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Background <p>Colonization with <i>C. difficile</i> is common in patients with inflammatory bowel disease (IBD). The use of antibiotics can trigger the transition from colonization to active infection, complicating IBD management and presenting challenges for the diagnosis of <i>C. difficile</i> infection (CDI). This study aims to investigate the effects of metronidazole-exposed outer membrane vesicles (EVs) derived from a toxigenic <i>C. difficile</i> strain with no detectable production of toxins A (TcdA) and B (TcdB) under the tested conditions on both the parental strain and host cells.</p> Methods <p>EVs were isolated from <i>C. difficile</i> (ATCC 43255) by ultracentrifugation. EVs from untreated bacteria were compared with those from bacteria exposed to subinhibitory concentrations of metronidazole (1/4 and 1/8 MIC); the latter were designated as metronidazole-induced EVs (Mi-EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and ELISA. Data-independent acquisition (DIA) proteomics was used to identify differentially produced proteins among the EV<sub>1/4MIC</sub>, EV<sub>1/8MIC</sub>, and EV<sub>con</sub> groups. Functional assays included: biofilm formation (crystal violet staining), host cell viability and migration (proliferation assays, flow cytometry, scratch assay), and pro-inflammatory cytokine secretion (ELISA).</p> Results <p>Metronidazole exposure significantly promoted EVs secretion. Notably, only EV<sub>1/4MIC</sub> significantly enhanced <i>C. difficile</i> biofilm formation. Regarding host cell viability, Mi-EVs promoted the proliferation of Caco-2 cells in a concentration-dependent manner, accompanied by mild apoptotic effects. Furthermore, Mi-EVs (especially EV<sub>1/4MIC</sub>) significantly inhibited Caco-2 cell migration. Meanwhile, Mi-EVs induced the secretion of TNF-α and IL-6 by macrophages in a dose-dependent manner.</p> Conclusions <p>Sub‑inhibitory concentrations of metronidazole promote the secretion of <i>C. difficile</i> EVs, which in turn enhance bacterial biofilm formation, host cell viability, and inflammatory responses in a concentration‑dependent manner, while also inhibiting cell migration in a concentration‑dependent manner. These findings offer insights into the complex interplay between antibiotics, colonizing <i>C. difficile</i> strains, and the host in the context of IBD.</p>

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Sub-inhibitory concentrations of metronidazole alter the proteomic profile and biological effects of extracellular vesicles from Clostridioides difficile

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月12日

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  • Zhaoxia Ran,
  • Xiaojie Ruan,
  • Yafei Pan,
  • Liru Wang,
  • Gang Li

摘要

Background

Colonization with C. difficile is common in patients with inflammatory bowel disease (IBD). The use of antibiotics can trigger the transition from colonization to active infection, complicating IBD management and presenting challenges for the diagnosis of C. difficile infection (CDI). This study aims to investigate the effects of metronidazole-exposed outer membrane vesicles (EVs) derived from a toxigenic C. difficile strain with no detectable production of toxins A (TcdA) and B (TcdB) under the tested conditions on both the parental strain and host cells.

Methods

EVs were isolated from C. difficile (ATCC 43255) by ultracentrifugation. EVs from untreated bacteria were compared with those from bacteria exposed to subinhibitory concentrations of metronidazole (1/4 and 1/8 MIC); the latter were designated as metronidazole-induced EVs (Mi-EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and ELISA. Data-independent acquisition (DIA) proteomics was used to identify differentially produced proteins among the EV1/4MIC, EV1/8MIC, and EVcon groups. Functional assays included: biofilm formation (crystal violet staining), host cell viability and migration (proliferation assays, flow cytometry, scratch assay), and pro-inflammatory cytokine secretion (ELISA).

Results

Metronidazole exposure significantly promoted EVs secretion. Notably, only EV1/4MIC significantly enhanced C. difficile biofilm formation. Regarding host cell viability, Mi-EVs promoted the proliferation of Caco-2 cells in a concentration-dependent manner, accompanied by mild apoptotic effects. Furthermore, Mi-EVs (especially EV1/4MIC) significantly inhibited Caco-2 cell migration. Meanwhile, Mi-EVs induced the secretion of TNF-α and IL-6 by macrophages in a dose-dependent manner.

Conclusions

Sub‑inhibitory concentrations of metronidazole promote the secretion of C. difficile EVs, which in turn enhance bacterial biofilm formation, host cell viability, and inflammatory responses in a concentration‑dependent manner, while also inhibiting cell migration in a concentration‑dependent manner. These findings offer insights into the complex interplay between antibiotics, colonizing C. difficile strains, and the host in the context of IBD.