Benzalkonium Susceptibility Assays (MIC and MBC Determination)

To determine the susceptibilities to BZK (SIGMA-ALDRICH) displayed by the isolates under study, a modified broth minimum inhibitory concentration (MIC) method was performed, using 96-well polystyrene microplates (Greiner Bio-One, Germany). Cell cultures grown in liquid LB (37°C, 250 rpm) until mid-exponential phase were centrifuged (10 min, 7,000 rpm, 20°C), resuspended in Mueller-Hinton (MH; Fluka Analytical) broth, and adjusted to a standardized OD₆₄₀ of 0.011. Of these cell cultures, 190 μL were used to inoculate the wells of 96-well microplates, together with 10 μL of BZK solutions at final concentrations of 16, 32, 64, 128, 192, 256, 384, and 512 μg/mL. The 96-well microplates were incubated at 23°C, in the dark, for 72 h. The lowest BZK concentration sufficient to inhibit bacterial growth was defined as the MIC or susceptible concentration for each Bcc isolate. These tests were performed in three biologically independent experiments.

The minimum bactericidal concentration (MBC) was also determined. After the above described 72 h incubation period, 4 μL of cell culture corresponding to each BZK concentration, were removed from the 96-well microplates and spotted in TSA 1/10 solid medium, followed by incubation for 24 h at 37°C. The MBC value was defined as the lowest BZK concentration for which bacterial growth was no longer observed.