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.

FIG 1

Morphology of P. aeruginosa aggregates. (A) Aggregation of strain PAO1 in vitro when combined with neutrophil debris for 48 h, demonstrating incorporation of extracellular material into the aggregate structure. (B) Representative sputum sample from 5 independent samples from a CF patient confirmed to be infected with P. aeruginosa. Intact neutrophils are apparent within the P. aeruginosa aggregate. (C) Planktonic culture of P. aeruginosa strain PAO1. (D) Neutrophil debris prepared by freeze-thawing, in the absence of bacteria. Samples were Gram stained and viewed with a 60× oil objective; length of size bar, 30 μm.

FIG 2

Viable bacteria are present within P. aeruginosa aggregates formed in the presence of lysed neutrophils. Strain PAO1-GFP was combined with neutrophil lysates for 48 h. Samples were stained with propidium iodide to distinguish nonviable bacteria. (A and D) Viable bacteria (green) occur in clusters within a larger aggregate (A) but are dispersed by DNase treatment (D). (B and E) Nonviable bacteria and extracellular DNA identified by propidium iodide (red) in aggregates (B); treatment with DNase reduces propidium iodide staining (E). (C and F) Overlay of red fluorescent and green fluorescence bacteria. (D to F) Samples treated with DNase, resulting in near complete disruption of the aggregates. Viewed with a 40× objective; length of size bar, 10 μm. Representative images from 9 independent experiments.

FIG 3

Distribution of aggregate size in the presence and absence of neutrophil lysates. Size of aggregates (μm²) plotted on a log₂ scale formed within the initial inoculum (○) and aggregates formed after 48 h in the absence (△) or presence (▽) of neutrophil products. (A and D) Strain PAO1; (B and E) early CF strain; (C and F) isogenic late CF strain; (D to F) larger aggregates were measured in the top quartile of aggregate size. Red bars represent the median for each condition. Analysis of variance performed for each strain. ****, P < 00001; *, P < 0.05, by Kruskal-Wallis test.

FIG 4

P. aeruginosa growth is not altered by the presence of neutrophil lysates. Viable bacteria after 48 h in the absence (△) or presence (▽) of neutrophil (PMN) products for PAO1 and early and late CF isolates. Aggregates were disrupted, and bacteria were quantified by colony counts following serial dilution and plating. Initial inoculum was approximately 5 × 10⁵ CFU. Red bars represent the median for each condition, which were not different by Kruskal-Wallis test.

FIG 5

Quorum sensing gene expression in aggregates and sputum. Expression of genes involved in quorum sensing in PAO1. Early and late CF isolates of planktonic cultures (open circles) were compared to P. aeruginosa grown for 48 h in the absence (blue circles) or presence (green circles) of neutrophil products and sputum samples from CF patients (red squares). Solid lines represent the mean. (A) Expression of lasA normalized to expression of rpoD; (B) expression of rhlA normalized to expression of rpoD; (C) expression of ureB normalized to expression of rpoD. *, P < 0.05; **, P < 0.01, by Wilcoxon signed-rank test. †, P < 0.0001, by unpaired t test comparing planktonic bacteria. a, P < 0.05; b, P < 0.01, by Mann-Whitney test for sputum samples compared to planktonic PAO1.

FIG 6

Aggregate structure allows for antibiotic resistance to tobramycin. (A) Planktonic bacterial survival when exposed to tobramycin (TOB; open bars) and tobramycin and DNase (hatched bars). (B) Survival of P. aeruginosa aggregates formed in the presence of neutrophil products and exposed to tobramycin (open bars) and tobramycin and DNase (hatched bars). Survival (%) was calculated by dividing the CFU of each treatment by the CFU of an untreated sample. The means ± standard errors of the means (SEM) are depicted from 8 independent experiments. Mann-Whitney t test between aggregates treated with tobramycin and those treated with tobramycin plus DNase. *, P < 0.05.

FIG 7

Antagonistic effect of azithromycin toward tobramycin-induced killing of P. aeruginosa aggregates. (A) Planktonic bacteria survival when exposed to tobramycin (TOB), azithromycin (AZM), or the combination. (B) Survival of P. aeruginosa aggregates formed for 48 h in the presence of neutrophil products from PAO1, and in the early and late CF strains, and exposed to tobramycin (TOB), azithromycin (AZM), or the combination. Survival (%) was calculated by dividing CFU from each treatment by CFU of cultures from the respective strain prior to antibiotic treatment. The means ± SEM are depicted from 4 to 8 independent experiments. Lines represent Dunn's multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.