## 2 Results
### Analysis of Persister Cells
#### 2.4.2 Effect of Chlorophylllin and Polyethylenimine on _P. aeruginosa_ Persister Cells  
Because of the higher susceptibility of the persister and growing cells to CHL and PEI in the M9 minimal medium compared to that in the BHI medium and the faster rate of degradation of CHL in M9 compared to BHI (Figures 2 and S1), the exposure time was reduced to 15, 30 and 60 min. The lethal effect (4 log\({}_{10}\) reduction) of the tested combination appeared after 30 min of exposure to red light and lower concentrations  
Figure 5: Influence of SHX addition on growth of _P. aeruginosa_. Bacteria were grown in M9 minimal medium without and with the addition of 500 \(\upmu\)g mL\({}^{-1}\) SHX. Figure 6: Induction of the stringent response. The normalized gene expression relative to the reference gene _rpo_S of the treated and untreated samples using the \(2^{-\Delta\Delta\text{CL}}\) method. Data represent the mean value of three biological replicates with three technical replicates (\(n=9\)). Error bars represent the standard deviations. \({}^{*}\)_t_-test: _p_-value < 0.05.  
exerted the same effect by doubling the exposure time (Figures 7 and 8). Unilluminated CHL, PEI alone and the red-light source had no lethal effect under the tested conditions, making the synergistic effect of the tested combinations more obvious (Figures 7 and 8). The dose-response matrices in Figure 7 showed that 800 \(\upmu\)g mL\({}^{-1}\) PEI and 125 \(\upmu\)g mL\({}^{-1}\) CHL were required to eradicate the growing cells after 30 min of exposure, whereas half of the concentration of the PEI (400 \(\upmu\)g mL\({}^{-1}\)) and double the concentration of the CHL (250 \(\upmu\)g mL\({}^{-1}\)) were required to exert the same effect on the persister cells (Figure 8). After 60 min of exposure, the eradication rate increased with the lower tested concentrations of CHL/PEI 31.25/100 \(\upmu\)g mL\({}^{-1}\) for the growing cells and CHL/PEI 15.62/100 \(\upmu\)g mL\({}^{-1}\) for the persister cells (Figures 7 and 8). This result suggests that persister cells are more vulnerable to CHL and PEI than the growing cells, and CHL alone requires more exposure time to exert its antimicrobial action. Figure 7: Influence of illuminated chlorophyll (CHL) and polyethyleneimine (PEI) on _P. aeruginosa_ growing cells. The dose-response matrices show the effect of the double serial dilution of CHL and PEI individually and in combination. ZIP (zero interaction potency) synergy score matrices depict the synergistic effect between CHL and PEI. The matrices were plotted using RStudio SynergyFinder R package. The results are an average of four biological replicates. In order to prove the result of the dose-response matrices, the efficacy of 100 \(\upmu\)g mL\({}^{-1}\) PEI and different concentrations of CHL were tested via viable cell count. The results in Figure 9 corroborated that the persister cells are more sensitive than the growing cells to the lowest tested concentrations of 100 \(\upmu\)g mL\({}^{-1}\) PEI and 31.25 \(\upmu\)g mL\({}^{-1}\) CHL with 1.3 and 1.4 \(\log_{10}\) reductions after 15 and 30 min, respectively. The growing cells were reduced by 0.4 \(\pm\) 0.1 \(\log_{10}\) after 15 min of exposure and significantly (\(p\) < 0.05) reduced by 1.4 \(\pm\) 0.17 \(\log_{10}\) after 30 min exposure at CHL/PEI 31.25/100 \(\upmu\)g mL\({}^{-1}\), whereas the persister cells were significantly (\(p\) < 0.05) reduced by 1.7 \(\pm\) 0.12 and 2.8 \(\pm\) 0.31 \(\log_{10}\), respectively, under the same conditions. It is also noted that the CHL at concentration 1000 \(\upmu\)g mL\({}^{-1}\) in presence of PEI 100 \(\upmu\)g mL\({}^{-1}\) is not lethal for the growing cells while significantly (\(p\) < 0.05) reducing the persister cells by 1.6 \(\log_{10}\) after 30 min of exposure to aPDI, indicating a non-linear response. These data, in conjunction with the previous  
Figure 8: Influence of illuminated chlorophyllin (CHL) and polyethyleneinimine (PEI) on _P. aeruginosa_ persister cells.