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Reviewed by:  Liu Xuyu, Clarence Sim, Hashmath Fatimah, Samantha Quah, Sreelakshmi Cheruvalli, Hana Marican and Viduthalai Rasheedkhan Regina

Singapore Centre for Environmental Life Science Engineering, Nanyang Technological University, Singapore

Summary:

The paper discusses plasma activated water (PAW) as a pre-treatment strategy for chronic wounds infected with biofilm forming bacteria and presented findings based on E. coli biofilm formed on glass as well as on fixed keratinocyte monolayer. To investigate the effect of PAW, an in-vitro biofilm model with keratinocyte monolayer was treated with PAW. Routine clinical antiseptics eg polyhexamethylene biguanide (PHMB), povidone iodine (PI), and a commercially available medical-grade manuka honey were tested after the PAW pretreatment and compared with sterile water. [V(1]  While the PAW pre-treatment has shown very effective in killing bacteria in biofilm, the choice of experimental system for this study is raising some interesting questions and will require further background and justification.

Major comments:

Pg 3: Keratinocyte culture experiment: fixing keratinocyte and using that as a wound model appears to be a new way of studying in vitro wound system, which is not widely reported. This requires further justification, either in the experimental section or discussion section. Furthermore, it also raises the question of potential toxicity of PAW on host cells due to its low pH. Authors may also consider using alternative systems eg 3D skin model or animal model to address the effect of PAW on skin cells. Perhaps, the reasons for not working with such alternatives such as cost etc. can also be discussed in the discussion section. Alternatively, since the mode of action has been identified to be RONS generation and the low pH of PAW does not have an effect on bacterial viability, the PAW could be adjusted to a neutral form in order to minimise toxicity on host cells.

Pg 8: graph can be represented better by explaining the experimental controls. For example, indicate that the blue line (control) is the starting concentration of cells.

The authors may want to discuss what is the difference between using other ROS producing antiseptics VS PAW. Perhaps, the strength of alternatives VS PAW can be discussed in the discussion section.  

Minor comments:

Pg 6: The experimental set up requires that PAW was removed after 15 mins and applied antiseptics. It will be interesting to explore if PAW can induce killing after being applied for a prolonged period of time.

Figure 3 can be split into 3A, 3B and 3C for clarity. There is formatting issue with the asterisk in figure.

Fig 4A, the error bar cannot be clearly seen. Fig 4B, the shapes used to represent different groups could not be seen clearly. 

Since nitrogen species has the highest quantities out of all RONS, the authors may explain if one of the scavengers used can scavenge RNS.

Figure 4D: the SEM images might not be a good tool to convey the information the authors want to show. Figure A and C strongly suggested there is membrane perturbation, however, this is not represented in the SEM images. We suggest the authors may use confocal imaging by using membrane dyes.

The effectiveness of PAW on polymicrobial models to capture the context of chronic wound can also be discussed as a potential next step in this research.

Competing interests

The author declares that they have no competing interests.