Understanding factors influencing Listeria monocytogenes biofilms aid in developing more effective elimination/prevention strategies. This study examined the effect of temperature (4 °C, 21 °C, 30 °C), materials (stainless steel 316 L with 2B and 2 R finishes, glass, and polypropylene), and slope (0°/horizontal or 90°/vertical) on mono- and dual-species biofilms using two L. monocytogenes strains and one Pseudomonas aeruginosa strain. All biofilms were grown in 10% TSB for 24 h and analyzed using culture-based methods. Additionally, the architecture of monospecies biofilms was studied using fluorescence microscopy. Overall, P. aeruginosa showed higher biofilm formation potential (6.2 log CFU/cm 2 ) than L. monocytogenes (4.0 log CFU/cm 2 ). Temperature greatly influenced P. aeruginosa and varied for L. monocytogenes. The slope predominantly influenced L. monocytogenes monospecies biofilms, with cell counts increasing by up to 2 log CFU/cm 2 . Surface material had little impact on biofilm formation. The study highlights the varying effects of different parameters on multispecies biofilms and the importance of surface geometry.
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