Intraperitoneal infection of mice.

A clinical isolate of P. aeruginosa (strain Q502) was grown overnight in nutrient broth at 37°C with constant agitation. The bacteria were centrifuged at 2,000 × g and washed three times in sterile, endotoxin-free PBS. The bacteria were resuspended in sterile, injection-grade saline, and the inoculum was adjusted to an OD of 0.5 (A₅₅₀). Female adult (8- to 12-week-old) C57BL6 mice were infected i.p. with 100 μl of the bacterial suspension. Subsequent growth of the inoculum on nutrient agar demonstrated that each animal received 10⁶ CFU. Six mice per treatment were used. This was determined by GraphPad StatMate 2.0 to ensure 80% power to detect statistically significant effects between antibiotic-treated and untreated animals at a significance level (alpha) of 0.05, two tailed. The actual power was >99%. Mice were selected at random from open stock cages (10 per cage), earmarked to allow individual identification, and then sequentially placed into treatment groups. During the course of the experiment, mice were housed in individually ventilated cages. This method of assigning animals to groups ensures an approximately equal distribution of mice from different stock cages in each group to minimize the influence of cage-to-cage variability. At the time of inoculation, the mice were treated with the standard pediatric PmB dose of 20,000 U/kg (n = 6), PmB and 100 µl of 25 µM BCN (n = 6), BCN only (n = 6), or a saline control (n = 6) by i.p. injection. The individual components injected into mice were added to the same syringe immediately before i.p. injection. Animals were culled by cervical dislocation 4 h postinoculation. That time point was selected because by that time point and under the infection conditions used, the untreated mice reach the humane endpoint and need to be culled, as defined within the UK Home Office license under which the experiments were carried out (PLL 2700). Because of the virulence of the clinical isolate and the dose of the bacterial inoculum and because the bacteria are delivered i.p., the mice rapidly succumb to the infection. In contrast, those given effective antibiotic treatment rapidly clear the infection and remain perfectly healthy. The vast divergence between the responses seen in this model provides us with the statistical power to robustly assess the microbial response to antibiotic therapies without requiring the use of a very large number of mice per group. We are therefore adhering to the reduction principle of the three R’s. The peritoneal cavity was lavaged with 3.5 ml of ice-cold, sterile, endotoxin-free PBS, and the volume recovered was recorded. Serial dilutions of the lavage fluid were plated onto cetrimide agar; and bacterial colonies were counted after 24 h of growth at 37°C. Harvesting of the samples and quantification of the bacterial burdens in the mice were done while blinded to the treatment groups. Data were not normally distributed, and there was no equal variance between groups; therefore, a nonparametric Kruskal-Wallis test was used. The mouse infection experiments carried out were assessed by the Queen’s University Belfast animal welfare and ethical review body (AWERB) committee and conducted under a license issued by the UK Home Office under the Animals (Scientific Procedures) Act 1986, amended in 2012.