The effect of bacterial preparation and bead size on inflammation and infection in the rat chronic lung infection model

Layla Malt1, Donghui Yu1, Anna Trzasko1, Natasha Dekhtyar1, Stephanie Solazzo1, Armelle Grevot2, Akos Szilvasi1, Vishal Saxena1, Elizabeth Hardaker3, Chris Poll3, Ellena Growcott3, Kathy Banner3, Colin Osborne1

1Novartis Institute for Biomedical Research, Cambridge, USA, 2Novartis Institute for Biomedical Research, Basel, Switzerland, 3Novartis Institute for Biomedical Research, Horsham, UK

Pseudomonas aeruginosa (P.a.) lung infections are a major cause of morbidity and mortality in cystic fibrosis (CF). The chronic rat model of P.a.-induced infection and inflammation in which P.a. is embedded in agar beads is used to evaluate potential CF therapies. Our bacterial bead preparations have produced variable bead sizes resulting in concerns about model variability and, where large beads are generated, animal welfare issues. Aims of this study were to standardize bead size to 100 -150 μm by the addition of surfactant and evaluate the effect of ciprofloxacin. P.a. PAO1V strain (University of Colorado) was mixed with molten agar noble (2% w/v) and injected through a 23 gauge needle into heated mineral oil ± 0.1% v/v SPAN 80 to produce beads. Bead size was measured using a Malvern Mastersizer 2000. Male Sprague Dawley rats (200-250g, n=5-6) were inoculated with 105 colony forming units (cfu) of either bead preparation into the left lung via tracheotomy under ether anaesthesia. Sham animals were inoculated with sterile beads to act as controls. Animals inoculated with SPAN 80 beads (n=6/group) were dosed with saline vehicle or 25-100mg/kg ciprofloxacin, s.c., twice daily for 2 days. Animals were culled 2 days post infection. Cfu counts and histological scoring were performed on lung tissue. Cell counts were performed on bronchoalveolar (BAL) fluid. Data are presented as mean ± SEM. Statistical analysis was preformed using a Kruskal Wallis test followed by a Dunn’s post test where appropriate. Addition of surfactant produced a smaller homogeneous bead size (Table 1). Both preparations had similar cfu values in vitro and in in vivo left lung homogenates (Table 1). Treatment with ciprofloxacin (25, 50, 100mg/kg) significantly reduced cfu for SPAN 80 beads (100 and 50mg/kg cleared infection, P<0.001, 25mg/kg 1.86 ± 0.39 Log cfu/lung, P<0.01, n=6) compared to vehicle controls (5.06 ± 0.21 Log cfu,). Preparations differed in terms of lung penetration; larger original beads were found to block airways in some cases, while SPAN 80 beads were found in the alveoli. Both bead types elicited an inflammatory response with comparable mean severity scores. SPAN 80 and original beads produced comparable, significantly elevated total cell counts compared to their sham controls (P<0.01 and P<0.05, n=6) reflected by an increase in neutrophils (P<0.01 and P<0.05, n=5-6, Table 1). Empirical post operative observations indicated adverse events such as gasping appeared reduced using the SPAN 80 bead preparation. In conclusion SPAN 80 preparations maintain inflammatory responses of the chronic model 2 days post infection and do not affect the model’s response to ciprofloxacin. This modification reduces variability within the bead size and should improve animal welfare.