Abstract: 【Abstract】 Objective To evaluate the efficacy and safety of cold atmospheric plasma （CAP） in the treatment of non-open Staphylococcus aureus skin infections in mice. Methods In the in vitro antibacterial efficacy experiment, Staphylococcus aureus suspension was evenly inoculated onto 12 blood agar plates, which were randomly divided into 4 groups （3 plates in each group）: the group receiving no CAP irradiation served as the control group, and the other 3 groups were irradiated with CAP at intensity levels 3, 5, and 8 for 3 minutes. Colony counts were calculated after 24 hours of incubation. In the in vivo efficacy experiment, a subcutaneous abscess model was established in 30 specific pathogen-free （SPF） BALB/c mice by subcutaneous injection of 0.05 ml of a Staphylococcus aureus suspension, and successful modeling was confirmed 24 hours later by visible abscess formation. According to the abscess volume, 30 mouse models of subcutaneous abscesses were divided into 3 groups using a stratified randomization method （10 mice in each group）: a control group （without intervention）, a mupirocin group （treated with topical mupirocin ointment on the mouse back）, and a CAP group （treated with CAP irradiation）. Interventions were given once daily for 10 days. Abscess volume and appearance were observed every other day. Microbiological and histopathological examinations were performed after the end of treatment, with skin tissues from one untreated normal mouse serving as the histopathological control. In the safety assessment experiment, 12 SPF BALB/c mice were randomly divided into 4 groups to be irradiated with CAP at intensity level 8 for varying durations and distances. Skin reactions （such as erythema and edema） were recorded and scored. The Kruskal-Wallis H test was used for in vitro antibacterial efficacy analysis; in the in vivo experiment, changes in abscess volume were analyzed by repeated-measures analysis of variance, and bacterial loads in abscesses were compared using one-way analysis of variance; two-way analysis of variance was used in the safety analysis. Results In the in vitro antibacterial efficacy experiment, a significant difference in viable colony counts was observed among the 4 groups （H = 10.42, P = 0.015）, the level 8 CAP group showed significantly decreased viable colony counts （median ［Q1, Q3］: 20 ［0, 40］ colony-forming units ［CFU］/ml） compared with the control group （560 ［400, 600］ CFU/ml, P = 0.013）, while there were no significant differences in viable colony counts between the other groups （all P > 0.05）. After successful modeling, no significant difference in the initial abscess volume was found among the control group, mupirocin group, and CAP group （F = 0.14, P = 0.865）. The abscess volume in all groups showed an initial increase followed by a decrease over time, with a significant main effect of time （F = 426.50, P < 0.001, partial η2 = 0.94）, a significant main effect of treatment （F = 1.62, P = 0.020, partial η2 = 0.25）, and a significant time-treatment interaction effect （F = 2.69, P = 0.031, partial η2 = 0.17）; on days 6, 8, and 10, the abscess volume was significantly lower in the CAP group than in the control group （all P < 0.05）; on days 8 and 10, the abscess volume was significantly lower in the mupirocin group than in the control group （both P < 0.05）; there were no significant differences in the abscess volume between the CAP group and the mupirocin group at any of the aforementioned time points （all P > 0.05）. After 10 days, bacterial loads in abscesses were significantly lower in both the mupirocin group （［4.16 ± 0.18］ lg CFU/ml） and the CAP group （［3.75 ± 0.27］ lg CFU/ml） than in the control group （［4.98 ± 0.32］ lg CFU/ml, both P < 0.001）, and significantly lower in the CAP group than in the mupirocin group （P = 0.029）. After 10 days, histopathological examination showed obvious subcutaneous abscesses with massive neutrophil infiltration in the control group, mupirocin group, and CAP group compared with the histopathological control group （normal mouse skin tissues）, and the degree of infiltration was lower in the CAP group and mupirocin group than in the control group. In the safety analysis, CAP irradiation at level 8 and a distance of 0.5 cm for 5 or 10 minutes was found to be relatively safe, without marked skin irritation on the mouse dorsal skin; CAP irradiation at level 8 and a distance of 0 cm for 5 or 10 minutes could induce erythema and edema, and the irritant reactions worsened with the extension of exposure duration. All adverse reactions subsided within 24 hours. Conclusion CAP was safe and effective for the treatment of non-open Staphylococcus aureus skin infections in mice, suggesting its potential as an emerging therapy for non-open bacterial skin infections.

Key words: Skin diseases, bacterial, Abscess, Cold atmospheric plasma, Treatment, Efficacy, Safety