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Listeriosis is a food-borne illness caused by Listeria monocytogenes . Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MV AMP ), GEN (MV GEN ), or SXT (MV SXT ) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MV TSB ). MV AMP induced more expression of tumor necrosis factor ( TNF )- α gene than MV TSB , MV GEN and MV SXT , whereas MV TSB induced more expression of interleukin ( IL )- 1β and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

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Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro