oes not have any effect on dispersal, P. aeruginosa PAO1 biofilms were grown for 1 day in continuous flow microfermenters and exposed to either mannitol or glucose as a positive control at 20 mM and 100 mM. While 1963850 glucose at both concentrations PCI-32765 resulted in biofilm dispersal as previously reported, addition of mannitol at 40 mM had no effect on dispersal and addition of mannitol at 100 mM could induce some dispersal but to much reduced extent compared to glucose. To assess whether the reversion of persister cells by mannitol required energy, the proton-motive force inhibitor CCCP was added at the same time as mannitol to pregrown young biofilms that had been treated or not with tobramycin. 25653074 The addition of 100 M CCCP did not appear to increase the number of tobramycin persisters in the absence of mannitol. In the presence of CCCP, the addition of mannitol, even at 40 mM which was shown above to revert persister cells, could not further increase killing compared to treatments with tobramycin and CCCP alone. In contrast, addition of CCCP did not prevent NaCl to increase killing of biofilm bacteria by tobramycin. Further, the effect of mannitol was also tested on young biofilms of a PAO1 mtlD::Tn5 mutant strain, unable to express the mannitol dehydrogenase and thus metabolise mannitol. The results show that 40 mM mannitol only had a limited, not statistically significant, effect on the reversion of persister cells in biofilms of this strain, improving tobramycin killing by only 86.2% compared to 98.6% in PAO1 wild type biofilms. In contrast, addition of 40 mM glucose or NaCl reverted persisters to a similar extent as the wild type strain, increasing killing by 98.2% and 97.0%, respectively. Taken together, these results strongly suggest that mannitol reverts persister bacteria mainly by inducing metabolism and generating a PMF in these cells. Mannitol can reverse persister cell formation in a P. aeruginosa mucoid clinically relevant strain The results presented thus far strongly suggest that mannitol may be useful for the eradication of P. aeruginosa biofilm related infections. To further assess the efficacy of this treatment, we investigated its effect on two clinically relevant strains. The mucoid strain P. aeruginosa FRD1 was observed to respond similarly to tobramycin as the PAO1 strain with concentrations of tobramycin 80 g/ml unable to cause further reduction in CFU indicating persister cells, and the addition of mannitol could alleviate the persister phenotype. When the CF isolate P. aeruginosa 18A was tested, exposure to tobramycin up to 1,600 mg/L, thus 80 MIC, showed only a 2 log reduction in CFU, indicating that biofilms of this strain were highly tolerant to tobramycin relative to PAO1 and FRD1, which is consistent with a higher MIC of tobramycin in this strain compared to PAO1 and FRD1. The addition 
of mannitol to 18A biofilms had no significant effect on the antibiotic resistant population at any concentration tested. Reversion of persister cells by mannitol is not linked to biofilm dispersal and requires energy The sudden supplementation of nutrients to established biofilms was previously found to induce dispersal and induction of biofilm dispersal has been linked to increased antibiotic susceptibility. Because mannitol can be used for metabolism in P. aeruginosa, we tested whether its addition to biofilms may also cause dispersal. In the absence of tobramycin, neither the addition of mannitol, glucose nor NaCl resulted in decr
