Pseudomonas aeruginosa aggregation and Psl expression in sputum is associated with antibiotic eradication failure in children with cystic fibrosis

Abstract

We previously demonstrated that P. aeruginosa isolates that persisted in children with cystic fibrosis (CF) despite inhaled tobramycin treatment had increased anti-Psl antibody binding in vitro compared to those successfully eradicated. We aimed to validate these findings by directly visualizing P. aeruginosa in CF sputum. This was a prospective observational study of children with CF with new-onset P. aeruginosa infection who underwent inhaled tobramycin eradication treatment. Using microbial identification passive clarity technique (MiPACT), P. aeruginosa was visualized in sputum samples obtained before treatment and classified as persistent or eradicated based on outcomes. Pre-treatment isolates were also grown as biofilms in vitro. Of 11 patients enrolled, 4 developed persistent infection and 7 eradicated infection. P. aeruginosa biovolume and the number as well as size of P. aeruginosa aggregates were greater in the sputum of those with persistent compared with eradicated infections (p < 0.01). The amount of Psl antibody binding in sputum was also greater overall (p < 0.05) in samples with increased P. aeruginosa biovolume. When visualized in sputum, P. aeruginosa had a greater biovolume, with more expressed Psl, and formed more numerous, larger aggregates in CF children who failed eradication therapy compared to those who successfully cleared their infection.

Figure 1

Patients with persistent infection had greater P. aeruginosa biovolume and greater number of larger aggregates compared to those with eradicated infection. P. aeruginosa visualization in initial sputum sample from negative controls (Negative PA, n = 3), positive controls (Chronic PA, n = 3), CF children who eradicated infection (Eradicated, n = 7) and CF children who developed persistent infection (Persistent, n = 4). Represented median (horizontal line) P. aeruginosa (A) average biovolume intensity (µm³)/sputum slice, (B) average number of aggregates/sputum slice, (C) average maximum aggregate size (µm)/sputum slice. **p < 0.01 using Mann–Whitney test.

Figure 2

Representative 3D confocal images of P. aeruginosa in the sputum of CF patients showing more numerous, larger aggregates in those with persistent compared to eradicated infection. P. aeruginosa structural differences are shown using a fluorescently-labelled molecular probe specific for P. aeruginosa (green), anti-Psl mAb (magenta), DAPI nucleic acid stain (blue) molecular probes and merged image, with ×100 oil objective lens.

Figure 3

Greater overall anti-Psl antibody staining of P. aeruginosa in sputum from patients with persistent compared to eradicated infection. Initial sputum sample from negative controls (Negative PA, n = 3), positive controls (Chronic PA, n = 3), children with CF for whom infection was eradicated (Eradicated, n = 7) and children with CF who developed persistent infection (Persistent, n = 4). Horizontal line indicates the median. (A) average anti-Psl antibody binding intensity (µm³)/sputum slice, (B) average Psl antibody binding per 100,000 µm³ P. aeruginosa biovolume/sputum slice, (C) average proportion of anti-Psl antibody co-localized with P. aeruginosa represented by the M2 coefficient, (D) representative images from sputum from a patient with Persistent, Eradicated and Chronic infection, green: P. aeruginosa, magenta: anti-Psl antibody, white: Psl antibody co-localized with P. aeruginosa. *p < 0.05, ns: not significant using Mann–Whitney test.

Figure 4

Greater overall anti-Psl antibody staining in glass slide chamber of P. aeruginosa isolates from patients with persistent compared to eradicated infection. Eradicated (n = 7) and persistent (n = 4) P. aeruginosa isolates grown for 48 h as biofilms in glass slide chamber and then labelled with fluorescent anti-Psl antibody for 3 h then visualized with confocal microscopy. Horizontal line, median. (A) average anti-Psl antibody binding intensity (µm³)/well, (B) average Psl antibody binding per 100,000 µm³ P. aeruginosa biovolume/well, (C) average P. aeruginosa biovolume intensity (µm³)/well. *p < 0.05, ns: not significant using Mann–Whitney test.

Figure 5

Differences in tobramycin response and aggregation between persistent and eradicated P. aeruginosa isolates in glass slide chamber. Eradicated (n = 7) and persistent (n = 4) P. aeruginosa isolates grown for 24 h as biofilms in glass slide chamber and then treated with 1000 µg/mL tobramycin for an additional 24 h then visualized with confocal microscopy. LB: Lysogeny Broth alone; + T: plus Tobramycin; SS: in presence of 5% pooled CF Sputum Supernatant. Horizontal line, median. (A) average P. aeruginosa biovolume intensity (µm³)/well, (B) average luminescence in relative light units (RLU) of ATP assay/well, (C) average degree of aggregation measured by surface to biovolume ratio/well. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant using Mann–Whitney test.