EnhancedIn VitroFormation and Antibiotic Resistance of Nonattached Pseudomonas aeruginosa Aggregates through Incorporation of Neutrophil Products

Abstract

ABSTRACTPseudomonas aeruginosais a major pathogen in cystic fibrosis (CF) lung disease. Children with CF are routinely exposed toP. aeruginosafrom the natural environment, and by adulthood, 80% of patients are chronically infected.P. aeruginosain the CF airway exhibits a unique biofilm-like structure, where it grows in small clusters or aggregates of bacteria in association with abundant polymers of neutrophil-derived components F-actin and DNA, among other components. These aggregates differ substantially in size and appearance compared to surface-attachedin vitrobiofilm models classically utilized for studies but are believed to share properties of surface-attached biofilms, including antibiotic resistance. However, little is known about the formation and function of surface-independent modes of biofilm growth, how they might be eradicated, and quorum sensing communication. To address these issues, we developed a novelin vitromodel ofP. aeruginosaaggregates incorporating human neutrophil-derived products. Aggregates grownin vitroand those found in CF patients' sputum samples were morphologically similar; viable bacteria were distributed in small pockets throughout the aggregate. ThelasAquorum sensing gene was differentially expressed in the presence of neutrophil products. Importantly, aggregates formed in the presence of neutrophils acquired resistance to tobramycin, which was lost when the aggregates were dispersed with DNase, and antagonism of tobramycin and azithromycin was observed. This novel yet simplein vitrosystem advances our ability to model infection of the CF airway and will be an important tool to study virulence and test alternative eradication strategies againstP. aeruginosa.