Pbs solution

HDPE beads coated by ZnO were kept in the dark before the experiment. Tests of the viability of bacteria were carried out in 15 mL of phosphate-buffered saline (PBS) solution (Roth, Karlsruhe, Germany) in thermostated vessels with stirring (333 rpm) at 22 °C. Before adding mixtures of bacteria, 1 g of HDPE beads coated by ZnO was activated for 15 min under visible-light irradiation (Solis-3C, 5700 K, wavelength range of approximately 400–800 nm, Thorlabs, Dachau, Germany) at 65 mW/cm². During the experiments, the concentration of S. Typhimurium and M. Luteus was 0.00001 and 0.00009 of OD₆₀₀, respectively. After 2.5 h of treatment by visible-light irradiation (65 mW/cm²), 250 µL of the sample was taken from the vessel and diluted 5 times. Bacteria viability tests were performed by a spread-plate technique using 50 µL of the diluted sample which were spread by glass beads on LB–Agar in a Petri dish (Copacabana Method, see [52 (link)]). The viability of S. Typhimurium was assessed after 18–22 h of incubation at 37 °C without agitation and of M. luteus, after 36–44 h.

The mice were euthanized by injection of 100 mg/kg of ketamine and 10 mg/kg of xylazine. Subsequently, the abdomen and thorax were opened and the left common iliac artery was cut through followed by a puncturing of the left ventricle with 20 ml of a PBS solution (Carl Roth, Karlsruhe, Deutschland). Approximately 10 min before, the mice were injected intraperitoneally with a 200 µl 0.2 molar heparin-PBS solution. All organs were removed and the aorta dissected. The surrounding perivascular fat was carefully removed from the aorta and excised almost 1 mm below the aortic arch and 1 mm above the bifurcation of the aorta.

Biofilms were collected by swab method (Copan Diagnostics Inc., Murrieta, CA, USA) from standard household siphons (bathroom sink, kitchen sink, shower 1, and shower 2) in Kleve, Germany. The biofilm was transferred into a 50 mL reaction tube (Sarstedt AG & Co. KG., Nümbrecht, Germany) containing 25 mL of PBS solution (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). After vigorous vortexing for 2 min, the suspension was mixed 1/1 with 80% glycerol. The biofilm suspension was homogenized by end-over-end rotation (IKA-Werke GmbH & Co. KG, Staufen, Germany) for 5 min. Additionally, 1 mL aliquots were prepared and stored at −80 °C.