This study was approved by the Ethics Committee of The Third Xiangya Hospital, Central South University (Changsha, China) and was carried out in accordance with the approved guidelines. All patients provided written informed consent.

The bacterial strains used in this study were S. aureus (ATCC 6538) and MRSA (ATCC 43300). Bacterial culture medium was purchased from Zhengzhou An Tu Biological Engineering Co., Ltd. (Zhengzhou, China). Dimethylsulfoxide (DMSO) was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany. Ozonated oil at the peroxide value of 2,000 to 2,200 mmol-equiv/Kg [provided by Hunan Health Care Technology Co., Ltd. (Hunan, China)] was obtained from the chemical reaction between ozone and camellia oil because of its high composition of unsaturated fatty acids. Ozonated water was created by Ozone Water Generating Instrument (from Hunan Health Care Technology Co., Ltd.) at the Dermatology Department of the Third Xiangya Hospital.

The bacterial strains were diluted with PBS to get the concentration reaching at 10⁸ CFU/ml.

For ozonated oil: The test oil suspension was constituted by 400 µl ozonized oil mixes, 50 µl DMSO and 50 µl microorganism suspension. Control oil suspension was constituted by 400 µl basal oil mixes 50 µl DMSO and 50 µl microorganism suspension. Both groups were incubated in 37°C incubator for 1, 5, 10, 15, 20, and 30 min respectively. Then the suspension was plated into Petri dishes and grew at 37°C for 24 h. Then the number of colonies in each agar plate was calculated. The control suspension constituted by 400 µl normal saline mixes 50 µl DMSO and 50 µl microorganism suspension was used as control to calculate the killing rate. Killing Rate (%)=(control colony number-oil colony number)/control colony number).

For ozonated water: 1.0 ml of bacteria liquid was mixed with 4.0 ml of ozonated water or PBS. 0.5 ml mixture was added into 4.5 ml neutralizer (Phosphate buffer solution including sodium thiosulfate) after oscillation for one minute. After that the sample was planted into Petri dishes and cultured in an incubator chamber at 37°C. The number of colonies in each agar plate was calculated after growing for 24 h. Killing Rate (%)=(control colony number-tested colony number)/control colony number).

The bacteria samples prepared above were dropped on sterile cotton swabs. The surface was lightly and uniformly inoculated by cotton swab on agar plate. Then the scrips impregnated with ozonated oil or control oil were pasted into the agar plate, followed by incubation at 37°C for 16–18 h. The inhibitory ring test was performed. If the inhibition zone diameter was bigger than 7 mm, it was considered effective; otherwise, it was not.

The Ethnic Committee of Third Xiangya Hospital approved the study, and the informed consents of all the participants were obtained. Two patients with skin MRSA infection were recruited in this study. In addition to skin infections, no other diseases were present in the two patients. The skin lesions were washed or debrided by ozonated water in our therapy room once a day, followed by application of ozonated oil twice per day at home. Antibiotics and other drugs were not administrated during the ozone therapy.

The lesions of patients were inoculated in the blood plate, then cultured in an incubator chamber with 5% CO₂ concentration at 35°C for 12 h. If there was colony formation of microorganism, the microorganism colony was stained by Gram staining. After confirming the gram-positive bacterial by the microscope examination, three bacterial colonies were added into physiological saline and were prepared with 0.5 turbidity ratio in VITEK2 system (VITEK2 gram-positive identification card) by electronic turbidimeter (DensiCHEK Plus, BioMérieux, Durham, NC, USA). Then bacterial species and drug sensitivity results of the samples were detected by Automatic Microorganisms Identification System (VITEK2-compact). The drug sensitivity results were further confirmed by VITEK2 AST-GP67 Test kit according to the manufacturer's protocol. Following the preliminary result of MRSA infection detected by Automatic Microorganisms Identification System, the microorganism colony was further confirmed as MRSA infection by the MRSA Quantitative Standards kit (no. Z-DD-0096-B; Liferiver Bio-Tech, San Diego, CA, USA) according to the manufacturer's protocol in CFX96^™Red-Time System.

The data were analyzed in SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). t-test was used to assess statistical differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

The killing rate of ozonated oil on S. aureus was much higher than the control. Almost 100% S. aureus were killed in 5 min. For MRSA, the killing rate of ozonated oil was also much higher than the control oil. Almost 100% MRSA were killed within 15 min (Fig. 1). The killing rates over time between ozonated oil and control for S. aureus and MRSA were presented in Tables I and II. The ozonated water (1 mg/l) can sterilize 100% S. aureus and 100% MRSA in one minute (Fig. 2).

The inhibition zone diameters of ozonated oil for S. aureus and MRSA were 17 and 13 mm respectively, which were significantly much larger than the control (Fig. 3).

Practicing treatment

A 21-year-old male patient presented to the Dermatology Department of our hospital complaining of a painful abscess on his left calf muscle for approximately 20 days. Denied any systemic diseases or health issues. Skin examination revealed induration, bleeding and surrounding erythema in the left upper Achilles tendon (Fig. 4A). The overlying skin had become thin and felt fluctuant with spontaneous pus secretion. Previous treatments included systemic antibiotics, cleaning and dressing changes for more than 20 days without any visible effect. Tests for diabetes and immune-suppression syndrome were negative. Bacteria culture, drug sensitive test and PCR test confirmed skin MRSA infection (Fig. 5A and C). The strain was resistant to clindamycin, erythromycin, cefoxitin, oxacillin, penicillin-G, piperacillin, and azithromycin (Fig. 5A). After diagnosis of MRSA infection, the patient voluntarily was put on topical application of ozone therapy. Ozonated water was used to wash the lesion for 10 min immediately, followed by soak the lesion for 20 min after debridement if necessary in our therapy room every day. The application of topical ozonated oil twice per day was carried out at home by the patient. Four days after treatment, the lesion was cleaned to remove necrotic tissue and pus secretion (Fig. 4B). Affected areas were reduced by more than 70% during the first month (Fig. 4C) and was almost healed by the end of second month (Fig. 4D). Bacteria culture test revealed that tissues from the lesion did not develop bacteria one month after the topical ozone treatment.

An 8-year-old male patient presented to our department complaining of a big blister on the dorsal aspect of the left hand. Denied any systemic diseases and any treatment at the moment. On skin examination, a 2×2 cm ulcerated lesion with yellow crusts was seen with surrounding erythema and fluid exudation on the dorsal aspect of the left hand. Laboratory tests confirmed MRSA skin infection, resistant to clindamycin, cefotetan, erythromycin, oxacillin, penicillin-G, and trimethoprim/sulfa-methoxazole (Fig. 5B and D). A complete healing from the lesion was achieved after 12 days of treatment with a combination of ozonated water and ozonated oil using the same remedy as we described in the first case (Fig. 6). Bacteria culture test revealed that scales from the lesion did not develop bacteria 12 days after the topical ozone treatment.