Figure 5

Mucin-dependent colony dispersion and macrophage uptake using motile pathogens S. typhimurium and E. piscicida. (A) The effect of mucin on colony dispersion/prevention of biofilms was investigated using the motile bacterium S. typhimurium and E. piscicida. Porcine mucin at variable concentrations was incorporated into soft agar and allowed to solidify. Mid-log phase bacterial culture was spotted on the agar. After 12 h incubation at 28 °C for E. piscicida and 37 °C for S. typhimurium, the colony diameter was measured. ** and *** indicate a significant difference compared to the non-treated control. (B) The mouse macrophage RAW264.7 cell line was seeded in 24-well plates at 5 × 10⁴ cells/well. Cell media was supplemented with porcine mucin at variable concentrations. S. typhimurium and E. piscicida were inoculated at MOI 40 and 100. After 1 h of co-incubation at 37 °C, the cells were washed with PBS three times, treated with gentamycin, and lysed with 0.05% Triton X-100 PBS. The whole lysate was collected and serially diluted for plating on agar. Internalized bacterial numbers were determined by CFU counting. The relative uptake was calculated based on the uptake of the control. ** and *** indicate a significant difference compared to the non-treated control. The level of significance was set at p < 0.05.