Real-time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes

Extracellular matrix (ECM) is the protein- and polysaccharide-rich backbone of bacterial biofilms that provides a defensive barrier in clinical, environmental and industrial settings. Understanding the dynamics of biofilm formation in native environments has been hindered by a lack of research tools. Here we report a method for simultaneous, real-time, in situ detection and differentiation of the Salmonella ECM components curli and cellulose, using non-toxic, luminescent conjugated oligothiophenes (LCOs). These flexible conjugated polymers emit a conformation-dependent fluorescence spectrum, which we use to kinetically define extracellular appearance of curli fibres and cellulose polysaccharides during bacterial growth. The scope of this technique is demonstrated by defining biofilm morphotypes of Salmonella enterica serovars Enteritidis and Typhimurium, and their isogenic mutants in liquid culture and on solid media, and by visualising the ECM components in native biofilms. Our reported use of LCOs across a number of platforms, including intracellular cellulose production in eukaryotic cells and in infected tissues, demonstrates the versatility of this optotracing technology, and its ability to redefine biofilm research. Opto-tracing: Watching biofilms grow Biofilm formation can be studied as it happens using chemicals that fluoresce at different wavelengths as their conformation changes. However, understanding the dynamics of biofilm formation has been hindered by the lack of suitable research tools. Agneta Richter–Dahlfors and colleagues at the Karolinska Institutet in Sweden, with co-workers at Linköping University, addressed this limitation using chemicals called oligothiophenes. The optical properties of these “chemical chameleons” vary as their conformation is affected by their environment. Changes in oligothiophene fluorescence monitored the formation of two key protein and polysaccharide components of the extracellular matrix of biofilms. The scope of the technique was demonstrated by characterizing biofilms of various forms of Salmonella bacteria. The work also revealed wider applications by studying cellulose formation inside cells of higher organisms. The researchers believe their ‘opto-tracing’ procedure could redefine biofilm research.