We are disappointed that Dr Brelles-Marino feels that her work was not given sufficient prominence in our recent publication in PloS One. We are aware of her contributions to this area of research and cite several of her publications in our paper.

However, we feel we have not made the broad based claims Dr Brelles-Marino seems to find within our paper. In our paper we make it very clear that we were completely focused on the use of kHz driven atmospheric plasma jets operating in He/O2 mixtures with low (~ 2l/min) gas flows and on interpreting our results in the context of the unique physical and chemical environments created in such jets. We therefore focused our referencing to address that environment.

Whilst we agree that we have used techniques in this paper, which Dr Brelles-Marino has also employed in her work, for any biofilm research group, such as ours, these are standard techniques, which are widely employed across the entire spectrum of our published biofilm research. Any suggestion that her group have ‘pioneered’ these techniques is misleading. Adopting appropriate standard techniques (which we correctly reference throughout and for which we make no claims of novelty) is a widely accepted, normal practice in such studies. We note several fundamental differences in our experimental set-up and, in our opinion; the comments made on the lack of novelty in our work are both incorrect and inappropriate.

Regarding the Zelaya et al., 2010 paper, which we are accused of ignoring, we found there only a very brief description of a commercial plasma jet device operating at 13.56 MHz in He/N2 at a flow rate of 20.4 L/min. There was no discussion of the nature of the jet. The plasma jet described in the reference to which the reader was directed (their reference 19) was of a completely different design to that described in Zelaya 2010. There was therefore insufficient information provided to allow us to assess the physical and chemical environment generated with that plasma jet and thus its relevance to our work. The biofilm growth models are also entirely different. We employ both a dedicated biofilm device (MBEC assay, an ASTM-approved method for biocide biofilm susceptibility testing) and a continuous flow biofilm growth chamber, and confocal laser scanning microscopy to establish 3-D images which illustrate extent and distribution of cell kill, biofilm thickness and effect of plasma exposure on the overall 3-D structure of the exposed biofilms.

It is also difficult to directly compare time-kill data between our study and that of Zelaya et al., 2010, since loss in viability is plotted as % survived cells on a normal-scale y-axis and so does not allow direct comparison of actual viable cell reduction. The term “almost 100% inactivation” makes comparison to our work difficult, since extent of reduction in viable count is impossible to assess. The lack of gas only control in this work also makes comparison of time-kill data difficult. One would fully expect that gas flow in excess of 20 L/min, (over ten times that used in our study), would physically remove a proportion of the biofilm resulting in apparent loss of viability, as noted by other groups in the field.

Finally, regarding Dr Brelles-Marino’s minor criticisms of our data interpretation: specifically, the lack of gas-only control for the bacterial growth inhibition zones experiment. We did not deem a gas-only control necessary in this experiment since we simply sought to visually define the extent (diameter) of the zone of bactericidal species generated, as we clearly stated. Nevertheless, we included the gas-only control in the Live/Dead-Confocal experiment (Fig. 7), observing negligible effects on P. aeruginosa viability over all exposure times tested. We wholeheartedly agree with Dr Brelles-Marino on the importance of appropriate gas-only controls, especially at high gas flow rates where mechanical removal of the biofilm is likely. Finally in response to the comment “the authors cannot conclude whether the increase in biofilm eradication is due to the change in frequency or to a combination of both frequency and temperature”. Perhaps it is worth stating that we did not attempt to make any such conclusion, nor to claim that the increased temperature alone was the cause of increased eradication rate, a point we addressed directly in our discussion. This is a subject of ongoing investigation in our group.

We hope these comments adequately address the criticisms and comments made by Dr Brelles-Marino regarding our work.