An In Vitro Evaluation of the Biocidal Effect of Oregano and Cloves’ Volatile Compounds against Microorganisms Colonizing an Oil Painting—A Pioneer Study
and Annalisa Balloi 2,3,*Round 1
Reviewer 1 Report
I carefully went through the manuscript by Gatti et al. dealing with the evaluation of the use of oregano and cloves EOs against microorganisms growing on oil paintings.
This is a relevant and interesting topic, and EOs are a really interesting resource in the conservation of Cultural Heritage. However the paper results a bit confused, not well balanced and and some revisions should be necessary to improve the robustness of the results.
1. First of all I wonder if the manuscript submitted is the right version: some elements are missing. In the list (page 12 lines 322-334) are cited an “Appendix A” and an “Appendix B” but these two not are presents in the manuscript, as well as Table 3 and Table 4 (that are really important for the comprehension of the results). Authors should add missing parts in the revised version.
2. The declared aim of the study was to test the suitability of oregano and clove EOs, but ample space (too ample) is given to the characterization of the microorganisms sampled on the artwork “Studio di nudo”. Furthermore the morphological characterization of microbial community is poor (and maybe the sampling method is not perfect). The morphological characterization of isolated strains is not not well presented, a description is not presented, not even for the most common. This part is not no so relevant if the aim is testing the microbial inhibition activity of EOs. Authors can move this part (figure 2, table 1 and table 2) in the Supplementary Materials in order to better balance the paper?
3. Both “contact test” and “contactless test” have a design with a “negative” control but in the tables negative control values are not shown. In my opinion 70% ethanol, is a particular case of “negative” control because ethanol had an antimicrobial activity too, if paper not report the ethanol values it is unclear if ethanol is a “negative” or “positive” control. Any isolated strain is inhibited by ethanol? If 70% ethanol is a negative control then a positive control is missing: poor growth of the isolated strains (i.e. in the contact less test) could be due to unfavorable incubation conditions. Authors can clarify, and show control results in table 3 and table 4?
4. As authors remarked, in the “contactless test” oregano EOs had performed better than in "contact test", this is interesting but also unexpected, this is worth of a more ample discussion (10μL in contact test and 100μL in contactless test can be relevant?) and maybe of a new experiment, also with few strains but with a positive and negative controls.
A list of comments is subsequently reported to detail those points of the text which should/may be re-checked to improve the clarity of the contents.
Title.
The expression “cleaning procedure” can generate a misunderstanding: the tests have been carried out on the “biocide effect” not on the “cleaning procedure”. The treatment with a biocide is only a propedeutic treatment for a cleaning procedure.
Abstract.
page 1 lines 13-14: “Featured Application: We hypothesize an effective and color respectful method” but in the present study there are no tests about any color changes. May the authors mitigate the claim?
Introduction
page 2 line 47 = “well known for their bactericidal and fungicidal properties” = some references should be anticipated here, e.g. Fidanza & Caneva, 2019.
Materials and Methods
page 3 lines 92-99 = all the 18 samples were collected in correspondence with visible biological attack? No control sampling point?
page 4 lines 136-139 = The formal botanical names of plants and fungi should be reported in the complete form (binomial name and authors) at least once: this is the correct paragraph for this.
page 4 line 141 “was inoculated in Petri dishes” = It is not clear how many replicates for each experimental thesis.
page 4 lines 145-148 = An evaluation at naked eye could be replaced by a image analysis program. Measurements of inhibition halo with this method, maybe, can be effective also in the case of fungi strains. In any case could authors provide a table with images as examples for representing different inhibitions scale degree?
Page 4 line 151 “One fungal or bacterial strain for each morphological cluster” = this mean no replicates?
Page 5 lines 172-173 “In general the microbial count on both obverse and reverse sides of the canvas case study was not particularly high.” = a reference is needed.
Page 5 lines 174-175 “and the possibility that not all the fungal strains might have been cultured” = as a general rule fungi are more easily cultivated in lab conditions than bacteria (cfr Sterflinger, 2010) why authors suppose that in this particular case fungi are non so easy to cultivate?
Page 5 lines 176-181 = the discussion of the microbiological colonization of this particular artwork is outside the scope of this study; this part can be tuned here and discussed better in the Supplementary materials section (were authors can add also a morphological description of more common clusters (I, VII, X, V for bacteria and IV, IX, II, XIII for fungi).
Page 5 Figure 2. The legends with the list of colors for each cluster are pleonastic because next to each column there are already labels indicating the cluster numbers. Perhaps it is better to add the absolute numbers alongside the percentage? I think it explains why so many clusters have equal percentages.
Page 5 line 190 “Phylogenetic characterization” = or “molecular characterization”?
Page 6 Table 1 & Table 2
-There are some missing data (e.g. IV a, V a, V) Can authors indicate in the caption the reason?
-The formal binomial names should be reported in the complete form here
- in the captions of Table 2 “ITS gene se”=”se”?
Page 9 table 3
A columns with control values should be added
Page 10 table 4
Column with control values should be added and in the caption of Table 4 “oregano and cloves essential oils” but in this test was used only oregano essential oil
page 11 lines 305-308
“An effective method for EOs application could be to flow a thin film of EOs onto an evaporating surface and to place it close to the painting using some supports, so that the vapors of the EOs may reach homogeneously the painting surface, therefore avoiding direct contact of EOs with the pigments.” = a suggestion: EOs on the lids of petri dishes and plates overturned it is a litte different: it is a confined environment.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
The antimicrobial properties of essential oils have been known for many centuries. The authors evaluated biocide activity of selected EOs against fungal and bacterial collections that isolated from canvas. While research topic is potentially interesting work, but the data is lacking rigorous statistical analysis and controls.
Suggestions:
- The novelty of this article is unclear. Please revise it in Introduction section.
- The concentration of EOs is absent in the present manuscript.
- The employed EOs is not easy to obtain. Authors should compare the activity of commercial EOs and employed EOs.
- Authors should address some biocide activity comparisons in Discussion section. For example,
- Conservation Issues of Modern Oil Paintings: A Molecular Model on Paint Curing Acc Chem Res 2019 17; 52(12): 3397-3406.
- Physiology of biodeterioration on canvas paintings. J Cell Physiol 2018, 233(4): 2741-2751.
- The references should be updates. 38/54 cited papers before 2016.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
Essential oils (EOs) from plant derivates with their biocides have been recently used to control microbial colonization on artifacts. However, applying EOs directly on the surface might pose unpredictable reactions to the artifacts. In this paper, the authors identified microorganisms associated with discolorations on a canvas and investigated the biocide activity of two plant derivatives (oregano and cloves essential oils on microorganisms in contact and contactless tests using cultivation and molecular methods. Penicillium, Aspergillus, and Cephaloteca fungal and Bacillus genus dominated the oil painting. Oregano EOs showed a stronger inhibition effect than cloves in both contact and contactless tests. The novelty of this work is to show inhibition effects of the volatile components of oregano EOs on biodeteriogens on oil paintings, which opens new ways to conserve oil paintings.
Overall, the manuscript is very well written. Study design and methods are appropriate and described adequately. Results appear to have been analyzed and interpreted clearly and appropriately.
I think this paper is a great fit for the Special Issue "Microbial Communities in Cultural Heritage and Their Control".
The only comment I have for the author is in Section 3.4. I think the contactless test showed the novelty of this work. The antimicrobial inhibition of oregano EOs has been studied widely. It would be great to have some discussion on the key volatile components of the oregano EOs, the inhibition mechanism, and the potential link between these components and identified bacteria/fungi here. Some of the information might explain the varied effects observed in this study. In terms of application, it would be helpful to have some discussion on the minimum inhibitory concentration and expose period. Do lighting and temperature conditions affect the efficacy of these EOs?
Author Response
Please see the attachment
Round 2
Reviewer 1 Report
The revised version of the paper by Gatti et al. has been deeply improved, and the authors have clarified the “missing points” of the previous version.
I think that this new version is suitable for publication with a few minor revision in the Supplementary materials:
- The Table S1 did not shown all the degree of the scale: [–] = no inhibition is missing
- Caption of Table S2: Cauterization = characterization?
- Caption of Table S2: Cauterization = characterization? Images in the column “Morphology” are useful, but a scale bar is needed.
- In Table S4 and Table S5 formal binomial names should be reported: in the case, of course, of a successful identification: Bacillus sp. is correct, but Bacillus subtilis subsp. subtilis is not in the complete form; the author's names are needed as reference.
-Figure S1 authors wrote: “For each cluster it is indicated the number of the isolated strains.” but in the figure the number is not shown neither in fig1a nor in fig1b. Wrong image? The cluster XIII in fig1a shloud be labelled as “5% (n=4)”
Author Response
Reviewer 1
- The Table S1 did not shown all the degree of the scale: [–] = no inhibition is missing
Answer:
Table S1 shows examples of contactless test representing different inhibition halo values. As, in this test all the fungi were inhibited by EOs, we did not indicate the symbol of no inhibition.
- Caption of Table S2: Cauterization = characterization?
Answer:
Page1 lines 7-8: Cauterization was changed with Clusterization
- Caption of Table S2: Cauterization = characterization?
Answer:
Page2 line 15: Cauterization was changed with Clusterization
- Images in the column
“Morphology” are useful, but a scale bar is needed.
Answer:
We thanks the Reviewer 2 for this observation. We added the scale bar
- In Table S4 and Table S5 formal binomial names should be reported: in the case, of course, of a successful identification: Bacillus sp. is correct, but Bacillus subtilis subsp. subtilis is not in the complete form; the author's names are needed as reference.
Answer:
Where it was possible, we added author's names as identification reference for the isolates. Where it was no possible to reach the author's names because it was no reported in NCBI database, we indicate the abbreviation NA= any author has been reported in NCBI database.
-Figure S1 authors wrote: “For each cluster it is indicated the number of the isolated strains.” but in the figure the number is not shown neither in fig1a nor in fig1b. Wrong image? The cluster XIII in fig1a should be labelled as “5% (n=4)”
Answer:
We added all the numbers related to the abundance of the isolates.
Reviewer 2 Report
English editing for this manuscript is suggested.
Author Response
Reviewer 2
English language revision is required.
Answer:
A more thorough review of the language was carried out. All corrections are visible with the track change.
