title: Enhanced in vitro formation and antibiotic resistance of nonattached Pseudomonas aeruginosa aggregates through incorporation of neutrophil products.
abstract: 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.
authors.affiliation: Department of Medicine, National Jewish Health, Denver, Colorado, USA.
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Caceres
authors.affiliation: Department of Medicine, National Jewish Health, Denver, Colorado, USA Department of Medicine, University of Colorado, Denver, Aurora, Colorado, USA malcolmk@njhealth.org.
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Malcolm
authors.affiliation: Department of Medicine, National Jewish Health, Denver, Colorado, USA Department of Medicine, University of Colorado, Denver, Aurora, Colorado, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Taylor-Cousar
authors.affiliation: Department of Medicine, National Jewish Health, Denver, Colorado, USA Department of Pediatrics, National Jewish Health, Denver, Colorado, USA Department of Medicine, University of Colorado, Denver, Aurora, Colorado, USA.
authors.forename: David P
authors.initials: DP
authors.lastname: Nichols
authors.affiliation: Department of Medicine, National Jewish Health, Denver, Colorado, USA Department of Medicine, University of Colorado, Denver, Aurora, Colorado, USA.
authors.forename: Milene T
authors.initials: MT
authors.lastname: Saavedra
authors.affiliation: Department of Pediatrics, National Jewish Health, Denver, Colorado, USA.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Bratton
authors.affiliation: Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Moskowitz
authors.affiliation: Division of Pediatric Infectious Disease, Department of Pediatrics, Seattle Children's Hospital, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Jane L
authors.initials: JL
authors.lastname: Burns
authors.affiliation: Department of Medicine, National Jewish Health, Denver, Colorado, USA Department of Medicine, University of Colorado, Denver, Aurora, Colorado, USA.
authors.forename: Jerry A
authors.initials: JA
authors.lastname: Nick
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017963:Azithromycin / Q000494:pharmacology; D018441:Biofilms; D003550:Cystic Fibrosis / Q000150:complications; D024901:Drug Resistance, Multiple, Bacterial; D006801:Humans; D045726:Metalloproteases / Q000096:biosynthesis* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009504:Neutrophils / Q000276:immunology*; D011552:Pseudomonas Infections / Q000150:complications / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D053038:Quorum Sensing / Q000235:genetics*; D013183:Sputum / Q000382:microbiology; D014031:Tobramycin / Q000494:pharmacology; D037521:Virulence Factors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D037521:Virulence Factors; D017963:Azithromycin; C548166:LasA protein, Pseudomonas aeruginosa; D045726:Metalloproteases; D014031:Tobramycin