(428) Pseudomonas aeruginosa lung infection in cystic fibrosis rats

Background: Recurrent bacterial infections with Pseudomonas aeruginosa result in chronic airway inflammation, lung damage and eventual respiratory failure, and are the major cause of morbidity and mortality in people with CF. Animal models are essential for understanding disease progression and assessing potential treatments in the presence of infection. Numerous animal models of CF have been developed and recapitulate different aspects of CF pathophysiology, however many do not exhibit lung disease. Administering bacteria into the lungs of wild-type (WT) and CF animal models is a method commonly used to mimic lung infections for CF. Embedding bacteria into agar beads can prevent bacterial clearance, thus generating a persistent infection, potentially replicating the chronic infection associated with clinical disease. The aim of this study was to develop CF-like lung infection in rats with two different CFTR mutations, and measure how responses to P. aeruginosa infection vary between the genotypes.

Methods: Agar beads containing P. aeruginosa (isolate 20844 mucoid) were prepared to an average suspension of 2.6 x 105 CFU/50 μl. Wild-type, knockout (KO) and Phe508del rats were deeply anaesthetized using isoflurane and suspended by their incisors. A miniature bronchoscope was used to deliver 50 µl of P. aeruginosa agar beads to the right main bronchus. Rats were allowed to recover and monitored daily for the study duration. The effects of infection were measured at 7, 14 or 21 days using flexiVent and X-ray Velocimetry assessments to quantify lung function. Rats were then humanely killed and were split into two groups for further assays. Group one had the right lobe excised and mechanically homogenized and plated for bacterial counts (CFU). Group two had bronchoalveolar lavage (BAL) performed on the right lobe with 1.5 ml of warmed PBS. BAL fluid was centrifuged to collect the cells, cytospun, and stained with Giemsa to evaluate the percentage of immune cells. The lung from the BAL group was then paraffin embedded, sectioned, stained and examined histologically.

Results: Preliminary results show that the method of embedding P. aeruginosa into agar beads retains the infection in the lung of all genotypes of rats for at least 21 days. CF rats exhibited a greater inflammation response compared to WT 7 days post inoculation and changes in lung function were observed over the period of infection. Delivering to a single lobe has enabled a localized infection without compromising the whole lung, thus reducing associated mortality, and improving welfare of the rats. Currently final data is being collected for all time points.

Conclusions: The agar embedded bead method was used to develop and maintain a chronic P. aeruginosa lung infection in rats. Our preliminary results suggest there are differences in the course of infection between the WT and CF rats. A successful lung infection model enables the ability to investigate whether the presence of bacterial infections will affect airway gene-addition therapy efficacy in future studies.

Acknowledgements: Cystic Fibrosis Foundation Grant DONNEL21GO

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