**5. Conclusions**

In this work, experimental design using the Taguchi method with a L9 orthogonal array was confirmed by S/N ratios and ANOVA, to optimize the operating parameters of CAPJ, achieving the best antimicrobial efficacy against *E. coli*. Parameters obtained via the Taguchi method were confirmed by 100% antimicrobial activity, with the final parameters of 8.5 kV CAPJ application voltage, 10 mm CAPJ-sample distance, 500 sccm Ar gas flow rate, and 300 s CAPJ treatment time. These parameters were further applied to wounds created on a rat model and showed a marked decrease in microbial load compared to an untreated wound, suggesting CPAJ have safe and effective application in vivo. As the intensity of hydroxyl radical produced by CAPJ is positively correlated to its antimicrobial efficiency, reactive species likely play a significant role for the plasma sterilization in this study. According to the observation of both the DNA damage assay and the bacterial cell wall integrity test after CAPJ treatment, the antimicrobial mechanism of CAPJ works through cell wall destruction and further DNA damage, thus ensuring antimicrobial activity. This makes CAPJ a promising and effective antimicrobial technique.

**Author Contributions:** B.-S.L., C.-H.L., and J.-W.L. designed the experiment, analyzed data, and wrote the paper. J.-H.H. designed CAPJ device. T.-P.C. and Y.-M.S. employed in vitro experiments. C.-M.C., C.-W.H., and P.-Y.C. performed in vivo experiment.

**Funding:** The authors gratefully acknowledge the financial support of the Ministry of Science and Technology, Taiwan, through contract nos. MOST 108-2218-E-030 and MOST 106-2218-E-131-003. The financial support from the Chang Gung Memorial Hospital through contract no. CMRPD5H0032 to B.S. Lou is also acknowledged.

**Conflicts of Interest:** The authors have no conflicts of interest relevant to this article.
