An Invertebrate Burn Wound Model That Recapitulates the Hallmarks of Burn Trauma and Infection Seen in Mammalian Models

Abstract

The primary reason for skin graft failure and the mortality of burn wound patients, particularly those in burn intensive care centers, is bacterial infection. Several animal models exist to study burn wound pathogens. The most commonly used model is the mouse, which can be used to study virulence determinants and pathogenicity of a wide range of clinically relevant burn wound pathogens. However, animal models of burn wound pathogenicity are governed by strict ethical guidelines and hindered by high levels of animal suffering and the high level of training that is required to achieve consistent reproducible results. In this study, we describe for the first time an invertebrate model of burn trauma and concomitant wound infection. We demonstrate that this model recapitulates many of the hallmarks of burn trauma and wound infection seen in mammalian models and in human patients. We outline how this model can be used to discriminate between high and low pathogenicity strains of two of the most common burn wound colonizers Pseudomonas aeruginosa and Staphylococcus aureus, and multi-drug resistant Acinetobacter baumannii. This model is less ethically challenging than traditional vertebrate burn wound models and has the capacity to enable experiments such as high throughput screening of both anti-infective compounds and genetic mutant libraries.

Keywords: Acinetobacter baumannii; Galleria mellonella; MRSA; Pseudomonas aeruginosa; biofilm; burn; infection.

FIGURE 1

Schematic outline of burn procedure: (1) Heating up the burn device in the middle of the flame of a Bunsen burner until the metal element is red/white-hot. (2) Cooling down the burn device near the flame for 4 s. (3) Applying the hot metal element to the middle segments of Galleria mellonella back for 4 s and slowly lifting it. (4) Applying 10 μl of 1:10 dilution of over-night bacterial culture onto the fresh wound. (5) Larvae are incubated at 37°C for 120 h and monitored every 24 h. The darkness of Galleria signifies the intensity of infection (Wojda, 2017). Created with BioRender.

FIGURE 2

Hallmarks of burn trauma: (A) Total burn surface area (TBSA) correlates with survival: it is well established in burn patients that the larger the TBSA, the greater the risk of mortality. We demonstrate that this central dogma can be recapitulated in the G. mellonella burn wound model. 1x = 2 mm² Burn, 2x = 2 × 2 mm² Burn, 3x = 3 × 2 mm² Burns. 1x vs 2x, Log Rank p < 0.001, 1x vs 3x Log Rank p < 0.001, 2x vs 3x Log Rank p < 0.001. n ≥ 30 per condition. (B) Fluid resuscitation: administering larvae with fluid after burn trauma can significantly increase their survival prognosis Log Rank p < 0.001. n ≥ 30 per condition.

FIGURE 3

Visualization of burn wound pathogenicity over time: (A) Following the burn wound procedure and subsequent infection, the tissue at site of the burn will turn necrotic and evidence of bacterial dissemination is seen through the melanization of the larvae and loss of motility. Representative images of each stage shown. (B) Percentage survival of larvae after infecting the burn site with PA14ΔexsA which has an inactive T3SS and Wild Type PA14 against the uninfected control group burn. PA14 vs Burn Only Log Rank p < 0.001, Burn Only vs PA14ΔexsA Log Rank p < 0.001, PA14ΔexsA vs Burn Only Log Rank p < 0.001. n ≥ 40 per condition. (C) Hemolymph (larval blood) was aspirated and the bacterial numbers enumerated by plating serial dilutions n = 25 larvae, with SEM. Mann–Whitney U-test ***p < 0.001.

FIGURE 4

Burn wound pathogenicity: (A) Percentage survival rate of larvae infected with A. baumannii AB5075 and the burn only control. A. baumannii AB5075 vs Burn Only Log Rank p < 0.001. (B) Percentage survival rate of larvae infected with MRSA and the non-burn wound pathogen S. epidermidis. MRSA vs Burn Only Log Rank p < 0.001, MRSA vs S. epidermidis Log Rank p < 0.001, S. epidermidis vs Burn Only Log Rank p < 0.14. n ≥ 30 per condition.