D-066. Disruption of Pseudomonas Aeruginosa Biofilms by Utilizing Strategies to Depolymerize F-Actin and DNA

Q. M. Parks1,2, R. L. Young2, K. C. Malcolm1, K. R. Poch1, M. L. Vasil2, J. A. Nick1,2;
1Natl. Jewish Med. and Res. Ctr., Denver, CO, 2Univ. of Colorado, Denver, CO.

In childhood, the cystic fibrosis airway is characterized by persistent inflammation with high quantities of neutrophils. Previously, we have shown a significant neutrophil dependent enhancement of P. aeruginosa biofilm density that is greatest for low initial concentrations of P. aeruginosa. Biofilm enhancement is facilitated via neutrophil DNA and F-actin, which form a framework that P. aeruginosa can exploit for growth. These polymers associate via positively charged molecules such as histones. P. aeruginosa then affixes to this matrix, which results in increased early biofilm density, displaying an increase orders of magnitude over P. aeruginosa in the absence of neutrophils at 24 hours. Our hypothesis is that compounds that disperse DNA and/or F-actin can disrupt neutrophil-enhanced biofilms. Poly(Aspartic acid), a negatively charged amino acid chain with the capacity to disrupt F-actin, was examined singly and in combination with DNase by various methods as well as clinical antibiotics. A Nunc-TSP system was used to allow for high throughput assessment of biofilm formation in the presence of neutrophils and the effect of various agents in disrupting the biofilm. P. aeruginosa strains tested were two isogenic CF isolates recovered from an initial infection (Early), and following established infection ~4 years later (Late), as well as PAO1. Biofilms were formed by incubation of P. aeruginosa with human neutrophils for 24 to 48 hours. The results indicate that antibiotics were incapable of significantly disrupting biofilms. Poly(D) significantly disrupted neutrophil-induced biofilms. DNase also disrupts a biofilm formed in the presence of human neutrophils. The combination of poly(D) + DNase resulted in an increase in biofilm disruption for a 24 hour biofilm. Biofilms allowed to form for 48 hours were more resistant to disruption by DNase and poly(D) as single agents, but the combination of both demonstrated a synergistic effect. A similar effect was observed when PAO1 was compared to Early and Late strains. We conclude that disruption of biofilms by targeting F-actin, DNA or linker proteins results in increased disruption in vitro.