Journal Article

Bactericidal, Anti-Biofilm, and Stress-Response Modulatory Effects of Lacticaseibacillus rhamnosus ATCC 9595 Cell-Free Supernatant Against Listeria monocytogenes

Rotta, Isabela Sguilla, Perini, Hugo Felix, Pinto, Uelinton Manoel, Ferreira Machado, Alessandra Barbosa, de Almeida, Felipe Alves, Rezende, Sthefânia Dalva da Cunha, Naves, Emiliane Andrade Araujo, da Silva, Marcos Vinicius, Paiva, Aline Dias, Sabino, Yasmin Neves Vieira

2025

Overview

This study evaluated the antagonistic activity of the cell-free supernatant of ATCC 9595 ( -CFS) against , a major foodborne pathogen, that represents a challenge to food safety, due to its remarkable tolerance to environmental stresses and strong biofilm-forming ability. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of -CFS against were defined as 31.25 and 62.5 mg/mL, respectively. Time-kill assays revealed dose- and time-dependent bactericidal effects. At sub-MICs, -CFS significantly reduced biofilm formation, disrupted preformed biofilms and decreased cell viability (80.3-96.7%), effects that were confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence microscopy. Transmission electron microscopy showed cell wall damage, cytoplasmic leakage, and morphological alterations consistent with bactericidal effects. Additionally, exposure to 1x and 2x MIC of -CFS induced reactive oxygen species (ROS) accumulation, indicating oxidative stress as part of the mechanism by which -CFS exerts its antimicrobial activity. Gene expression analysis revealed upregulation of stress and virulence-associated genes ( , , , , , , , and ) upon exposure to 0.5x MIC suggesting a complex cross-talk network between adaptive mechanisms and environmental stresses. Although initiates a stress response, it appears unable to counteract the damage induced by -CFS, resulting in cell death. These findings highlight the antimicrobial and anti-biofilm properties of -CFS against . Given its in vitro efficacy, -CFS emerges as a promising biocontrol agent to improve food safety by mitigating the persistence of in food processing settings.

• Publisher: MDPI AG
• Subject: Analysis / Antimicrobial activity / Antimicrobial agents / biofilm / Biofilms / Biological control / Cell death / Cell viability / Cell walls / Damage / Environmental stress / FlaA protein / Fluorescence / Fluorescence microscopy / Food / Food contamination & poisoning / Food processing / Food safety / foodborne pathogen / Foodborne pathogens / Gene expression / Genes / lactic acid bacteria / Lacticaseibacillus rhamnosus / Listeria / Listeria monocytogenes / Microorganisms / Microscopy / Minimum inhibitory concentration / Oxidative stress / Pathogens / Pests / Reactive oxygen species / Safety and security measures / Time dependence / Transmission electron microscopy / Virulence / Virulence (Microbiology) / virulence gene expression