The Impact of the hly Gene Deletion on Biofilm Formation and Antibiotic Sensitivity in Listeria monocytogenes

Listeria monocytogenes ( L. monocytogenes ) is a pathogenic bacterium that poses a significant threat in food safety due to its ability to form resilient biofilms, contributing to cross-contamination risks in food processing environments. This study examines the role of the hly gene biofilm formation and antibiotic resistance in L. monocytogenes . By generating a hly deletion mutant (Lm-Δ hly ), we investigated how the absence of this gene affects bacterial behavior and biofilm development. Our results revealed that hly deletion did not impact bacterial growth but significantly impaired biofilm formation. The Lm-Δ hly strain exhibited a reduced biofilm biomass and a looser biofilm structure compared to the wild-type (WT) strain. Microscopic analysis, including SEM and CLSM, confirmed that biofilm architecture was compromised, with more viable cells in the WT biofilms and a substantial decrease in extracellular polymeric substances (EPS) in the mutant strain. Furthermore, the Lm-Δ hly strain displayed reduced motility, auto-aggregation, and surface hydrophobicity, indicating a reduced ability to adhere and disseminate. Gene expression analysis revealed downregulation of key virulence factors such as prfA , sigB , and quorum sensing (QS) genes in the Lm-Δ hly strain, suggesting that hly plays a role in their regulation. Antibiotic susceptibility testing revealed that the Lm-Δ hly strain was more sensitive to ribosome-targeting antibiotics, including tetracycline and roxithromycin, correlating with impaired biofilm development under antibiotic stress. These findings emphasize the importance of hly in biofilm development, antibiotic resistance, and virulence regulation in L. monocytogenes . Targeting hly or its associated pathways may be a promising strategy to combat persistent L. monocytogenes contamination in food-related environments. Further investigation into hly ’ interactions with broader regulatory networks is needed to fully elucidate its role in L. monocytogenes pathogenesis.