2.4. Biocide Susceptibility Testing

Susceptibility of the E. coli isolates was tested against the two biocides formaldehyde (FA, Carl Roth, Karlsruhe, Germany) and chlorocresol (p-chloro-m-cresol, PCMC, Merck KGaA) used for C&D on the farms under study and five biocides commonly applied in farm hygiene, namely the quaternary ammonium compounds benzalkonium chloride (BAC, Sigma Aldrich, Steinheim, Germany) and didecyldimethylammonium chloride (DDAC, Merck KGaA), hydrogen peroxide (HP, Carl Roth), peroxyacetic acid (PAA, VWR, Dresden, Germany), and acetic acid (AA, Carl Roth). Biocides were serially diluted in 2-fold steps just before the experiment using standardized hard water as defined in EN 1276. The following final concentration ranges were tested: 320 to 5 mg/L BAC, 40 to 0.3 mg/L DDAC, 640 to 5 mg/L FA, 1024 to 8 mg/L HP, 2000 to 16 mg/L PAA, 16,384 to 128 mg/L AA, and 4000 to 63 mg/L PCMC.

Biocide MICs were determined using broth microdilution. Overnight cultures grown on tryptic soy agar (TSA; Merck KGaA) were adjusted to about 10⁶ CFU/mL in 2-fold concentrated tryptic soy broth (TSB; Merck KGaA). In 96-well microtiter plates (Greiner Bio-One, Frickenhausen, Germany), 50 µL of the bacterial suspension was added to 50 µL of the double-concentrated biocide solution. Plates were 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 tryptic soy broth, Thermo Fisher Scientific) and a ΔOD_{595 nm} of 0.08 was applied as the cut-off value. The MIC was defined as the lowest concentration of a biocide at which no growth was observed. Three independent experiments were performed on different days and the median was considered as the final MIC.

The MBC of each strain and biocide was determined by broth microdilution according to Knapp et al., with minor modifications [28]. Dey-Engley neutralizing broth (Sigma-Aldrich) was used to quench biocidal effects for MBC testing. Neutralizer efficacy and toxicity were tested before [36]. The MBC was defined as the lowest concentration of a biocide, which revealed no visible colonies after subculture on tryptic soy agar (TSA, Thermo Fisher Scientific). The reference strain E. coli ATCC 25922 was used as internal quality control in both MIC and MBC tests and showed comparable results throughout the experiments.

To distinguish between biocide susceptible isolates and isolates with reduced susceptibility, the MIC (or MBC) that encompassed 95% of all MIC (or MBC) values in the distribution was designated as MIC₉₅ (or MBC₉₅).