The presence of drug tolerant, persister bacteria in biofilms causes major health concerns leading to chronic infections, which require frequent hospital visits for intensive therapy or at times cannot be controlled. Here we found that mannitol, one of the most abundant sugar alcohols in nature, could revert the persister phenotype of P. aeruginosa biofilms and increase, by several orders of magnitude, the efficacy of the antibiotic tobramycin. This effect of mannitol was consistent with published data obtained with persisters of other bacterial strains [21]. Because another metabolic substrate, glucose, similarly improved the outcome of antibiotic therapy and the effects of mannitol were blocked by a PMF inhibitor, it is likely that the primary effect is metabolic. Further, NaCl at osmolarities higher than those used here for mannitol, also showed a positive outcome on the efficacy of tobramycin but to a lesser extent than observed for the mannitol treatments. Taken together, the data suggest that mannitol acts primarily through a metabolic effect and an increase in osmolarity may also contribute to its impact on the tobramycin sensitivity in biofilms.

Treatments with increasing concentrations of tobramycin revealed that P. aeruginosa biofilms harbour a high number of persister bacteria. Young, growing biofilms, which were covered with bacteria and small clusters of cells or microcolonies ( Figure 4B ), were found to typically contain 5 × 10 4 CFU cm -2 persisters, representing 0.1% of the total biofilm CFU. Established, starving biofilms, which contained lower total CFU ( Figure 3 ) and had less cells on the wells bottom surface compared to young biofilms ( Figure 4B ) due to dispersal events [ 37 ], were far more resistant to tobramycin treatment compared to young biofilms. They harboured 1 × 10 5 CFU cm -2 persisters, representing up to 21% of the total population. The biofilm-associated persisters occur in much higher numbers than has been observed for mid-log phase cultures, where persisters typically represent 0.001% of the overall bacterial cells [ 2 ]. In biofilms, the presence of steep nutrient gradients can lead to nutrient depletion even in growing biofilms [ 40 - 42 ], which supports a link between low nutrient availability, starvation and the persister phenotype. While it remains unclear if the formation of persisters was induced upon treatment with the antibiotic in our experiments, as it was observed previously using ciprofloxacin [ 43 ], or if persisters were already present in biofilms, our data strongly suggest that mature biofilms favour the generation of persisters. The failure of higher concentrations of tobramycin to clear the persister cells from the biofilm demonstrates that increasing the concentrations of antibiotics is unlikely to have any beneficial effect for the removal of biofilms. Moreover, caution should be taken as exposure to tobramycin alone has previously been found to result in an increase in biofilm formation in several CF isolates of P. aeruginosa [ 44 ].

Mannitol has a positive effect on biofilm clearance by antibiotics

Mannitol increased the efficacy of tobramycin against P. aeruginosa biofilms in a concentration dependent manner, and up to 3 log at 40 mM. Dry powder mannitol is used as a mucus-clearing agent in CF patients. While the concentration of mannitol is constantly changing in the airway surface liquid due to its water absorbing activities initial concentrations of mannitol are well above 100 mM. Hence the concentrations studied here appear to be well within the therapeutically relevant and achievable range.

The osmolarity of fresh biofilm M9 medium containing 5 mM glucose is 250 mOsm/L and the addition of 40 mM mannitol increases the osmolarity of the medium by 40 mOsm/L, which is the same as for 20 mM NaCl. In this system, increases in osmolarity by 80-100 mOsm/L using higher concentrations of NaCl resulted in improved killing by tobramycin. This effect appeared to be independent of a PMF as it was not reverted by addition of CCCP (Figure 5A), which suggests that it likely resulted from increased drug uptake induced by osmotic changes. This observation is consistent with recent findings that the addition of osmolytes prevented persister formation in E. coli [45]. However, lower concentrations of NaCl had little effect. This suggests that osmotic stress may contribute to antibiotic mediated killing of persisters but was not the main factor underlying the effect of mannitol in these experiments. In contrast, the metabolic substrate glucose, at concentrations similar to mannitol, also had a strong impact on persisters. While glucose was already present in the growth medium, the addition of supplementary glucose likely alleviated nutrient depletion due to gradients within the biofilm and increased metabolic activity in cells previously experiencing low glucose levels. Further, the effect of mannitol appeared to be dependent on increased metabolic activity as its effects were abolished in the presence of the PMF inhibitor CCCP. It should be noted that exposure of the biofilm to tobramycin in the presence of CCCP did not change the number of persister cells identified, approximately 105 CFU cm-2 compared to tobramycin alone treatments. The importance of a metabolic effect of mannitol on the reversion of persister cells was further supported by the observation that biofilms of a mannitol dehydrogenase mutant strain were not significantly affected by mannitol but still showed increased sensitivity to tobramycin after exposure to glucose and to a lesser extent NaCl (Figure 5B). This strongly suggests that at 40 mM (~0.7% w/v) mannitol reverts the persister phenotype in P. aeruginosa biofilms mainly by inducing a metabolic pathway and generating a PMF, in a similar fashion as it was demonstrated in planktonic E. coli bacteria [21]. The downstream effect on tobramycin toxicity may be via increased uptake of the aminoglycoside as was suggested in E. coli [21]. Another possibility is that exposure to mannitol in P. aeruginosa persisters may inhibit starvation-induced defences such as the stringent response, thus leading to decreased production of antioxidant such as catalase. In turn, reduced antioxidant defence could increase the potency of antibiotics such as tobramycin that are known to induce oxidative stress [46,47]. Mannitol is known to have antioxidant properties [48] but may not have any impact on tobramycin-mediated oxidative stress.

In our experiments, beneficial effects of mannitol on tobramycin sensitivity could be observed in biofilms of two strains tested, P. aeruginosa PAO1 and FRD1, however, no effect was found when mannitol was added to biofilms of the P. aeruginosa strain 18A which was recently isolated from a CF patient. The resistance to tobramycin of P. aeruginosa 18A biofilms suggests that P. aeruginosa 18A may also have a multi-drug resistant phenotype, consistent with an MIC for tobramycin more than 15 fold higher compared to that of PAO1. This finding supports the assumption that a multi-drug resistance phenotype may be the consequence of a genetically encoded mechanism, such as increased expression of efflux pumps or enzymatic deactivation of antibiotics, the efficacy of which are unlikely to be influenced by the presence or absence of a metabolite. In contrast, the persister phenotype is a transient state that develops over the growth of the bacterial population, does not affect all the cells and can be reverted by exposure to mannitol. Other chemical compounds have recently been found that are able to revert the persister phenotype. The synthetic brominated furanone (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one, a known quorum sensing inhibitor, was found to increase susceptibility of P. aeruginosa towards tobramycin as well as the fluoroquinolone ciprofloxacin, and the effect was suggested to involve direct activation of transport systems rather than operate via a quorum sensing pathway [49]. In another study, a screen of 6,800 compounds from a chemical library identified 3-[4-(4-methoxyphenyl)piperazin-1-yl]piperidin-4-yl biphenyl-4-carboxylate, a polycyclic small molecule, as a potent treatment effective at reverting E. coli persisters into fast growing cells and restoring their susceptibility to the fluoroquinolone norfloxacin [50]. Therefore several strategies may be useful in order to combat persister cells and antimicrobial resistance, and increasing metabolic activity of persister cells appears to be a key element to improve antibiotic therapy outcomes.

Overall our results, using clinically relevant strains and the standard aminoglycoside tobramycin, suggest that the use of mannitol as an adjuvant to antibiotic therapy may improve the clearance of recalcitrant biofilm infections in addition to improving lung function. In a recent phase III efficacy study of mannitol in CF patients, there was no evidence that the reduction of pulmonary exacerbation incidence observed in the mannitol group was associated with a change in bacterial load in sputum after several weeks exposure to mannitol [26]. However mannitol was given in addition to best care, which includes regular antibiotic treatment and sputum collections were not synchronised with antibiotic applications. Furthermore, the analysis of microorganisms dispersed in sputum may not accurately reflect the efficacy of treatments against biofilms still residing in the lungs. Further studies will be needed to better understand the impact of combined treatments of mannitol and antibiotics for the removal of biofilm infections in vivo.