Combined Biocidal Effect of Gaseous Ozone and Citric Acid on Acinetobacter baumannii Biofilm Formed on Ceramic Tiles and Polystyrene as a Novel Approach for Infection Prevention and Control

Round 1

Reviewer 1 Report

Dear authors the article describes a simple study on developing an effective method for A. baumannii biofilm reduction. The manuscript is well written with good English and results presentation structure. The comment that could improve quality of manuscript are listed below.

 

Comments;

1.       The reason of choose of 15% of citric acid concentration and 10 minutes of exposure time for surface treatment should be clearly explained in materials and methods and discussion section.

2.       The figures 1 and 2 could be formatted by omitting the horizontal lines and frame.

Author Response

Response to Reviewer #1 of the manuscript ID: processes- 1889076 entitled: Combined Biocidal Effect of Gaseous Ozone and Citric Acid on Acinetobacter baumannii Biofilm Formed on Ceramic Tiles and Polystyrene as a Novel Approach for Infection Prevention and Control” by Kaća Piletić, Bruno Kovač, Matej Planinić, Vanja Vasiljev, Irena Brčić Karačonji, Jure Žigon, Ivana Gobin and Martina Oder

 

General response:

The Authors would like to thank Reviewer #1 for her/his valuable comments on our manuscript. We have responded to all of the comments and revised the paper accordingly. Detailed responses to each comment are provided below.

Comment 1:  The reason of choose of 15% of citric acid concentration and 10 minutes of exposure time for surface treatment should be clearly explained in materials and methods and discussion section.

Response: Authors would like to thanks for this comment and emphasise that 15% citric acid was used in mentioned concentration because it was available on the market in that concentration and all marketed biocides proposed this concentration for cleaning purposes. In order to go in line with manufacturers propositions authors used mentioned concentration. Exposure time in duration of 10 minutes was chosen due to it’s practical application and usual time for achieving biocidal activity for majority of available disinfectants. According to Reviewer’s comment, manuscript was changed in sections Materials and methods and Discussion with appropriate information regarding concentration and exposure time for citric acid.

Comment 2: The figures 1 and 2 could be formatted by omitting the horizontal lines and frame.

Response: Authors would like to thanks for the comment. Manuscript was changed in a way that Figure 1 and Figure 2 was depicted without horizontal lines and frames.

Author Response File: Author Response.docx

Reviewer 2 Report

Acinetobacter baumannii interesting has ability to form biofilm on abiotic and biotic surfaces. This Gram-negative opportunistic pathogen form biofilm. Because of biofilm formation, A. baumannii can survive long stretches of time in harsh environment like hospital inanimate surfaces. Biofilm represents bacterial congregation embedded in self-produced extracellular polymeric substance (EPS) that serves as shield and protects bacteria within from environmental influence.

In this manuscript, the authors describe different protocols for applying ozone and citric acid biocides to A. baumannii biofilm on ceramic and polystyrene surfaces. The authors found that the most effective protocol that treats surfaces with the sequence citric acid-ozone-citric acid.

The research in this paper is very significant in terms of developing an effective biocide (degradable into non-toxic components) for healthcare facilities and containing relatively inexpensive components.

The experiment is well designed and the results are accompanied by adequate figures of experimental outcomes.

Minor.

In the introductory part, the Authors could show EPC in a little more detail and if there is a chemical formula for this polymer, then they could show it.

In the discussion part, the Authors could provide a schematic representation of the action of ozone and citric acid: for example, the site of action, whether they destroy EPCs, penetration through individual bacterial membranes, action on certain biochemical pathways, etc.

What do the authors think if, in addition to this combination, a combination with some surfactants that destroy or at least increase the permeability of the lipid membrane would be applied?

Author Response

Response to Reviewer #2 of the manuscript ID: processes- 1889076 entitled: Combined Biocidal Effect of Gaseous Ozone and Citric Acid on Acinetobacter baumannii Biofilm Formed on Ceramic Tiles and Polystyrene as a Novel Approach for Infection Prevention and Control” by Kaća Piletić, Bruno Kovač, Matej Planinić, Vanja Vasiljev, Irena Brčić Karačonji, Jure Žigon, Ivana Gobin and Martina Oder

General response:

The Authors would like to thank Reviewer #1 for her/his valuable comments on our manuscript. We have responded to all of the comments and revised the paper accordingly. Detailed responses to each comment are provided below.

Comment 1:  In the introductory part, the Authors could show EPC in a little more detail and if there is a chemical formula for this polymer, then they could show it.

Response: Authors would like to thanks for this comment. Extracellular polymeric substances represent complex mixture of interlaced biological polymers, primarily polysaccharides, proteins and extracellular DNA. Each bacterial biofilm differs in the content of EPS. Authors wish to emphasise that it was not the intent of this study to determine content of A. baumannii biofilm EPS before and after the treatments, only to determine combined biocidal effect of ozone and citric acid on the number of bacteria due to the lack of chemicals needed for determination of sugars in EPS. Also, authors wish to emphasise that determination of EPS content after the treatments will be a part of further investigation into the ozone/citric acid mechanism of action when funds and chemicals will be available.

Comment 2: In the discussion part, the Authors could provide a schematic representation of the action of ozone and citric acid: for example, the site of action, whether they destroy EPCs, penetration through individual bacterial membranes, action on certain biochemical pathways, etc.

Response: Authors would like to thank for the comment. Authors changed manuscript in the way of providing schematic representation of gaseous ozone and citric acid combined effect on A. baumannii biofilm in the Discussion section.

Comment 3: What do the authors think if, in addition to this combination, a combination with some surfactants that destroy or at least increase the permeability of the lipid membrane would be applied?

Response: Authors would like to thank for the comment. Yes, Reviewer 2 is right when suggesting that application of surfactants before disinfection could enhance disinfection efficacy regarding changes in the permeability of cell membrane. Detergents, especially stronger ones can serve as surfactants before the disinfection protocol, so all protocols stress out the need for typical cleaning before disinfection. Authors have change manuscript in Discussion part adding wording detergents as surfactants and emphasising need for mechanical cleaning with detergents before the treatments.

Author Response File: Author Response.docx