# Shikonin inhibits MRSA biofilm formation to alleviate periprosthetic joint infection

## TL;DR

Shikonin reduces MRSA biofilm formation in joint infections, offering a potential new treatment for drug-resistant bacterial infections.

## Contribution

Shikonin's ability to inhibit MRSA biofilm formation and its mechanism related to cysteine metabolism are newly demonstrated.

## Key findings

- High doses of shikonin reduced biofilm formation in MRSA-infected rats and improved tissue repair.
- Shikonin inhibited MRSA biofilm formation at sublethal concentrations and removed existing biofilms.
- The mechanism involves regulation of cysteine metabolism in MRSA, validated by transcriptomic and metabolomic analyses.

## Abstract

To alleviate periprosthetic joint infection (PJI) with methicillin-resistant Staphylococcus aureus (MRSA), shikonin (SKN) had been used to intervene the biofilm formation of MRSA in vivo and in vitro, which provides theoretical support and practical foundation for SKN as a novel drug against drug-resistant bacterial infection.

The rat model of periprosthetic joint infection was established, utilizing techniques such as scanning electron microscopy and pathology test to evaluate the MRSA inhibitory of bacterial load and biofilm formation effects of SKN. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments were conducted to assess the antibacterial activity of SKN. The crystal violet staining method was employed to evaluate the effects of SKN on MRSA biofilm formation and eradication. Transcriptomic and amino acid metabolomics analyses were used to investigate the mechanism of SKN inhibition in MRSA biofilm formation. Total thiol detection was used to assess the impact of SKN on the intracellular cysteine levels in MRSA. Finally, MIC and crystal violet staining were used to evaluate the antibacterial effects and biofilm eradication efficacy of SKN against clinical MRSA strains.

In vivo experimental results demonstrated that high doses of SKN significantly reduced the biofilm formation in MRSA PJI in rats, improved local inflammatory responses, and promoted tissue repair. Observations using scanning electron microscopy confirmed that SKN effectively inhibited the formation of biofilms on implant surface. MIC experiments revealed that the lowest inhibitory concentration of SKN was 70 μM, indicating significant antibacterial activity, although no direct bactericidal effects were observed. Results of crystal violet staining showed that SKN could significantly inhibit biofilm formation of MRSA at sublethal concentrations and exhibited efficacy of biofilm removal. Transcriptomic and acid amino metabolomic analyses prompted that the inhibition of MRSA biofilm formation by SKN might be related to regulate the cysteine metabolism in MRSA. Total thiol detection was used to validate the omics findings in vitro. Finally, SKN intervention in MRSA clinical strains showed that the SKN could inhibit MRSA clinical strains and remove biofilm.

SKN inhibits MRSA by suppressing biofilm formation, effectively alleviating periprosthetic joint infection by MRSA, and the mechanism of SKN antibacterial activity may be related to regulate the cysteine metabolism in MRSA.