Evaluation of short synthetic antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infections present a serious challenge because of the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, antimicrobial peptides are novel alternatives to traditional antibiotics for tackling the issue of bacterial multidrug resistance. Herein, we investigated the antibacterial activity of two short novel peptides (WR12, a 12 residue peptide composed exclusively of arginine and tryptophan, and D-IK8, an eight residue β-sheet peptide) against multidrug resistant staphylococci. In vitro, both peptides exhibited good antibacterial activity against MRSA, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis. WR12 and D-IK8 were able to eradicate persisters, MRSA in stationary growth phase, and showed significant clearance of intracellular MRSA in comparison to both vancomycin and linezolid. In vivo, topical WR12 and D-IK8 significantly reduced both the bacterial load and the levels of the pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in MRSA-infected skin lesions. Moreover, both peptides disrupted established in vitro biofilms of S. aureus and S. epidermidis significantly more so than traditional antimicrobials tested. Taken together, these results support the potential of WR12 and D-IK8 to be used as a topical antimicrobial agent for the treatment of staphylococcal skin infections.

Figure 1. The kinetics of killing of peptides and antibiotics against logarithmic, persister cells and stationary phase of MRSA USA300.

(a) Logarithmic phase of MRSA USA300 exposed to peptides (WR12, D-IK8) at 5X and 10X MIC or (b) antibiotics at 10X MIC. (c) The kinetics of killing of persister cells and (d) stationary phase of MRSA USA300 exposed to peptides (WR12, D-IK8) and antibiotics at 10X MIC. In Fig. 1c, CIP means treatment of MRSA with ciprofloxacin at 10X MIC for 6 hr then the surviving persisters were exposed to peptides or antibiotics at 10X MIC as the arrow pointed. Untreated samples served as a control. Abbreviations, Van, vancomycin; Lin, linezolid; Cip, ciprofloxacin. The results are given as means ± SD (no = 3); data without error bars indicate that the SD is too small to be seen.

Figure 2

The effect of peptides (WR12, 0and LL-37) and antibiotics (vancomycin & linezolid) on 24 hr (a,c) and 48 hr (b,d) old biofilms of S. aureus (a,b) and S. epidermidis (c,d). The adherent biofilm stained by crystal violet, then the dye was extracted with ethanol, measured at 595 or 490 nm absorbance and presented as percentage of biofilm reduction compared to untreated wells “control”. All experiments were done in triplicate for statistical significance. The two tailed Student t test, was used to determine statistical significance between two groups. One asterisk (*) indicates statistically different than control (p < 0.05). Symbol (#) indicates statistically different than the antibiotic treated wells (p < 0.05). Detailed P values are listed: (a): WR12 vs control, 0.0032; D-IK8 vs control, 0.0058; LL-37 vs control, 0.0166; linezolid vs control, 0.0072; vancomycin vs control, 0.0047. (b) WR12 vs control, 0.0033; D-IK8 vs control, 0.0031; LL-37 vs control, 0.0251; linezolid vs control, 0.2122; vancomycin vs control, 0.2284; WR12 vs LL-37, 0.0015; WR12 vs linezolid, 0.0015; WR12 vs vancomycin, 0.0014; D-IK8 vs LL-37, 0.0007; D-IK8 vs linezolid, 0.0015; D-IK8 vs vancomycin, 0.0011. (c) WR12 vs control, 0.0016; D-IK8 vs control, 0.0006; LL-37 vs control, 0.0025; linezolid vs control; 0.0194 vancomycin vs control, 0.0283; WR12 vs LL-37, 0.0466; WR12 vs linezolid, 0.0079; WR12 vs vancomycin, 0.0064; IK8-D vs LL-37, 0.0008; D-IK8 vs linezolid, 0.0079; D-IK8 vs vancomycin, 0.0011. (d) WR12 vs control, 0.0012; D-IK8 vs control, 0.0014; LL-37 vs control, 0.0188; linezolid vs control, 0.1763; vancomycin vs control, 0.2786; WR12 vs LL-37, 0.0009; WR12 vs linezolid, 0.0026;WR12 vs vancomycin, 0.0024; D-IK8 vs LL-37, 0.0027; D-IK8 vs linezolid, 0.0026; D-IK8 vs vancomycin, 0.0025.

Figure 3

Toxicity (a) and intracellular anti-staphylococcal activity (b) of peptides in human keratinocyte (HaCat). (a) Cytotoxicity assay showing the percent mean absorbance at 490 nm after incubating human keratinocyte (HaCat) with peptides (WR12 and D-IK8) at different concentrations. Sterile water (peptide diluent) served as negative control. Cell viability was measured by MTS assay. EC₅₀ is the half maximal effective concentration which equal 128 μM for WR12 and >256 μM for D-IK8. Results are expressed as means from three measurements ± standard deviation. (b) The effect of (WR12 and D-IK8) and antibiotics (vancomycin & linezolid) to kill MRSA USA300 (left panel) and MSSA ATCC 6538 (right panel) inside HaCat cells after treatment with 4 X MIC for 24 hr in DMEM + 10% FBS. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P values of <0.05 were considered significant. One asterisk (*) indicates significance from control negative. Two asterisk (**) indicates significance from control antibiotics. Detailed P values are listed: (MRSA USA300): vancomycin vs control, 0.3139; linezolid vs control, 0.0071; WR12 vs control, 0.0047; D-IK8 vs control, 0.00006; WR12 vs vancomycin, 0.0063; WR12 vs linezolid, 0.1336; D-IK8 vs vancomycin, 0.00003; D-IK8 vs linezolid, 0.00002; D-IK8 vs WR12, 0.00029. (MSSA ATCC 6538): vancomycin vs control, 0.3294; linezolid vs control, 0.0094; WR12 vs control, 0.0051; D-IK8 vs control, 0.0013; WR12 vs vancomycin, 0.0022; WR12 vs linezolid, 0.0712; D-IK8 vs vancomycin, 0.0009; D-IK8 vs linezolid, 0.0032; D-IK8 vs WR12, 0.0079. Experiments were done in three biological replicates per each treatment.

Figure 4

Efficacy of peptides and control antibiotics in bacterial load (a) and level of pro-inflammatory cytokines (b,d) in a murine model of MRSA skin infection. (a) Mouse was injected with the highly virulent MRSA USA300. After injection, mouse developed an abscess at the local site of injection over the back which further developed into an open wound within 48 hr from infection. Two days after the start of infection, mice were treated twice daily for 3 days either topically with fusidic acid (2%), D-IK8 (2%), WR12 (2%) formulated in petroleum jelly; or orally with linezolid (25 mg/kg). Petroleum jelly alone served as negative control. Figure 4a show the average log count of MRSA after treatment. Figure 4b,c show the effect of peptides on production of anti-inflammatory cytokines (TNF-α and IL-6) in MRSA skin lesions. Tissue homogenate supernatants were examined for cytokine production using ELISA. Cytokine levels were expressed as percent change relative to negative control. The two tailed Student t test, was used to determine statistical significance between two groups (a P value of < 0.05 was considered significant). One asterisk (*) indicates significance from control negative. Symbol (#) indicates statistically different than the antibiotic treated mice (p < 0.05). Detailed P values are listed: (a) linezolid vs control, 0.0009; fusidic acid vs control, 0.00005; D-IK8 vs control, 0.0001; WR12 vs control, 0.0001. (b) linezolid vs control, 0.6824; fusidic acid vs control, 0.0410; WR12 vs control, 0.0070; WR12 vs linezolid, 0.0168; WR12 vs fusidic acid, 0.0079; D-IK8 vs control, 0.0095; D-IK8 vs linezolid, 0.0235; D-IK8 vs fusidic acid, 0.0171. (c) linezolid vs control, 0.9328; fusidic acid vs control, 0.0230; WR12 vs control, 0.0046. WR12 vs linezolid, 0.0047; WR12 vs fusidic acid, 0.0188; D-IK8 vs control, 0.0024; D-IK8 vs linezolid, 0.0026; D-IK8 vs fusidic acid, 0.0040.