Minimum inhibitory concentrations (MIC)

Frozen plates were thawed at room temperature and inoculated with 100 μl of 10⁵ cells ml^-1. The plates were then covered with a sterile lid and incubated for 18–24 h at 37°C. Growth of bacteria was observed visually; turbid wells indicated growth and clear wells were recorded as no growth (Fig 1). The MIC was recorded as the lowest concentration of biocide at which no visible bacterial growth was observed. The MIC₅₀ (median MIC) and MIC₉₀ represented the MIC at which ≥50% and ≥90% of all of the isolates were susceptible, respectively. The median MIC₅₀ for all isolates was defined as the breakpoint for each chemical, the median used as the MIC is a interval-censored variable and not a continuous variable. A difference of one 2-fold dilution factor MIC (+/-) from the breakpoint was considered allowable variation, therefore, isolates with ≥ two 2-fold dilutions MICs higher than the breakpoint was considered to have reduced susceptibility (“resistance”) to that compound; isolates with MICs ≤ two 2-fold dilutions lower than the breakpoint were considered hyper susceptible.Each batch of plates was tested with negative control strains LT2, CT18, and ATCC4931, and also SRS positive control strains L1, L2, and L3 which are resistant to DC. Any batch of plates that did not give the expected results +/- one 2-fold dilution with these control strains were discarded and all assays were repeated with new plates. This step was done to detect pipette error during dilutions and/or replicate plating and to detect the decay of any of the compounds during storage. Any batch of plates with greater than one 2-fold dilution MIC change for any chemical for the control strains were discarded, after the developmental stage, no plate batches failed this test. Each experimental isolate was tested two to four times with different plate batches, on different days to ensure reproducibility. Data was accepted only if the two assays determined that the MIC differed by less than one 2-fold dilution.