# Antimicrobial Activity of L-Lysine and Poly-L-Lysine with Pulsed Electric Fields
## 3 Results  
To test the effect of Lys and PLL on the growth of _E. coli_, _S. aureus_, _C. albicans_ and _T. rubrum_ microorganisms, the disk diffusion test was performed. First, the antimicrobial efficacy of Lys and PLL was determined using the same concentration of 1 mg/mL. Obtained results are presented in Figure 1. No inhibition zone diameters of L-lysine at 1 mg/mL concentration were observed on tested microorganisms. As it can be observed, the antimicrobial efficiency on tested microorganisms was stronger using PLL at a concentration of 1 mg/mL (Figure 1). Since microorganisms show different sensitivity, the minimal inhibitory concentration (MIC) was performed to find the right concentration for each substance. The effective concentrations are presented in Table 1.  
PLL exhibited very strong antimicrobial activity on all tested microorganisms. However, Lys had no effect on the growth of _C. albicans_ and _T. rubrum_. According to these results, the selected concentration to use with PEF treatment for Lys was 500 mg/mL and for PLL 0.5 was \(\upmu\)g/mL. Since the highest activity was observed on _T. rubrum_, the first treatment using PEF and Lys/PLL was analyzed and results are presented in Figure 2. Indeed, all the data was normalized to non-treated control. As can be seen in Figure 2A, both Lys and PLL can be effectively used with electroporation for inactivation of _T. rubrum_. PLL features better antimicrobial properties. The duration of the PEF pulses was varied, and different efficacies of the treatment were acquired. The energy of the bursts was the same independent on the pulse duration, however, the 500 ns and 1 \(\upmu\)s treatments showed significantly weaker effects compared to the 10 and 100 \(\upmu\)s bursts. When the number of pulses was increased (2.5-fold energy increase), the efficacy of the treatment was observed. However, the increase of PEF amplitude to 30 kV/cm significantly improved the inactivation efficacy (Figure 2B). The viability drop was more than 3 log reduction for the highest energy burst (100 \(\upmu\)s \(\times\) 25). Similar to the 15 kV/cm treatment, the 500 ns and 1 \(\upmu\)s protocols were less effective. The differences between Lys and PLL were not as reflective as in 15 kV/cm (except the highest energy burst). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Microorganisms** & **Lys** & **PLL** \\ \hline  & & MIC \\ _E. coli_ & 500 mg/ml & 250 mg/ml \\ _S. aureus_ & 125 mg/ml & 2.4 ng/ml \\ _C. albicans_ & NI * & 1.5 ng/ml \\ _T. rubrum_ & NI & 0.3 ng/ml \\ \hline \hline \end{tabular}  
* NI—no inhibition at 1g/mL concentration of L-lysine.  
\end{table} 
Table 1: Minimal inhibitory concentration of L-lysine (Lys) and poly-L-lysine (PLL) on microorganisms. Figure 1: The antimicrobial efficacy of L-lysine ((Lys) (disk 1 mg/mL)) and poly-L-lysine ((PLL) (disk 1 mg/mL)) on microorganisms. Further, the susceptibility of _C. albicans_ to the treatment was analyzed. The results are summarized in Figure 3.  
Figure 2: The antimicrobial efficacy of L-lysine and poly-L-lysine with pulsed electric field treatment against _T. rubrum_, where (**A**) 15 kV/cm treatment; (**B**) 30 kV/cm treatment. Asterisk (*) represents statistically significant (_p_ < 0.05) difference versus Lys or PLL treatment, respectively. CTRL—samples with Lys or PLL without PEF treatment. As can be seen in Figure 3, a similar tendency was observed, i.e., the 500 ns and 1 us treatments are the least effective. _C. albicans_ demonstrated higher susceptibility to the combination of PEF + Lys compared to _T. rubrum_. Also, when combined with PEF, the PEF + Lys treatment was more effective than PEF + PLL treatment. In general, the response of _C. albicans_ to PEF + PLL was significantly weaker (_p_ < 0.05) versus _T. rubrum_. Further, the inactivation of bacteria was also studied. The results for _S. aureus_ are presented in Figure 4.  
Figure 3: The antimicrobial efficacy of L-lysine and poly-L-lysine with pulsed electric field treatment against _C.