Composition comprising an oxidoreductase enzyme and its antibacterial use

Abstract

A composition comprising an oxidoreductase enzyme and a substrate for the enzyme for use in treating or preventing biofilm formation by a population of pathogenic bacteria. The composition can be incorporated into a wound dressing for use in promoting wound healing.

Claims

1. A method for the treatment or prevention of biofilm formation by a population of pathogenic bacteria, comprising administration of a therapeutic amount of a composition comprising an oxidoreductase enzyme and a substrate to a patient in need thereof, wherein the composition does not comprise lactoperoxidase.

2. The method of claim 1, wherein the oxidoreductase enzyme is glucose oxidase and the substrate is glucose.

3. The method of claim 1, wherein the population of pathogenic bacteria comprises Pseudomonas aeruginosa, Staphylococcus aureus and/or Staphylococcus epidermidis.

4. The method of claim 1, wherein the population of pathogenic bacteria comprises Pseudomonas aeruginosa.

5. The method of claim 1, wherein the composition consists of the enzyme, substrate and a pharmaceutically acceptable carrier.

6. The method of claim 1, wherein the composition is administered to a wound on the patient.

7. The method of claim 1, comprising a preceding step of identifying a wound in the patient that contains, or is at risk of infection by, a population of biofilm-forming pathogenic bacteria.

8. The method of claim 7, wherein the wound is a chronic wound.

9. The method of claim 7, further comprising administering negative pressure therapy to the wound.

10. The method of claim 1, wherein the oxidoreductase enzyme in the composition has a concentration of 0.2 U/mL to 60 U/mL.

11. The method of claim 1, wherein the pH of the composition is 4.5-6.5.

Description

FIGURES

(1) The present invention will now be further described by way of reference to the following Example and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to the following Figures in which:

(2) FIG. 1 illustrates the effect of 24 hour honey, glucose oxidase and honey/glucose oxidase exposure against 24 hour old P. aeruginosa biofilms;

(3) FIG. 2 illustrates the effect of 24 hour honey, glucose oxidase and honey/glucose oxidase exposure against 24 hour old S. aureus biofilms.

EXAMPLES

Example 1 Biofilm Model

(4) Method

(5) Stationary phase cultures of S. aureus or P. aeruginosa grown in TSB were adjusted to 0.5 McFarland (c. 1.510.sup.8 CFU/mL) and diluted a further 1:4 ratio (1 mL inoculum with 3 mL 0.1% Bactopeptone). A viable count was taken of each inoculum preparation. Sterile discs (6 mm diameter1.0 mm) housed in 24 well culture plates were inoculated with 80 L of culture. 0.2 ml of a test media was then added, the assay plates were parafilmed and incubated for 24 h at 37 degrees Celsius with 80 rpm shaking to allow biofilm formation. Unused wells were filled with PBS to increase humidity and prevent evaporation.

(6) After 24 hours incubation, cellulose discs/biofilms were rinsed with PBS to remove any adherent vegetative cells and transferred to 150 l of fresh media.

(7) For the honey (as the source of glucose) and Prontosan, 30 l was added to each test disc.

(8) For the glucose oxidase, a 100 U/ml stock solution was made as follows; glucose oxidase is supplied as 100,000-250,000 U/g freeze dried powder. Assuming the lowest concentration (100,000 U/g), 3.5 mg (350 U) was added to 3.5 ml of DPBS to give a 100 U/ml working stock which was filter sterilised.

(9) For the Pseudomonas experiment, the working stock was diluted in PBS to give a 10 U/ml solution-30 l was added to a disc/biofilm.

(10) For the S. aureus experiment, 100 l and 300 l of stock was diluted in 5 ml volumes of PBS to prepare low and high glucose oxidase alone solutions30 l added to a disc/biofilm.

(11) For the Honey/Glucose Oxidase Mixture

(12) Two honey/glucose oxidate mixtures were tested: A2 U/g (2 U/ml) referred to as Low5 g honey+100 l of glucose oxidase stock (100 U/ml) B6 U/g (6 U/ml) referred to as High5 g honey+300 l of glucose oxidase stock (300 U/ml)

(13) 30 l of each was added to a disc/biofilm.

(14) The assay plates were parafilmed and incubated for 24 hours at 37 C. with 80 rpm shaking to allow biofilm disruption. Unused wells were filled with PBS to increase humidity and prevent evaporation.

(15) After incubation (with shaking) at 37 degrees, discs/biofilms were removed and placed in 665 L DE neutralising broth (1:20 dilution) for 10 mins, vortexed vigorously for 3 minutes then subjected to serial dilution and viable counting onto TSA by Miles and Misra (1:10 serial dilutions, 10 l spots in triplicate onto agar).

(16) Results

(17) Both the compositions comprising honey and glucose oxidase demonstrated significant inhibition of 24 hour old single species biofilms of P. aeruginosa and S. aureus, whereas compositions comprising honey alone and glucose oxidase alone showed no inhibition of the biofilm. Prontosan gel was highly active against S. aureus biofilms but had less activity against P. aeruginosa biofilms (see FIGS. 1 and 2).