*2.4. Taguchi Method*

Four control factors of the Taguchi experiment including (A) application voltage (kV), (B) CAPJ downstream-sample distance (mm), (C) Ar gas flow rate (sccm), and (D) CAPJ treatment time (s) were applied to analyze the influence of the sterilization parameters on *E coli.* Three levels were considered for each factor and shown in Table 1. Table 2 represented the Taguchi design and structure of L9 orthogonal array reflecting the parameters and levels in Table 1. The higher the better concept was applied to estimate the significant parameters of CAPJ sterilization treatment by data analysis and signal-to-noise (S/N) ratios. The contribution percentages of individual parameters were determined by analysis of variance (ANOVA) [26]. The S/N ratio is defined as follows:

$$\frac{S}{N} = -10 \times \log \left[ \frac{1}{n} \sum\_{i=1}^{n} \frac{1}{y^2} \right]$$

where *n* is the number of experiments and *y* is the observed data.

A confirmation test was carried out to validate the Taguchi's optimization approach. The summary statistic S/N at optimal conditions was calculated after the sanative parameters, which can be used to determine the optimal condition.

$$(\text{S/N})\_{\text{opt}} = \text{m} + (\text{m}\_{\text{x}} - \text{m}) + (\text{m}\_{\text{y}} - \text{m})$$

where m is the overall mean, and mx and my are the mean effect sensitive parameters at the optimal level [19]. The L9 orthogonal array for sample designation and detailed CAPJ treated parameters was tabulated in Table 3, which was the designed L9 orthogonal array in Table 2 filled with the corresponding parameters assigned in Table 1.


**Table 3.** The Taguchi L9 sample designation and detailed CAPJ parameters.

#### *2.5. Bacterial Viability Assay*

The LIVE/DEAD Bacterial Viability Kit (Thermo Fisher Scientific, Camarillo, CA, USA) was subjected to analyze the viability of bacterial populations based on the membrane integrity [27]. Two nucleic acid fluorescent dyes, SYTO9 and propidium iodide (PI), were used to determine the bacterial viability. The *E. coli* bacteria suspensions, which were untreated (S0) and CAPJ treated under S10, were collected and washed with PBS. The prepared samples were stained with SYTO9 and PI, according to the manufacturer's instructions. The stained bacteria were then analyzed by a confocal laser scanning microscope (LSM 780; Carl Zeiss, Göttingen, Germany). The quantification of fluorescence intensity was performed by using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

### *2.6. DNA Damage Assay*

A plasmid DNA, pGL3 (2 μg/μL) (Promega, Madison, WI, USA) was untreated (S0) or S10 CAPJ treated. Both the S0 and S10 CAPJ treated plasmids were then dissolved in DNA suspension bu ffer. DNA solution was loaded on 1.0% agarose gel for electrophoresis. Ethidium bromide-stained DNA was visualized under UV light. Photograph was taken by using a UV transilluminator (Azure Biosystems c400; Dublin, CA, USA).

#### *2.7. Field Emission Scanning Electron Microscope Analysis*

The morphologies of untreated S0 and S10 CAPJ treated bacteria were analyzed using a field-emission scanning electron microscope (FE-SEM, JSM 6701F, JEOL, Akishima, Japan). The bacteria samples were fixed in slides with 2% glutaraldehyde for 2 h, followed by washing with saline solution, and then exposed to 25%, 50%, and 75% of ethanol for 20 min, respectively, and finally immersed in 100% of ethanol for one hour. The slides were dried using a critical point dryer for 2 h afterwards. The bacteria samples were coated with a thin platinum layer around 5 nm thick by a sputter system (JFC 1600, JEOL, Akishima, Japan).

#### *2.8. In Vivo Evaluation*

Animal experiments were performed in accordance with the ethical standard approved by the Institutional Animal Care and Use Committee, Chang Gung University (Approval No. CGU105-032). Three male Sprague-Dawley (SD) rats with body weight range of 550 ± 30 g were study. The animals were housed under controlled conditions of temperature of 21–22 ◦C, relative humidity of 55% to 65%, and a 12 h light/dark cycle with artificial lighting. The animals received a standard feed and water ad libitum and were acclimatized under the aforementioned conditions before wounding experiment to create one full-thickness wounds with 17 mm in diameter on each side of rat's shoulder as described in literature [28,29]. The right-side wounds were treated with S10 CAPJ, and the left side wounds were kept untreated as control (S0). Two bacterial swabs were immediately taken from each superficial wound site right after CAPJ treatment on day 0 and day 4. Swabs were immediately immersed in 3 mL PBS and inoculated 200 μL sample on LB agar plates. After incubation at 37 ◦C for 24 h, the CFUs were counted.
