**4. Discussion**

Infectious disease remains a grea<sup>t</sup> challenge in the field of public health as it is among the top 10 causes of death and also leads the cause of disability [32]. Inadequate antibiotic therapy worsens the control of pathogenic microbes and leads to drug resistance. The discovery of new and effective antibacterial agents or methods is quite a challenge because of its time and financially consuming nature. In addition, poor sanitation and increasing international travel have made transmission of infectious diseases easier.

This study focused on the development and optimizations of a sterilization technique using CAPJ utilizing the Taguchi method to shorten to experimental time spent on trial and error. In general, a design of orthogonal arrays of Taguchi analysis provides a maximum number of main factors to be estimated to minimize experimental trials. S/N ratios are transferred from the responses in the bactericidal activity by CAPJ treatment. ANOVA can further determine the contribution of CAPJ operating parameters on the preferable efficiency of antimicrobial condition. The effectiveness of Taguchi's optimization approach is evaluated by antimicrobial effect, where 100% was obtained with the CAPJ using parameters obtained from the Taguchi method. As determined by the Taguchi method, the optimal antimicrobial activity is achieved with CAPJ parameters of application voltage 8.5 kV, APJ-sample distance 10 mm, Ar gas flow rate 500 sccm, and treatment time 300 s. We found CAPJ treatment time affected the plasma's efficacy the most, and it required 5 min of treatment to achieve 100% bactericidal activity. By extension, the principles and concepts of Taguchi approach can also benefit industry reducing experimental trials of the performance, quality, and cost [22,33].

A number of studies represent very promising results for infection control by atmospheric pressure plasma [25]. In this work, we demonstrated that CAPJ built in our lab inhibited bacterial growth. The beneficial e ffects of plasma in sterilization are not ye<sup>t</sup> fully understood, so we used our CAPJ to explore proposed mechanisms described in the literature. The observed temperatures of this CAPJ clearly indicated that consecutive plasma treatment by CAPJ remains below human body temperature for 30 min without causing thermal injury by utilizing the application voltage lower than 8.5 kV (Figure 2), which is an important requirement for biological applications. Previous studies reported that reactive oxygen, hydrogen peroxide, and UV photons produced by cold plasmas might target cell membrane and cell wall for antibacterial activity [34,35]. From the plasma diagnostics by OES (Figure 3), there is nearly no emission in the germicidal UV-C region around 254 nm [36]. The e ffect of heat and UV radiation as the main antimicrobial mechanism for plasma component, therefore, is unlikely.

On the other hand, The Ar mixed into He-based plasma described in this study contains OH and NO radicals, nitrogen and oxygen species, and metastable species of He and Ar (Figure 3), which probably interact with biological organisms to generate further reactive species [37]. Previous reports demonstrated ROS from di fferent technologies might target di fferent components of bacterial cells [38], which subsequently leads to the destruction of bacterial cell wall to achieve antimicrobial strategies. For example, ROS attacks the polyunsaturated fatty acids of the fatty acid membrane to initiate a self-propagating chain reaction [39], induces lipid peroxidation in Gram-negative bacteria [40], decomposes macromolecules such as DNA and protein [41], and breaks important C–O, C–N and C–C bonds of the peptidoglycan structure [42]. The distinguished emission at 309 nm in Figure 3 is a measurement for substantial amount of OH radicals, which are produced by plasma chemical reactions of dissociation and excitation of water molecules present in the air and are likely the part of plasma that is lethal toward living bacterial cells [43,44]. The intensity of OH produced by CAPJ is influenced by the feed gas mixture and increases with increasing Ar flow rate similar to a previous report [45]. The amount of OH radicals positively correlates to the antimicrobial e fficiency as well. Deleterious OH radicals are the dominant reactive species and play a significant role in cell death [46,47].

It has been known that bacterial colonization of wounds exacerbated inflammation around the injury sites and slowed the skin healing. A previous study in bacteria infected skin diseases shows that *E. coli* is one of the four main Gram-negative bacteria among 90 isolated bacteria cultured from skin ulcers [48]. In the present study, we first assessed whether CAPJ possessed bactericidal e ffects on pathogenic *E. coli* and validated the bactericidal activity by using the animal experiment. We showed that S10 CAPJ treatment dramatically decreased bacterial load on day 4 as compared to untreated rat. However, we did not identify the bacterial species that were presented on the wounds. Our results are in line with previous in vitro studies, which showed that cold atmospheric plasma can decrease bacterial load independent of the strains [49,50]. Although CAPJ can e ffectively reduce bacterial loads in the animal study, further identification of bacterial species is worth studying in the future research.

According to the observation of both the DNA damage assay (Figure 7) and the morphology of bacteria after CAPJ treatment by FE-SEM (Figure 8), CAPJ disrupts bacterial cell walls and induces DNA damage to exercise its antimicrobial e ffect in plasma mediated reactions. Figure 10 is a schematic that suggests the antimicrobial mechanism of CAPJ treatment noted in this study.

**Figure 10.** Antimicrobial mechanism of CAPJ treatment. The mechanism of the antimicrobial efficiency by CAPJ is suggested to kill the bacteria by destroying the cell wall of *E. coli*, damaging its DNA structure.
