A recent study has provided new insight into the genetic and phenotypic adaptations that enable Listeria monocytogenes to form robust biofilms.

The ability of L. monocytogenes to form biofilms on surfaces such as stainless steel, plastic, and glass has long been recognized as a key factor in its survival and contamination potential in food production environments. In this context, researchers from Wageningen University and Research (WUR) sought to better understand the evolutionary mechanisms driving L. monocytogenes strains’ enhanced biofilm formation ability, which, up until this point, have remained largely unexplored.

The study was published in Microbiological Research.

Using an experimental evolution model, the researchers isolated evolved variant (EV) strains (i.e., strains that have developed a specific advantage through evolution) from two L. monocytogenes backgrounds: EGDe (a reference strain) and FBR16 (a hypermutator food isolate). To isolate the EV strains, the original EDGe and FBR16 strains underwent repeated cycles of surface colonization, biofilm formation, dispersal, and reattachment. The resulting EV strains demonstrated up to a seven-fold increase in biofilm production compared to their ancestral counterparts.

Phenotypic assays revealed that increased cell surface hydrophobicity was a dominant trait among EV isolates, correlating with stronger attachment to hydrophobic surfaces such as polystyrene and stainless steel. Proteomic analysis identified two significantly upregulated proteins, Lmo1798 and Lmo1799, as key contributors to the enhanced biofilm phenotype.

Genomic analysis of the EGDe EV strain pinpointed a single-nucleotide insertion upstream of lmo1799 and a 42-nucleotide in-frame deletion within the gene. These mutations were associated with elevated expression of lmo1799, which in turn influenced cell surface properties and biofilm capacity. Mutants lacking lmo1799 or the upstream insertion exhibited reduced biofilm formation and lower hydrophobicity, underscoring the functional importance of these genetic changes.

Interestingly, a similar upstream insertion was identified in the FBR16 EV strain, reinforcing the role of this mutation in regulating the lmo1798–1799 operon. The study also highlighted that the impact of lmo1799 on biofilm formation is linked to its overexpression rather than just its presence, aligning with previous findings that deletion of lmo1799 alone does not significantly alter biofilm capacity.

The Lmo1799 protein, characterized by its LPXTG cell wall anchor motif and low negative charge, may alter the surface charge of L. monocytogenes, facilitating stronger attachment to surfaces. Its structural similarity to known biofilm-associated proteins further supports its role in biofilm architecture and persistence.

While the study focused on biofilm-related phenotypes, the mutations observed may also influence stress resistance and virulence. Notably, research has linked lmo1799 expression to stress adaptation pathways, and lmo1799 expression has been shown to be upregulated in blood and during bacteriophage exposure.

The WUR study marks the first application of experimental evolution to study biofilm enhancement in L. monocytogenes, offering a foundational understanding of how certain mutations can drive significant phenotypic shifts in this pathogen, posing an important microbiological threat to food safety.