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

Abstract

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.

Biofilm formation – what we can learn from recent developments

“…On the other hand, Congo red can bind to both, exopolysaccharides and amyloid fibers [22][23][24][25]. In the search for alternative compounds, luminescent conjugated oligothiophene derivatives were recently identified through compound library screening approaches to selectively interact with curli amyloid fibres and cellulose, respectively [26]. If successfully broadened, this approach could aid the in-depth characterization of biofilm formation in vitro and in vivo.…”

Organic bioelectronics in medicine

“…). So‐called luminescent conjugated oligothiophenes (LCOs) have been synthesized to visualize and study distinct conformational variants of protein aggregates , components of the bacterial extracellular matrix and carbohydrates .…”

“…Further, based on the spectral shift (Fig. b), it was possible to discriminate between Aβ plaques and intracellular neurofibrillary tangles (NFTs) as well as between early and mature plaques (Fig. a) using a single LCO molecule.…”

“…The kinetics of cellulose digestion were dependent on the macroscopic cellulose structure; microcrystalline cellulose breakdown was slow, whilst digestion was more than 10 times faster with nanocellulose‐based materials. As cellulose and bacterial amyloid proteins (curli) play a major role in bacterial biofilm formation, LCOs were investigated as probes for biofilm detection .…”

“…Choong et al . showed that LCOs can dynamically detect and differentiate between amyloid curli fibres and cellulose in developing bacterial biofilms both in vitro and in vivo . The authors were able to discern fluorescent bacteria from cellulose and curli present within the extracellular matrix by means of the optical signature of the applied LCO (Fig.…”