## 4 Materials and Methods
### CCCP Induction of P. aeruginosa Planktonic Persisters and Evaluation of LRM-CFS Activity  
Overnight cultures of PaCF1 and PaCF4 in Luria Bertani broth (LB, Oxoid, Basingstoke, Hampshire, UK) were incubated with CCCP 200 \(\upmu\)g/mL for 3 h at 37 \({}^{\circ}\)C with shaking in an Eppendorf ThermoMixer\({}^{\otimes}\)C at 550 rpm, as previously described [20]. A control sample was prepared by adding the same amount of DMSO, used to dissolve CCCP, and incubating it under the same conditions. After exposure to CCCP, _P. aeruginosa_ persister cells were exposed to LRM-CFS diluted at 1:4, 1:6, and 1:8 for 3 h at 37 \({}^{\circ}\)C. At this aim, persister bacteria were washed twice with PBS to remove CCCP by centrifugation at 1700\(\times g\) for 10 min, and then they were resuspendedin ASM in the presence of LRM-CFS diluted at 1:4, 1:6, and 1:8. To confirm the persistence condition of bacteria, in each set of experiments, two control samples were prepared: CCCP-pretreated bacteria were incubated with ciprofloxacin 5 ug/mL (20 x MIC) or with MRSB at pH = 4 (at the same volume of LRM-CFS). Samples were incubated for 3 h in shaking conditions at 550 rpm, at the temperature of 37 \({}^{\circ}\)C in an Eppendorf ThermoMixer(r) C (Eppendorf, Hamburg, Germany). After incubation, samples were centrifuged and washed again as described before, serially diluted, and plated on TSA. TSA plates were incubated for 24-72 h for CFU enumeration. Tobramycin Selection of P. aeruginosa Planktonic Persisters in ASM and Evaluation of LRM-CFS Activity  
Overnight cultures of PaCF1 and PaCF4 grown in LB were centrifuged at 1700\(\times\)\(g\) for 10 min, and the bacterial pellet was re-suspended in ASM. Tobramycin was added to the ASM at concentrations of 64 ug/mL (64 x MIC), 128 ug/mL (128 x MIC), and 256 ug/mL (256 x MIC). Samples were then incubated at 37 \({}^{\circ}\)C in shaking conditions at 550 rpm in a ThermoMixer (Eppendorf). A control condition was prepared by adding water instead of tobramycin and incubated in the same conditions. At time 0, 8 h, and 24 h of incubation with tobramycin, the samples were centrifuged at 2200\(\times\)\(g\) for 10 min, and the bacterial pellet was washed two times with PBS, diluted, and plated on TSA. TSA plates were incubated for 24-72 h for CFU enumeration. To ensure that the observed phenomenon was not due to the development of resistance, a standard MIC test was performed after the second tobramycin exposure, with the method previously described in Section 4.5. _P. aeruginosa_ persisters obtained after exposure to tobramycin were exposed to LRM-CFS diluted at 1:4, 1:6, and 1:8 for 24 h. Persister bacteria were washed twice with PBS to remove tobramycin by centrifugation at 2200\(\times\)\(g\) for 10 min, and then they were re-suspended in ASM in the presence of LRM-CFS diluted at 1:4, 1:6, and 1:8. To confirm the maintenance of the persistent status, in each set of experiments, persisters obtained with tobramycin 128 ug/mL were incubated again with tobramycin at the concentration of 128 ug/mL. An additional control was represented by persisters incubated with MRSB at pH = 4 at the same volume of LRM-CFS. Samples were incubated for 24 h in shaking conditions at 550 rpm, at the temperature of 37 \({}^{\circ}\)C in an Eppendorf ThermoMixer(r) C. After incubation, samples were centrifuged and washed again as described before, serially diluted, and plated on TSA. TSA plates were incubated for 24-72 h for CFU enumeration. Ciprofloxacin Selection of P. aeruginosa Biofilm Persisters in ASM and Evaluation of LRM-CFS Activity  
Overnight cultures of PaCF1 and PaCF4 grown in LB were diluted at 1:50 in ASM and seeded at the volume of 100 \(\upmu\)L into wells of a polystyrene 96-well microtiter plate. The microtiter plate was incubated in static conditions at 37 \({}^{\circ}\)C to let biofilms grow.