Quantifying Bacterial Survival in Blood

To measure bacterial replication and survival ex vivo, 0.5 ml of freshly drawn mouse or human blood anticoagulated with 0.005 mg desirudin per ml was incubated with 50 µl of a bacterial suspension containing 5 × 10⁵ CFU (mouse) or 5 × 10⁶ CFU (human). Where indicated, human blood was processed to generate desirudin-plasma or serum. Where indicated, 5% Alexa Fluor 488–conjugated human fibrinogen (Life Technologies), CD (0.04 mM), or purified mouse monoclonal antibodies (∼10 µg ml⁻¹ final concentration) were added to the samples. After incubation at 37°C for 0, 30, or 60 min, 0.5 ml of PBS with 0.5% saponin or 0.5 ml agglutination lysis buffer (0.5% saponin, 200 U SK K, 100 µg trypsin, 2 µg DNase, 10 µg RNase per ml PBS) were added to each sample for 10 min at 37°C before plating on agar for enumeration of CFU. Treatment with agglutination lysis buffer is annotated as +SK in the figures. Statistical analysis was performed by two-tailed Student’s t test. For flow cytometry analysis, samples were incubated first with lysostaphin (10 µg ml⁻¹) for 5 min to lyse extracellular bacteria and next with erythrocyte lysis buffer (QIAGEN) for 30 min on ice. Blood leukocytes were recovered after centrifugation at 400 g, washed three times, and suspended in PBS containing 1% FBS. Cells were stained with allophycocyanin-conjugated α-GR1 and analyzed using a FACSCanto (BD). The data were analyzed with the two-tailed Student’s t test. Human volunteers were enrolled under a protocol that was reviewed and approved by the University of Chicago's Institutional Review Board.