*2.2. Eggshells Contamination with Salmonella spp.*

Fresh eggs of class M (medium eggs, with weight ranging from 53 to 63 g) were wiped with a fine damp cloth to remove visible soil. Next, eggs were sterilized with a high-energy electron beam (20 kGy) in the Institute of Nuclear Chemistry and Technology in Warsaw, Poland. The efficacy of disinfection was confirmed by taking swabs from the egg surface and plating them onto Columbia Agar with 5% blood sheep (bioMérieux, Marcy-l'Etoile, France) (37 ◦C, 24 h).

This study used bacterial suspensions of three different densities, 10<sup>8</sup> CFU/mL (ranged from 8.43 log CFU/mL to 8.60 log CFU/mL), 105 CFU/mL (ranged from 5.68 log CFU/mL to 5.76 log CFU/mL) and 10<sup>3</sup> CFU/mL (ranged from 2.70 log CFU/mL to 2.83 log CFU/mL), representing heavy, medium and light contamination, respectively. This step was used to establish the initial number of bacteria in prepared suspensions with which the eggs were contaminated, therefore the number is referred to as CFU/mL.

The 0.5 McFarland's bacterial suspension (8.43 log to 8.60 log CFU/mL) was prepared as 108 CFU/mL (initial bacterial contamination level). Then, this initial suspension was diluted to obtain the inoculum of 103 or 10<sup>5</sup> CFU/mL. Another variant of the experiment was the contamination of the egg with organic pollution. The microbial suspensions were mixed in a volumetric ratio (v/v) 1:1 with fresh poultry manure (from a hen farm in Poland, collected in March 2021). Poultry manure was stored at 4 ◦C until testing.

Each side of the egg surface was contaminated with hundred drops (5 μL each) of bacterial suspension and dried (20 min in a laminar chamber (1 m3) per one side of the egg). The drops did not flow freely down the egg. Experiments were conducted at 4 ◦C (fridge, standard size) and 20 ◦C (hermetic chamber, 1 m3). We used 324 eggs for each strain, including all variants and repetitions.

Figure 1 shows the design of the experiment.

**Figure 1.** Scheme of experiment variants.
