## 3 Discussion  
Generation of persisters in vivo allows subpopulations of bacteria to survive under antibiotic treatment and may account for the recalcitrance of most chronic infections and antibiotic treatment failure [22]. Persisters formation is a complex survival strategy and is correlated with various factors that include growth phase, expression of stringent response genes, SOS response, quorum sensing and toxin-antitoxin systems [23]. In this study, we aimed to test the ability of LRM-CFS to kill persister cells of _P. aeruginosa_ both in planktonic and biofilm models of growth in ASM, resembling the CF lung environment. Persister-enriched cultures were first obtained by chemical treatment of stationary phase cultures with CCCP, a membrane uncoupling agent [20]. We have previously demonstrated that CCCP-treated cultures undergo a general reduction of metabolic activity, as assessed by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry [20]. Furthermore, we have  
Figure 4: Cytotoxic effect of the LRM-CFS diluted at 1:4, 1:6, and 1:8 in complete RPMI medium was tested in vitro against NCI-H441 cell line grown on the surface of collagen scaffolds in the 3D lung epithelium model. Cells were stained for 5 min with 0.1 \(\upmu\)g/mL propidium iodide at room temperature before performing flow cytometric acquisition. (**a**) Percentage of live NCI-H441 cells after exposure to LRM-CFS for 7 and 24 h. (**b**) Overlay histogram data from a representative experiment showing PI-positive NCI-H441 percentages (dead cells). CTRL: MRSB diluted at 1:4 in complete RPMI medium. Results are shown as mean \(\pm\) standard error of the mean values (\(n=3\)). Statistical significance was evaluated by one-Way ANOVA followed by the Tukey–Kramer post hoc test. *** \(p<0.001\). reported that after CCCP removal, induced persisters show a lag phase before the resumption of normal growth and a reversion to an antibiotic-sensitive phenotype, following metabolic reactivation [20]. All these observations are compatible with the acquisition of a CCCP-induced persister-like status that has been linked to the inhibition of ATP synthesis and the consequent reduction in bacterial metabolic activity [24]. Interestingly, by employing the same procedure in the present study, we demonstrated the ability of LMR-CFS to exert a bactericidal effect against CCCP-generated persister cells of _P. aeruginosa_ in ASM. In contrast, ciprofloxacin, an antibiotic commonly used for the therapy of CF lung infections, was inactive when tested at very high concentration (20 x MIC). Several activities have been ascribed to supernatants from lactic acid bacteria, including antibacterial, antibiofilm, antioxidants, antivirulence, and wound-healing activity [18; 25; 26], but to the best of our knowledge, anti-persister activity has never been reported before. In the attempt to mimic the in vivo generation of persister cells in the lungs of CF patients, in this study we further explored the anti-persister activity of LMR-CFS against persister-enriched populations of _P. aeruginosa_ obtained through exposure to high antibiotic concentrations in ASM. It has been demonstrated that upon antibiotic exposure, the stringent response (_relA_ and _spoT_) is induced, leading to an increase in (p)ppGpp, which in turn arrests the cell growth (by directly interacting with RNA polymerase and inhibiting the transcription) providing a fitness advantage in stressful conditions [27]. Thus, in this study, we also selected persisters directly in ASM after exposure of _P. aeruginosa_ to high doses (128 x MIC) of tobromycin, an antibiotic commonly used for the therapy of lung infections by _P. aeruginosa_ in CF patients. We observed that tobramycin at very high concentrations (up to 256-fold MIC) in ASM caused only a mild decrease in viable bacteria from stationary-phase cultures of both _P.