In the case of E. coli ATCC 35318, E. coli
5539, and P. aeruginosa ATCC 27853, the MICs of PE1 and PE2 were higher than that of polymyxin B. Interestingly, P. aeruginosa 5215, a pan-drug resistant clinical isolate, was highly sensitive to PE1 and PE2, with MICs of 2 μg/mL that was slightly lower than that of polymyxin B. Table 1 The minimum inhibitory concentrations (MICs) of lipopeptide antibiotics (PE1 and PE2) produced by Paenibacillus ehimensis B7 Indicator strain MIC (μg/mL) PE1 PE2 polymyxin B Staphylococcus epidermidis CMCC 26069 1 1 4 Staphylococcus aureus ATCC 25923 8 8 64 Staphylococcus aureus ATCC 43300 4 4 32 Escherichia coli ATCC 35318 8 8 2 Escherichia coli 5539 4 4 1 Pseudomonas aeruginosa ATCC 27853 8 4 2 Pseudomonas aeruginosa 5215 2 2 4 Candida albicans ATCC 10231 8 8 64 Time-kill assays To further evaluate the growth inhibition effect of newly isolated PF-02341066 chemical structure antibiotics, killing experiments of PE1 and PE2 against S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 were performed. The time-kill curves of PE1 against both strains were similar to PE2 (Figure 4). In the case of P. aeruginosa ATCC 27853, all of the tested antibiotics at 4 × MIC rapidly reduced the number of selleck viable cells of this strain by at least
3 orders of magnitude over the first 3 h of exposure, and no bacteria could be detected after a 24 h incubation. In the case of S. aureus ATCC 43300, the number of viable cells counted also dramatically decreased within a period of 3 h following the addition of these two compounds, although substantial re-growth occurred after 24 h. Thus, PE1 and PE2 were determined to be bactericidal at high concentrations, which is consistent new with the characteristics of other cationic cyclic lipopeptides [21, 22]. Figure 4 Growth curves of Pseudomonas aeruginosa ATCC
27853 and Staphylococcus aureus ATCC 43300 treated with 4 × MIC peptide antibiotics. The curves are viable cell concentrations plotted against time. In two panels, non-antibiotic control, open diamond; 4 × MIC PE1, filled circle; 4 × MIC PE2, filled triangle; 4 × MIC polymyxin B, filled diamond. For the two strains in the present study, time-kill assays were independently performed 3 times and similar results were obtained. Mean values of the triplicate cfu/mL measurements from a single experiment are plotted. Effect of divalent cations on antibacterial activity To determine the effect of divalent cations on the antibacterial activity of the lipopeptides that are produced by P. ehimensis B7, the MICs of PE1 against S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 were determined in MH medium with 10 mM Ca2+ or Mg2+. In normal medium, the MICs of PE1 for S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 were 4 and 8 μg/mL, respectively. However, the MICs of PE1 for S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 increased to 8 and >64 μg/mL, respectively, when 10 mM CaCl2 was added to the test medium.