Figure 1.
Biofilm-associated persister bacteria in P. aeruginosa.
(A) Young P. aeruginosa biofilms pre-grown for 5 h in the absence of antibiotic before being treated with tobramycin at 0-160 mg/L for 1-3 h. Biofilm viability was analysed by the drop plate method and CFU counts. Error bars indicate standard error of the geometric mean (SEM, n = 3). (B) Same as in (A) but established P. aeruginosa biofilms were pre-grown for 20 h in the absence of antibiotic before being treated with tobramycin. Error bars indicate standard deviation (SD, n = 4).
Figure 2.
Mannitol prevents the formation of persister cells in P. aeruginosa biofilms.
(A) Biofilms were grown in multiwell plates for 5 h in M9 minimal medium with glucose as a carbon source, with or without mannitol at 5-40 mM. Tobramycin was then added to the culture medium to 80 mg/L and the plates were incubated for a further 2 h before enumerating CFU. Error bars indicate SEM (n = 3). (B) To check for substrate or osmotic effect, mannitol was replaced with NaCl or additional glucose at various concentrations. Error bars indicate SD (n = 4). Asterisks indicate statistically significant difference of combination treatments versus tobramycin only (*, P < 0.05; ***, P < 0.001).
Figure 3.
Exposure to mannitol reverses the persister phenotype in P. aeruginosa biofilms.
(A) Young, 5 h old biofilms grown in multiwell plates were first treated with or without 80 mg/L tobramycin for 1 h to select for persister cells. Then mannitol was added to the culture and the plates were incubated for a further 1-2 h, before enumerating CFU. Glucose and NaCl were used in place of mannitol to check for substrate or osmotic effects. (B) Established, starving biofilms grown for 20 h in the absence of treatment were exposed or not to tobramycin for 1 h. Mannitol, glucose or NaCl were then added the cultures for a further 1-3 h, before analysing CFU. Error bars indicate SEM (n is indicated in parentheses for each set of samples). Asterisks indicate statistically significant difference of combination treatments versus tobramycin only (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Figure 4.
Mannitol alone does not induce biofilm dispersal or decrease in biofilm CFU counts.
(A) Biofilms were pre-grown for 5 h in microtitre plates in the absence of any treatment. Then individual treatments with mannitol, glucose, NaCl or tobramycin as a positive control were added to the wells and the plates incubated for further 2 h before analysing biofilm viability by the drop plate method and CFU counts. Error bars indicate SD (n = 4). (B) Microscopic images of LIVE/DEAD stained P. aeruginosa biofilms grown at the bottom of 24-well plates for 6 h or 24 h in the presence or absence of 40 mM mannitol. Live cells appear green, dead cells appear red. Note that the DEAD stain is also known to bind extracellular DNA in the biofilm matrix [51]. Bar, 20 µm. (C) P. aeruginosa biofilms were cultivated in continuous flow microfermenters with glucose at 2 mM. After 1 day of growth, the medium inlet was supplemented (arrow) with glucose at 20 mM (opened circles), 100 mM (opened squares), or mannitol at 20 mM (filled upright triangles) or 100 mM (filled inverted triangles) and the release of dispersal cell was monitored by measuring the OD600 of the effluent runoff.
Figure 5.
Mannitol reverts persister P. aeruginosa in biofilms mainly by increasing their metabolic activity.
(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, mtlD, mutant strain. mtlD::Tn5 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 (P ≥ 0.1) and asterisks indicate statistically significant difference (*, P < 0.05; **, P < 0.01) of combination treatments versus tobramycin and CCCP only in panel A, or versus tobramycin alone only in panel B.
Figure 6.
Mannitol increases the efficacy of tobramycin against clinically relevant strain P. aeruginosa FRD1, but not tobramycin resistant P. aeruginosa 18A, which was isolated from a CF patient.
(A) Biofilms were grown in multiwell plates for 5 h (FRD1) or 24 h (18A) at which time tobramycin was added to the cultures at various concentrations. The plates were incubated for a further 2 h before enumerating CFU. (B) Biofilms were grown for 5 h (FRD1) or 24 h (18A) in the absence of antibiotic, before being treated with or without tobramycin at 80 mg/L (FRD1) or 400 mg/L (18A) for 1 h to select for persisters. Then mannitol was directly added to the wells at 0-40 mM, and the plates were incubated for a further 2 h before analysing CFU. Error bars indicate SD (n = 4). Asterisks indicate statistically significant difference of combination treatments versus tobramycin only (**, P < 0.01; ***, P < 0.001).