Effective decolonization strategy for mupirocin-resistant Staphylococcus aureus by TPGS-modified mupirocin-silver complex

Abstract

The widespread utilization of mupirocin to treat methicillin-resistant Staphylococcus aureus (MRSA)-caused infectious diseases has led to the emergence of mupirocin-resistant Staphylococcus aureus (MuRSA), posing a serious global medical threat. In order to counteract MuRSA, we develop a d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) modified mupirocin and silver complex (TPGS/Mup-Ag) to combat MuRSA. The surfactivity of TPGS endows Mup-Ag with a homogeneous and small particle size (∼16 ​nm), which significantly enhances bacterial internalization. Silver ions are released from the mupirocin-Ag complex (Mup-Ag) to exert a synergistic antibacterial activity with mupirocin. Results manifest that our strategy reduces the concentration of mupirocin that induces 50% bacterial death from about 1000 ​μmol/mL to about 16 ​μmol/mL. In vitro bacterial infection model suggests that TPGS/Mup-Ag can not only eliminate both intracellular and inhibit bacterial adhesion, but also living cells are not affected. Results of in vivo experiments demonstrate that TPGS/Mup-Ag can effectively inhibit the progression of skin infection and accelerate wound healing, as well as alleviate systemic inflammation in both the subcutaneous infection model and the wound infection model. Furthermore, this study may contribute to the development of therapeutic agents for antibiotic-resistant bacteria and offer ideas for silver-based bactericides.

Keywords: Drug resistance; Mupirocin; Silver; Skin and tissue infection; Staphylococcus aureus.

Graphical abstract

Fig. 1

Characterization of the Mup-Ag complex and TPGS/Mup-Ag. (A) Schematic illustration showing Mup-Ag and TPGS/Mup-Ag preparation. (B) ¹H NMR spectra of Mup (bottom) and Mup-Ag (top). (C) DSC thermograms for Mup (bottom) and Mup-Ag (top) with a heating rate of 10 ​°C/min (D) A TEM image of Mup-Ag. (E) Low magnification SEM image of Mup-Ag. (F) High magnification SEM image of Mup-Ag. (G) Energy dispersive X-ray (EDX) analysis showing the elementary composition of Mup-Ag. (H) Size distribution and polydispersity index (PDI) of TPGS micelle and a TEM image. (I) Size distribution and polydispersity index (PDI) of TPGS/Mup-Ag micelle and a TEM image.

Fig. 2

Evaluation of the antibacterial capacity of TPGS/Mup-Ag towards MuRSA and MuSSA in vitro. (A) MIC and (B) MBC susceptibility semiquantitative profiles of TPGS/Mup-Ag against MuRSA and MuSSA obtained by the microplate broth dilution method (n ​= ​3). (C) Growth curves of MuRSA and MuSSA incubated with different preparations (n ​= ​3). (D) Representative TEM images and (E) SEM images of MuRSA and MuSSA after incubating with different preparations for 6 ​h. (F) Fluorescence staining images of MuRSA and MuSSA after incubating with different preparations for 4 ​h. Live and dead bacteria were stained with calcein-AM (green fluorescence) and dead bacteria were stained with PI (red fluorescence). (G, H) Crystal violet staining and determination of the relative biofilm formation rate of MuRSA and MuSSA using a microplate reader upon OD₅₉₀. Data are presented as the mean ​± ​SD: ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.

Fig. 3

In vitro antibacterial capacity in bacterial infection model. (A) Fluorescence images of HaCaT cells infected by MuRSA for 4 ​h, followed by treating with different preparations for 12 ​h. Live and dead cells were respectively stained with calcein-AM (green fluorescence) and dead cells were stained with PI (red fluorescence). (B) Representative TEM images of MuRSA-infected HaCaT cells (MOI ​= ​20) after being treated with different preparations for 12 ​h. (C) The ratio of PI-positive cells to total cells in (A). (D) Survival rate of intracellular MuRSA cells after being treated with different preparations for 12 ​h (n ​= ​3). (E) Rate of MuRSA adhesion to HaCaT after exposure to different preparations for 12 ​h (n ​= ​3). Data are presented as the mean ​± ​SD: ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.

Fig. 4

Mechanism of overcoming drug resistance. (A) Confocal fluorescent images of MuRSA after 2 ​h and 6 ​h of incubation with TPGS/Mup-Ag and Mup-Ag. (B) Quantitative analysis of fluorescence in (A) with imageJ. (C) Expression levels of NorA mRNA transcripts of MuRSA treated with different preparations for 20 ​h assessed by RT-PCR. Untreated MuRSA was used as a control. (D) Confocal fluorescent images of MuRSA after exposure to EB for 4 ​h, following pretreatment with TPGS and CCCP (n ​= ​3). (E) Quantitative analysis of the increase in EB accumulation compared with the negative control groups (n ​= ​3). Data are presented as the mean ​± ​SD: ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.

Fig. 5

In vivo antibacterial capacity on mice subcutaneous infection model. (A) Experimental design and treatment protocols (created with BioRender.com). (B) Photographs of the MuRSA-infected abscesses of mice that were treated with different preparations for 11 consecutive days. (C) Curves showing wound areas after various treatments (wound area on the day after infection was used as 100%). (D) Curves show the body weight throughout the treatment process. (E) IL-6 and (F) TNF-α levels in wound tissue on the ninth day (n ​= ​6). (G) Lymphocyte count, (H) red blood cell count, and (I) neutrophil percentage in mice blood at the ninth day (n ​= ​6). (J) Photographs of bacterial colonies obtained from infected tissues of mice on the ninth day and bacteria colonies were counted by Image J (K) (n ​= ​6). (L–M) Representative images of wound tissue after H&E and Masson staining. (N) Fluorescent images of wound tissue after staining with anti-MPO antibody, in which the green and blue indicate neutrophil infiltration and nucleus respectively. Data are presented as the mean ​± ​SD: ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.

Fig. 6

In vivo antibacterial capacity in mice wound infection model. (B) Experimental design and treatment protocols (created with BioRender.com). (A) Photographs of the MuRSA-infected wounds of mice that were treated with different preparations for nine consecutive days. (C) Curves showing wound areas after various treatments (wound area on the day after infection was used as 100%) (n ​= ​6). (D) Photographs of bacterial colonies obtained from infected tissues of mice on the ninth day. (E–F) Representative images of wound tissue after H&E and Masson staining. (G) Fluorescent images of wound tissue after staining with anti-F4/80 antibody, in which the red and blue fluorescent signals indicate macrophage and nucleus respectively. (H) TNF-α and (I) IL-6 levels in wound tissue on the ninth day (n ​= ​6). Data are presented as the mean ​± ​SD: ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.