Bacteriophage therapy reduces Staphylococcus aureus in a porcine and human ex vivo burn wound infection model

Abstract

Burn wounds are a major burden, with high mortality rates due to infections. Staphylococcus aureus is a major causative agent of burn wound infections, which can be difficult to treat because of antibiotic resistance and biofilm formation. An alternative to antibiotics is the use of bacteriophages, viruses that infect and kill bacteria. We investigated the efficacy of bacteriophage therapy for burn wound infections, in both a porcine and a newly developed human ex vivo skin model. In both models, the efficacy of a reference antibiotic treatment (fusidic acid) and bacteriophage treatment was determined for a single treatment, successive treatment, and prophylaxis. Both models showed a reduction in bacterial load after a single bacteriophage treatment. Increasing the frequency of bacteriophage treatments increased bacteriophage efficacy in the human ex vivo skin model, but not in the porcine model. In both models, prophylaxis with bacteriophages increased treatment efficacy. In all cases, bacteriophage treatment outperformed fusidic acid treatment. Both models allowed investigation of bacteriophage-bacteria dynamics in burn wounds. Overall, bacteriophage treatment outperformed antibiotic control underlining the potential of bacteriophage therapy for the treatment of burn wound infections, especially when used prophylactically.

Keywords: Staphylococcus aureus; bacteriophage therapy; burn wound infection; ex vivo models; human; porcine.

Fig 1

Single phage treatment of burn wound infections. S. aureus LUH14616 was added to burn wounds on either (A) porcine skin or (B) human skin. The burn wound infection was treated once with either phage ISP or RPCSa2 at different concentrations, fusidic acid (FA), or PBS (growth control, GC). As a negative control (NC), burned skin without the addition of bacteria was included. Each condition was tested in triplicate. Colony forming units (CFU) were determined 2, 4, and 24 h after treatment. The bacterial load of each treated sample was compared with the GC using a t-test, and significant decreases in CFU are indicated with (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig 2

Successive phage treatment of burn wound infections. S. aureus LUH14616 was added to burn wounds on either (A) porcine skin or (B) human skin, which were treated with phage ISP or RCPSa2 at different concentrations of fusidic acid (FA). For successive treatment, the burn wounds were treated successively at 1, 4, and 7 h after the application of S. aureus. An untreated control as growth control (GC) and burned skin without the addition of bacteria as negative control (NC) were included. Each condition was tested in triplicate. CFU were determined after 24 h. Bacterial load of each treated sample was compared to the GC using a t-test, and significant decreases in CFU are indicated with (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig 3

Prophylactic phage treatment of burn wound infections. S. aureus LUH14616 was added to burn wounds on either (A) porcine skin or (B) human skin, which were treated once with phage ISP or RCPSa2 at different concentrations of fusidic acid (FA). For prophylactic treatment, the skin was incubated with either phage ISP or RPCSa2 at different concentrations for 1 h, after which S. aureus was added to the burn wound. An untreated control as growth control (GC) and burned skin without the addition of bacteria as negative control (NC) were included. Each condition was tested in triplicate. CFU were determined after 24 h. Bacterial load of each treated sample was compared with the GC using a t-test, and significant decreases in CFU are indicated with (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig 4

Gram-staining of porcine and human skin after phage treatment or prophylaxis. Images of Gram-stained skin tissue 24 h after phage treatment or prophylaxis with 10⁸ pfu/mL of phage ISP or RPCSa2. A positive growth control (untreated) and negative control (no bacteria added) were included for comparison. Bacteria are indicated with black arrows. Images were made at 1000× magnification.

Fig 5

Two-dimensional confocal microscopy images of porcine skin 24 h after phage treatment or prophylaxis Images were made of porcine skin tissue 24 h post-treatment or prophylaxis. Skin was incubated with S. aureus and was (A) left untreated as growth control, (C) treated or (D) prophylactically treated with 10⁸ pfu/mL ISP, or (E) treated or (F) prophylactically treated with 10⁸ pfu/mL RPCSa2. Skin without bacteria was included as (B) negative control. Dead bacterial and eukaryotic cells were stained with propidium iodide (red). Living bacteria (~1 µm) or eukaryotic nuclei (~10 µm) were stained with acridine orange (green). Additionally, WGA-594 (red) stained N-acetyl glucosamine, a sugar often produced in biofilms. All images were made at 20× magnification. B, bacteria; n, eukaryotic nuclei.

Fig 6

Two-dimensional confocal microscopy images of human skin 24 h after phage treatment or prophylaxis. Images were made of human skin tissue 24 h post-treatment or prophylaxis. Skin was either incubated with S. aureus and was (A) left untreated as growth control, (C) treated or (D) prophylactically treated with 10⁸ pfu/mL ISP, or (E) treated or (F) prophylactically treated with 10⁸ pfu/mL RPCSa2. Skin without bacteria was included as (B) negative control. Dead bacterial and eukaryotic cells were stained with propidium iodide (red). Living bacteria (~1 µm) or eukaryotic nuclei (~10 µm) were stained with acridine orange (green). Additionally, WGA-594 (red) stained N-acetyl glucosamine, a sugar often produced in biofilms. All images were made at 20× magnification. B, bacteria.

Fig 7

Fluorescent confocal microscopy images of burn wound infections 1 h after incubation with S. aureus. S. aureus was applied to (A, C) porcine skin or (B, D) human skin. After 1 h of incubation, planktonic cells were removed by washing, and dead bacterial and eukaryotic cells were stained with propidium iodide (red). Living bacteria (~1 µm) or eukaryotic nuclei (~10 µm) were stained with acridine orange (green). Additionally, WGA-594 (red) stained N-acetyl glucosamine, a sugar often produced in biofilms. All images were made using a 60× magnification objective. For clarity, two-dimensional cross-sections (C and D) are shown from the original three-dimensional images (A and B). B, bacteria; n, eukaryotic nuclei.