International Wound Journal ISSN 1742-4801

ORIGINAL ARTICLE

Topical simvastatin promotes healing of Staphylococcus aureus-contaminated cutaneous wounds Chia-Chi Wang1,2 , Po-Wei Yang1 , Sheau-Fang Yang3,4,5 , Kun-Pin Hsieh1 , Sung-Pin Tseng6 & Ying-Chi Lin1,2 1 School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 2 PhD Program of Toxicology, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan 4 Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 5 Department of Pathology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan

Key words Staphylococcus aureus; Statin; Topical; Wound healing

Wang C-C, Yang P-W, Yang S-F, Hsieh K-P, Tseng S-P, Lin Y-C. Topical simvastatin promotes healing of Staphylococcus aureus-contaminated cutaneous wounds. Int Wound J 2015; doi: 10.1111/iwj.12431

Correspondence to

Abstract

Y-C Lin, PhD School of Pharmacy, College of Pharmacy Kaohsiung Medical University N612, 100 Shih-Chuan 1st Road Kaohsiung 80708 Taiwan E-mail: [email protected]

Cutaneous wounds are prompt to be contaminated by bacteria, but the clinical benefits of applying antibiotics and antiseptics in wound management have not been proven. Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors commonly used to lower cholesterol levels. Studies indicated that statins, especially simvastatin, promote wound healing in experimental models. As Staphylococcus aureus is one of the most important microorganism responsible for wound infections, the aims of this study were to characterise the anti-staphylococcal activity of simvastatin and to evaluate the application of simvastatin as a topical therapy for S. aureus-contaminated wounds. In the present study, simvastatin was bacteriostatic against S. aureus at sub-inhibitory concentrations up to 8 hours after exposure. Further increased concentrations of simvastatin above the minimal inhibitory concentration (MIC) did not enhance the growth inhibitory effect. By contrast, the ability of simvastatin to inhibit S. aureus biofilm formation was concentration dependent. Topical application of simvastatin at its MIC against S. aureus accelerated the healing and bacterial clearance of S. aureus-contaminated wounds in an excisional mice wound model. This effective concentration is well below the safe concentration for topical use. Collectively, topical application of simvastatin has the potential as a novel modality for managing wound infections and promoting wound healing.

Background

Wounds, resulting from surgery, trauma events, burns, diabetes or chronic pressure, are prevalent clinical problems and are a burden to both the patients and the society (1). Wounds are vulnerable to bacterial insults, which disturb the healing process by inducing inflammation and tissue damage (2). The clinical benefit of using antimicrobials and antiseptics on open wounds is still controversial. Systemic or topical antibiotics have not been shown to promote wound healing, and the frequent applications of these agents can lead to the emergence of drug-resistant microorganisms (1,3). On the other hand, topical antiseptics, such as povidone-iodine, chlorhexidine, alcohol, hydrogen peroxide and silver compounds, show toxicity to host cells, which © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd doi: 10.1111/iwj.12431

potentially impedes the wound healing (3–5). Therefore, there is a need for better wound care modalities, which ideally comprise strategies both to decrease the bacterial contamination in the wound to prevent wound infections and to promote wound healing.

Key Messages

• topical simvastatin application has potential as a wound care modality for preventing or treating Staphylococcus aureus wound infections

1

C.-C. Wang et al.

Topical simvastatin for S. aureus-contaminated wound management

• in vitro bacterial growth inhibitory curve and microtitre biofilm assays were used to characterise anti-staphylococcal effect of simvastatin. A mice excisional wound infection model was applied to evaluate the effect of simvastatin on S. aureus-contaminated wounds • simvastatin exhibits bacteriostatic effect against S. aureus and its anti-staphylococcal effect is independent of HMG-CoA reductase inhibitory activity • simvastatin inhibit S. aureus biofilm formation, which is dependent on its bacterial growth inhibitory effect • topical simvastatin application promotes healing and bacterial clearance of S. aureus-contaminated wounds

bacteria-contaminated wounds. These studies provide important supporting evidence that the antimicrobial effect of statins, especially simvastatin, may offer additional clinical potential in managing infected skin wounds. Given that S. aureus is one of the most prevalent causes of surgical wound infections (17–21), the objectives of the present study were to further characterise the anti-staphylococcal activity of simvastatin and to examine the effect of topical application of simvastatin on cutaneous wounds contaminated with S. aureus. Materials and methods Chemicals and bacterial strains

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, which are well known for their ability to lower serum cholesterol levels (6–8). The ability of statins to block the synthesis of many important non-steroidal isoprenoids, while inhibiting mevalonate formation, has been suggested to account for the pleotropic effects of statins (9). Animal and observational studies have shown that statins are beneficial in promoting the rate of wound healing and wound strength (9,10). A recent randomised, double-blind, placebo-controlled trial in patients with venous ulcers further demonstrated the beneficial effects of simvastatin on healing disorders in humans, in which patients given 40 mg simvastatin orally everyday had significant shorter wound healing time compared with the control patients (11). These data indicated that statin treatment has the potential to be a novel wound care modality, for healing disorders such as diabetic foot ulcers and venous ulcers. Statins have also been associated with reduced mortality in sepsis and infections in retrospective observational studies such as clinical cohort and case–control studies (12). This association, however, has not been confirmed in prospective randomised controlled trials. Experimental data on whether or not statin has therapeutic potential in managing infection are also limited. In vitro minimal inhibitory concentration (MIC) values have provided experimental evidence on the direct antimicrobial properties of statins (13,14). Masadeh et al. found that atorvastatin and simvastatin were potent against methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and other microorganisms commonly identified from wounds, such as Acinetobacter baumannii, Staphylococcus epidermidis and vancomycin-susceptible and -resistant enterococci (VSE and VRE) (13). Fluvastatin and rosuvastatin, by contrast, did not exhibit comparable antimicrobial activity against MSSA and MRSA isolates as did simvastatin (13,14). The mean MIC values of simvastatin against MSSA and MRSA reported in these studies were 29⋅2 and 75 μg/ml, respectively (14). Given the serum concentration of simvastatin of about 0⋅02 μg/ml, these MICs were inapplicable systemically in vivo (15). Rego et al. reported that topical simvastatin microemulsion (10 mg/ml) reduced bacterial loads, wound inflammation and neutrophil infiltration of polymicrobial-contaminated skin wounds in a rat excisional model (16). To our knowledge, this study was the only direct assessment on the effects of statins on 2

S. aureus ATCC 29213 strain, a methicillin-susceptible isolate originated from wound, was obtained from Bioresource Collection and Research Center, Taiwan (BCRC11863). Simvastatin, dimethyl sulfoxide (DMSO) and other chemicals used in this study were purchased from Sigma Aldrich (St. Louis, MO). Simvastatin stock was dissolved in 100% DMSO and stored in −20 ∘ C as 25 mg/ml aliquots. For the bacterial experiments, the simvastatin stock was either added directly into the experimental groups or serial diluted with culture medium to obtain the designated concentrations. The vehicle control group contained the DMSO concentration as the highest simvastatin-treatment group. For the in vivo experiments, the stock solution was first diluted 1:200 with PBS to prepare 125 μg/ml working solution in 0⋅5% DMSO, and then diluted with 0⋅5% DMSO solution to 62⋅5 μg/ml. The solubility of simvastatin 125 μg/ml in 0⋅5% DMSO was verified by no visual precipitation. MIC and bacterial growth inhibitory assays

The MIC of simvastatin against S. aureus was determined by broth microdilution method following the guidance from the Clinical and Laboratory Standards Institute (CLSI) (22). For bacterial growth inhibitory curves, bacterial macrodilution method was used. Briefly, one single colony of S. aureus was inoculated into Todd Hewitt broth and cultured at 37 ∘ C with shaking at 200 rpm overnight. The bacterial pellet from the overnight culture was centrifuged with 16 000 g for 1 minute to pellet the bacteria. After removal of culture supernatant, the bacteria were then re-suspended in fresh tryptic soy broth (TSB; Bacto, BD, Franklin Lakes, NJ) to 106 CFU/ml. After the exposure of simvastatin, the bacteria were cultured at 37 ∘ C with shaking at 200 rpm. The effects of simvastatin on bacterial growth were examined at times 0, 2, 8 and 24 hours by plating. Antibiofilm assay

The biofilm protocol was adopted from Smeltzer and coworkers (23). Briefly, overnight culture of S. aureus was pelleted and reconstituted into biofilm-conditioned broth (TSB with 0⋅5% glucose and 3% NaCl). The bacterial suspension was then diluted 1:200 with the biofilm-conditioned broth and placed at a concentration of 100 μl/well into 96-well tissue culture microtitre plates pre-coated with 10% human plasma (Sigma, © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd

C.-C. Wang et al.

Topical simvastatin for S. aureus-contaminated wound management

the fields and then normalised by the observed length of the wounds in the sections.

St. Louis, MO). To assess the effect of simvastatin on biofilm formation, the drug was added into the wells 1 hour after bacterial inoculation. After 24 hours of drug exposure, the biofilms were gently washed two times with 200 μl PBS to remove non-adherent cells using a multi-channel pipette. The biofilm was fixed with 100% ethanol for 10 minutes and then stained with 0⋅41% crystal violet in 12% ethanol for 2 minutes at room temperature. The stained wells were washed three times with PBS and air-dried for 20 minutes before adding 100% ethanol to solubilise the crystal violet. The liquid was transferred to another microtitre plate and the absorbance at 595 nm was recorded by an ELISA plate reader.

Statistical data were analysed and graphed by using GraphPad Prism Software Version 5.01 (GraphPad Software Inc., La Jolla, CA). One-way ANOVA with Dunnett’s multiple comparison test was used to compare the results in groups. For comparing the size of the wounds in the same mice, paired Student’s t-test was used. Data were represented as mean ± SEM. Statistical significant was set as P value

Topical simvastatin promotes healing of Staphylococcus aureus-contaminated cutaneous wounds.

Cutaneous wounds are prompt to be contaminated by bacteria, but the clinical benefits of applying antibiotics and antiseptics in wound management have...
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