Biocide susceptibility testing.

(i) MICs. The MICs of the biocides under study were determined by broth microdilution assays. An overnight culture of each isolate grown on tryptic soy agar (TSA; Merck, Darmstadt, Germany) was adjusted to about 10⁶ CFU/ml 2-fold concentrated tryptic soy broth (TSB; Merck). In a 96-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 50 μl of the bacterial solution was added to 50 μl of the double-concentrated biocide. The plate was incubated at 37°C for 20 ± 2 h. Optical density at 595 nm (OD₅₉₅) was measured after 5 s of shaking using the Mithras² multimode reader (Berthold Technologies, Bad Wildbad, Germany; Software MikroWin 2010 v5.18, German UI). Bacterial growth was compared to a negative control (microtiter well containing biocide solution and TSB), and a ΔOD₅₉₅ of 0.1 was considered the cutoff value. The MIC was defined as the lowest concentration of a biocide at which no growth was observed. Biological replicates derived from two independent experiments were conducted on different dates. A MIC variation of one dilution step between the two experiments was accepted. The lower value was defined as the MIC. In case of higher variation, the test was repeated once more, and the median was considered the final MIC.