Characterisation of Lactobacillus plantarum single and multi-strain biofilms

Biofilms consist of microorganisms attached to a surface and embedded in a protective matrix of extracellular polymeric substances. Within a biofilm, micro-organisms are protected from harsh environmental conditions including those resulting from cleaning and disinfecting agents leading to food safety and quality issues after dispersal of life biofilm cells and subsequent contamination of foods. In this thesis, single and multi-strain biofilm formation by Lactobacillus plantarum isolates was characterised including the model strain L. plantarum WCFS1 and food spoilage isolates. Analysis of the L. plantarum single strain biofilm formation showed a role for proteins and/or proteinaceous material in surface colonization and extracellular DNA as components of the biofilm matrix. The relevance of lysis for the build-up of the biofilm matrix with eDNA was demonstrated using L. plantarum WCFS1 mutants affected in the production of cell wall polysaccharides resulting in altered cell surface composition and mutants lacking cell wall lytic enzymes that showed decreased cell lysis. Dual and multi-strain biofilms were studied using quantitative PCR and next generation sequencing based on detection of strain specific alleles in competitive planktonic and surface-attached biofilm growth models. In multi-strain cultures, the performance of individual strains generally correlated with their performance in pure culture, and relative strain abundance in multi-strain static biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The next generation sequencing approach provided additional insights in the performance of twelve individual L. plantarum strains in static and dynamic flow competitive biofilm models and showed that environmental stresses such as absence of Mn(II) and increased temperature affected not only the relative abundance of each strain both in planktonic and static biofilm growth but also the release of eDNA. The strains dominating the biofilms in static conditions were not the same as those dominating in biofilms developed in dynamic flowing conditions. The genome content of the dominating strains was explored to identify genetic factors that potentially contribute to strain specific competitive-biofilm forming capacity under dynamic flowing conditions, providing leads for further research. All the single, dual and multi-strain biofilms contained a considerable number of viable L. plantarum cells, representing a potential source of contamination. The developed tools and insights obtained in L. plantarum biofilm formation capacity may assist development of strategies to prevent (re)contamination from biofilms in food processing environments.