Investigations of inter and intraspecies bacterial interactions for probiotic improvement by in vivo and in vitro model systems

Microbial interactions influence animal health, animal production and consequently shape human and environmental health. The overuse of antibiotics as growth promoters in animal husbandry has contributed to the overspread of antibiotic resistance in important pathogens, which is of global concern, hence antibiotic’s use for animal growth promotion has been banned by EU regulation. Probiotics, which are gaining in popularity are considered a green solution to the problem and demand for probiotics as feed supplements in animal’s husbandry is increasing. However, despite increasing usage of probiotics the understanding and knowledge of mechanisms driving probiotic-pathogen and probiotic- host interactions are limited. We combine our state-of-the-art on social microbiology (expertise of Mandic Mulec group) with our recent discovery of the potential Bacillus subtilis probiotic strain to address through extended collaboration with other partners this gap in knowledge, which is important for improvement of probiotics, for the control of food borne pathogens and for advancement of animal health. To address this problem we will investigate bacterial interactions in bacterial cocultures and biofilms that we here refer to as in vitro model systems. Moreover, to asses the effect of bacterial interactions on host we will apply eukaryotic cell cultures and an animal model (broilers) that we here refer to as in vivo model systems. We will focus on interactions of three bacterial species: 1) Bacillus subtilis PS-216, which is a potent antagonist of C. jejuni and broiler's growth promoter; and on two pathogens: 1) Campylobacter jejuni and 3) Salmonella Typhimurium, which are both the predominant causative agents of gastrointestinal infectious disease in EU/USA. The two pathogens are transmitted though infected chicken meat and are therefore of high concern in relation to animal (broilers) husbandry, food safety and human health. This highly collaborative project, which brings together three partner institutions: UL-BF, UL-VF and NIB, who will combine their expertise and methodologies in microbiology, recombinant strain engineering, confocal microscopy, advanced omics, tissue culture and know how on broilers health to investigate microbial interactions. In vitro microbial interaction models will be complemented by cell tissue culture (Coco-2) and in animal (broilers) models to address the mechanisms and consequences of intra- and interspecies interactions. The main objectives of the project, organized into five work packages, are to characterize: 1) the role of molecular determinants (e.g. antibiotics, biofilm matrix components, peptidoglycan, signaling molecules) during pathogen-probiotic competition in biofilms/cocultures; 2) the effect of kin discrimination mediated interactions between B. subtilis strains on competitive exclusion of pathogens by B. subtilis PS-216; and determine 3) the effect of B. subtilis PS-216 spores added as feed/water supplements on broiler’s weight, health, immune status and microbiota. The project is hypotheses driven and predicts that bacteria are capable of competition sensing that regulates transcription of adaptive genes (controlling attack and defense) and the fitness of interacting species. Hence, the project will bridge the gap in the understanding of the molecular and evolutionary logic underlying the regulation of the competition sensing and bacterial attack. Secondly, the project will generate the new knowledge in relation to microbe – pathogen and microbe-host interactions, which will contribute to the development of safer, more efficient and reliable probiotics.