## 3 Results  
Firstly, the FEM model was used to characterize the treatment and the expected PEF spatial distribution. The spatial distribution of the electric field when a 3 kV voltage was applied between the electrodes is shown in Figure 4.  
It can be seen that the distribution of the electric field is non-homogeneous, with the field being higher near the tips of the electrodes. In the simulation, it is assumed (and based on available knowledge) that the first few pulses in the burst trigger irreversible electroporation (Figure 4A), which results in a significant increase in the conductivity of the separate layers (refer to Table 1). As a result, the spatial distribution of PEF is altered (Figure 4B). It can be seen that the upper layers of the skin in the middle of the skin lump are subjected to PEF exceeding 6 kV/cm, while PEF up to 25 kV/cm is reached in close proximity to the electrodes. In general, the treatment is superficial, with low penetration (<1 mm) to deeper tissue (i.e., muscle). Figure 4: The spatial distribution of the electric field during pulsed electric field treatment, where (**A**) tissue conductivities are nominal and not affected by PEF; (**B**) tissue electrical conductivities are increased due to permeabilization triggered by PEF; (**C**) Joule heating due to PEF application. A 3 kV voltage is applied between the terminals (gap 1.2 mm). The electric field spatial distribution data are shown in kV/cm. The Joule heating effect was analyzed after the 1000 \(\times\) 750 ns pulse burst delivered at 15 kHz (Figure 4C). It can be seen that in close proximity to the electrodes, the temperature rises up to 61 \({}^{\circ}\)C, which is a 24 \({}^{\circ}\)C increase. Such an increase in temperature is in agreement with established knowledge; however, it is indicative that a further increase in the input energy is not possible. Following the FEM analysis, the mice were treated with two nanosecond protocols (1.8 kV \(\times\) 750 ns \(\times\) 1000 pulses and 3 kV \(\times\) 750 ns \(\times\) 1000 pulses) and acetic acid (1%). In both cases, the gap between tweezer electrodes was 1.2 mm. A typical response to the treatment is shown in Figure 5.  
It can be seen that AA with a concentration as low as 1% does not result in a successful treatment. The luminescence signal increased almost eight-fold on day 3, indicating an increased number of bacterial cells, which is proportional to the luminescence. At the same time, PEF + AA treatment results in the effective killing of bacteria. Shortly after the treatment, the luminescence signal was beyond the detection limits of our infrastructure. On day 3, in some experimental mice, a weak signal could be detected; however, in general, it was lower when compared to the AA-only treatment. The quantified results with both nanosecond protocols are shown in Figure 6. It can be seen that there is a positive response to both treatments; however, the 3 kV treatment is more effective. The standard deviation is also significantly lower when the 3 kV protocol is employed, which results in a statistically significant (\(p<0.001\)) decrease in the luminescence signal (i.e., bacterial viability) when compared to PEF-untreated mice. It should be also noted that the variation in response may partly be dependent on the human factor and the accuracy of electrode repositioning. A total of 14 \(\pm\) 5 repositions of the tweezer electrodes was required to cover the whole treatment area. In order to determine the immune response to bacterial infection, IgM and IgG (IgG1, IgG2a, IgG2b, IgG3) antibodies against _P. aeruginosa_ surface and intracellular antigens were evaluated. Based on the results presented in Figure 6, the study was limited to the 3 kV protocol. The data are summarized in Figure 7.  
Figure 5: The mice with only the AA (1%) treatment (CTRL) and the mice treated with PEF (3 kV voltage is applied between 1.2 mm gap electrodes) + 1% AA treatment. Red circle indicates region of interest (ROI) where the luminescence signal was quantified.