Bactericidal and anti-quorum sensing activity of repurposing drug Visomitin against Staphylococcus aureus

Abstract

With the growing antibiotic resistance in Staphylococcus aureus, it is imperative to develop innovative therapeutic strategies against new targets to reduce selective survival pressures and incidence of resistance. In S. aureus, interbacterial communication relies on a quorum sensing system that regulates gene expression and physiological activities. Here, we identified that Visomitin, an antioxidant small molecule, exhibited bactericidal efficacy against methicillin-resistant S. aureus and its high tolerance phenotypes like intracellular bacteria and persister cells without inducing resistance. Critically, sub-minimal inhibitory concentrations (sub-MICs) of Visomitin could serve as a potent quorum-quencher reducing virulence production (such as haemolysin and staphyloxanthin), along with inhibiting biofilm formation, self-aggregation, and colony spreading of S. aureus. These effects were probably mediated by interfering with the S. aureus accessory gene regulator quorum sensing system. In summary, our findings suggest that Visomitin shows dual antimicrobial effects, including bactericidal effects at the concentrations above MIC and quorum sensing inhibition effects at sub-MICs, which holds promise for treating MRSA-related refractory infections.

Keywords: Methicillin-resistant Staphylococcus aureus; Visomitin; antimicrobial; biofilm; haemolysis; quorum sensing.

Graphical abstract

Figure 1.

Antimicrobial activity of visomitin against S. aureus. (a) The structural formula of visomitin. (b) Disc diffusion assay of visomitin against MRSA ATCC 43,300. The discs contain 20 μg, 40 μg, 80 μg, 120 μg, 160 μg of visomitin, respectively. DMSO (8 μL) was used as a control. (c) Time-dependent killing of visomitin against MRSA ATCC 43,300 at 1/4 - 2 × MIC (0.5 - 4 μg/mL). L.O.D., limit of detection. (d) Resistance development of S. aureus ATCC 43,300 and MW2 by visomitin or ciprofloxacin (CIP) at the concentration of 1/2 × MIC (1 μg/mL) in duplicate (P1 and P2). (e) Dose-dependent growth inhibition of visomitin against the cip-induced highly resistant S. aureus ATCC 43,300 and MW2.

Figure 2.

Bactericidal activity of visomitin against S. aureus intracellular cells and persister cells. (a) Representative images of MRSA USA300 endocytosed by Raw264.7 observed by gram staining. The extracellular bacteria were eliminated by GEN (100 μg/mL) treatment. Magnification: 400 × . (b) Intracellular killing activity against MRSA USA300 by visomitin. VAN and LZD were used as controls. (c) Bactericidal effects of visomitin against S. aureus persister cells of MRSA ATCC 43,300, MSSA MW2, and MRSA USA300. VAN (32 μg/mL) was used as a control.

Figure 3.

Inhibition of haemolytic activity of S. aureus by Visomitin at the sub-MICs. (a) Bacterial count after treatment with Visomitin at the concentration of 1/4 × MIC (0.5 μg/mL). (b) Representative images of dose-dependent inhibition of haemolytic activity against S. aureus MW2 by Visomitin on blood plates. (c) Quantitative analysis of haemolytic activity inhibition by Visomitin. (d) Representative images of the haemolysis inhibition in a 96-well cell plate. ****: p < 0.0001.

Figure 4.

Inhibition of visomitin on staphyloxanthin production. (a) Representative images of pigment extraction using methanol. (b) Qualitative assessment of staphyloxanthin production of S. aureus strains after sub-MICs of Visomitin treatment. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Figure 5.

Effects of Visomitin on biofilm formation of S. aureus. (a) Dose-dependent biofilm inhibition effect of Visomitin determined by crystal violet staining. (b) Representative fluorescence images of the biofilm inhibition effects of Visomitin by SYTO9/PI staining. (c) Fluorescent quantitative analysis by image J. **: p < 0.01; ****: p < 0.0001.

Figure 6.

Additional inhibitory effects of visomitin against S. aureus. (a) Quantitative analysis of S. aureus aggregation after sub-MICs of visomitin treatment. (b) Inhibition of visomitin on colony spreading of S. aureus. *: p < 0.05; **: p < 0.01; ****: p < 0.0001.

Figure 7.

Quantitative expression of genes associated with biofilm and virulence including hla, agrA, and RNAIII. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Figure 8.

Cytotoxicity determination of Visomitin. (a) Haemolytic activity of Visomitin to human erythrocytes. (b) Representative images of human erythrocytes in the presence or absence of Visomitin. 1% (vol/vol) triton X-100 and 0.1% (vol/vol) DMSO were used as positive and negative controls, respectively. Magnification: 400 × . (c) The cell viability of HeN1, HaCaT, AC16, A2780, MDA-1, HEK293T, A549, and BT549 after treatment with Visomitin for 24 h determined by CCK-8 assay. (d) Cell viability observation of HEK293T by calcein-AM and PI staining. Scale: 20 μm. Green and red fluorescence represent the live and dead cells, respectively. ****: p < 0.0001.

Figure 9.

Antibacterial efficacy of visomitin against ATCC 43,300 in vivo. (a) The viable bacterial loads of the abscess after the treatment with visomitin (20 mg/kg). (b) Representative images of H&E staining of the skin abscesses treated or untreated with visomitin. ***: P  < 0.001.