(463) Psl glycosyl hydrolase (PslGh) improves eradication of CF Pseudomonas aeruginosa biofilms when combined with antibiotics

Background: Pseudomonas aeruginosa (Pa) lung infection in cystic fibrosis is characterised by a biofilm mode of growth, wherein a matrix of self-secreted molecules provides physical protection against antimicrobials. PslG hydrolase (PslGh) has the ability to degrade the Psl glycan, an exoplysaccharide involved in the biofilm structure of Pa. In this study, we used in vitro biofilm cultures from clinical Pa strains to determine whether there was benefit in combining PslGh with a variety of antibiotics routinely used as nebulized agents. 

Methods: We selected 14 different Pa strains isolated from children with cystic fibrosis. We grew biofilms in 96-well plates for 24 hours in Luria Broth before removing planktonic growth and adding new media with aztreonam (ATM), colistin (CST) or tobramycin (TOB) alone and with PslGh for 18 hours before staining with crystal violet (CV) for biofilm quantification. For all antibiotics, we used peak concentrations achievable in sputum [1] and for the PslGh, we used a fixed concentration of 0.05mg/mL. To further investigate, we selected an eradicated (PA50) and a persistent (PA380) Pa strain [2], and PAO1 to examine under confocal microscopy using the same treatment parameters in 8 well glass chamber slides stained with Syto 9 (Sigma Aldrich) to quantify live cell biovolume. Colony forming unit (CFU/mL) analysis was performed to confirm reduction in signal intensity correlated with reduction in viable cells. We performed our experiments in biological triplicate and then used paired t-tests between treatment groups (percent biofilm reduction compared to controls) to discern significance (p< 0.05). 

Results: Overall, for the CV assay, there was an additional reduction in biomass of 18.3% (12.8-23.8, 95% CI), 6.6% (-2.5-15.7, 95% CI), and 10.0% (0.7-19.2, 95% CI) when PslG hydrolase was added to TOB, CST, and ATM respectively. At the level of individual strains, 64% (9/14) strains showed significant reduction in biofilm biomass when PslGh was combined with TOB, 29% (4/14) strains with CST, and 14% (2/14) with ATM. Using confocal microscopy analysis, a significant reduction in live cell biomass was seen for TOB in the eradicated strain PA50, for CST in PA01, and for ATM in PA01 as well as the persistent strain PA380. Changes in biomass correlated with reduction in CFU/mL. Results of the CV assay did not always correlate with the confocal microscopy or colony counts as, particularly with ATM, where there was a high level of CV staining with dead filamentous matter, but fewer viable cells, as confirmed by Syto 9 staining and CFU analysis.   

Conclusions: Our results demonstrate that the addition of PslGh potentiates the action of all 3 antibiotics tested against clinical Pa isolates grown in biofilm culture. The greatest additional biofilm eradication was seen with TOB, although there were bacterial strain differences for all antibiotics evaluated. TOB-PslGh combination may be a novel anti-biofilm agent for nebulization to optimize patient outcomes.

Acknowledgements:

References: [1] Chmiel JF et al. Antibiotic management of lung infections in cystic fibrosis. II. Nontuberculous mycobacteria, anaerobic bacteria, and fungi. Annals of the American Thoracic Society. 2014 Oct;11(8):1298-306.
[2] Morris AJ, Jackson L, CW Yau Y, Reichhardt C, Beaudoin T, Uwumarenogie S, Guttman KM, Lynne Howell P, Parsek MR, Hoffman LR, Nguyen D. The role of Psl in the failure to eradicate Pseudomonas aeruginosa biofilms in children with cystic fibrosis. npj Biofilms and Microbiomes. 2021 Aug 4;7(1):1-8.