Predictive Modeling of UV-C Inactivation of Microorganisms in Glass, Titanium, and Polyether Ether Ketone

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I am not sure what is the novelty of this research? UVC germicidal lights are so far used very successfully for surface sterilization in hospitals and other medical applications. As a result we got overview of susceptibility of different causative agents of nosocomial infections to different UVC dosage and applied time. And also, we got some insight into surface topography on the efficacy of UVC treatment. But I don't see any solutions how to overcome problems of nosocomial infections in hospital environments. Also, microbial strains used in this research are those from ATCC Collection. I think that authors should consider using original isolates from hospital environment or some other clinical isolates, and in that case ATCC strains could make a nice control group. 

Overall, this is a study that has an applied impact but does not help to resolve the problems of implant device-associate infections. However, the study is very well done technically and very good presented, and it will surely find its readers, but it does not bring any novel knowledge to the topic,

 

Author Response

Dear Professor Hana Li, Editor of Microbiology Research

The authors would like acknowledge the careful and insightful review performed by all the Reviewers. We carefully analysed all comments and did our best to meet the Reviewers expectations. In our opinion, thanks to the Reviewers suggestions, we improve the clarity, quality and impact of the manuscript.

Reviewers responses are displayed in blue. The revised manuscript will display the changes highlighted in yellow.

We would like to highlight that all plots units that possessed an hyphen were updated to minus.

Finally, Amira Chorudi e-mail updated her personal e-mail to the Institutional e-mail.

 

Reviewer 1:

Reviewer Comment 1: “I am not sure what is the novelty of this research? UVC germicidal lights are so far used very successfully for surface sterilization in hospitals and other medical applications. As a result we got overview of susceptibility of different causative agents of nosocomial infections to different UVC dosage and applied time. And also, we got some insight into surface topography on the efficacy of UVC treatment. But I don't see any solutions how to overcome problems of nosocomial infections in hospital environments. Also, microbial strains used in this research are those from ATCC Collection. I think that authors should consider using original isolates from hospital environment or some other clinical isolates, and in that case ATCC strains could make a nice control group.”

 

Response 1: We would like to acknowledge the insightful comments of the Reviewer. We completely agree with the Reviewer: 1. UV-C as germicidal light application is already extensively used for decades. 2. There are some insights on the surface topography influence. 3. If this work included clinical isolates would have a greater impact. Nevertheless, our intended purpose was to exhibit the reduction rates models, that may be useful to provide a tailored dose of UV-C, which would ensure the disinfection of the biomaterial without or with minimal impact by the UV-C.  We selected the ATCC microorganisms as solid starting point. Future work will include methicillin resistant Staphylococcus aureus (MRSA), at least one bacteriophage as a virus model, and clinical isolates. However, we consider relevant the immediate report of the findings. We hope that the Reviewer agrees with our point of view.

 

 

 

 

Reviewer comment 2: “Overall, this is a study that has an applied impact but does not help to resolve the problems of implant device-associate infections. However, the study is very well done technically and very good presented, and it will surely find its readers, but it does not bring any novel knowledge to the topic.”

 

Response 2: We are deeply grateful the Reviewers complements to our work. Moreover, we would like to underscore that we believe that the precise analysis of the reduction models will represent a pivotal role in tackling future UV-C sterilization of current and future implants which are/will be hindered by extensive (standard) doses of UV-C.

 

Considering the Reviewer included the following sentence:

Line: 442 to 444: “Finally, the precise development of viability reduction models for a preselected range of microorganisms will be useful to safely tailor the UV-C dosage treatment for a spe-cific implant, with minimal impact on the material.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The issue of biomaterial disinfection is really, very relevant. The authors have done a great job of studying the resistance of microorganisms to UV-C radiation.

As noted in the introduction, UV-C radiation does not penetrate deeply into materials. This means that it can interact with the surface, being absorbed, reflected, scattered (if the surface is rough).

There was a typo in the description of samples/controls, lines 210-211: «Where Control corresponds to the microorganism concentration inoculated in the sample without UV-C exposure, and UV-C exposed represents the microorganism concentration inoculated in the sample without UV-C exposure.

It turns out that there were samples with/without UV-C irradiation, but there were no inoculate without slides or discs. For example, application of inoculate to culture plastic with/without UV-C exposure could be used as a control.

The manuscript does not contain absorption spectra for the materials studied. I'm guessing they're all very opaque to UV-C. Then that part of the light that could be absorbed by the materials can be converted into heat, and the sample can be heated to a certain temperature, determined by the heat capacity and thermal conductivity. It is necessary to analyze the possible materials heating and make experimental heating measurements of slides or discs depending on the radiation dose.

In the Table 1. Different UV-C dosage applied to the samples inoculated with the distinct microorganisms - the resulting radiation dose values are presented. Irradiation time is also an important parameter, please provide it as the second column. This way it will be possible to have an idea of the radiation power density. The radiation dose is important. In particular, it may depend on the surface roughness due to scattering properties. Similar to photodynamic therapy of thin layers of bacterial films, for example in G. Meerovich et al.: Photodynamic Action in Thin Sensitized Layers: Estimating the Utilization of Light Energy / doi: 10.18287/JBPE21.07.040301 (https://jbpe.ssau.ru/index.php/JBPE/article/view/3426)

It seems that the antibacterial effect in the experiment may partly depend not only on the destructive effect of UV-C radiation on biological molecules, but also on the heating of the samples during irradiation. It is necessary to include an experiment on measuring temperature and a discussion on this topic.

Author Response

Dear Professor Hana Li, Editor of Microbiology Research

The authors would like to acknowledge the careful and insightful review performed by all the Reviewers. We carefully analyzed all comments and did our best to meet the Reviewers expectations. In our opinion, thanks to the Reviewers suggestions, we improve the clarity, quality and impact of the manuscript.

Reviewers responses are displayed in blue. The revised manuscript will display the changes highlighted in yellow.

We would like to highlight that we revised the caption of Y-axis of Figures 3 and 5 and all plots units that possessed an hyphen were updated to minus.

Finally, Amira Chorudi e-mail updated her personal e-mail to the Institutional e-mail.

 

Reviewer 2:

Reviewer comment 1: The issue of biomaterial disinfection is really, very relevant. The authors have done a great job of studying the resistance of microorganisms to UV-C radiation.

Response 1: Please allows us to deeply acknowledge the Reviewers complement.

 

 

Reviewer comment 2: As noted in the introduction, UV-C radiation does not penetrate deeply into materials. This means that it can interact with the surface, being absorbed, reflected, scattered (if the surface is rough).

Response 2: We are in complete agreement with the Reviewer statement, thus the following sentence was added to the manuscript:

Line 355 to 356: It should be noted that UV-C has a very low penetration depth of solely approximately 2 µm [60].

 

Reference [60]: Meinhardt, M.; Krebs, R.; Anders, A.; Heinrich, U.; Tronnier, H. Wavelength-dependent penetration depths of ultraviolet radiation in human skin. J Biomed Opt 2008, 13, 044030, doi:10.1117/1.2957970.

 

 

 

 

Reviewer comment 3: There was a typo in the description of samples/controls, lines 210-211: «Where Control corresponds to the microorganism concentration inoculated in the sample without UV-C exposure, and UV-C exposed represents the microorganism concentration inoculated in the sample without UV-C exposure.

Response 3: Thank you for your through review, the typo revised:

Line 219 to 221: “Where Control corresponds to the microorganism concentration inoculated in the sample without UV-C exposure, and UV-C exposed represents the microorganism concentration inoculated in the sample subjected to UV-C exposure”.

 

Reviewer comment 4: It turns out that there were samples with/without UV-C irradiation, but there were no inoculate without slides or discs. For example, application of inoculate to culture plastic with/without UV-C exposure could be used as a control.

Response 4: The reduction was always calculated according to the inocula concentration, that was not exposed to UV-C. Thus, the viability reductions are very precise, in our point of view. However, the Reviewer is correct in stating that no inocula was tested with UV-C on culture plastic, as that would be another substrate that we did not analyze. However, we did not considered polystyrene as an interesting substrate to be tested. Polystyrene, in our point of view, is not a relevant material for implant applications, mainly due to its inherent stiffness and relative brittleness.

 

Reviewer comment 5: The manuscript does not contain absorption spectra for the materials studied. I'm guessing they're all very opaque to UV-C. Then that part of the light that could be absorbed by the materials can be converted into heat, and the sample can be heated to a certain temperature, determined by the heat capacity and thermal conductivity. It is necessary to analyze the possible materials heating and make experimental heating measurements of slides or discs depending on the radiation dose.

 In the Table 1. Different UV-C dosage applied to the samples inoculated with the distinct microorganisms - the resulting radiation dose values are presented. Irradiation time is also an important parameter, please provide it as the second column. This way it will be possible to have an idea of the radiation power density. The radiation dose is important. In particular, it may depend on the surface roughness due to scattering properties. Similar to photodynamic therapy of thin layers of bacterial films, for example in G. Meerovich et al.: Photodynamic Action in Thin Sensitized Layers: Estimating the Utilization of Light Energy / doi: 10.18287/JBPE21.07.040301 (https://jbpe.ssau.ru/index.php/JBPE/article/view/3426)

It seems that the antibacterial effect in the experiment may partly depend not only on the destructive effect of UV-C radiation on biological molecules, but also on the heating of the samples during irradiation. It is necessary to include an experiment on measuring temperature and a discussion on this topic.

Response 5: We understand and acknowledge the Reviewers insightful set of comments. We are very grateful for these highly relevant point of views that we missed. Thus, we included several analyses that we believe that meet the Reviewers interesting points:

 

Temperature:

We have analyzed the temperature variation to verify if the heating of the samples during irradiation would be a factor that would affect the reduction of the viability.

In the Material and Methods section we added a novel subsection numbered as 2.3. named: Temperature variation during irradiation. The following method is described:

Line 185 to 189: “The UV-C treatments were performed at room temperature that was approximately 22 °C. Nevertheless, to discard the temperature influence during UV-C treatment temperature at the sample holder position was monitored using an alcohol thermometer after: 5, 15, 30 and 60 minutes. To enhance the temperature variation resolution, ImageJ (1.54g, National Institutes of Health, Bethesda, MD, USA) software was used [33].”

Reference [33]:

The temperature variation was analyzed, and the results were displayed in the supplementary file. The temperature variation rate was estimated, and it is considered as negligible as stated in the Results and Discussion section:

Line 235 to 241: “The recorded temperature variation approximately followed a linear increase of 0.04 °C.min-1 (Supplementary file 1). The maximum exposure time was 1.5 min, thus the foreseen temperature increase is of approximately 0.06 °C. This temperature difference if far from the optimal temperature for the tested microorganism that ranged between 30 to 37 °C. Therefore, temperature was not considered as factor that interfered with the microorganisms viability, despite the different absorption and reflectance profiles exhibited by GLS, Ti and PEEK”.

 

In the supplementary file 1, the following supplementary Figure 1 was added:

Supplementary Figure 1. Temperature variation during UV-C exposure. The linear equation fitted to the results: y = 0.004x + 0.1103, r² = 0.9654.

 

Absorbance:

The section: 2.4. Absorbance and reflectance was added, describing the following:

Line 190 to 193: “The absorbance and reflectance of the GLS, Ti and PEEK were analyzed in a UV-visible light spectrometer Shimadzu UV-2600 (Kyoto, Japan), using a wavelength range between 200 to 800 nm, and a resolution of 1 nm.”

 

The following results were included:

Line 242 to 255: “The UV-C absorbance and reflectance spectra of GLS is displayed in Figure 2.

 

 

Figure 2. GLS absorbance and reflectance spectra.

 

It is clear the minimal absorbance of GLS until 327 nm (UV-A), it sharply increas-es, reaching its maximum at 283 nm (UV-B). At 254 nm, the maximum of irradiation provided by the low-pressure mercury lamp used, the absorbance is considerable [35]. The reflectance at 254 nm is solely 1.0 %. Overall, the maximum recorded reflectance was nearly 3.4 %. Thus, reflectance may be considered negligible, with nearly all UV-B and UV-C irradiation being absorbed by GLS. This is accordance to the literature that states the opaque nature of GLS below near UV-B irradiation wavelengths. Only at these lower  wavelengths the photons possess the required energy to excite the silica (the main GLS material) across the band gap, leading to its absorption [36]”.

Reference [36]: Doremus, R.H. Glass Science; Wiley: 1994.

 

And:

Line 350 to 380: “The absorption and reflectance spectra of Ti and PEEK are depicted in Figure 4.

 Figure 4. Ti and PEEK absorbance and reflectance spectra.

 

It should be noted that UV-C has a very low penetration depth of solely approximately 2 µm [61].

The absorption of TI and PEEK was unexpectedly higher in the visible region, in comparison to GLS due to their opacity. The absorbance starts to rise further in Ti at 249 nm and reaches its maximum at 216 nm. Nevertheless, exhibited lower absorption levels then GLS. PEEK absorbance starts to increase at 241 nm and spikes at 205 nm. It exhibits a similar absorbance level similar to GLS. The reflectance of Ti, that is nearly 32 % for the higher wavelength tested, consistently decreases, and further drops at nearly at 350 nm stabilizing at approximately 300 nm. PEEK reflectance, that was nearly two-fold lower than Ti at the highest wavelength, also consistently slightly de-creased until approximately 425 nm. At lower wavelengths, the PEEK reflectance low-ers more rapidly until nearly 5 % from 385 nm to 200 nm. The discrepancy between the absorption and reflectance profiles may highlight the higher scattering in Ti in com-parison to GLS. Therefore, the Ti may effectively scattered light until the UV-C region where this irradiation is able to excite electrons from the valence band to the conduction band leading to its absorbance [62,63]. Nevertheless, the absorbance range of UV-C light seems to occur at a slightly lower wavelength than the 254 nm provided by the low-pressure mercury lamp used. Thus, based on these results the UV-C absorbance by Ti may not be directly correlated with microorganism’s loss of viability. Nevertheless, a transmittance spectra that exhibits nearly full absorption of UV light below nearly 380 nm may be found in the literature [64]. These differences may be based on the different surface treatments underwent by the Ti. PEEK, is described to nearly ab-sorb the entire irradiation between 300 and 380 nm [65]. This is corroborated by the reflectance profile. However, the absorbance profile does not display the same tendency as described in the literature, that is inversely proportional to the observed reflectance curve [65,66]. This may be due the very high absorbance values throughout the entire UV-visible light spectra.”

References:

[62] Lu, S.-y.; Wu, D.; Wang, Q.-l.; Yan, J.; Buekens, A.G.; Cen, K.-f. Photocatalytic decomposition on nano-TiO2: Destruction of chloroaromatic compounds. Chemosphere 2011, 82, 1215-1224, doi:https://doi.org/10.1016/j.chemosphere.2010.12.034.

[63]     Nakhaei, K.; Ishijima, M.; Ikeda, T.; Ghassemi, A.; Saruta, J.; Ogawa, T. Ultraviolet Light Treatment of Titanium Enhances Attachment, Adhesion, and Retention of Human Oral Epithelial Cells via Decarbonization. Materials 2021, 14, 151.

[64]     Liu, B.; Zhao, X.; Zhao, Q.; He, X.; Feng, J. Effect of heat treatment on the UV–vis–NIR and PL spectra of TiO2 films. Journal of Electron Spectroscopy and Related Phenomena 2005, 148, 158-163, doi:https://doi.org/10.1016/j.elspec.2005.05.003.

[65]     Giancaterina, S.; Rossi, A.; Rivaton, A.; Gardette, J.L. Photochemical evolution of poly(ether ether ketone). Polymer Deg-radation and Stability 2000, 68, 133-144, doi:https://doi.org/10.1016/S0141-3910(99)00181-0.

[66]     Švorčı́k, V.; Prošková, K.; Rybka, V.; Vacı́k, J.; Hnatowicz, V.; Kobayashi, Y. Changes of PEEK surface chemistry by ion irradiation. Materials Letters 1998, 36, 128-131, doi:https://doi.org/10.1016/S0167-577X(98)00030-5.

 

We hope these two responses are in line with the requested performed by the Reviewer.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I accept authors response to my criticism, and hence all my concerns regarding this manuscript are eliminated. So, I agree with the publication... 

Author Response

Dear Professor Hana Li, Editor of Microbiology Research

The authors would like kindly acknowledge the inputs performed by the Reviewers in this second round. We thoroughly analyzed all comments and performed further improvements, that, in our opinion, enhanced the quality of the manuscript.

The English was carefully revised.

Reviewers responses are displayed in blue. The revised manuscript will display the changes highlighted in light blue for this second round of review.

 

Reviewer 1:

Reviewer Comment 1: “I accept authors response to my criticism, and hence all my concerns regarding this manuscript are eliminated. So, I agree with the publication...”

 

Response 1: We would like to express our deep gratitude for the positive comments performed by the Reviewer. Foremost for the insightful suggestions.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks to the authors for carrying out additional measurements. Understanding the possible heating of samples is indeed very important. The authors are great for trying to measure the temperature of the holder position, but I’m not sure that an alcohol thermometer is suitable for this. Also, the question is rather about how hot the materials under study are during sterilization. The ideal solution to answer the question would be to use a thermal camera (or a thermocouple hidden under the sample) to determine the surface temperature of all three samples. And this does not take into account the absorption of radiation by microbes, which also exists.

There is still a question with the radiation power density, which could be calculated from Table 1 if the time were indicated there. I believe this can be resolved by enabling power density [mW/cm²] as a unit of measurement on the radiometer. Then the total radiation dose [J/cm²] will easily be equal to the product of power density and time. Equation 1 on line 182 is not needed. It is better to simply indicate the radiation power density in the sample area.

Author Response

 

 

Dear Professor Hana Li, Editor of Microbiology Research

The authors would like kindly acknowledge the inputs performed by the Reviewers in this second round. We thoroughly analyzed all comments and performed further improvements, that, in our opinion, enhanced the quality of the manuscript.

The English was carefully revised.

Reviewers responses are displayed in blue. The revised manuscript will display the changes highlighted in light blue for this second round of review.

 

Reviewer 2:

Reviewer comment 1: Thanks to the authors for carrying out additional measurements. Understanding the possible heating of samples is indeed very important. The authors are great for trying to measure the temperature of the holder position, but I’m not sure that an alcohol thermometer is suitable for this. Also, the question is rather about how hot the materials under study are during sterilization. The ideal solution to answer the question would be to use a thermal camera (or a thermocouple hidden under the sample) to determine the surface temperature of all three samples. And this does not take into account the absorption of radiation by microbes, which also exists.

 

Response 1: The Reviewer is absolutely correct, so we followed the Reviewer suggestion. We maintained the results from the thermometer and complemented with the data collected using a thermographic camera directly pointed at the samples. Added text in the Materials and Methods section:

Line 189 to 192 : “Furthermore, to analyze the temperature variation of GLS, Ti and PEEK, their temperature when exposed to UV-C was monitored using a thermographic camera (testo 876, Titisee-Neustadt, Germany) and recorded at 5, 15, 30, 45 and 60 minutes.”

 

The following text was added to the manuscript:

Line 238 to 254: “The recorded temperature variation is exhibited in Figure 2.

 

 

Figure 2. Temperature variation: within the air flow chamber (thermometer), GLS, Ti and PEEK (thermographic camera) during UV-C exposure

 

The temperature increase ratio followed a nearly linear increase (Supplementary file 1).  The air flow chamber increased approximately 0.04 °C.min-1 (Supplementary file 1). The maximum exposure time was 1.5 min, thus the foreseen temperature in-crease is of approximately 0.06 °C. The temperature variation of the tested materials displayed: 0.01, 0.03 and 0.06 °C min-1 for GLS, Ti and PEEK, respectively. The maxi-mum exposure time was 1.5 min, thus the predicted temperature increase would be: 0.01, 0.04 and 0.08 °C for GLS, Ti and PEEK, respectively. The expected temperature increase is far from the optimal temperature for the tested microorganism that ranged between 30 to 37 °C. Therefore, temperature was not considered as factor that inter-fered with the microorganisms viability. However, it should be noted that PEEK exhib-ited a 1.5-fold higher rate than the environmental temperature. Whereas, GLS exhib-ited a nearly 4-fold lower temperature increase rate in comparison to the ambient temperature increase.

Line 269 to 270: “These results corroborate the lower temperature increase by GLS in comparison to Ti and PEEK.”

Line 395: “, which may result in the higher temperature variation”

Line 461 to 462: “The short UV-C exposure did not increase considerably the tested material temperature, nevertheless, this factor should not be disregarded.”

 

The following data was added to the Supplementary File 1:

Supplementary Table 1: Temperature variation during UV-C exposure estimated linear equations.

	Linear equation	r2
Air flow chamber	y = 0.004x + 0.1103	0.965
GLS	y = 0.01x – 0.02	0.907
Ti	y = 0.03x + 0.11	0.966
PEEK	y=0.06x + 0.13	0.962

 

 

 

 

Reviewer comment 2: There is still a question with the radiation power density, which could be calculated from Table 1 if the time were indicated there. I believe this can be resolved by enabling power density [mW/cm2] as a unit of measurement on the radiometer. Then the total radiation dose [J/cm2] will easily be equal to the product of power density and time. Equation 1 on line 182 is not needed. It is better to simply indicate the radiation power density in the sample area.

 

Response 2: Please allow us to respectfully disagree. Radiation dosage is, in our point of view, the most accurate and reader friendly way to describe how much energy is involved in the process. The use of power density, in our opinion, would only force the reader to do the math and figure out how much radiation dose is being used. This opinion is also vastly present in the literature. Please, check the following references:

1. Bernardy, C.; Elardo, N.; Trautz, A.; Malley, J.; Wang, D.; Ducoste, J. Effects of UV-C Disinfection on N95 and KN95 Filtering Facepiece Respirator Reuse. Applied and Environmental Microbiology 2022, 88, e01221-01222, doi:doi:10.1128/aem.01221-22.
2. Li, P.; Koziel, J.A.; Zimmerman, J.J.; Zhang, J.; Cheng, T.-Y.; Yim-Im, W.; Jenks, W.S.; Lee, M.; Chen, B.; Hoff, S.J. Mitigation of Airborne PRRSV Transmission with UV Light Treatment: Proof-of-Concept. Agriculture 2021, 11, 259.
3. Maguluri, R.K.; Nettam, P.; Chaudhari, S.R.; Yannam, S.K. Evaluation of UV-C LEDs efficacy for microbial inactivation in tender coconut water. Journal of Food Processing and Preservation 2021, 45, e15727, doi:https://doi.org/10.1111/jfpp.15727.
4. Boegel, S.J.; Gabriel, M.; Sasges, M.; Petri, B.; D’Agostino, M.R.; Zhang, A.; Ang, J.C.; Miller, M.S.; Meunier, S.M.; Aucoin, M.G. Robust Evaluation of Ultraviolet-C Sensitivity for SARS-CoV-2 and Surrogate Coronaviruses. Microbiology Spectrum 2021, 9, e00537-00521, doi:doi:10.1128/Spectrum.00537-21.
5. Schleusener, J.; Lohan, S.B.; Busch, L.; Zamudio Díaz, D.F.; Opitz, N.; Sicher, C.; Lichtenthäler, T.; Danker, K.; Dommerich, S.; Filler, T.; et al. Irradiation of human oral mucosa by 233 nm far UV-C LEDs for the safe inactivation of nosocomial pathogens. Scientific Reports 2023, 13, 22391, doi:10.1038/s41598-023-49745-3.
6. Lindblad, M.; Tano, E.; Lindahl, C.; Huss, F. Ultraviolet-C decontamination of a hospital room: Amount of UV light needed. Burns 2020, 46, 842-849, doi:https://doi.org/10.1016/j.burns.2019.10.004.
7. Santana-Jiménez, A.Z.; Quintero-Ramos, A.; Sánchez-Madrigal, M.Á.; Meléndez-Pizarro, C.O.; Valdez-Cárdenas, M.d.C.; Orizaga-Heredia, M.d.R.; Méndez-Zamora, G.; Talamás-Abbud, R. Effects of UV-C Irradiation and Thermal Processing on the Microbial and Physicochemical Properties of Agave tequilana Weber var. azul Extracts at Various pH Values. Processes 2020, 8, 841.
8. Eadie, E.; Hiwar, W.; Fletcher, L.; Tidswell, E.; O’Mahoney, P.; Buonanno, M.; Welch, D.; Adamson, C.S.; Brenner, D.J.; Noakes, C.; et al. Far-UVC (222 nm) efficiently inactivates an airborne pathogen in a room-sized chamber. Scientific Reports 2022, 12, 4373, doi:10.1038/s41598-022-08462-z.
9. Biasin, M.; Bianco, A.; Pareschi, G.; Cavalleri, A.; Cavatorta, C.; Fenizia, C.; Galli, P.; Lessio, L.; Lualdi, M.; Tombetti, E.; et al. UV-C irradiation is highly effective in inactivating SARS-CoV-2 replication. Scientific Reports 2021, 11, 6260, doi:10.1038/s41598-021-85425-w.
10. Sabino, C.P.; Sellera, F.P.; Sales-Medina, D.F.; Machado, R.R.G.; Durigon, E.L.; Freitas-Junior, L.H.; Ribeiro, M.S. UV-C (254 nm) lethal doses for SARS-CoV-2. Photodiagnosis and Photodynamic Therapy 2020, 32, 101995, doi:https://doi.org/10.1016/j.pdpdt.2020.101995.
11. Sharma, A.; Mahmoud, H.; Pendyala, B.; Balamurugan, S.; Patras, A. UV-C inactivation of microorganisms in droplets on food contact surfaces using UV-C light-emitting diode devices. Frontiers in Food Science and Technology 2023, 3, doi:10.3389/frfst.2023.1182765.
12. Sliney, D.H.; Stuck, B.E. A Need to Revise Human Exposure Limits for Ultraviolet UV-C Radiation†. Photochemistry and Photobiology 2021, 97, 485-492, doi:https://doi.org/10.1111/php.13402.
13. Kohli, I.; Lyons, A.B.; Golding, B.; Narla, S.; Torres, A.E.; Parks-Miller, A.; Ozog, D.; Lim, H.W.; Hamzavi, I.H. UVC Germicidal Units: Determination of Dose Received and Parameters to be Considered for N95 Respirator Decontamination and Reuse. Photochemistry and Photobiology 2020, 96, 1083-1087, doi:https://doi.org/10.1111/php.13322.
14. Sun, W.; Jing, Z.; Zhao, Z.; Yin, R.; Santoro, D.; Mao, T.; Lu, Z. Dose–Response Behavior of Pathogens and Surrogate Microorganisms across the Ultraviolet-C Spectrum: Inactivation Efficiencies, Action Spectra, and Mechanisms. Environmental Science & Technology 2023, 57, 10891-10900, doi:10.1021/acs.est.3c00518.
15. Bhardwaj, S.K.; Singh, H.; Deep, A.; Khatri, M.; Bhaumik, J.; Kim, K.-H.; Bhardwaj, N. UVC-based photoinactivation as an efficient tool to control the transmission of coronaviruses. Science of The Total Environment 2021, 792, 148548, doi:https://doi.org/10.1016/j.scitotenv.2021.148548.
16. de la Rocha, C.; Caprara, C.d.S.C.; Poester, V.R.; Xavier, M.O.; Porte, A.F.; Galarça, M.M.; Filgueira, D.d.M.V.B.; Votto, A.P.d.S.; Ramos, D.F. Highly effective decontamination in a hospital environment: An easy-to-operate, low-cost prototype. Photochemistry and Photobiology n/a, doi:https://doi.org/10.1111/php.13945.
17. Ruetalo, N.; Berger, S.; Niessner, J.; Schindler, M. Inactivation of aerosolized SARS-CoV-2 by 254 nm UV-C irradiation. Indoor Air 2022, 32, e13115, doi:https://doi.org/10.1111/ina.13115.
18. Adhikari, A.; Parraga Estrada, K.J.; Chhetri, V.S.; Janes, M.; Fontenot, K.; Beaulieu, J.C. Evaluation of ultraviolet (UV-C) light treatment for microbial inactivation in agricultural waters with different levels of turbidity. Food Science & Nutrition 2020, 8, 1237-1243, doi:https://doi.org/10.1002/fsn3.1412.

 

Thus, no modifications to the manuscript were performed to the manuscript.

Author Response File: Author Response.pdf