## 4 Discussion  
In this work, we have developed an in vivo superficial bacterial infection model using clinically relevant bacteria (_P. aeruginosa_) and showed the application potential of nanosecond electroporation for the killing of bacteria. We have improved the recent concept presented by [PERSON] et al. [28] and provided experimental data on bacterial growth dynamics throughout the week, which was not possible with a previous model [5]. The proposed nanosecond methodology does not require us to create a skin fold, which, in many clinical cases, is impossible due to the complex morphology of the wound and surrounding tissue. We have shown that the tweezer electrodes can induce a sufficient electric field for the inactivation of bacteria when combined with low concentrations of acetic acid. This methodology is a hybrid of the tissue ablation [42,43] and electrochemotherapy [44] methods, which are used in the cancer treatment context. In our work, based on FEM simulation, we conclude that the small gap (1.2 mm) between the electrodes ensures the superficial permeabilization of cells without affecting the deeper tissues. The inhomogeneity of the electric field should be compensated by a higher field intensity (in our case, 3 kV at 1.2 mm gap) or unsatisfactory bacterial killing efficiency will be triggered (Figure 6A). The input energy of the proposed PEF protocols is lower compared to available research on this topic [19,28]. Minimizing the energy is advantageous since a non-thermal procedure can be ensured, while the application of bursts of microsecond pulses with a high-intensity electric field results in significant Joule heating [45]. In our work, the temperature rise based on FEM is >20 \({}^{\circ}\)C, which indicates the need to reduce the input energy even further to ensure a non-thermal procedure. Additionally, moving towards the shorter pulse duration range reduces muscle contractions [46] and potentially pain [47]. We have also used low-concentration acetic acid (1%), showing a synergistic effect with electroporation (i.e., electrochemotherapy), which supports our preliminary in vitro [29] and in vivo data [5]. It should be noted that we have modeled a superficial infection. In essence, the skin was mechanically irritated for the bacteria to colonize. However, in the case of chronic wounds, the methodology should be further optimized to account for the possible non-homogeneity of the PEF spatial distribution due to the complex morphology of the treatment area. Additionally, the method can be supplemented with various patching and wound dressing techniques [48,49]. In terms of pulsing, the methodology can be also improved. We have used a 3 kV charging voltage (limitation of our infrastructure) to induce up to a 25 kV/cm electric field, which is sufficient to kill the bacteria with acetic acid; however, the application of a higher-intensity electric field (but a shorter pulse duration and smaller number of pulses) should potentially even further improve the input energy management [50]. The PEF alone, without any chemotherapeutic agents, can be also used; however, the intensity of PEF should be increased to ensure irreversible electroporation. As a matter for future work, the application of high-frequency bipolar or MHz-range unipolar pulses should be considered [51,52,53]. High-frequency bipolar pulses enable the mitigation of the impedance changes and a more uniform treatment [54], while compressing the pulses to a MHz burst significantly lowers the electroporation threshold [55,56]. However, in this case, the effects of Joule heating should be considered and a trade-off between the input energy and the resultant Joule heating should be sought. In this work, _P. aeruginosa_ was used as a cell model. Gram-negative bacteria are one of the most resistant types of microorganisms to PEF due to the specific structure of the cell wall [57].