Quantity of bacteria associated with Porcine Nasal Organoids (PNOs) after 2 h (adhesion) and overnight (colonization) incubation. PNOs coming from two animals were incubated with approximately 10 5 CFU/ml of R. nasimurium ( Rn , in blue), 10 3 CFU/ml of M. pluranimalium ( Mp , in red), 10 3 CFU/ml of non-virulent G. parasuis (nv Gp , in green) or 10 6 CFU/ml of virulent G. parasuis (v Gp , in yellow) for 2 h to assess the bacterial adhesion capacity (plain pattern) or overnight to evaluate the colonization (o/n; dotted pattern). After incubation, non-attached bacteria were eliminated by washing, and attached bacteria were quantified by dilutions and plating after lysis of the cells with saponin. Three independent experiments were performed with duplicate wells and each dot in the plot represents individual well results. A Non-differentiated PNOs, ( B ) Differentiated PNOs. C Presence of mucus (Muc5A gene (indicating mucus secretion by goblet cells) and ZO-1 gene (tight junctions) after 2 h of infection in non-differentiated and differentiated PNOs, respectively. Significant differences are shown in the graph as p values using KruskalWallis multiple comparison with Benjamini, Krieger and Yekutieli post-hoc test
