Efficacy of short novel antimicrobial and anti-inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection

The therapeutic efficacy of two novel short antimicrobial and anti-inflammatory peptides (RR and RRIKA) was evaluated in a mouse model of staphylococcal skin infection. RR (2%) and RRIKA (2%) significantly reduced the bacterial counts and the levels of proinflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, in methicillin-resistant Staphylococcus aureus USA 300-0114 skin lesions. Furthermore, the combined therapy of RRIKA (1%) and lysostaphin (0.5%) had significantly higher antistaphylococcal and anti-inflammatory activity compared to monotherapy. This study supports the potential use of these peptides for topical treatment of methicillin-resistant Staphylococcus aureus skin infections.


Introduction
Bacterial infections, especially those caused by Staphylococcus aureus, are the most significant complication encountered in the management of wounds. 1 Furthermore, multidrug-resistant S. aureus strains and their secreted toxins are responsible for interfering with the wound-healing process and creating portals of entry for systemic complications in affected patients. 2 With the increasing incidence of staphylococcal resistance to topical antimicrobials, such as mupirocin and fusidic acid, [3][4][5] there is a pressing need to develop novel antimicrobials and new approaches to circumvent this burgeoning problem. Recently, there has been increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics, due to their high potency, broad spectrum of activity, and reduced potential for resistance development. 6,7 In addition to the potent bactericidal activity of AMPs, the recognized anti-inflammatory response of certain AMPs should be an advantage in the treatment of S. aureus skin infections. 8 In a recent study, we described two novel short peptides -RR (WLRRIKAWLRR) and RRIKA (WLRRIKAWLRRIKA) -with potent bactericidal activity in vitro against multiple clinical isolates of methicillin-resistant S. aureus (MRSA). 6 In particular, the peptides were active against the highly virulent MRSA USA 300-0114, a communityassociated strain responsible for outbreaks of staphylococcal skin and soft-tissue infections in the US. 9 Moreover, RR and RRIKA were superior in reducing adherent biofilms of both S. aureus and Staphylococcus epidermidis when compared to conventional antibiotics. Furthermore, both RR and RRIKA enhanced the antistaphylococcal activity of lysostaphin in vitro more than 1,000-fold. 6 Although lysostaphin demonstrated potent efficacy against MRSA infections in different animal models, 10-12 its therapeutic potential was hampered by the emergence of bacterial resistance. 10,13 In light of our previous results, showing enhancement of the antimicrobial effectiveness of lysostaphin against MRSA when combined with AMPs in vitro, 6 we moved forward with an in vivo experiment in a mouse model of MRSA skin infection.

Bacterial isolate
We obtained the community-acquired MSRA strain NRS384 (MRSA USA 300-0114) isolated from a wound from a patient in Mississippi, USA. The strain is resistant to erythromycin and tetracycline; positive for mec (subtype IV); pvl +/− ; multilocus sequence type 8 − ; eGenomic spa type 1, eGenomic spa repeats YHGFMBQBLO; Ridom spa type t008; agr grp I.

Peptides and antibacterial agents
RR and RRIKA were synthesized by GenScript (Piscataway, NJ, USA). Recombinant lysostaphin was purchased from Ambi Products (Lawrence, NY, USA). Linezolid and fusidic acid were purchased from Chem-Impex International (Wood Dale, IL, USA).

Mouse infection
Female BALB/c mice (6-8 weeks old) were obtained from Harlan Laboratories (Indianapolis, IN, USA). All procedures were approved by the Purdue University Animal Care and Use Committee (1207000676). The murine model of MRSA skin infection has been described before. 14,15 Eight groups of mice (n=5) were inoculated with 40 µL of MRSA USA 300 (3×10 7 CFU) intradermally. Forty-eight hours after infection and formation of an open wound, six 6 groups were treated topically with either 2% fusidic acid, 2% RR, 2% RRIKA, 1% RRIKA, 0.5% lysostaphin, or 1% RRIKA plus 0.5% lysostaphin formulated in 20 mg petroleum jelly. One group received vehicle only (petroleum jelly), and the last group was treated orally with linezolid (25 mg/kg). All groups were treated twice a day for 3 days. Twenty-four hours after the last treatment, mice were humanely killed, the area around the wound lightly swabbed with 70% ethanol, and the wound (around 1 cm 2 ) excised for bacterial counting after homogenization in 1 mL tryptic soy broth.

Cytokine detection
Enzyme-linked immunosorbent assay (ELISA) development kits for detection of cytokines were purchased from R&D Systems (Minneapolis, MN, USA). Homogenized skin lesions after bacterial counting were centrifuged at 15,000 rpm for 10 minutes. The supernatants were removed and kept at −20°C until analyzed. The supernatants were examined for cytokine production: tumor-necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β using ELISA as described before. 16 Cytokine levels were expressed as percentage change relative to negative control.

Statistical analyses
Data are presented as means ± standard deviation. Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). P-values were calculated by the two-tailed Student's t-test. P-values ,0.05 were considered significant.

Results and discussion
As shown in Figure 1, all treatments significantly reduced the mean bacterial counts compared with the control group (P0.01). Among groups treated with monotherapy, the group treated with 2% RRIKA had the highest reduction in CFU (2.08±0.20 log 10 ), followed by 2% fusidic acid (1.94±0.36 log 10 ), 2% RR (1.83±0.30 log 10 ), and linezolid (1.74±0.53 log 10 ). Groups treated with 0.5% lysostaphin or 1% RRIKA achieved a significant reduction of 1.79±0.56 log 10 and 1.08±0.36 log 10 , respectively, when compared with  (Figure 1). The combination therapy (0.5% lysostaphin with 1% RRIKA) was significantly more effective than treatment with RRIKA or lysostaphin alone, and achieved statistically significant bacterial reduction of 2.65±0.44 log 10 compared to the negative control (P0.01). The bacterial reduction in the combined therapy was also statistically significant compared to monotherapy (P,0.05) or control antibiotics (P,0.05). Previously, RR and RRIKA showed potent immunomodulatory activities in vitro through inhibition of mitogen-activated protein kinase (MK2). 17,18 In this study, topical treatment with RRIKA and RR significantly reduced TNFα and IL-6 production in MRSA skin lesions. As shown in Figure 2A and B, 2% RR and 2% RRIKA reduced the TNFα level by 49% and Figure 2 The effect of peptides on cytokines TNFα (A), IL-6 (B), and IL-1β (C) production in methicillin-resistant Staphylococcus aureus skin lesions. Tissue homogenate supernatants were examined for cytokine production using the enzyme-linked immunosorbent assay. Cytokine levels were expressed as percentage change relative to negative control. Data are presented as means ± standard deviation from duplicates consisting of four mice per group. Statistical analysis was calculated by the two-tailed Student's t-test. P-values ,0.05 were considered significant (*). Abbreviations: TNF, tumor necrosis factor; IL, interleukin. 56%, respectively, while the IL-6 level was reduced by 29% and 60%, respectively. Treatment with 0.5% lysostaphin and 2% fusidic acid caused 39% and 29% reduction of the TNFα level, respectively, and 13% and 25% reduction of the IL-6 level, respectively, which is in agreement with previous findings. 19,20 There was a synergistic anti-inflammatory response observed when 0.5% lysostaphin was combined with 1% RRIKA. The combined therapy significantly reduced TNFα and IL-6 levels by 62% and 67%, respectively. On the other hand, none of the treatments interfered with IL-1β production, which is necessary for S. aureus clearance in cutaneous infections ( Figure 2C). [21][22] In this study, topical application of RR and RRIKA was shown to be very effective in reducing the bacterial load in MRSA skin lesions. Moreover, peptides reduced the release of TNFα and IL-6, which might benefit the healing of infected wounds. [23][24][25][26][27] In addition, the combination of RRIKA with lysostaphin was significantly more effective in the treatment of MRSA skin lesions than treatment with either peptide alone. This combination therapy is also expected to overcome some of the limitations associated with lysostaphin monotherapy through hindering the emergence of bacterial resistance and lowering the required therapeutic dose.
In conclusion, our findings in MRSA skin lesions should significantly impact and inform efforts to use a combination of anti-inflammatory and AMP therapies as novel topical treatment options for multidrug-resistant pathogens.