The bactericidal effect of enzymes on planktonic cells of each strain from each of S. aureus and P. aeruginosa species was independently evaluated as described previously [10 (link)]. The experiment was done using the minimum effective concentration of each enzyme and lower concentrations (Trypsin: ≤ 1 μg/ml, DNase I: ≤ 150 U/ml, β-glucosidase: ≤ 8 U/ml).
Briefly, 50 μl of Mueller Hinton broth (Merck, Germany) was added to each microtiter plate well (Tissue culture plate 96 wells, SPL, Korea). The enzyme was loaded to each well. Finally, 50 μl of bacterial suspension with a final inoculum of 106 CFU/ml was added to each well. The microtiter plate was then incubated for 20 h at 37° C. Plates were inspected based on bacterial growth. The lowest enzyme concentration that visibly inhibited microbial growth was defined as the minimum inhibitory concentration (MIC) [36 ]. The minimum bactericidal concentration (MBC) was determined by pipetting 10 μl of each well with a clear suspension onto a TSA. After incubation at 37° C for 24 h, the plates were inspected for the presence of colonies. Inoculated MHB without enzyme and MHB plus enzyme with no bacteria were considered as control groups.
Briefly, 50 μl of Mueller Hinton broth (Merck, Germany) was added to each microtiter plate well (Tissue culture plate 96 wells, SPL, Korea). The enzyme was loaded to each well. Finally, 50 μl of bacterial suspension with a final inoculum of 106 CFU/ml was added to each well. The microtiter plate was then incubated for 20 h at 37° C. Plates were inspected based on bacterial growth. The lowest enzyme concentration that visibly inhibited microbial growth was defined as the minimum inhibitory concentration (MIC) [36 ]. The minimum bactericidal concentration (MBC) was determined by pipetting 10 μl of each well with a clear suspension onto a TSA. After incubation at 37° C for 24 h, the plates were inspected for the presence of colonies. Inoculated MHB without enzyme and MHB plus enzyme with no bacteria were considered as control groups.
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