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

Abstract

e Pseudomonas aeruginosa is a major pathogen in cystic fibrosis (CF) lung disease. Children with CF are routinely exposed to P. aeruginosa from the natural environment, and by adulthood, 80% of patients are chronically infected. P. aeruginosa in 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-attached in vitro biofilm 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 novel in vitro model of P. aeruginosa aggregates incorporating human neutrophil-derived products. Aggregates grown in vitro and those found in CF patients' sputum samples were morphologically similar; viable bacteria were distributed in small pockets throughout the aggregate. The lasA quorum 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 simple in vitro system advances our ability to model infection of the CF airway and will be an important tool to study virulence and test alternative eradication strategies against P. aeruginosa.

Microbial Biofilms in Pulmonary and Critical Care Diseases

“…Bacteria form similar biofilm aggregates within high-density gels when bacterial motility is restricted, and conditions associated with CF sputum and chronic inflammation, including the presence of neutrophil elastase, DNA, and amino acids, are sufficient to promote biofilm aggregate formation (41,122,123). Importantly, this nonattached biofilm growth also confers resistance to antibiotics and neutrophil killing in vitro (41).…”

Impact of azithromycin on the clinical and antimicrobial effectiveness of tobramycin in the treatment of cystic fibrosis

“…Concomitant use of the two agents is now common, but has not been tested prospectively. Several independent in vitro studies have reported antagonism between azithromycin and tobramycin against P. aeruginosa 4–6 . We have hypothesized that combined therapy with these two medications may unexpectedly provide less rather than more clinical improvement.…”

Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis

“…In chronic CF airways, P. aeruginosa does not form; however, these flat, thick biofilms covering the airway surface but reside instead in three-dimensional biofilms also called suspension biofilms or non-attached aggregates [212,213,214,215,216]. These bacterial aggregates accompanied by large amounts of PMNs represent the typical clinical image found in the airways of CF patients chronically infected with P. aeruginosa (Figure 1) [212,213,214,215,216]. …”

“…ecDNA is a main component of P. aeruginosa aggregates, and ecDNA obtained from necrotic PMNs has been shown to support aggregate growth of P. aeruginosa [214]. NET formation can likely be the source of PMN DNA in CF airways, since P. aeruginosa elicits robust NET release in PMNs [122,128,170,205].…”