In this study, Signstek Portable High Frequency Machine (Signstek, Full Rise LLC, Wilmington, DE/USA) carrying CE labeling (approved for use in humans in Europe) was used as a technical device consisting of a handheld with an intensity regulator on the bottom and an output for plugging in different glass tubes filled with helium below atmospheric pressure. Four different glass tubes are supplied: a comb tube for treating the scalp, a tongue tube for sensitive areas (such as dark circles beneath the eyes, nose, or lips), a mushroom tube for large areas (forehead, back), and a bent tube with a spherical tip for regular areas like cheeks (Fig. 1a).

Fig. 1.

Plasma source and mechanism effects. (a) Plasma device consisting of a handheld with intensity adjust and different glass tubes to plug in. Formation of orange colored plasma when turned on. (b) Pictures taken with a thermal imaging camera after 0, 1, 2 and 4 minutes without any sign of heat development. (c) Ozone formation during time measured by an O3 detector demonstrating an increase of O3 concentration after 10 minutes already. The dashed line indicates the odor threshold concentration for ozone (20‒40 µg/m3; 0.01‒0.02 ppm).

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In all experiments here, the bent tube was used at a working frequency of 50 Hz. The intensity was regulated at 50%.

Current strength was measured for three conditions (low, medium, and full power) using a standard ammeter (Multimeter ABB Metrawatt 2012). Applied voltage corresponding to the electric outlet (in Germany: 230 V).

For further investigations on the precise mechanism of action of our plasma source, a thermal imaging camera (FLIR MS Technology C3, FLIR® Systems, Inc., Wilsonville, Oregon, USA) was utilized to detect potential heat development. Pictures were taken after 1, 2, and 4 minutes of HF treatment of an agar plate to resemble an experiment on bacteria.

Ozone (O3) concentration was measured using an O3 detector (Leory PM 2.5 O3 Ozondetektor TVOC, Thingnovation, Shenzhen, China). Our plasma source was switched on to 100% in a room with locked windows and doors of approximately 10.5 cubic meters. Measurement time points were 10, 20, 30, 40 and 50 minutes.

Skin flora from three different body areas was isolated on blood agar plates and incubated for 24 h at 37 °C before HF treatment.

P. acnes, as an anaerobic bacterium, was cultivated in an anaerobic vial (BD BACTEC™ Lytic/10 Anaerobic/F Culture Vial) until an approximate cell density of 3.0 × 108 CFU/mL referring to McFarland Standard nº 1 was reached, then following HF treatment on a blood agar plate. After treatment, plates were incubated for 7d at 37 °C in anaerobic conditions using BD GasPak™. Fungal species were cultivated on BD Dermatophyte Agar for 7d at 37 °C before treatment.

After incubation, each investigated colony was resuspended in approximately 0.5 mL of sterile 0.9% saline solution. For skin flora, the authors chose five morphologically different appearing colonies for further evaluation. Thereafter, 1 µL of the microbial solution was applied centrally to the appropriate agar plates in pairs. Next, the HF device with an attached, wipe-disinfected bent tube was fixed on a stand at a distance of 5 mm above the plate. Of each pair, one plate was treated for 1 minute, and the other one served as a control to determine the relative difference. In the case of P. acnes, treatment periods varied between 1 and 8 minutes (1, 2, 4 and 8 minutes of HF treatment). Subsequently, the microbial solution of treated and untreated plates was spread using sterilized glass beads followed by an incubation period of 1d for skin flora and 7d for P. acnes and fungi at 37 °C. After incubation, on plates with more than approximately 1000 colonies, three 1 cm² areas on each agar plate were counted, and mean values were multiplied by the size of the agar plate (57 cm²) to obtain the absolute number of colonies per plate. Plates with 100 to 1000 colonies were divided into quarters and one counted quarter was multiplied by four, plates with less than 100 colonies were counted completely.

Bacterial colonies of each plate pair were further characterized by performing a PCR (HotStarTaq Master Mix Kit, Qiagen) as described previously.9

Primers 27f (5’-AGA GTT TGA TCA TGG CTC A-3’) and 1492 r (5’-TAC GGT TAC CTT GTT ACG ACT T-3’) partially amplified the 16S rRNA gene using a thermocycler (Biometra Professional Basic, Analytik Jena GmbH, Jena, Germany).

The DNA products were visualized by 1.0% agarose gel electrophoresis to confirm a successful amplification. Sanger sequencing was performed by Eurofins Genomics (Ebersberg, Germany) using the 27f primer. Obtained sequences were aligned using the online tool BLASTn to identify the species (https://blast.ncbi.nlm.nih.gov/Blast.cgi; NCBI).10

For visualization and statistical analyses (unpaired Student’s t-test and row test), GraphPad Prism (version 9.0.0, GraphPad Software Inc, San Diego, CA/USA) was used and data was visualized in logarithmically scaled graphs. All experiments were performed in triplicates. The following values are indicated by mean ± standard deviation. The significance level was set at p < 0.05 (*), p < 0.01 (**), and p < 0.001 were considered highly significant (***).

To evaluate electrical current strength during treatment, a multimeter was used showing a current ranging from 0.28 (full power) to 0.32 ampere (low power) at a voltage of 230 V.

Heat development after 1, 2, and 4 minutes of HF treatment were not observed. Pictures taken with a thermal imaging camera did not show changes in temperature during the investigated period (Fig. 1b).

Ozone formation with a concentration of 0.015 ppm was observed after 10 minutes of a fully switched-on device. After 20 minutes, O3 concentrations almost doubled (c = 0.027 ppm). In the further course, concentration levels increased (c = 0.032 after 30 minutes, c = 0.040 after 40 minutes, c = 0.090 after 50 minutes) (Fig. 1c).

To assess the efficacy of HF therapy against common skin flora, blood agar plates with and without HF treatment were counted after the incubation period followed by a PCR to specify bacterial species. In broad terms, for almost every identified species a significant reduction in colony count was observed. More precisely, on the forehead, the authors detected Micrococcus yunnanensis twice, Aerococcus viridans, Staphylococcus epidermidis and Staphylococcus capitis. M. yunnanensis was reduced by 67.5% (31267.0 ± 81.7 vs. 1016.3 ± 57.1; p < 0.001) and on the second plate by 12.5% (6405.0 ± 18.5 vs. 5605.3 ± 19.4; p < 0.001), A. viridans by 26.1% (9339.0 ± 72.1 vs. 6906.3 ± 58.8; p < 0.001), S. epidermidis by 83.43% (3785.7 ± 68.7 vs. 627.3 ± 18.2; p < 0.001) and S. capitis by 41.9% (1488.0 ± 70.4 vs. 864.3 ± 30.3; p < 0.001).

Likewise for bacteria of the forearm: B. cereus was reduced by 75.9% (11396.3 ± 757.7 vs. 2750.7 ± 127.9; p < 0.001), S. capitis by 63.9% (2700.3 ± 104.0 vs. 973.7 ± 25.1; p < 0.001), A. urinaeequi by 95.1% (16201.0 ± 757.9 vs. 800.0 ± 35.0; p < 0.001), M. yunnanensis by 99.0% (7209.3 ± 344.8 vs. 75.0 ± 12.8; p < 0.001) and M. luteus by 3.1% (6280.0 ± 443.3 vs. 6087.7 ± 119.3; p = 0.5).

Regarding bacterial species of the genital region, S. capitis was reduced by 81.6% (31699.3 ± 763.7 vs. 5839.7 ± 111.3; p < 0.001), S. lugdunensis by 97.6% (15214.3 ± 405.1 vs. 363.3 ± 16.2; p < 0.001), M. luteus 17.9% (957.0 ± 37.6 vs. 785.3 ± 22.3; p = 0.002), S. haemolyticus 99.0% (6684.0 ± 273.0 vs. 70.0 ± 7.2; p < 0.001), M. yunnanensis 70.3% (4322.0 ± 252.3 vs. 1284.3 ± 86.4; p < 0.001) (Fig. 2a).

Fig. 2.

Antimicrobial effects of HF therapy. (a) HF treatment shows a significant decrease of bacterium and (b) fungus count in vitro. (c) CFU of P. acnes are significantly reduced after 1 minute, continuing the treatment leads to further reduction.

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The next step of this study was to further evaluate the effects of HF treatment against a random selection of dermatophytes. The total number of all dermatophytes’ colonies was significantly reduced by 58.0% after HF treatment (55.8 ± 33.6 vs. 18.2 ± 6.5; p = 0.040). More precisely, in T. mentagrophytes colonies were reduced by 61.5% (69.3 ± 4.0 vs. 26.7 ± 3.1; p < 0.001), in T. violaceum by 78.6% (103.0 ± 6.6 vs. 22.0 ± 3.0; p < 0.001), in T. benhamiae by 45.2% (17.7 ± 1.5 vs. 9.7 ± 1.5; p = 0.003), in M. canis by 69.1% (65.7 ± 2.1 vs. 20.3 ± 1.5; p < 0.001) and in T. rubrum by 61.6% (31.3 ± 1.5 vs. 12.0 ± 2.0; p < 0.001) (Fig. 2b).

To further address the question of whether HF treatment is beneficial against P. acnes, P. acnes was treated by HF over time (Fig. 2c). CFU on different blood agar plates was counted before and after 1, 2, 4, and 8 minutes of HF treatment. Significant results were reached after 1 minute of treatment (7288.0 ± 145.9 vs. 30.0 ± 3.6; p < 0.001). Continuing the treatment for 2 minutes, 4- and 8-minutes lead to a further decrease in the amount of CFU. When comparing the values after 2 and 4 minutes of treatment a further significant reduction of P. acnes CFU was observed (4.7 ± 0.6 vs. 1.3 ± 1.5; p = 0.020). Prolonged treatment over 8 minutes seemed not to have further benefits (1.3 ± 1.5 vs. 1 ± 0; p = 0.700) (Fig. 2c).