Discovery and Characterization of Small Molecule Inhibitors of Biofilm Formation in Pseudomonas Aeruginosa

Abstract

Bacterial biofilms are thought to aid in the survivability of a variety of intractable infections in humans. Specifically, biofilm production in Pseudomonas aeruginosa has been shown to play a significant role in chronic infection of cystic fibrosis (CF) patients. Unfortunately, no clinically effective inhibitors of biofilm formation are available. Through the development and application of convenient high-throughput screening methods for non-antibiotic biofilm inhibitors, it was discovered that simple iron salts are potent inhibitors of biofilm formation in P. aeruginosa. These salts function in a dose-dependent manner to cause biofilm detachment in both static and flow chamber experiments. P. aeruginosa strains taken from the sputum of 17 CF patients showed a similar response to elevated iron levels. Because free iron salts have toxic effects that likely preclude their wide use as clinical anti-biofilm agents, I tested response of P. aeruginosa PA14 biofilms to elevated iron concentrations, utilizing nine structurally varied iron chelators as iron delivery agents in a microtiter plate assay for biofilm production. Picolinic acid and acetohydroxamic acid-chelated iron were able to stymie biofilm production at moderate concentrations in P. aeruginosa PA14 and 17 Pseudomonas strains from CF patients. Chelated iron sources showed outstanding distributive properties in an Andersen cascade impactor model of lung-targeted drug delivery, highlighting the potential of these chelators as nebulized agents to combat P. aeruginosa in Cystic Fibrosis. Previous research showed that production of excess rhamnolipids in P. aeruginosa PA01 caused dispersal of its biofilms. To identify small molecule inducers of rhamnolipid biosynthesis, in-house chemical libraries were screened using P. aeruginosa PS36, a strain possessing a chromosomally-encoded rhlA::gfp reporter, for molecules that would effect increased transcription of rhlA. Compound ML1275 was found to upregulate transcription of rhlA in a potent, dose-dependent fashion. Subsequent assays confirmed ML1275 induction of rhamnolipid overproduction in the wild-type P. aeruginosa PA14 strain, and that ML1275 has modest anti-biofilm activity in P. aeruginosa PA14. Through these studies, several novel inhibitors of biofilm formation were discovered and characterized, hopefully an important step toward improving treatments for P. aeruginosa infections in cystic fibrosis patients.