Department of Food Science and Technology
Type of project
Type of project
01.05.2020 - 30.06.2022
Project manager at BFKovačec Eva
Scientific background: In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture and systems biology, since biofilm formation is the most common multicellular behaviour exhibited by bacteria in nature. Microbial sociality can be cooperative or competitive, resulting in changed volume and function of biofilms both qualitatively and quantitatively. Influence of social interactions on the spatial distribution and mechanical properties of biofilms are crucial to understand the physical stability of biofilms, to help improve cleaning procedures or develop better therapeutics strategies. Probiotic supplements containing Bacillus sp. are gaining in popularity as they show good potential to combat enteropathogens (e.g. Salmonella), and to improve growth, survival and health status of animals. Although Bacillus probiotics are already in use, the knowledge on mechanisms that make them effective probiotics are poorly understood. Therefore this project aims to investigate the competitive mechanisms between Bacillus subtilis and Salmonella enterica by addressing fitness consequences, adhesion, spatial segregation and mechanical changes of mixed biofilms. Moreover, we will investigate molecular mechanisms behind these interactions. The major focus of this project will be on two types of social interactions: interference competition and kin discrimination (KD). KD is a fascinating social behaviour of B. subtilis strains that differentially behave towards more and less related strains. However, it is not known how KD between B. subtilis strains influences B. subtilis - S. enterica interactions/coexistence in mixed species groups and will be addressed in this project. Moreover, bacteria in multi-species biofilms exhibit specific physiologies associated with increased tolerance/resistance to antimicrobials and we plan to investigate the role of KD in it.
Problem identification: Outbreaks in the EU due to Salmonella are on the rise again, with S. enterica causing one in six food-borne disease outbreaks in 2016. The intensive use of antibiotics has also driven the evolution of multi-resistant strains that are no longer susceptible to most commonly used antibiotics, posing need to develop alternative antimicrobials, such as probiotics. Among bacilli B. subtilis is known to have the highest number of reported antibiotics with 4–5% of its genome allocated to the production of antimicrobials. Although not a new idea, B. subtilis spores can reduce S. enterica infections in poultry. However, the data on the molecular mechanisms that drive this combat are missing. To our knowledge the mechanisms of their interactions in mixed biofilms have not been studied before. We know even less why multispecies biofilms show increased tolerance against antimicrobial agents. We will test different antimicrobials (antibiotic and antiseptic) on the Bacillus – Salmonella mixed-species biofilms to unravel effects of social interactions on antimicrobial tolerance, and the mechanisms behind this.
Objectives of the proposed project: The project aims to investigate interspecies social interactions between B. subtilis and S. enterica, focusing on the role of B. subtilis antagonistic factors and extracellular matrix components in competitiveness, adhesion, spatial segregation and mechanical properties of mixed biofilms containing both species. Moreover, a fascinating discovery of kin discrimination within B. subtilis species has never been addressed in a multispecies context. We aim to apply a model system that mimics conditions of human intestinal epithelium, which will allow us to investigate B. subtilis -S. enterica interactions in more realistic setting. In addition, the antimicrobial susceptibility of mixed species biofilms will be study to assess whether B. subtilis has a negative effect on S. enterica biofilm formation and consequently it can be useful in its removal.
THE PHASES OF THE PROJECT AND THEIR REALIZATION
WP1: Fitness consequences and molecular mechanisms that drive the competitive interactions between Bacillus and Salmonella (M 1-12)
The objective of WP1 is to reveal the molecular mechanisms of interactions between B. subtilis in S. enterica and the consequences of these interactions (stress response/antibiotic susceptibility). We will investigate whether mixed-species biofilms have different susceptibility to antimicrobial treatments than monospecies biofilms. We will test the susceptibility of mixed biofilms, composed of S. enterica and B. subtilis strains. Additionally we will add more than one B. subtilis strain that differ in their relatedness to mixed species communities as recent publications suggest that genetic diversity may enhances the tolerance of microbial communities against antimicrobial treatment.
WP2. Investigations of adhesion, spatial distribution and mechanical properties of mixed species biofilms (M 10-16)
In this WP we will test whether interference competition promotes spatial segregation of two species with benefits for the probiotic bacterium. We will use test the effect on adhesion and whether mixing of two species influences viscoelastic properties of biofilms. We will apply fluorescent stereomicroscope to monitor biofilms’s growth and swarming dynamics and localization/segregation of each species in a mixed setting versus monocultures over time.
WP3. Investigations of the role of B. subtilis kin discrimination on interactions with Salmonella (M 18-24)
The objective of WP3 is to reveal the role of kin discrimination and its potential effect on competitiveness of B. subtilis against S. enterica. In this WP we will test if interference competition between non-kin B. subtilis weakens their competitiveness against S. enterica, due to investment in fighting each other. We will evaluate segregation of strains/species and the speed on colonisation of the agar surface. The results will give us new insights into the surface colonization of heterogeneous microbial communities and most importantly allow us to test whether cooperative/antagonistic interactions between B. subtilis strains have influence on their total effectiveness against S. enterica.