coli expression system and purified using a 2-step ion-exchange chromatography procedure selleck products . Susceptibility to P128 determined by MIC and MBC assay Determination of MIC and MBC is a commonly used method to assess susceptibility to antimicrobial agents. We determined the MIC and MBC of P128 for a panel of 31 globally represented strains of S. aureus using modified CLSI methods . Microtiter plate wells were pre-coated with BSA before adding P128 to minimize nonspecific adherence and loss of protein to the polypropylene surface. The MIC of P128 for the various strains of S. aureus ranged from 1 to 64 μg/mL (Table
1). The MIC at which 50% of the strains tested were inhibited (MIC50) was 8 μg/mL. The MBC of P128 across S. aureus strains tested also ranged from 1 to 64 μg/mL; and the MBC50 was found to be 16 μg/mL (Table 1). MIC selleck screening library and MBC of check details Vancomycin were determined using the same procedure that was used in case of P128. For the reference strain, S. aureus ATCC 25923 MIC and MBC of Vancomycin was found
be 0.5 μg/mL and 2 μg/mL respectively. These values correlate with the reported MIC and MBC of Vancomycin for this strain, validating the assay used in this work. Vancomycin was also tested on a panel of S. aureus strains that represented the MIC range of P128 (1 to 64 μg/mL). MIC of Vancomycin for these strains ranged from 0.5 to 1 μg/mL and MBC ranged from 1 to 4 μg/mL (Table 2). Table 2 MIC and MBC of Vancomycin against a panel of S. aureus isolates Sl.
No. Strain Vancomycin MIC (μg/mL) MBC (μg/mL) 1 BK#9918 0.5 2 2 BK# 2926 1 1 3 BK#19069 1 4 4 BK#9897 1 4 5 BK#8374 1 4 6 BK#2394 1 4 7 USA500/2 1 4 8 S. aureus, ATCC 25923 0.5 2 MIC was determined by modified broth microdilution method following the CLSI procedure. Vancomycin test concentration was in the range of 256 to 0.125 μg/mL. S. aureus ATCC 25923 was used as the control strain. MBC was determined following the CLSI procedure by plating 100 μL from the MIC, MIC × 2, MIC × 4 and MIC × 8 wells on LB agar and incubating the plates at 37°C overnight. The strains used here span the MIC range of P128. Strains 1-6 were selected from a globally represented panel of distinct, typed clinical isolates (MSSA, strain 1; MRSA, strains 2-7) obtained from The Public Health Research Institute, Idoxuridine New Jersey, USA; strain 7 is USA500/2, and 8 is S. aureus, ATCC 25923 Since MIC relates to growth inhibition activity of an antimicrobial agent, MBC may be a more appropriate measure of activity of P128 which is bactericidal in action. Time-kill curve studies Time-kill assays were performed in accordance with the CLSI guidelines, with a starting inoculum of 5 × 104 CFU/mL and, various multiples of the MICs. The objective of this assay was to evaluate concentration-dependent bactericidal activity. In order to find the optimal concentration required to achieve and maintain > 99.99% killing upto 24 h, sub-MIC levels were not considered.