# Anti-Bacterial Action of Plasma Multi-Jets in the Context of Chronic Wound Healing
## 3 Results
### Pilot Tolerance Study on the Use of Plasma Therapy for Wound Treatment  
This preliminary study was performed at Orleans Regional Hospital (CHRO) in the department of Tropical and Infectious Diseases under the supervision of Dr [PERSON]. The study was approved by the CHRO ethical committee on the base of bibliographic data published by different teams reporting clinical trials with non-thermal plasma devices and following a tolerance study performed at Institut Gustave Roussey (Villejuif, France) under the supervision of Dr [PERSON] [62], with the multi-jet applicator technology described in this work applied to both nude and hairy mice. It was measured that neither severe nor irreversible skin disorders were induced following minutes long multi-jet delivery in a single fraction. The tolerance study performed at CHRO included five hospitalized patients bearing superinfected wounds having diameters ranging from 3 to 15 cm. A single jet low voltage device was used to deliver plasma during one minute for each half square centimeter surface unit, as shown in Figure 12. The full time exposure of the wound treated zone, which was not always covering the whole ulcerated tissue, ranged from 15 to 30 min. The plasma device was handheld and operated either by the coauthors or the medical staff of the hospital. Each of the five patients was plasma treated either daily or each other day, reaching at the end a median five-day fraction of plasma treatment. The patients were preventively grounded by means of an antistatic bracelet so to better control their potential and the plasma treatment. Tolerance was assessed by scoring the patient feelings during and after the plasma delivery fractions. It was also systematically and carefully investigated by nurses in charge of the treatment that no necrotic, inflammatory and cutaneous stress appeared at the place where the plasma jet was applied. As reported in Figure 12, the temperature of the treated area was monitored live by means of an infrared camera (Fluke Ti480 by Fluke). The surface of the wound is in general colder due to the presence of liquid and the consequent cooling caused by evaporation. At the place where the plasma jet was applied a temporary small increase (about +3-5 \({}^{\circ}\)C) of the surface temperature was observed. The recorded temperature always remained below 40 \({}^{\circ}\)C, so any adverse effect due to excessive heating can be discarded.  
The tolerance study was overall scored as excellent. The scoring, associated with any local or systemic pain felt during or after the plasma fraction, remained at the same value as that expressed before plasma treatment for each of the patients or for all of the plasma fractions. This testifies to the absence of pain, burn or any unpleasant feeling by the patient during the plasma delivery on injured skin. No inflammation, necrosis or degradation of the ulcerated tissue was reported.  
Figure 13 presents the _Staphylococcus aureus ATCC9144_ and _Pseudomonas aeruginosa ATCC9027_ colony reduction versus the plasma exposure duration. For the two bacteria, three different initial concentrations were exposed in triplicate: respectively about 170, 90 and 50 and 160, 55, 30 colonies/membrane were used for Staphylococcus aureus and Pseudomonas aeruginosa respectively. These colony concentrations were selected as being representative of typical infection levels in the hospital context following the baseline decontamination protocols using disinfectants. In Figure 13, the colony reduction was plotted after applying a normalization for three experiments (realized in triplicate) for each of the bacterial strains. It is worth noting that whatever the initial colony concentration, the same trends are measured, leading to an efficient reduction after a 10-min-long exposure for both bacterial strains.