Mannitol reverts persister <i>P. aeruginosa</i> in biofilms mainly by increasing their metabolic activity.

<p>(A) Addition of the PMF inhibitor CCCP blocks the effect of mannitol on tobramycin susceptibility. Biofilms were grown in multiwell plates for 5 h as described in Figure 3A except that after treatment with tobramycin for 1 h, CCCP was added at 100 µM at the same time as mannitol to the cultures, and the plates were incubated for a further 1-2 h before enumerating CFU. Error bars indicate SEM (n = 3). (B) Mannitol has limited effect on biofilms of a mannitol dehydrogenase, <i>mtlD</i>, mutant strain. <i>mtlD</i>::Tn<i>5</i> mutant biofilms were grown as described above and treated with tobramycin for 1 h, then mannitol, glucose or NaCl were added to the wells and the plates incubated for a further 2 h before enumerating CFU. Error bars indicate SD (n = 4). ns indicates no significant difference (<i>P</i> ≥ 0.1) and asterisks indicate statistically significant difference (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01) of combination treatments versus tobramycin and CCCP only in panel A, or versus tobramycin alone only in panel B.