Polygonum tinctorium extract suppresses the virulence of methicillin-resistant Staphylococcus aureus by disrupting its extracellular vesicles.

Ethnopharmacological Relevance: Methicillin-resistant S. aureus (MRSA) is a significant global health concern, causing both hospital- and community-acquired infections. The extracellular vesicles released by S. aureus (SaEVs) contain essential factors related to the bacterial survival and pathogenicity. Polygonum tinctorium is traditionally used as a natural dye (indigo) and for treating various infectious diseases caused by microorganisms. However, the effect of P. tinctorium extract (Indigo Ex) and its mechanism on SaEVs is unknown.

Aim Of The Study: We investigated the effect and mechanism of Indigo Ex on SaEVs, which could be used in controlling S. aureus, especially MRSA infection.

Materials And Methods: Indigo Ex was prepared from pesticide-free P. tinctorium, which was dried, powdered, and extracted with d-limonene. SaEVs were isolated and purified from MRSA culture supernatant by step-gradient ultracentrifugation. The effect of Indigo Ex on SaEVs morphology was observed by both transmission and scanning electron microscopy after incubating the Indigo Ex and SaEVs under shaking conditions. The cytotoxicity of Indigo Ex was performed using mouse macrophage cell line, RAW 264.7. In addition, the ability of Indigo Ex-treated SaEVs to stimulate the immune response and cytotoxicity in RAW 264.7 cells were evaluated by ELISA and WST-1 assay, respectively.

Results: SaEV particles were disrupted when treated with undiluted Indigo Ex in a time-dependent manner. For the cytotoxicity of Indigo Ex on RAW 264.7 cells, over 50% of the cell viability decreased when diluted Indigo Ex 1000-fold and no cytotoxic effect was observed at a 25,000-fold dilution of Indigo Ex. Interestingly, the Indigo Ex-treated SaEVs showed less cytotoxic effect than SaEVs alone. Similarly, SaEVs treated with Indigo Ex reduced stimulation of pro-inflammatory cytokines (TNF-α and IL-6) and anti-inflammatory cytokine (IL-10) in RAW 264.7 cells compared to untreated SaEVs. Our results indicate that Indigo Ex disrupted SaEV particles, resulting in reduced virulence and stimulation of immune response.

Conclusions: This study reveals that the low concentration of Indigo Ex can suppresses the virulence of SaEVs without causing cytotoxicity to the host cells. Therefore, Indigo Ex may have the potential to be used to control S. aureus infection.