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

This study evaluated the antagonistic activity of the cell-free supernatant of <i>Lacticaseibacillus rhamnosus</i> ATCC 9595 (<i>Lcr</i>-CFS) against <i>Listeria monocytogenes</i>, 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 <i>Lcr</i>-CFS against <i>L. monocytogenes</i> were defined as 31.25 and 62.5 mg/mL, respectively. Time-kill assays revealed dose- and time-dependent bactericidal effects. At sub-MICs, <i>Lcr</i>-CFS significantly reduced <i>L. monocytogenes</i> 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 <i>L. monocytogenes</i> cell wall damage, cytoplasmic leakage, and morphological alterations consistent with bactericidal effects. Additionally, exposure to 1x and 2x MIC of <i>Lcr</i>-CFS induced reactive oxygen species (ROS) accumulation, indicating oxidative stress as part of the mechanism by which <i>Lcr</i>-CFS exerts its antimicrobial activity. Gene expression analysis revealed upregulation of stress and virulence-associated genes (<i>sigB</i>, <i>prfA</i>, <i>degU</i>, <i>flaA</i>, <i>motA</i>, <i>hlyA</i>, <i>pclA</i>, and <i>actA</i>) upon exposure to 0.5x MIC suggesting a complex cross-talk network between adaptive mechanisms and environmental stresses. Although <i>L. monocytogenes</i> initiates a stress response, it appears unable to counteract the damage induced by <i>Lcr</i>-CFS, resulting in cell death. These findings highlight the antimicrobial and anti-biofilm properties of <i>Lcr</i>-CFS against <i>L. monocytogenes</i>. Given its in vitro efficacy, <i>Lcr</i>-CFS emerges as a promising biocontrol agent to improve food safety by mitigating the persistence of <i>L. monocytogenes</i> in food processing settings.