Mouse Models of Pneumonia and In Vivo S. aureus Gene Expression Analysis

All studies conformed to National Institutes of Health guidelines and were approved by the Animal Care and Use Committee at Montana State University, Bozeman (MSU). Male and female BALB/c mice (8–10 weeks old) were either bred at the MSU Animal Resources Center or were commercially acquired.

All challenges with S. aureus were performed on day 6 post-IAV infection. WSN-infected mice were challenged via intranasal administration of S. aureus with 5 × 10⁸ for gene expression assays and for morbidity and mortality studies. PR8-infected mice were challenged via intratracheal administration of S. aureus at 5 × 10⁸ colony-forming units (CFU)/50 µL for gene expression, or 2.5 × 10⁸ for morbidity and mortality assays. Doses were determined by pilot studies and are in accordance with trends observed in Miller et al [8].

Mouse models of intranasal infections were adapted from Lee et al [3]. Mice were slightly anesthetized with isoflurane and scruffed in an upright position while a 50-µL volume was administered directly to mouse nares. After aspiration, mice were held upright for an additional 60 seconds to ensure distribution into the lower respiratory tract. For intratracheal infection, mice were deeply anesthetized with isoflurane and suspended from front incisors at a 60°–70° angle. A blunt-tipped, bent 18-g needle was inserted through the trachea and suspended above the carina to administer 50 µL directly into the lungs. Mouse nostrils were then blocked until a large gasp was observed. Mice were euthanized using a morbidity scale based on activity, feeding, and respiratory distress in combination with weight loss exceeding 20% of initial body mass.

Mice were euthanized at 4, 6, and 8 hours post–S. aureus challenge and lungs were homogenized in 2 mL of PBS. Aliquots (100 µL) were used for CFU determination and the remaining sample was centrifuged at 4000 rpm (3724 × g) for 10 minutes at 4°C. Supernatant was removed and pelleted lung tissue was resuspended in buffer RLT (Qiagen). Staphylococcus aureus RNA was purified as described previously [9]. Purified RNA from individual mice within each treatment group (3–4 per experiment) was pooled and adjusted to a final concentration of 5 ng/µL and subjected to Quantigene 2.0 assays (Affymetrix) or TaqMan reverse-transcription polymerase chain reaction (RT-PCR) (Supplementary Table 1) [7].