## 2 Results  
### Bactericidal Activity of LRM-CFS in ASM against CCCP-Generated P. aeruginosa Persisters  
To explore the LRM-CFS therapeutic potential in conditions closely resembling the CF lung, we assessed the anti-persisters activity of LRM-CFS in ASM, which mimics the nutritional environment of the CF lung. To obtain _P. aeruginosa_ persisters, two CF clinical isolates (PaCF1 and PaCF4), exhibiting a non-mucoid and a mucoid phenotype, respectively, were exposed to CCCP for 3 h in LB medium, according to a previously described procedure [20]. The ability of CCCP to induce persistence was confirmed by exposing CCCP-pretreated cultures to ciprofloxacin at very high concentration (5 ug/mL corresponding to 20 x MIC) in ASM. Ciprofloxacin displayed a modest effect against CCCP-pretreated bacteria of both _P. aeruginosa_ strains, whereas it showed a marked bactericidal activity against mock-pretreated bacteria (Figure 1a,b), confirming the acquisition of a ciprofloxacin-tolerant status of CCCP-pretreated bacteria. The bactericidal activity of LRM-CFS against CCCP-generated persisters of _P. aeruginosa_ was then evaluated after 3 h of incubation in ASM. LRM-CFS used at dilutions of 1:6 and 1:4 eradicated persister-enriched cultures to the limit of detection (5 CFU/mL) for both _P. aeruginosa_ strains (PaCF1 and PaCF4) (Figure 1a,b). When tested at the dilution 1:8, LRM-CFS caused a statistically significant decrease in the CFU/mL number of both _P. aeruginosa_ strains, accounting for approximately 1.5-log compared to the number of persister bacteria surviving to ciprofloxacin treatment (Figure 1a,b). Figure 1: Killing activity of LRM-CFS on CCCP-generated persisters of PaCF1 (**a**) and PaCF4 (**b**) in ASM. PaCF1 and PaCF4 persisters were generated by exposure of stationary-phase cultures to CCCP 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for 3 h in LB. LRM-CFS were diluted at 1:4, 1:6, and 1:8 in de Man, Rogosa, and Sharpe broth (MRSB) and tested against persisters of PaCF1 (**a**) and PaCF4 (**b**) strains in ASM. After 3 h of incubation, samples were serially diluted and plated on solid medium for CFU counts. Samples denominated “mock treatment” represent control bacteria not pre-incubated with CCCP. CTRL: untreated bacteria incubated in ASM with De Man-Rogosa-Sharpe broth (MRSB) pH 4 diluted at 1:4; Ciprofloxacin was tested at 5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (20 x MIC for both strains). Results are shown as mean \(\pm\) standard error of the mean values (\(n=4\)). Statistical significance was evaluated by one-way ANOVA followed by the Tukey–Kramer post hoc test. * \(p\) < 0.05, *** \(p\) < 0.001. 8 h of incubation, tobramycin at all three concentrations tested caused a slight decrease in the CFU number of both _P. aeruginosa_ strains. Such numbers remained almost constant at 24 h irrespective of the antibiotic dose used. The characteristic biphasic killing curves obtained, reaching a plateau at increasing exposure times, were indicative of the presence of persister cells. To verify that the observed tolerance of persister cells to tobramycin was not due to the acquisition of resistance mechanisms, MIC values of tobramycin towards both _P. aeruginosa_ strains were determined before persistence induction and after removal of the antibiotic and resuspension of the persistent population in fresh medium. For both bacterial strains, MICs of tobramycin either before or after persister reactivation were 1 ug/mL, confirming that the tobramycin tolerance of _P. aeruginosa_ strains was transient and not due to resistance mechanisms. To test the antimicrobial effect of LRM-CFS on tobramycin-tolerant _P. aeruginosa_, bacteria pre-exposed to 128 ug/mL of tobramycin for 24 h in ASM were further exposed for 24 h to LRM-CFS (diluted at 1:8, 1:6, and 1:4) or to tobramycin 128 ug/mL in fresh ASM. The choice of 24 h as the exposure time of _P. aeruginosa_ to LRM-CFS in ASM was due to the inefficacy of supernatants at earlier times (data not shown). Both _P.