Breaking the Mold: Towards Rapid and Cost-Effective Microbial Contamination Detection in Paints and Cosmetics Using ATP-Bioluminescence
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript describes the study of a potential tool for determining microbial contamination in paints and cosmetics products using an ATP- based assay, with treatments of a mix of four bacterial species into four distinct matrices.
It is an interesting approach for developing strategies to identify microbial contamination in some industrial sectors. The manuscript is clear and possesses some interesting points, but some aspects must be improved before acceptance.
I suggest including a synthesis of the strategies, other than culture-based methods, explored to detect microorganisms in cosmetics and/or painting products as in these papers:
https://doi.org/10.1016/j.aca.2020.06.069
https://doi.org/10.1039/C2AY25833A
https://doi.org/10.3390/cosmetics7020038
https://doi.org/10.1007/s13762-020-02801-1
And, I suggest retaking this point to discuss about the pros and cons comparisons of the different methods and how the ATP-based method could be an optimal option.
L109: Eliminate the equal sign into the parenthesis.
L124: Do you mean µM and fM???
L144-145: Why did the authors not assay a lower concentration? Similar to the detection limit for dipslide assays (10^2)
L143-151: What time was spent since the bacteria inoculation and the processing of the samples? I mean, it is important considering the microorganisms survival in the cosmetic and paints matrices, taking in account that if microorganisms could establish, they can develop biofilm structures that could difficult detach the cells, or even to die in these matrices giving a bias in the detection of living cells.
L157: Please homologue the format O/W and W/O with that of the line 92-93 and 173
L218-220: Are there any reports of microbial communities in some cosmetic or paint products? This could be useful as a reference point. Also, I suggest including each bacterial species' response in the graphic, and not only the mean data to show the response of each species with respect to the mix.
L257-260: Authors say, “On a positive note, alkaline pH-values did not interfere with the applicability and efficacy of the ATP assay, which is important considering that most commercially available, biocide-free coatings are high pH formulations with pH values up to 11,” but the paint and cosmetics samples ranked around neutral to slightly alkaline pH (8 the highest), so it is uncertain that this actually occurs, at least that they show any reference about that.
L265-283: Authors attribute the higher ATP-based cell count using WO emulsion to the cells’ growth phase, but in all the assays, the bacteria were in the stationary phase when they were mixed with matrix; the literature shows that ATP secretion is lower in the stationary phase than in exponential. Additionally, in the methods, it does not look like the bacteria had time for any growth after the mixture with the matrix.
I suggest improving the writing in this section. It is confusing. Are the authors trying to explain why the ATP levels were lower than cultures counting in three of the treatments? This is opposite to the result observed with WO.
The cells were in the same conditions in all the treatments; they didn’t spend any time after the mixture preparation. The only difference between treatments was the matrix used, so the composition of these products could be the factor that determines the observed results.
L311-313: How much time did the bacteria spend in the matrix to grow??? Methods do not mention any time to let growing bacteria. This is important data; the authors showed the low survival of bacteria after incubating them for a “few days” (please specify the time) in the WS and OW matrices; it is difficult to know how this could affect the sensitivity of ATP detection. Why did the authors not perform the ATP-based detection in these assays? This could be useful in better knowing the bacterial growth phase effect.
Author Response
The authors would like to thank the reviewer for the constructive feedback and time. As far as possible all suggestions were incorporated in the revised manuscript. All comments were answered point by point (highlighted in bold letters) with listed changes and corresponding line numbers (associated with the clean version). Please see attachment. A revised manuscript version including “track changes” and a clean version were uploaded.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsMeasuring ATP is used as a means of evaluating sanitation and determining if organic load is still present. It is not used to determine if bacteria are present.
The introduction is missing a discussion of specific culture independent diagnostic tests (CIDT) which is what is used in most cases, including high sensitivity with very low detection limits like digital PCR.
The authors seem to misrepresent ATP detection as used to detect microbial contamination at low levels (line 44), but really ATP is detecting organic load without specificity.
Why was CASO agar used and what does CASO stand for. It is selective? Why not use LB or a traditional minimal media like TSB?
Why was PCR not used as a CIDT comparative assay to determine the levels detected and also compare for detection of non-viable bacteria. ATP also detects non-viable bacteria. PCR is in the discussion, but should be included before this point.
It is not clear of the paints and cosmetics were diluted as much as possible to reduce the artificial reads of the samples with the organic materials included. This could answer the questions regarding the matrix-related quenching.
Why was an organic material not included as a control or perhaps heat-killed bacteria?
Did the authors confirm the production of spores? Why include this conversation in the discussion? There seems not to be enough time in the environment for cells to form spores? But it is not clearly described here.
Author Response
The authors would like to thank the reviewer for the constructive feedback and time. As far as possible all suggestions were incorporated in the revised manuscript. All comments were answered point by point (highlighted in bold letters) with listed changes and corresponding line numbers (associated with the clean version). Please see attachment. A revised manuscript version including “track changes” and a clean version were uploaded.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have made satisfactory revisions and addressed the concerns from the previous version. However, minor corrections must be made to the manuscript before it can be accepted.
Please correct redaction details and typos:
Text format L140-141
L297 Space after “C).”
L306 Revise extra punctuation
Author Response
The authors would like to thank the reviewer again for the valuable time and accurate reading of the manuscript. All suggestions were incorporated in the revised manuscript. All comments were answered point by point (highlighted in bold letters) with listed changes and corresponding line numbers (associated with the clean version) and a revised manuscript version including “track changes” were uploaded.
Author Response File: Author Response.pdf
