2.3. Infection with P. aeruginosa

The clinical mucoid strain P. aeruginosa M57-15, a gift from Anna Van Heeckeren, PhD, Case Western University, was used in these experiments. The bacteria were grown in trypticase soy broth (TSB) to late log phase or early stationary phase. The method for incorporation of the bacteria into agarose beads, essential to induce prolonged infection in mice, was adapted from previously described methods (Nacucchio et al., 1984; van Heeckeren et al., 2002). P. aeruginosa-laden agarose beads were diluted to achieve an inoculum of 1 to 2x10⁵ CFU per 100µl, which is approximately the lethal dose 10 percent (LD10). Fourteen days after Pneumocystis inoculation as described above, P. aeruginosa-laden beads were instilled intratracheally using a blunted 24-gauge curved inoculation needle while the animals were under isofluorane anesthesia. To confirm the actual inocula given, an aliquot of the bead preparation was homogenized and plated on Pseudomonas selection agar immediately after infection and counted after overnight incubation.

Mice were humanely euthanized on day 0 prior to infection with P. aeruginosa and on postinfection days 3 and 10. Bronchial alveolar lavage and lung digest samples were obtained as described above. An aliquot was plated to assess bacterial burden by manual CFU counting on Pseudomonas selection agar. This agar was used to avoid contamination from upper airway flora. We have verified that the lavage procedure does not significantly affect bacterial counts of homogenized lung tissue.