Research Projects

Abstract

It is well known that the microbial biofilms pose a significant risk for human health and economy. The increased resistance of microbes in biofilms against antimicrobial agents presents a major challenge for microbial biotechnology. The current research identifies the extracellular matrix that can vary in its exopolymeric (EPS) composition significantly even in the same bacterial species, as the decisive component responsible for mechanical and antimicrobials resistance of biofilms. Therefore, it is crucial to understand how reduced efficiency of antibiofilm strategies depends on EPS composition, structure and interaction. In the last decade synthetic biology has made a quick progress, nevertheless there has been precious little engineering in biofilm research. The microbial biotechnology engineers are frustrated by the lack of standardized biofilm components. Our proposal is to create, for the first time, the synthetic biofilms composed of multiple native exopolymeric compounds (EPS) with bacterial strains of adjustable cell physiology. We intend to isolate and structurally characterize EPS components such as polysaccharides (e.g. levan), amyloid proteins (e.g. TasA), polyanions (γ-polyglutamic acid and eDNA) from bacterial biofilms. We also intend to isolate and characterize new EPS components. By mixing these intrinsically different polymers we will be able to construct synthetic biofilms with tailored mechanical and diffusion properties designing a novel research platform. In synthetic EPS matrix of various composition the wild-type cells will be integrated.

The constructed synthetic biofilms will then be exposed to mechanical and antimicrobials stress, which are the current strategies to eradicate biofilms. The most important mechanical properties of the gel like structures like biofilm are visco-elastic parameters that define the structural response to the external shear force, which will be measured by rotational and micro rheology. The diffusion properties of biofilm will be measured by fluorescence techniques as FRAP/FLIP. The visco-elastic parameters are related to the porosity and diffusion properties of the biofilm therefore affecting the penetration of antimicrobials. Currently, we do not have a clear picture on how different EPS components affect mechanical and diffusional properties of biofilms and how this is related to antibiofilm efficiency. Therefore our designed platform for synthetic biofilms, with controllable mechanical and diffusion properties offers a unique opportunity to improve our understanding of persistent, health threatening and corrosive biofilms. The identification of “weak” spots in the biofilm construction will not only enhance our abilities to find better antibiofilm strategies, but also offer us ideas how to construct synthetic super-resistant biofilms. Based on the obtained knowledge we will be able to create super-resistant biofilms from synthetic resistant EPS and pre-selected resistant cells. These “worst case scenario” engineered biofilms will then be exposed to the action of conventional and novel antimicrobial agents- as phytocannabinoids and DNA gyrase inhibitors that were recently discovered and can be chemically modified to maximize biofilm destruction. By confocal laser scanning microscopy and differential staining we will observe both the viability of individual cells and their position in the biofilm. Based on the analysis of the collected data, we will be able to improve the existing antimicrobials and synthesize new that will be retested on synthetic super-resistant biofilms and native biofilms. The new approach will provide essential guidance for rational design of antimicrobials with improved anti biofilm activity.

Researchers

• Iztok Dogša
• Katja Molan
• Andi Erega
• Katarina Šimunovič
• Barbara Kraigher
• Ines Mandič Mulec
• David Stopar
• Nace Zidar
• Štefan Možina

The phases of the project and their realization

(WP1): EPS components isolation, characterization, synthetic biofilm formation, in progress

(WP2): synthetic biofilms in microfluidic devices, in progress

(WP3): testing of biofilms on mechanical stress and resistance against antimicrobials

(WP4): identification of “weak” or “problematic” EPS in biofilm resistance

(WP5): the creation of super-resistance biofilms and improved antimicrobials 

Citations for bibliographic records

• Iztok Dogša
• Katja Molan
• Andi Erega
• Katarina Šimunovič
• Barbara Kraigher
• Ines Madič Mulec
• David Stopar
• Nace Zidar
• Štefan Možina

Project partners

• UL Faculty of Pharmacy