Richard Janissen² Prasana Sahoo³ Duber Murillo¹ Moniellen Monteiro¹ Alessandro Cavalli⁴ Joao Hermes Clerici¹ Hernandes Carvalho¹ Carlos Cesar¹ Alessandra Souza⁵ Erik Bakkers⁴ Monica Cotta¹

2, Delft University of Technology, Delft, , Netherlands
3, University of South Florida, Tampa, Florida, United States
1, UNICAMP, Campinas SP, , Brazil
4, Eindhoven University of Technology, Eindhoven, , Netherlands
5, Instituto Agronômico de Campinas, Campinas, , Brazil

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Surface attachment of a planktonic bacteria, mediated by adhesins and hydrated extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Indeed, some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. However, identification of changes in EPS composition during biofilm life cycle, as well as their trigger mechanisms, are still challenging, particularly in early stages. In this work, we analyzed the entire biofilm formation process of the economically important phytopathogen Xylella fastidiosa at single-cell resolution, using several nanometer-resolution spectro-microscopy techniques. Our Scanning Probe Microscopy results reveal electrical and elastic signatures for spatial and temporal EPS distribution at different stages of the bacterial life cycle. Ex-vivo single-cell adhesion forces mediated by EPS were probed using a different approach, based on nanowire arrays. Larger adhesion forces at the cell poles and additional mechanical support from secreted EPS layers were shown for X.fastidiosa. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues.