Sequential attachment and <i>Listeria</i> predominance under turbulent flow

The exploration of multispecies biofilms has provided significant insight into how bacteria interact in natural environments. However, crucial information is still lacking when the assumption that all the bacteria approach the substrate simultaneously during multispecies biofilm formation is challenged. The objective of this study was to analyse the multispecies biofilm formed by industrially relevant bacteria, including <i>Pseudomonas fluorescens</i> and <i>Listeria monocytogenes</i>, focusing on sequential colonisation under turbulent flow and static conditions. Under flow conditions, the attachment of <i>Listeria</i> on preformed <i>Pseudomonas</i> biofilm was significantly higher (<i>p</i> < 0.001) and reached its highest cell concentration (8.9 log CFUcm^-2) earlier (by 24 h) compared to control (sterile stainless-steel surfaces) and co-inoculated (with <i>P. fluorescens</i>) conditions. The conditioning of stainless-steel surfaces with freeze-dried and rehydrated exopolysaccharides (0-27.5 µgml^-1) extracted from <i>P. fluorescens</i> did not affect <i>L. monocytogenes</i> attachment, indicating the importance of biofilm architecture. The cell concentration of <i>P. fluorescens</i> was not affected by either the flow rate or the colonisation order in a multispecies biofilm. Overall, the sequence of colonisation affects biofilm formation in multispecies biofilms under turbulent flow and is an important variable in studies of the community lifestyle of multispecies biofilms.