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

Jung-Hwa Woo,Ji-Hyun Shin,Shukho Kim,Je-Chul Lee,Taewon Lee

doi:10.3390/genes12030415

Jung-Hwa Woo, Ji-Hyun Shin + Show 3 more

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https://doi.org/10.3390/genes12030415

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Abstract

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 (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB 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.

Highlights

L. monocytogenes is a ubiquitous Gram-positive, non-spore-forming, facultative intracellular bacterium responsible for human listeriosis with high mortality in risk group, including pregnant women, the elderly, newborns, and immunocompromised individuals [1,2]
Cytotoxicity was induced in Caco-2 cells treated with ≥5 μg/mL of MVSXT, ≥10 μg/mL of MVGEN and MVTSB, and ≥15 μg/mL of MVAMP when the cytotoxicity was compared to untreated control cells (Figure 3B)
Membrane vesicle (MV) derived from L. monocytogenes cultured under three antibiotic stress conditions were more cytotoxic at ≥ 10 μg/mL than MVTSB at the same concentration

Summary

Introduction

L. monocytogenes is a ubiquitous Gram-positive, non-spore-forming, facultative intracellular bacterium responsible for human listeriosis with high mortality in risk group, including pregnant women, the elderly, newborns, and immunocompromised individuals [1,2]. Β-lactam antibiotics such as ampicillin (AMP), penicillin, and amoxicillin have been used for the treatment of listeriosis as the first-line option [3]. Effective antimicrobial therapy is essential to treat listeriosis. These antibiotics can penetrate into host cells where L. monocytogenes resides and bind to penicillin-binding protein 3 (involved in peptidoglycan biosynthesis) expressed by L. monocytogenes [4]. The antimicrobial dose is important when treating L. monocytogenes infections because penicillin exhibits bacteriostatic effects within cells only when cells are exposed to a high concentration. Trimethoprim/sulfamethoxazole (SXT) can be used to treat listeriosis as a second-line option [7,8]

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