In contrast, the gram-negative cultures tested were not affected by P128 (Figure 6a). Figure 6 Specificity and dose-dependent bactericidal activity of P128. (a) P128 (50 μg/ml) was tested on log phase cells of gram positive and gram negative bacterial species by the turbidity reduction assay. P128 showed activity only against Staphylococcus species, which lysed rapidly after addition of the protein. No activity was observed against gram-negative bacteria or the other gram-positive bacteria tested. (b) The bactericidal effects of P128 are dose-dependent and 0.5 μg/ml was sufficient

to reduce viable cell numbers by 90%. Reduction in viable cell numbers of over three orders of magnitude was observed in the concentration range of 2.5 – 25 μg/ml. The bactericidal activity of P128 against Staphylococcus see more strains was dose-dependent. The minimum concentration of P128 required achieve > 99.9% killing was determined by a bactericidal activity assay with the MRSA COL strain. We found that concentrations ≥ 2.5 μg/mL killed > 99.9% of the cells (Figure 6b). Activity against global panel of S. aureus strains To further characterize P128, its antimicrobial activity was tested on a panel of typed S. aureus strains, representing more than 3000 isolates worldwide. This panel included several MRSA strains and the clinically significant strains USA100, USA300, and USA400 (see additional file 1, Table S1). P128 reduced the cell numbers of these

strains by 99% to 99.99%, demonstrating its efficacy against isolates of clinical significance (Figure 7). Figure 7 Bactericidal activity of P128

MLN2238 clinical trial on a panel of distinct clinical isolates. Thirty globally represented S aureus clinical isolates consisting of MRSA and methicillin-sensitive S. aureus strains demonstrated sensitivity to P128 (10 μg/ml) with significant reduction in viable cell numbers. Blue bars represent cell controls and purple bars PLEK2 represent P128-treated cells. Experimental colonization of rat nares Before initiating MRSA colonization, we evaluated the commensal bacterial flora of the rat nares in several experiments. In these Wistar rats, we found Staphylococcus strains including S. sciuri subsp. rodentium, S. chromogenes, and S. equorum; however, S. aureus was not detected (data not shown). Multiple experiments were performed using a total of 50 rats to establish the rate and the degree of colonization of MRSA USA300 on day 3 after instillation. Overall, 47 of 50 animals (94%) were colonized, and the median CFU recovered from the colonized animals was 2 × 104 (additional file 5, Table S3). Evaluation of P128 efficacy in vivo At the time of treatment, one death had occurred in each of the placebo and P128 treatment groups. In the remaining rats, P128 hydrogel was found to completely decolonize four of nine (44.4%) animals (Table 1). Median CFU numbers recovered in the P128 hydrogel-treated group was two orders of magnitude lower than those of the other groups.