal pathogens, Candida albicans. Their development of microfkuidic chips combined with ONMD provides a rapid test platform to quantify cell responses in real time.

The last statement of the abstract may be a bit exaggerated that developing countries could use such a platform on a routine basis. Maybe this should be reconsidered.

Notes:

L134: DSY1024 (mutant for efflux systems): the genotype should be described clearly.

L270: There wasn’t: rather ther was not

L274: microwells

Fig. 9: a bar chart may be better to discern the differences of the 4 treatments. For me it is hard to see what the authors want to show.

 

L404: During the treatment of C. albicans DSY1024 strain with different concentrations of 404 caspofungin, i.e., 0.5 µg/ml, 1 µg/ml, and 100 µg/ml, we observed a significant decrease 405 Fermentation 2022, 8, x FOR PEER REVIEW 13 of 21 in the cellular displacements and total displacement

>>> I had to read up to page 13 to figure this out…This should be stated in the abstract as this is proving the usefulness of the technique…

 

Author Response

See attached file with our responses to the reviewer's comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

The microfluidic chip was designed to enable the easy exchange of liquids without flushing out the cells that are trapped in the microwells. Such a chip permits monitoring of physiological characteristics of single cells in different wells, and also to analyze tenths of cells per experiment. This manuscript is a continuation of the research already published by the same team in Fermentation 2022, 8, 195. https://doi.org/10.3390/fermentation8050195, the main authors also took part in research published in Willaert et al., Sci. Adv. 6, eaba3139 (2020).

There are three main parts in the Results of this manuscript: 1) development of the microfluidic chip, 2) monitoring characteristics of the nano motion of single cells, and 3) assessment of the real-time response of single C. albicans cells to fungicidal caspofungin treatment. The results presented in the first part are new, original, and clearly presented. The construction of the microfluidic chip is described in detail, interesting computational simulation of the fluid flow, and the chip setup is well described. Due to the creation of a vortex in the microwells, the same cell can be observed before and after exposure to an anti-fungal drug. This is very important, the major advantage of this new method. The assessment of a single-cell nano motion is clearly described, but its connection to the efficiency of caspofungin treatment, and the effect of this antifungal on the metabolic activity of single cells is unconvincing.

The reasons for the nano motion of the single yeast cell are not known, the attempts to find the correlations between some characteristics of the single cell nano motion and the sensitivity of the cells to caspofungin are barren. The manuscript does not provide any additional information or new ideas about the reasons for nano motion. 

Three caspofungin concentrations were studied: 0.5, 1.0, and 100 mg/L. Criteria for the selection of such concentrations should be better explained. We can find in the manuscript that MIC values of caspofungin for the strain CAF2-1 are 0.03 - 0.06 mg/L, and the strain DSY1024 is even more sensitive! 1 mg /L is already a high concentration, why 100 mg/L is needed?

The sensitivity of the individual Candida albicans cells to caspofungin was characterized by analyzing averaged cellular nano motion of the individual cells. “However, observing the single-cell nano motion of strain DSY1024 during the treatment with 100 mg/L caspofungin, we observed that many cells after 300 min stayed with similar nano movements as before the treatment.” (lines 427-429). 

The criterion that the decreases in the slopes of DSY1024 cells were larger than the ones for the wild-type strain, demonstrates that this strain is more susceptible and also is not strong enough. Standard methods to perform AFST rely on measuring fungal growth in the presence of antifungals. We need direct comparison results of the traditional antimicrobial susceptibility testing (disc diffusion, broth dilution, formation of colonies on the agar plates) with the results of the method described in the manuscript. I do not see any evidence, that the DSY1024 strain is hypersusceptible. Yes, it should be like this, but I do not see any evidence, that caspofungin is a substrate of efflux pumps. According to the structure and charge of caspofungin, it should be water-soluble. Is it lipophilic enough to interact with efflux pumps? 

Even if caspofungin is a substrate of efflux, the difference between wt and mutant strains should disappear at so high (100 mg/ml) concentrations of the antifungals. The efflux pumps would be oversaturated! Besides this, de-energization of the yeast cells should occur at so high concentrations of antifungals, and the efflux would be switched off. 

To my mind, experiments with heat-killed cells could help to find the answer about the reasons for the nano movement of yeasts.

Author Response

See attached document for our responses on the comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript “Single-Cell Optical Nanomotion of Candida albicans in Microwells for Rapid Antifungal Susceptibility Testing” described a new method based on microfluidic chip containing an array of microwells designed to trap the cells and perform rapid antifungal susceptibility tests using optical nanomotion detection.

 

General comments :

In comparison to the previous papers where the authors analyzed the response of fungi to antifungal using optical nanomotion detection (DOI: 10.1126/sciadv.aba3139) , the authors developed in this paper a microfluidic chip containing an array of microwells to trap the cells and analyzed the susceptibility of each yeast to antifungal. That is an interesting system but in this context the authors didn’t explain why this system is better that the previous one where the response of yeasts together was analyzed. They didn’t compare here the results between one and more one yeasts per well. How the authors interpreted the results when the yeasts had different responses?

The microwells microfluidic chip was developed here to detect the susceptibility of fungi to antifungal. The authors didn’t cite in the introduction the referees about the gold standard methods of antifungal susceptibility (10.1111/j.1469-0691.2012.03880.x; Clinical and Laboratory Standards Institute. M38-A2: Reference method for broth microdilutionantifungal susceptibility testing of filamenteous fungi. CLSI (editor). Wayne, PA, USA (2019), and the AFST article mentioned in the references was about bacteria AFST (5), however some papers about fungi could be cited (10.1080/14787210.2020.1760841 ; 10.3389/fcimb.2021.720609 ).

The authors didn’t use a yeast susceptible and resistant to antifungal as used in literature when a new antifungal test was developed. They used 2 strains of Candida, susceptible to echinocandin (capsofungin). They indicated that DSY1024 is a hyper susceptible strain but described in the literature as a hyper susceptible strain to azole, a different classe of antifungal drugs (not echinocandin class) used in the treatment of mycosis. It will be interesting to used 2 or more strains susceptible or resistant to capsofungin to validate the assay.

 

Point by point :

Materiels and methods :

Could the authors explain in this section how they determined a total displacement at time =0 min? T=0 is the first position ? not yet variation ?

L229 : the authors analyzed 100 x100 px.  A well represents how many pixels ?

L233 : The authors used x40 objective.  A pixel represents how many microns ?  why the authors used the pixels to express their results and not the microns as in the previous paper ?

 

Results :

L356-357 : The authors used 3 concentrations of capsofungin. The concentrations were elevated. In the usual AFST the concentrations were between 0,008 and 4 mg/ml.

L367 : the figure S4 was cited. was the Figure S3 cited in the text ?

L378 : the authors said “As a control experiment, ….the hypersusceptible C. albicans DSY1024 strain was monitored”. As explain above, it is not a control. It is an another strain susceptible to capsofungin.

Figure 10 , Figure 13… : X , Y were the position on the area analyzed or a displacement in X and in Y ?

The authors analyzed 20 cells. How they choose these cells. Is it only the well with the one cell ? The cells were analyzed in the different frame or sometimes in the same frame ? Why only 20 cells were analyzed. It is possible to increase the number of cells if the response of each cell was too different ?

Figure 14 , L434: the authors calculated the slopes to characterize the sensitivity of individual cells to capsofungin. Which averaged value at 10 min the authors found for each strain with capsofungin. 

L476 : “the diagonal lines correspond to the slope value of nanomotion of CAF2-1 cells for the same time points” I didn’t understand the difference with the box.

 

Conclusion :

L503: The authors said “The method allows the analysis of a large population of cells by conserving single-cell sensitivity.” Here they analyzed 20 cells it is not a large population and they showed that only 31% of well had one cell.

Author Response

See the uploaded file for our replies to the comments of the reviewer.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The changes made neither improved the manuscript nor made it worse. Additional data on the effect of fluconazole on the nano motion of DSY1024 strain cells did not improve the microbiological part of the paper. Not clear, why strain CAF2-1 cells were not tested, why not compared to DSY1024 cells? The reasons for the nano motion of the single yeast cell are still not known, and the attempts to find the correlations between the nano motion and sensitivity of the cells to antifungals are still barren. Very simple control experiments on the nano motion of heat-inactivated yeast cells would help a lot. Why the correlation of nano motion with the viability of the cells assayed using traditional methods are not tested? According to the summary, the equipment for nano motion studies, as “a simple and label-free technique could be further developed into a simple-to-use device that allows performing fast AFST as part of a routine hospital procedure”. I agree that authors have made substantial progress in the development of the technological part of the device, but this meaningless machine still cannot be used for medical purposes.

Author Response

See attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors modified the text and figures. As previously said its is an interesting method, but the authors didn't demonstrate  their assay could determine the sensitivity or resistance of strains. They didn't use a resistance strains to antifungal.  They used 2 strains with a different sensibility to fluconazole (sensitive and hypersensitive). The authors added  the results with fluconazole for the hypersensitive strain they didn't show in the article the results against fluconazole for the both strains to demonstrate different profils. Could the complete with a resistante strain ? or change the tille as for example Single-cell optical nanomotion of C.albicans in microwells for rapid  response analysis to molecules.

 

Author Response

See attached file.

Author Response File: Author Response.pdf