aureus click here strain BK#13237 cultured on LB agar: (a) 103 CFU/well, (b) 102 CFU/well. Well #1 represents the media control, and well #2 represents the cell control. In both (a) and (b), P128 gel preparations (100-1.56 μg/mL) were added to wells #3-9; P128 protein formulated in physiological saline (100 μg/mL) was added in well #10 as a positive control; buffer gel was added to well #11 as a negative control. INT dye was added to the visualize growth of the surviving bacteria. Bactericidal activity of P128 in simulated nasal fluid Activity of P128 was tested in a buffer that simulated the ionic composition of nasal fluid. The simulated nasal fluid (SNF) contained 0.87% NaCl, 0.088% CaCl2. 2H20, 0.31% KCl, and 0.636% BSA .
The S. aureus COL strain was subcultured in LB medium from an overnight culture PI3K Inhibitor Library and grown at 37°C and 200 rpm until the OD600 reached 1.0 to 1.5 (5 × 108 CFU/mL). 100 μL of this cell suspension (5 × 107 CFU) was centrifuged at 3000 × g for 10 min and the cell pellet was suspended in 100 μL of SNF. 100 μL of P128 prepared in SNF (1.5 μg/mL) was added to the cells. As a positive control, P128 contained in physiological saline was added to cells suspended in physiological 4EGI-1 purchase saline. After addition of P128, tubes were incubated for 1 h in a shaker incubator at 37°C, 200 rpm. Cells were then pelleted
and resuspended in 1 mL LB, and 10-fold dilutions were plated on LB agar and incubated at 37°C overnight. Cells treated with SNF or saline served as untreated cell controls. Efficacy of P128 gel on nasal Staphylococci in their native physiological state Nasal commensal Staphylococci of 31 healthy people were characterized and evaluated for sensitivity to P128. A dry swab (Copan Diagnostics) was inserted into
each nostril, rotated six times to cover the entire mucosal surface of the anterior nare, and slowly withdrawn. The swab from one nostril of each individual was immersed in a vial containing 200 μL P128 hydrogel (40 μg/200 μL), and a swab from the other nostril was immersed in a vial containing 200 μL buffer gel (control). The vials were placed in a biosafety cabinet for 1 h at ambient temperature (about Gemcitabine ic50 25°C). The entire vial contents were then spread on blood agar plates and incubated overnight at 37°C. CFUs recovered were characterized in terms of colony morphology, hemolysis on blood agar, Gram stain, and a HiStaph identification kit (Himedia). Results and discussion P128 is a bacteriophage derived staphylococcal cell-wall degrading enzyme. This protein is under development in our laboratory for topical therapeutic use in humans. In this study, we tested the bactericidal activity of P128 protein on globally prevalent S. aureus clinical strains. We assessed the biological activity of P128 using various in vitro assays and under conditions designed to simulate physiological conditions. P128 protein preparations used in this study were of > 95% purity.