Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogenic bacterium known for causing nosocomial infections in immunocompromised individuals. The ability of P. aeruginosa to develop biofilms contributes to its resistance to antimicrobial agents and immune defenses, making the treatment of infections more difficult. Recent strategies to combat biofilm infections include enzymatic treatments to degrade the biofilm extracellular polysaccharide matrix (EPS), allowing for better diffusion of antibiotics. While it is crucial to understand biofilm degradation, current methods for quantitatively measuring enzymatic degradation of the extracellular matrix are time-consuming and not amendable to high-throughput screening of suitable enzyme candidates. Herein, we present biofilm-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on biofilm EPS. The biofilms are harvested and used to suspend the hydrophobic SWCNT via tip sonication. The extent of biofilm EPS degradation is quantified by monitoring quenching of the SWCNT fluorescence caused by changes to the SWCNT local dielectric upon enzymatic degradation. We used this platform to screen thirteen hydrolases with varying bond breaking selectivity against a panel of wild type P. aeruginosa and various of its mutants deficient in one or more EPS polymer or altered in the amount or type EPS produced. Our results indicate that the enzymes with selectivity towards α-(1,4), β-(1,4), and β-(1,3) glycosidic linkages, such as lyticase and amyloglucosidase, can most effectively disrupt the EPS. Next, we performed concentration-dependent studies of enzymes on two of the most common strains found in Cystic Fibrosis (CF) environments (PDO300-alginate overproducer and MJK8-small colony variant) and extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model. We found a good model fit (R² ranging from 0.71 to 0.97) of the dynamic data from six enzymes tested at five different concentrations each using the same five parameter model. Reaction rates (turnover rate) are dependent on enzyme concentration and ranges from 2.16e-9 to 2.80e-3 *s^-1 per mg/ml of enzyme. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of the hydrolase panel. This platform should enable faster discovery and design of enzyme-based therapies.

Sparsh Agarwal

Graduate Research Assistant, Iowa State University

Appearances