Alberto Bombelli (Wageningen University & Research)
Summary of presentations:
Evolution of Listeria monocytogenes to a strong biofilm producer phenotype
Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a severe invasive illness characterised by one of the highest mortality rates of bacterial foodborne infections. Biofilm formation is key in Listeria monocytogenes’ transmission and persistence in food processing environments. However, possible strategies for the evolution of L. monocytogenes towards higher biofilm formation capacity had not yet been investigated.
To further understand the mechanisms contributing to biofilm formation, an experimental evolution system was used to isolate strong biofilm-producing strains of L. monocytogenes EGDe (reference strain) and FBR16 (hypermutator food isolate).
After cycles of plastic surface colonisation, biofilm formation, dispersal and attachment to new surfaces, evolved variants (EV) strains were isolated and found to produce up to seven-fold more biofilm and improved attachment to different surfaces than their respective ancestral (AN) strains. Phenotypic assays revealed an increase in cell surface hydrophobicity as a shared dominant feature of EV isolates. Proteomic analysis showed protein Lmo1799, a putative peptidoglycan binding protein with 226 Ala-Asp tandem repeats, to be the most upregulated protein in EV strains compared to the AN strains. Genomic analysis of the EGDe EV strain identified a single-nucleotide insertion in the upstream region of lmo1799 and an in-frame deletion of 42 nucleotides in lmo1799, conceivably resulting in high-level expression of lmo1799. To evaluate the impact of Lmo1799 on the EV phenotypes and the overall biofilm capacity of L. monocytogenes, EGDe EV mutants lacking lmo1799 and/or the upstream insertion were constructed. Notably, both constructed mutants showed reduced biofilm formation and lower surface hydrophobicity compared to the EV strain, indicating the importance of these mutations for the strong biofilm capacity.
Overall, this study demonstrated the ability of L. monocytogenes to evolve and adapt its phenotype to become a strong biofilm producer and further explored the mechanisms contributing to biofilm formation.
Understanding the mode of action of antimicrobial prenylated isoflavonoids through an integrated approach
Prenylated isoflavonoids are a class of natural compounds extracted from plants of the Leguminosae/Fabaceae family, known for their remarkable antimicrobial activity against a broad range of microorganisms. Members of this phytochemical class have also been reported for their potential use as food preservatives and surface disinfectants.
To support their potential application in the food sector and beyond, the understanding of their mode of action is essential. In this study, the mode of action of antimicrobial prenylated isoflavonoids was investigated using a combined phenotypic and proteomic approach in bacterial cells, complemented by an integrated in silico–in vitro analysis on a model system.
Our phenotypic and proteomic analyses demonstrated that the tested prenylated isoflavonoids target the bacterial cell envelope. Fluorescence and transmission electron microscopy revealed membrane permeabilization upon treatment with lethal concentrations. Comparative proteomic analysis of control and sub-lethally treated cells showed upregulation of proteins involved in the cell envelope stress response, such as two-component systems. Liposome permeabilization assays confirmed the lipid bilayer as the primary target of the compounds. Furthermore, molecular dynamics (MD) simulations were employed to study their intercalation and favourable localization within the phospholipid bilayer.
Collectively, this comprehensive analysis confirmed the bacterial cell envelope as the primary target of antimicrobial prenylated isoflavonoids. This work provides valuable insights into their mode of action and offers leads for future applications of these novel antimicrobial compounds.