Atmospheric Pressure Plasma Chemical Vapor Deposition of Carvacrol Thin Films on Stainless Steel to Reduce the Formation of E. Coli and S. Aureus Biofilms
, Gabriela F. da Silva 3, Iolanda S. Duarte 3, Milton E. Kayama 4, Elidiane C. Rangel 1 and Nilson C. Cruz 1,*Round 1
Reviewer 1 Report
I felt that this paper did not discuss about biofilm matters. This is the biggest problem for the paper. The bioassay for biofilms in this paper was based just on the bacterial number on materials. The most characteristic matter for biofilm should be EPS (External polymeric substances). However, they didn't mention nor measure them at all. Therefore, the word "biofilm" should be deleted from the title and instead, the term "antibacterial effect" should be inserted. Or they could add their additional experiments relating to EPS newly.
In addition, I think authors should add or modify the following points.
1: Line 62: The indent should be deleted for the title of section.
2: Line 75. The equation should be centered in the line.
3. Section 2.2: Fig.1 is fine. However, the detailed explanation about "dielectric barrier discharge plasma reactor" is missing. I feel it is needed. The authors should add the detailed explanation about the principle and the merits for the method.
4. line 112: The centering is appropriate? Please confirm this.
5. In Fig.2, the explanation mentioned in the text should be reflected. I mean, the authors should indicate the corresponding parts they described in the text.
6. The safety of Carvacrol was not explained. I presume the chemical would destroy the membrane of bacterial cells. The authors should explain about the mechanism in the discussion part and they should also mention its safety for human beings.
Author Response
Reviewer #1
Comment 1: I felt that this paper did not discuss about biofilm matters. This is the biggest problem for the paper. The bioassay for biofilms in this paper was based just on the bacterial number on materials. The most characteristic matter for biofilm should be EPS (External polymeric substances). However, they didn't mention nor measure them at all. Therefore, the word "biofilm" should be deleted from the title and instead, the term "antibacterial effect" should be inserted. Or they could add their additional experiments relating to EPS newly.
Reply: We have performed additional experiments and included SEM micrographs showing the substrates after bacterial incubation.
Comment 2: Line 62: The indent should be deleted for the title of section. Line 75. The equation should be centered in the line. Section 2.2: Fig.1 is fine. However, the detailed explanation about "dielectric barrier discharge plasma reactor" is missing. I feel it is needed. The authors should add a detailed explanation about the principle and the merits for the method. In Fig.2, the explanation mentioned in the text should be reflected. I mean, the authors should indicate the corresponding parts they described in the text. Line 112: The centering is appropriate? Please confirm this.
Reply: All suggested corrections have been made and are highlighted in red in the manuscript. The benefits of using dielectric barrier discharge plasmas include the fact that they operate at atmospheric pressure, which makes them easily scaled up for industrial applications of various film deposition schemes.
Comment 3: The safety of Carvacrol was not explained. I presume the chemical would destroy the membrane of bacterial cells. The authors should explain about the mechanism in the discussion part and they should also mention its safety for human beings.
Reply: The manuscript has been modified to include discussions on the mechanisms of bactericidal activity and health safety of carvacrol
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript “Atmospheric pressure plasma chemical vapor deposition of carvacrol thin films on stainless steel to reduce the formation of E. coli and S. aureus biofilms” reports a study on thin films from natural carvacrol deposited on stainless steel surfaces and their antimicrobial activity. The paper contains new and interesting experimental data, but there are some aspects to be improved:
1) Page 3 Line 124. The authors should provide a more detailed discussion on the formation of clusters. It is about "reactive species" from the essential oil or filaments? Does polymerization occur anywhere or only in the filament area and results in clusters?
2) Page 4 line 129 “t”is duration of the plasma deposition process?
3) Page 5 line 148 “…of atomic oxygen and other reactive species resulting from interactions between the plasma and the atmospheric air surrounding the samples…” What species? Regarding the further discussion about the densification of the film citing reference [20], maybe another example with a natural oil it might be more suitable than the one discussing the behavior of hexamethyldisiloxane.
4) Page 5 Chemical structural analysis – from the FTIR spectra of deposited film it is not possible to conclusion that a polymer was formed.
5) Page 6 What model was used for the computation of the surface energy and what is the reason to choose it?
6) Page 7 line 212 “....results in coatings highly cross-linked with good adhesion to the substrate.” No data is provided to support both part of the statement: about cross-linking and about adhesion.
7) Page 7 line 213 UV exposure does not means “harsh environment”.
8) Page 7 Line 213 The authors should provide some data to sustain the statement “plasma coatings are stable…”. The authors refer to chemical stability? The same for activity after 120 days.
Author Response
Reviewer #2
Comment 1: The authors should provide a more detailed discussion on the formation of clusters. It is about "reactive species" from the essential oil or filaments? Does polymerization occur anywhere or only in the filament area and results in clusters?
Reply: The clusters are formed during film growing. They can be formed when monomer fragments in the atmosphere interact, forming agglomerates excessively large to remain in the gas phase, or through the recombination of species adsorbed on the substrate. Although under our experimental conditions the discharges operate in filamentary mode, the coatings are deposited on the whole exposed area of the substrate, as confirmed by EDS mapping shown in Figure S6.
Comment 2: Page 4 line 129 “t” is duration of the plasma deposition process?
Reply: As defined in the manuscript, t is the duration of the depositions.
Comment 3: Page 5 line 148 “…of atomic oxygen and other reactive species resulting from interactions between the plasma and the atmospheric air surrounding the samples…” What species? Regarding the further discussion about the densification of the film citing reference [20], maybe another example with a natural oil it might be more suitable than the one discussing the behavior of hexamethyldisiloxane.
Reply: As included in the manuscript, the samples are surrounded by reactive oxygen species such as OH and O3. Furthermore, since, as long as we are aware, this is the first time that the deposition of coatings from natural oil using DBD discharge is reported, we could not find any reference regarding the densification of such films. In this sense, considering the similarity of the mechanisms involved on the densification of films deposited in oxygen containing plasmas, the cited reference on hexamethyldisiloxane film seems to be illustrative.
Comment 3: Page 5 Chemical structural analysis – from the FTIR spectra of deposited film it is not possible to conclusion that a polymer was formed.
Reply: The manuscript has been modified and the term “plasma polymerized” has been substituted by “plasma deposited” carvacrol films. However, it is important to stress that the deposition process of any organic material using plasmas is commonly referred to as “plasma polymerization” and, consequently, the material so produced is known as “plasma polymer”, even when the film does not have any repetitive unity.
Comment 4: Page 6 what model was used for the computation of the surface energy and what is the reason to choose it?
Reply: Surface energy has been calculated using the method proposed by Owens-Wendt based on harmonic mean, which has been considered the most universal procedure for such evaluation.
Comment 5: Page 7 line 212 “....results in coatings highly cross-linked with good adhesion to the substrate.” No data is provided to support both part of the statement: about cross-linking and about adhesion. Page 7 line 213 UV exposure does not means “harsh environment”. Page 7 Line 213 the authors should provide some data to sustain the statement “plasma coatings are stable”. The authors refer to chemical stability? The same for activity after 120 days.
Reply: We have evaluated the chemical and physical stabilities of the coatings exposing the samples to high power UV radiation, immersion in TSA culture media and storage in air for up to 120 days. As no significative modification has been observed after such tests, as illustrated, for instance, by the results in Figures S3 and S5 in the supplementary material, the samples can be considered stable.
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors report on the deposition of thin films from natural carvacrolextract using DBD plasma polymerization to inhibit bacterial adhesion and proliferation. Several experimental techniques were used to analyse the coatings. The authors showed that coating completely inhibited the adhesion of E. Coli and reduced up to 90% the formation of D. Aureus biofilm.
I have the following comments and suggestions for the revision of the manuscript which I list in the following:
There language needs to be corrected, grammar is bad and there are a lot of mistakes. The fact that plasma exposure enhances hydrophilicity is not new. But the question is is this hydrophilicity then stable for your coatings? Or do the coatings turn hydrophobic with time? How did this inhomogeniety of the coating with holes influence the adhesion of bacteria? Are there any SEM images available with the adhered bacteria on the coating?
If the authors respond and review the manuscript accordingly, I recommend it for publication in Materials.
Comments for author File: Comments.pdf
Author Response
Reviewer #3
Comment 1: There language needs to be corrected, grammar is bad and there are a lot of mistakes. The fact that plasma exposure enhances hydrophilicity is not new. But the question is, is this hydrophilicity then stable for your coatings? Or do the coatings turn hydrophobic with time? How did this inhomogeneity of the coating with holes influence the adhesion of bacteria? Are there any SEM images available with the adhered bacteria on the coating?
Reply: We have carefully revised the manuscript. As it can be observed in Figure S5, the aging for, at least 35 days, does not affect the wettability of the coatings.
EDS mapping revealed that the whole sample area, even the bottom part of the crater-like structures such as those observed in Figure 2, is covered by the films. In addition, we have not observed any influence of such structures on the bacterial adhesion to the samples. This is in agreement with the findings of several authors (see, for instance, Riedewald F., PDA J Pharm Sci and Tech, 2006, 60, 164-171) which have found that surface roughness in the range of 0.15 to 2.0 µm Ra, which is the case of our samples, has no significant influence on initial bacterial attachment.
We have included (Figure 7) SEM micrographs showing bacteria adhered (or the absence of bacteria) on surfaces as-received and after treatment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
The authors have responded properly. I recommend tha paper for publication.
