Poster: 84Large-scale aggregation of Pseudomonas aeruginosa in cystic fibrosis sputum despite reduced airway burden after CFTR modulator therapy

Abstract

Background

Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have transformed care for people with cystic fibrosis (pwCF), improving lung function and reducing airway bacterial burden. However, chronic Pseudomonas aeruginosa (Pa) infection often persists despite treatment, suggesting that factors beyond bacterial load contribute to infection chronicity. One such factor is bacterial aggregation, which is associated with increased antibiotic tolerance and the evasion of host immune defenses. Yet, clinical evaluations of CFTR modulator efficacy focus largely on bacterial quantity, often overlooking structural changes within the airway microbial community. We therefore evaluated the impact of CFTR modulation on Pa burden and aggregation directly in expectorated CF sputum.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have transformed care for people with cystic fibrosis (pwCF), improving lung function and reducing airway bacterial burden. However, chronic Pseudomonas aeruginosa (Pa) infection often persists despite treatment, suggesting that factors beyond bacterial load contribute to infection chronicity. One such factor is bacterial aggregation, which is associated with increased antibiotic tolerance and the evasion of host immune defenses. Yet, clinical evaluations of CFTR modulator efficacy focus largely on bacterial quantity, often overlooking structural changes within the airway microbial community. We therefore evaluated the impact of CFTR modulation on Pa burden and aggregation directly in expectorated CF sputum.

Methods

In this three-year prospective longitudinal study, pwCF with chronic Pa infection were enrolled at three Canadian CF centers (2022-2025). Sputum samples were embedded in hydrogels using the Microbial Identification after Passive CLARITY Technique (MiPACT), followed by visualization via fluorescence in situ hybridization (FISH) and three-dimensional confocal laser scanning microscopy. Image analyses quantified Pa biovolume, aggregate number, maximum aggregate size, and the proportion of large aggregates (>20 µm) relative to planktonic or small aggregates (0.5-20 µm). Mixed-effects models were applied to assess longitudinal and cross-sectional differences between participants receiving CFTR modulators and those not on therapy.

In this three-year prospective longitudinal study, pwCF with chronic Pa infection were enrolled at three Canadian CF centers (2022-2025). Sputum samples were embedded in hydrogels using the Microbial Identification after Passive CLARITY Technique (MiPACT), followed by visualization via fluorescence in situ hybridization (FISH) and three-dimensional confocal laser scanning microscopy. Image analyses quantified Pa biovolume, aggregate number, maximum aggregate size, and the proportion of large aggregates (>20 µm) relative to planktonic or small aggregates (0.5-20 µm). Mixed-effects models were applied to assess longitudinal and cross-sectional differences between participants receiving CFTR modulators and those not on therapy.

Results

Forty-two participates were included (30 adults and 12 children), of whom 27 (64%) were receiving CFTR modulator therapy. A total of 181 sputum samples were analyzed, and forced expiratory volume in 1 second (FEV₁) was recorded at the time of each collection. Longitudinal analyses showed significant reductions in Pa biovolume (p = 0.005) and aggregate number (p = 0.006) among modulator-treated participants, with no significant changes in untreated individuals. In contrast, cross-sectional analyses revealed no significant differences between those on versus off CFTR modulatory therapy with respect to Pa biovolume, aggregate size and the proportion of large versus small aggregates; although aggregate counts trended lower in participants receiving modulators. Maximum aggregate size and the proportion of large versus small aggregates remained stable over time and were not significantly affected by CFTR modulation; approximately 20% of the Pa subpopulations persisted as large aggregates in both groups.

Forty-two participates were included (30 adults and 12 children), of whom 27 (64%) were receiving CFTR modulator therapy. A total of 181 sputum samples were analyzed, and forced expiratory volume in 1 second (FEV₁) was recorded at the time of each collection. Longitudinal analyses showed significant reductions in Pa biovolume (p = 0.005) and aggregate number (p = 0.006) among modulator-treated participants, with no significant changes in untreated individuals. In contrast, cross-sectional analyses revealed no significant differences between those on versus off CFTR modulatory therapy with respect to Pa biovolume, aggregate size and the proportion of large versus small aggregates; although aggregate counts trended lower in participants receiving modulators. Maximum aggregate size and the proportion of large versus small aggregates remained stable over time and were not significantly affected by CFTR modulation; approximately 20% of the Pa subpopulations persisted as large aggregates in both groups.

Conclusions

CFTR modulator therapy reduced overall Pa burden but did not alter large-scale bacterial aggregation within CF sputum. The persistence of large aggregates, despite reductions in biovolume and aggregate number, suggests the presence of structurally resilient microbial communities that may contribute to ongoing chronic infection. These findings indicate that therapeutic strategies specifically targeting bacterial aggregation may be necessary to further improve Pa eradication and long-term respiratory outcomes in pwCF receiving CFTR modulator therapy.

CFTR modulator therapy reduced overall Pa burden but did not alter large-scale bacterial aggregation within CF sputum. The persistence of large aggregates, despite reductions in biovolume and aggregate number, suggests the presence of structurally resilient microbial communities that may contribute to ongoing chronic infection. These findings indicate that therapeutic strategies specifically targeting bacterial aggregation may be necessary to further improve Pa eradication and long-term respiratory outcomes in pwCF receiving CFTR modulator therapy.