Characterization of Biofilm Formation and Bacterial Resistance to Benzalkonium Chloride under Contrasting Cultivation Conditions

Benzalkonium chloride (BAC) is one of the most commonly used quaternary ammonium compounds in the pharmaceutical, cosmetic, and food industries. The aim of our study was to compare the physiological responses of Escherichia coli MSCL 332, Pseudomonas putida MCCL 650, and Staphylococcus epidermidis MSCL 333 on 50 mg/L BAC in rich and poor medium (100% and 5% tryptone soya broth (TSB)) in the temperature range from 8 °C to 37 °C, under static and shaking conditions. A high-throughput, 96-well microplate method was used to compare a broad range of cultivation conditions. The effect of BAC on growth, biofilm formation activity, and dehydrogenase and fluorescein diacetate hydrolysis activity was evaluated. Addition of BAC to 100% TSB inhibited biofilm formation at 37 °C by 2.4, 1.8, and 1.6 times for E. coli, P. putida, and S. epidermidis, respectively. In turn, BAC stimulated biofilm formation in E. coli in 5% TSB at 37 °C and 100% TSB at 8 °C, i.e., 1.4 and 1.3 times, respectively. Statistical optimization of broth composition with emphasis on biofilm formation and further testing under experimental conditions was performed with P. putida.