Abstract

Purpose: Biofilms have been recognized as causing more than 65% of human microbial infections. Mannitol is used in the treatment of cystic fibrosis as an osmotic agent and is also under investigation as an excipient for inhalation products. Previously, mannitol was reported to be able to increase the susceptibility of persister bacteria in PA01 biofilms to tobramycin. However, in clinical practice, mannitol is not always used in combination with tobramycin. Therefore it is important to determine the effect of mannitol itself on pseudomonas biofilms. The objective of this study is to evaluate the effect of a clinically relevant range of concentrations of mannitol on the growth of pre-formed Pseudomonas aeruginosa biofilms, in presence and absence of a subinhibitory dose of tobramycin. Methods: P. aeruginosa (PA01) was grown in BHI medium from a loopful of an overnight blood agar culture. Bacterial suspensions were standardized by spectrometry to a concentration of 1x105 cells/mL. One-hundred microliters of this standard suspension were added into each well of a 96-well plate and incubated at 37°C and 75 rpm for 1.5 h to promote bacterial adhesion. Then supernatant was discarded and each well was washed twice with PBS before adding 100 μL of BHI medium and reincubated for 24 h. After incubation, biofilms were washed with PBS and incubated in M9 medium supplemented with 10 mg/mL peptone as carbon source, and mannitol at concentrations from 2 to 1024 mM. Thirty μg/mL was used as a subinhibitory dose for tobramycin (showing less than 80% of reduction in proliferation). XTT cell proliferation assay was used to evaluate bacterial viability in biofilms after treatment. Each condition was tested at least 4 times. Statistical analysis was done in JMP® 10.0.2 software (p<0.05). Results: It was found that incubation of pre-formed Pseudomonas aeruginosa biofilms with mannitol for 24h caused a significant increase in biofilm growth respect control (Absorbance: 0.082 ± 0.024) at concentrations above 32 mM (p<0.01). However, when tobramycin was added together with mannitol, no significant differences were observed compared to tobramycin used alone (Absorbance: 0.028± 0.014). Conclusions: This preliminary study highlights the importance of the evaluation of new formulations containing mannitol on biofilm proliferation when is intended to be used in diseases with identified presence of P. aeruginosa biofilms. Further studies are necessary to assess the effect of mannitol association with different concentrations of tobramycin on prevention of biofilm formation and the effect of the aging of the biofilm.