The antimicrobial properties of OEO-PbH was evaluated by dilution method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), Clinical Laboratory and Standard Institute (CLSI), and other previous studies [42 (link),43 ,44 (link),45 (link),46 (link)].
Seven microorganisms were tested for their susceptibility to OEO-PbH. From each microbial strain, standardized suspensions were prepared to a concentration of 0.5 MacFarland, approximatively 108 CFU/mL (CFU—colony forming unit). Then, broth was added (Mueller–Hinton or Mueller–Hinton supplemented with defibrinated horse blood and β-NAD, bioMérieux, Craponne, France) and serial dilutions were obtained from OEO-PbH, obtaining a final microbial density of approximately 105 CFU/mL. After incubating at 35–37 °C for 24 h, MIC was read. MIC is defined as the lowest concentration of the test compounds without visible growth. To determine MBC or MFC, a volume of 1 µL from the test tubes with no visible growth was inoculated on solid culture media (Columbia agar + 5% sheep blood or Sabouraud Dextrose Agar, bioMérieux, France). The lowest concentration which killed 99.9% of the microorganisms was established after being incubated at 35–37 °C for 24 h. In the same mode OEO was also tested as positive control and the excipient as negative control.
Seven microorganisms were tested for their susceptibility to OEO-PbH. From each microbial strain, standardized suspensions were prepared to a concentration of 0.5 MacFarland, approximatively 108 CFU/mL (CFU—colony forming unit). Then, broth was added (Mueller–Hinton or Mueller–Hinton supplemented with defibrinated horse blood and β-NAD, bioMérieux, Craponne, France) and serial dilutions were obtained from OEO-PbH, obtaining a final microbial density of approximately 105 CFU/mL. After incubating at 35–37 °C for 24 h, MIC was read. MIC is defined as the lowest concentration of the test compounds without visible growth. To determine MBC or MFC, a volume of 1 µL from the test tubes with no visible growth was inoculated on solid culture media (Columbia agar + 5% sheep blood or Sabouraud Dextrose Agar, bioMérieux, France). The lowest concentration which killed 99.9% of the microorganisms was established after being incubated at 35–37 °C for 24 h. In the same mode OEO was also tested as positive control and the excipient as negative control.