Subinhibitory Metronidazole Enhances Production, Virulence Factor Loading, and Endothelial Cytotoxicity of <em>Porphyromonas gingivalis</em> Extracellular Vesicles

Objectives: Porphyromonas gingivalis (P. gingivalis), a key periodontal pathogen, has been linked to atherosclerosis development. The clinical failure of antibiotics to improve cardiovascular outcomes necessitates alternative explanations. This study examines how sub-minimal inhibitory concentrations (sub-MIC) of metronidazole affect the biogenesis and pathogenic potential of P. gingivalis extracellular vesicles (EVs) on human umbilical vein endothelial cells (HUVECs). Design: EVs were isolated from untreated (N-EVs) and sub-MIC metronidazole-treated (M-EVs) bacteria through ultracentrifugation. Characterization included TEM, nanoparticle tracking analysis, and Western blotting for virulence factors. HUVECs were evaluated using viability, migration, cell death assays, ROS detection, NF-κB activation imaging, and cytokine measurement. Results: Sub-MIC metronidazole increased EVs production by 2.3-fold and enriched M-EVs with virulence factors (lipid A LPS, Kgp, RgpA). M-EVs demonstrated significantly stronger cytotoxicity, causing greater impairment of HUVEC viability and migration, alongside increased cell death. Mechanistically, M-EVs induced elevated mitochondrial and cellular ROS, promoting NF-κB activation and enhancing secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). Conclusion: Sub-MIC metronidazole exacerbates endothelial injury by amplifying EV production and virulence factor loading in P. gingivalis, offering a mechanistic explanation for the limited cardiovascular benefits of antibiotic therapy in periodontitis patients.