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•Biofilm formation rates were monitored using interfacial rheology.•Viscoelastic properties were correlated with key matrix components production.•Biofilm formation kinetics ...determined the final biofilm composition.•V. cholerae inhibited the production of curli unless E. coli was given a head-start.
Biofilm is the dominant microbial form found in nature, in which bacterial species are embedded in a self-produced extracellular matrix (ECM). These complex microbial communities are responsible for several infections when they involve multispecies pathogenic bacteria. In previous studies, interfacial rheology proved to be a unique quantitative technique to follow in real-time the biofilm formation at the air-liquid interface.
In this work, we studied a model system composed of two bacteria pathogenic capable of forming a pellicle biofilm, V. cholerae and E. coli. We used an integrated approach by combining a real-time quantitative analysis of the biofilm rheological properties, with the investigation of major matrix components and the pellicle microstructure. The results highlight the competition for the interface between the two species, driven by the biofilm formation growth rate. In the dual-species biofilm, the viscoelastic properties were dominated by V. cholera, which formed a mature biofilm 18 h faster than E. coli. The microstructure of the dual-species biofilm revealed a similar morphology to V. cholerae alone when both bacteria were initially added at the same amount. The analysis of some major ECM components showed that E. coli was not able to produce curli in the presence of V. cholerae, unless enough time was given for E. coli to colonize the air-liquid interface first. E. coli secreted phosphoethanolamine (pEtN) cellulose in the dual-species biofilm, but did not form a filamentous structure. Our pathogenic model system demonstrated the importance of the biofilm growth rate for multispecies biofilm composition at the air-liquid interface.
In natural habitats, bacterial species often coexist in biofilms. They interact in synergetic or antagonistic ways and their interactions can influence the biofilm development and properties. Still, ...very little is known about how the coexistence of multiple organisms impact the multispecies biofilm properties. In this study, we examined the behaviour of a dual-species biofilm at the air-liquid interface composed by two environmental bacteria: Bacillus licheniformis and a phenazine mutant of Pseudomonas fluorescens. Study of the planktonic and biofilm growths for each species revealed that P. fluorescens grew faster than B. licheniformis and no bactericidal effect from P. fluorescens was detected, suggesting that the growth kinetics could be the main factor in the dual-species biofilm composition. To validate this hypothesis, the single- and dual-species biofilm were characterized by biomass quantification, microscopy and rheology. Bacterial counts and microscale architecture analysis showed that both bacterial populations coexist in the mature pellicle, with a dominance of P. fluorescens. Real-time measurement of the dual-species biofilms' viscoelastic (i.e. mechanical) properties using interfacial rheology confirmed that P. fluorescens was the main contributor of the biofilm properties. Evaluation of the dual-species pellicle viscoelasticity at longer time revealed that the biofilm, after reaching a first equilibrium, created a stronger and more cohesive network. Interfacial rheology proves to be a unique quantitative technique, which combined with microscale imaging, contributes to the understanding of the time-dependent properties within a polymicrobial community at various stages of biofilm development. This work demonstrates the importance of growth kinetics in the bacteria competition for the interface in a model dual-species biofilm.
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