An in vitro and ex vivo wound infection model to test topical and systemic treatment with antibiotics

Abstract

Aims: This study aimed to develop a wound infection model that could be used to test antibiotic-loaded electrospun matrices for the topical treatment of infected skin and compare the effectiveness of this treatment to systemically applied antibiotics.

Methods and results: 3D-printed flow chambers were made in which Staphylococcus aureus biofilms were grown either on a polycarbonate membrane or explanted porcine skin. The biofilms were then treated either topically, by placing antibiotic-loaded electrospun matrices on top of the biofilms, or systemically by the addition of antibiotics in the growth medium that flowed underneath the membrane or skin. The medium that was used was either a rich medium or an artificial wound fluid. The results showed that microbial viability in the biofilms was reduced to a greater extent with the topical electrospun matrices when compared to systemic treatment.

Conclusions: An ex vivo infection model was developed that is flexible and can be used to test both topical and systemic treatment of wound infections. It represents a significant improvement over previous in vitro models that we have used to test electrospun membranes.

Significance and impact of the study: The availability of a relatively simple wound infection model in which different delivery methods and dosage regimes can be tested is beneficial for the development of improved treatments for wound infections.

Keywords: Staphylococcus aureus; antibiotics; electrospinning; wound dressing; wound infection model.

FIGURE 1

Schematic image of 3D‐printed flow chamber with a 10° slope. Dimensions of the flow chamber are shown in a, and a side view of the setup showing the flow of medium through the chamber (arrows) is shown in b.

FIGURE 2

Histological images of punctured porcine skin placed on TSA (a, b, c and d), or AWFA (e and f) with/without Staphylococcus aureus MRSA252. Panels a, b, e, and f show infected wound beds; panel c shows infected skin but outside the area where the biopsy was made, and panel d shows uninfected skin. Black arrows show areas of skin with growth of S. aureus MRSA252. The scale bar is equivalent to 200 μm.

FIGURE 3

Viable counts of Staphylococcus aureus MRSA252 grown on porcine skin pieces, incubated on TSA or AWFA. Experiments were performed in triplicate with two technical repeats, and the error bars shown are standard deviations. An unpaired t‐test was used to compare the mean of the two groups (**p < 0.01).

FIGURE 4

Representative images of ortho‐ and side‐view images of Staphylococcus aureus MRSA252 biofilm grown on PC membranes in the flow system with TSB (a, b, and c) and AWF (d, e and f), where green indicates live cells and red indicates dead cells. The images were captured at 1 h (a, d), 4 h (b, e), and 8 h (c, f) of biofilm growth in the flow system. The scale bar is equivalent to 20 μm.

FIGURE 5

Effect of topically applied antibiotics on Staphylococcus aureus MRSA252 biofilms in the flow system. Biofilms were grown for 48 h in TSB (a) or AWF (b), and then electrospun matrices containing antibiotics were placed on top for 24 h. The number of viable cells is expressed as the % of viable cells compared to controls without antibiotics. The experiments were performed in triplicate, with two technical repeats in each, and the error bars shown represent standard deviations. Black bars: biofilms grown on PC membranes; grey bars: biofilms grown on porcine skin. Statistical analysis was performed using unpaired t‐tests with a False Discovery Rate correction (***p < 0.001).

FIGURE 6

Effect of mimicking systemic delivery of antibiotics to Staphylococcus aureus MRSA252 biofilms in the flow system. Biofilms were grown on PC membranes for 48 h in either TSB (a) or AWF (b) in the flow device, and then antibiotics were added to the growth medium that flows underneath the biofilm for 24 h. The number of viable cells is expressed as the % of viable cells compared to controls without antibiotics. The experiments were performed in triplicate, with two technical repeats in each, and the error bars shown represent standard deviations. Statistical analysis was performed using an unpaired t‐test (***p < 0.001).

FIGURE 7

Effect of systemic and topical treatment of infected porcine skin. The skin was inoculated with Staphylococcus aureus MRSA252 and grown for 2 days in the flow cells using either TSB (a) or AWF (b). The biofilms were then treated topically with gentamicin (drug‐loaded electrospun PCL/SF matrices) or systemically (by adding gentamicin to the growth medium flowing underneath the skin) for 24 h. The experiments were performed in triplicate, with two technical repeats, and error bars are standard deviations. Statistical analysis was performed using an unpaired t‐test (**p < 0.01).