## 3 Discussion
aeruginosa_ strains tested, and the killing curves were typically biphasic, suggesting the formation of persisters in the adopted conditions. The acquisition of a tobramycin tolerance state was confirmed by the marked survival of bacteria observed following a second exposure of the cultures at high dose of the same antibiotic. Despite the high number of tobramycin-tolerant bacteria, LRM-CFS (diluted at 1:6 and 1:4) were able to exert a strong bactericidal effect, causing a decrease in the CFU number under or close to the limit of detection. This point is of particular importance, as eradication of the entire bacterial population is mandatory to avoid the relapse of infections and minimize the chance of resistance development during prolonged antibiotic treatments. The antibacterial activity of CFS from lactobacillus is mainly ascribed due to the acidic pH caused by the presence of weak organic acids (WOAs) (i.e., lactic, acetic, and propionic acids) [28]. The presence of high levels of lactic acid (approximately 0.15 M) was confirmed in our LRM-CFS preparation. Previous studies have suggested several potential mechanisms of antimicrobial action for WOAs [29; 30], some of which could be involved in the anti-persisters activity of LRM-CFS observed in the present study. As WOAs are relatively hydrophobic, they can diffuse across the bacterial cell membranes, dissociate, and lower the cytoplasmic pH of the bacteria. Such a decrease in the cytoplasmic pH can also increase the osmolality, resulting in an influx of water and subsequent increase in turgor pressure [31]. Another suggested mechanism of action for WOAs is the permeabilization of the outer membrane of Gram-negative bacteria [32]. The lipophilic nature of some organic acids implies that they can migrate and intercalate in the lipid membranes of the bacterial cell envelope, with potentially toxic consequences [32]. The likely membrane-targeting mechanism of action of WOAs could explain the observed ability of LRM-CFS to kill dormant persister cells more efficiently than conventional antibiotics (e.g., tobramycin) that, instead, act mostly on actively replicating bacterial cells [20]. Nonetheless, we cannot exclude a contribution to the anti-persister activity of LRM-CFS of the bacteriocins. Indeed, the genome sequence of _L. rhamn_s GG strains (GenBank accession no. FM179322), which was used in this study, indicates the presence of bacteriocin genes biosynthetic proteins (LRHM_2289 to LRHM_2312). Moreover, we produced LRM-CFS in MRS broth at pH 6.2 and 37 \({}^{\circ}\)C, which were previously determined as the optimal conditions for bacteriocin production [33]. Of note, WOAs and bacteriocins might synergize against persister bacteria;in fact, WOAs might have a destabilizing effect on the outer membrane of Gram-negative bacteria, favoring bacteriocins reaching their main target represented by the cytoplasmic membrane [32]. The biofilm lifestyle represents a reservoir of high phenotypic diversity, and it is considered one of the most important adaptive mechanisms of _P. aeruginosa_ within the CF lung [34; 35]; yet, the transition towards the persister phenotype in _P. aeruginosa_ is not fully elucidated. Biofilms have a high tolerance towards antibiotics, which makes their eradication extremely challenging, urging the need to find new antibiofilm strategies [36; 37]. It has been demonstrated that certain antibiotics, such as gentamicin and ciprofloxacin, can induce the formation of highly tolerant dormant cells in biofilms, explaining, at least partially, the biofilm recalcitrance to antimicrobial treatments [23]. Thus, in this study, we lastly attempted to select _P. aeruginosa_ persisters in ASM, exposing biofilms of both PaCF1 and PaCF4 strains to high doses of ciprofloxacin. The biphasic killing curve obtained was indicative of a successful enrichment of ciprofloxacin-tolerant persister cells in the biofilm.