PREVENTION AND TREATMENT OF MICROBIAL INFECTIONS

Abstract

Compositions for prevention and treatment of microbial infectious, such as microbial infections that comprise a biofilm, or a microbe that is capable of forming a biofilm are described. The compositions comprise an enzyme that is able to convert a substrate to release hydrogen peroxide and a substance that includes a substrate for the enzyme. The enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance. The substance may be an unrefined natural substance or a substance that includes a purified substrate for the enzyme.

Claims

1. A composition for generating anti-microbial activity for use in the prevention or treatment of a microbial infection that comprises a biofilm, or a microbe that is capable of forming a biofilm, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

2. Use of composition for generating anti-microbial activity in the manufacture of a medicament for use in the prevention or treatment of a microbial infection that comprises a biofilm, or a microbe that is capable of forming a biofilm, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

3. Use according to claim 1 or 2, wherein the composition prevents or inhibits growth or seeding of the biofilm, or prevents or inhibits formation of a biofilm by the microbe.

4. Use according to any preceding claim, wherein the substance is an unrefined natural sugar substance.

5. Use according to claim 4, wherein the sugar substance is a honey.

6. Use according to claim 5, wherein the honey is an unpasteurised honey, preferably a creamed unpasteurised honey.

7. Use according to any preceding claim, wherein the enzyme is an oxidoreductase enzyme.

8. Use according to claim 7, wherein the oxidoreductase enzyme is a glucose oxidase.

9. Use according to any preceding claim, wherein the composition is a sterile composition.

10. Use according to any preceding claim, wherein the microbial infection is a chronic microbial infection.

11. Use according to any preceding claim, wherein the microbial infection is a wound infection, preferably a chronic wound infection.

12. Use according to claim 11, wherein the wound infection is a skin wound infection or a burn wound infection.

13. Use according to any preceding claim, wherein the microbial infection comprises a bacterium, preferably a Gram-negative bacterium, that is capable of forming a biofilm.

14. Use according to any preceding claim, wherein the biofilm comprises any of the following species of bacteria, or wherein the microbe is any of the following species of bacteria: Pseudomonas aeruginosa; Acinetobacter baumannii, Methicillin-resistant Staphylococcus aureus (MRSA), or Methicillin-susceptible Staphylococcus aureus (MSSA).

15. Use according to any of claims 1 to 10, wherein the microbial infection is a sinus infection, such as chronic rhinosinusitis (CRS).

16. Use according to any of claims 1 to 10, wherein the microbial infection is a microbial lung infection, such as a Mycobacterium tuberculosis infection, or a Pseudomonas aeruginosa infection in a subject with cystic fibrosis.

17. An in vitro method for preventing or inhibiting formation of a biofilm, which comprises contacting an effective amount of a composition for generating anti-microbial activity with a microbe that is capable of forming a biofilm, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

18. An in vitro method for preventing or inhibiting growth or seeding of a biofilm, which comprises contacting an effective amount of a composition for generating anti-microbial activity with a biofilm, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

19. A method of preventing or treating a microbial infection that includes a biofilm, or a microbe that is capable of forming a biofilm, wherein the method comprises administering an effective amount of a composition for generating anti-microbial activity to a site of the infection, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

20. A composition for generating anti-microbial activity, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; and a salt; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

21. A kit, which comprises a composition for generating anti-microbial activity, wherein the composition is a storage-stable composition that comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; and separately, a salt; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

22. An aqueous mixture, which comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance; and a salt.

23. A composition according to claim 20, a kit according to claim 21, or a mixture according to claim 22, wherein the salt comprises sodium chloride.

24. A composition according to claim 20, a kit according to claim 21, or a mixture according to claim 22, which further comprises a buffering agent, such as sodium bicarbonate.

25. A composition, kit, or mixture according to any of claims 20 to 24, which is sterile.

26. A composition according to claim 20, or a mixture according to claim 22, for use as a medicament.

27. A composition according to claim 20, or a mixture according to claim 22, for use in the prevention or treatment of a sinus infection, such as CRS.

28. Use of a composition according to claim 20, or a mixture according to claim 22, in the manufacture of a medicament for the prevention or treatment of a sinus infection, such as CRS.

29. A composition comprising: an enzyme that is able to convert a substrate to release hydrogen peroxide; an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance; and a polymer.

30. A composition according to claim 29, which is a sprayable or atomisable composition.

31. A composition according to claim 29 or claim 30, wherein the polymer is selected from polyethylene oxide, polyvinyl alcohol and polyvinylpyrrolidone.

32. A device for delivering a composition to a patient, the device comprising a composition, as defined in any of claims 29 to 31.

33. The device according to claim 32, which is a spraying or atomising device.

34. A kit comprising: i) a composition as defined in any of claims 29 to 31; and ii) a device for delivering a composition to a patient.

35. A method of applying or administering a composition to a patient, the composition as defined in any of claims 29 to 31, wherein the method comprises spraying the composition, injecting the composition, inhaling the composition or applying the composition to a patient's respiratory tract.

36. A composition for use as a medicament, the composition as defined in any of claims 29 to 31.

37. A composition for use, according to claim 36, wherein the composition is administered or applied by spraying, by injection, by inhalation, or by applying the composition to a patient's respiratory tract.

38. A composition for generating anti-microbial activity that comprises: an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a purified substrate for the enzyme; wherein the composition is a storage-stable composition that does not include sufficient free water to allow the enzyme to convert the substrate, and wherein the composition provides for sustained release of hydrogen peroxide at a level of less than 2 mmol/litre for a period of at least 24 hours following dilution of the composition.

39. An aqueous mixture, which comprises: an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a purified substrate for the enzyme; wherein the composition provides for sustained release of hydrogen peroxide at a level of less than 2 mmol/litre for a period of at least 24 hours.

40. A composition according to claim 38, or a mixture according to claim 39 that comprises sufficient enzyme and substrate to provide for sustained release of at least 0.1 mmol/litre hydrogen peroxide for a period of at least 24 hours.

41. A composition or mixture according to any of claims 38 to 40, which further comprises a salt.

42. A composition or mixture according to any of claims 38 to 41 for use as a medicament.

43. A composition or mixture according to any of claims 38 to 41 for use in the prevention or treatment of a microbial infection.

44. Use of a composition or mixture according to any of claims 38 to 41 in the manufacture of a medicament for the prevention or treatment of a microbial infection.

45. A method of preventing or treating a microbial infection, wherein the method comprises administering an effective amount of a composition or mixture according to any of claims 38 to 41 to a site of the infection.

46. Use according to claim 43 or 44, or a method according to claim 45, wherein the microbial infection includes a biofilm, or a microbe that is capable of forming a biofilm.

47. A composition according to any of claim 20, or 29 to 31, a mixture according to claim 22, a composition or mixture according to any of claims 38 to 41, or a storage-stable composition, for use in the prevention or treatment of an MRSA colonisation or infection, wherein the storage-stable composition comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

48. Use of a composition according to any of claim 20, or 29 to 31, a mixture according to claim 22, a composition or mixture according to any of claims 38 to 41, or a storage-stable composition, in the manufacture of a medicament for the prevention or treatment of an MRSA colonisation or infection, wherein the storage-stable composition comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

49. A method of preventing or treating an MRSA colonisation or infection, which comprises administering an effective amount of a composition according to any of claim 20, or 29 to 31, a mixture according to claim 22, a composition or mixture according to any of claims 38 to 41, or a storage-stable composition, to a subject in need of such prevention or treatment, wherein the storage-stable composition comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme; or an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that lacks catalase activity and that includes a substrate for the enzyme; wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance, and the composition does not include sufficient free water to allow the enzyme to convert the substrate.

50. A composition as defined in any of claim 20, 23-25, 29-31, 38 or 40-41, contained within a water-soluble sachet.

51. A wound dressing comprising a composition as defined in any of claim 20, 23-25, 29-31, 38 or 40-41; and a water-soluble layer enclosing the composition.

52. A composition as defined in any of claim 20, 23-25, 29-31, 38 or 40-41, which does not include any detectable hydrogen peroxide.

53. Use, kit, method, composition or device according to any of claims 1 to 19, 21, 26 to 28, 32 to 37 or 42 to 49, wherein the composition is contained within a water-soluble sachet.

54. Use, kit, method, composition or device according to any of claims 1 to 19, 21, 26 to 28, 32 to 37 or 42 to 49, wherein the composition is provided in a wound dressing, with a water-soluble layer enclosing the composition.

55. Use, kit, method, composition, device or wound dressing according to any of claim 1 to 19, 21, 26 to 28, 32 to 37, 42 to 49, 51, 53 or 54, in which the composition does not include any detectable hydrogen peroxide.

56. Use, kit, method, composition, device or wound dressing according to any of claims 1 to 21, 23 to 38, or 40 to 55, in which the composition comprises 12% or less (by weight) of water.

57. Use, kit, method, composition, device or wound dressing according to any of claims 1 to 21, 23 to 38, or 40 to 55, in which the composition comprises 10% or less (by weight) of water.

58. Use, kit, method, composition, device or wound dressing according to any of claims 1 to 21, 23 to 38, or 40 to 55, in which the composition comprises 5% or less (by weight) of water.

59. Use, kit, method, composition, device or wound dressing according to any of claims 1 to 21, 23 to 38, or 40 to 55, in which the composition comprises 3% or less (by weight) of water.

60. Use, kit, method, composition, device or wound dressing according to any of claims 56 to 59, wherein the composition comprises a processing aid, drying aid, bulking agent and/or anticaking agent.

61. Use, kit, method, composition, device or wound dressing according to any of claims 56 to 60, wherein the composition is obtained or is obtainable, by drying.

62. Use, kit, method, composition, device or wound dressing according to claim 61 wherein the drying comprises freeze-drying, vacuum-drying or spray-drying.

63. Use, kit, method, composition, device or wound dressing according to any of claims 56 to 62, in a solid form.

64. Use, kit, method, composition, device or wound dressing according to claim 63, wherein the solid form is a powder, flakes, granules, a lozenge or a tablet.

65. Use, kit, method, composition, device, mixture or wound dressing according to any preceding claim, wherein the composition or mixture provides for a sustained release of hydrogen peroxide at a level of 10 to 500 ppm for a period of at least 1 hour, 12 hours, 24 hours, 2 days or 4 days.

66. Use, kit, method, composition, device, mixture or wound dressing according to claim 65, wherein the composition or mixture provides for a sustained release of hydrogen peroxide at a level of 50 to 100 ppm for a period of at least 1 hour, 12 hours, 24 hours, 2 days or 4 days.

67. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 1 to 64, wherein the composition or mixture provides for a sustained release of hydrogen peroxide at a level of 2 to 50 ppm for a period of at least 1 hour, 12 hours, 24 hours, 2 days or 4 days.

68. Use, kit, method, composition, device, mixture or wound dressing according to claim 67, wherein the composition or mixture provides for a sustained release of hydrogen peroxide at a level of 5 to 10 ppm for a period of at least 1 hour, 12 hours, 24 hours, 2 days or 4 days.

69. Use, kit, method, composition, device, mixture or wound dressing according to any preceding claim, wherein the composition or mixture comprises a non-aqueous solvent.

70. Use, kit, method, composition, device, mixture or wound dressing according to claim 69, wherein the non-aqueous solvent is selected from the group consisting of ethanol, dimethyl sulphoxide, glycerol, ethylene glycol and propylene glycol.

71. Use, kit, method, composition, device, mixture or wound dressing according to claim 70, wherein the non-aqueous solvent is glycerol.

72. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 71, wherein the non-aqueous solvent is at least 10% by weight of the composition or mixture.

73. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 71, wherein the non-aqueous solvent is at least 20% by weight of the composition or mixture.

74. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 71, wherein the non-aqueous solvent is at least 50% by weight of the composition or mixture.

75. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 71, wherein the non-aqueous solvent is at least 75% by weight of the composition or mixture.

76. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 75, wherein the non-aqueous solvent is 50% or less by weight of the composition or mixture.

77. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 71, wherein the non-aqueous solvent is 1-50% by weight of the composition or mixture.

78. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 71, wherein the non-aqueous solvent is 50-90% by weight of the composition or mixture.

79. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 78, wherein the substance of the composition or mixture is, or comprises, honey.

80. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 78, wherein the substance of the composition or mixture is honey and the honey is present in an amount of at least 25% by weight.

81. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 78, wherein the substance of the composition or mixture is honey and the honey is present in an amount of at least 50% by weight.

82. Use, kit, method, composition, device, mixture or wound dressing according to any of claim 69-74 or 76-77, wherein the substance of the composition or mixture is honey and the honey is present in an amount of at least 75% by weight.

83. Use, kit, method, composition, device, mixture or wound dressing according to any of claims 69 to 78, wherein the substance of the composition or mixture is honey and the honey is present in an amount of 1-50% by weight.

84. Use, kit, method, composition, device, mixture or wound dressing according to any of claim 69-74 or 76-77, wherein the substance of the composition or mixture is honey and the honey is present in an amount of 50-90% by weight.

Description

[0317] Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings in which:

[0318] FIG. 1 shows the effect of: (A) neat SH1 Surgihoney; or (B) serial dilutions of SH1 Surgihoney on formation of biofilms by Pseudomonas aeruginosa strain PA01, and clinical burn wound isolate 1054; and (C) neat SH1 Surgihoney; or (D) serial dilutions of SH1 Surgihoney on formation of biofilms by Acinetobacter baumannii control strain AYE, and ACI clone NCTC_13420 (C59);

[0319] FIG. 2 shows the effect of neat Manuka honey on formation of biofilms by: (A) Pseudomonas aeruginosa strain PA01, and clinical burn wound isolate 1054; or (B) Acinetobacter baumannii control strain AYE, and ACI clone NCTC_13420 (C59); and (C) serial dilutions of Manuka honey on formation of biofilms by Pseudomonas aeruginosa strain PA01, and clinical burn wound isolate 1054 (upper part of FIG. 2(C)), or Acinetobacter baumannii control strain AYE, and ACI clone NCTC_13420 (C59) (lower part of FIG. 2(C));

[0320] FIG. 3 shows the effect of neat and serial dilutions of SH1, SH2, and SH3 Surgihoney on formation of biofilms by: (A) Pseudomonas aeruginosa strain PA01, and clinical burn wound isolate 1054; and (B) Acinetobacter baumannii control strain AYE, and ACI clone NCTC_13420 (C59);

[0321] FIG. 4 shows the effect of Surgihoney preparations SH1, SH2, and SH3, and Manuka honey (MH), on biofilm formation by a biofilm-producing isolate of Pseudomonas aeruginosa (PS_1586);

[0322] FIG. 5 shows the effect of Surgihoney preparations SH1, SH2, and SH3, and Manuka honey (MH), on biofilm formation by a biofilm-producing isolate of Pseudomonas aeruginosa (PA_6749);

[0323] FIG. 6 shows the effect of Surgihoney preparations SH1, SH2, and SH3, and Manuka honey (MH), on biofilm formation by a biofilm-producing isolate of Acinetobacter baumannii (ACI_19606);

[0324] FIG. 7 shows the effect of Surgihoney preparations SH1, SH2, and SH3, and Manuka honey (MH), on biofilm formation by a biofilm-producing isolate of Acinetobacter baumannii (ACI_060);

[0325] FIG. 8 shows the effect of Surgihoney SH1, SH2, and SH3 preparations, and Manuka honey (MH), on the prevention or reduction of the seeding of pre-formed biofilms produced by Acinetobacter baumannii ACI_C59;

[0326] FIG. 9 shows the effect of Surgihoney SH1, SH2, and SH3 preparations, and Manuka honey (MH), on the prevention or reduction of the seeding of pre-formed biofilms produced by Acinetobacter baumannii ACI_AYE;

[0327] FIG. 10 shows the effect of Surgihoney SH1, SH2, and SH3 preparations on prevention of biofilm formation by Pseudomonas aeruginosa (control strain PA01) in comparison with deactivated Surgihoney (DE), Manuka honey (MH: ‘Comvita Manukacare 18+’), acetic acid (AA), and several commercially available wound dressings, and wound creams;

[0328] FIG. 11 shows the effect of Surgihoney SH1, SH2, and SH3 preparations on prevention of biofilm formation by Acinetobacter baumannii (control strain AYE) in comparison with deactivated Surgihoney (DE), Manuka honey (MH: ‘Comvita Manukacare 18+’), acetic acid (AA), and several commercially available wound dressings, and wound creams;

[0329] FIG. 12 shows the effect of Surgihoney preparation SH1 and Manuka honey (MH) on biofilm formation by multi-drug resistant (MDR) biofilm-producing isolates of Pseudomonas aeruginosa (VIM positive) and CRE Klebsiella pneumoniae, and two biofilm-producing isolates of Acinetobacter baumanii (ACI_060 (1), ACI_C60 (2));

[0330] FIG. 13 shows the effect of Surgihoney treatment of chronic rhinosinusitis (CRS)-associated S. aureus planktonic populations at different concentrations of Surgihoney to determine the minimum inhibitory concentration (MIC) of Surgihoney. The first column for each treatment condition represents the result for a Methicillin-resistant Staphylococcus aureus (MRSA) isolate, the next four columns represent the results for different Methicillin-sensitive Staphylococcus aureus (MSSA) isolates;

[0331] FIG. 14 shows photographs of chocolate blood agar plates onto which planktonic Methicillin-resistant Staphylococcus aureus (MRSA) or Methicillin-sensitive Staphylococcus aureus (MSSA) have been subcultured with effective concentrations of Surgihoney to determine the minimum bactericidal concentration (MBC) of Surgihoney;

[0332] FIG. 15 shows the effect of Surgihoney treatment of CRS-associated S. aureus biofilms at different concentrations of Surgihoney. ** p=0.002;

[0333] FIG. 16A shows different hydrogen peroxide production rates for Surgihoney SH1, SH2, and SH3. FIG. 16B shows the relationship between phenol activity and maximum hydrogen peroxide activity in Surgihoney SH1, SH2, and SH3;

[0334] FIG. 17 shows a photograph of a Salter respiratory nebuliser with a nebuliser face mask tub attached. The reservoir contains a liquefied mixture of Surgihoney and saline;

[0335] FIG. 18 shows photographs of blood agar plates that have been cultured with paper discs inoculated with Staphylococcus aureus and contacted with nebulised Surgihoney for 5, 15, 20, or 30 minutes;

[0336] FIG. 19 shows the effect of Surgihoney treatment of CRS-associated S. aureus biofilms (MSSA) at different concentrations of Surgihoney;

[0337] FIG. 20 shows: (A) a photograph of a long-standing ischaemic ulcer chronically infected with Pseudomonas aeruginosa; (B) a photograph of the same ulcer after a 7 day treatment with Surgihoney;

[0338] FIG. 21 shows: (A) a photograph of a paediatric MRSA wound infection; (B) a photograph of the same wound infection after a 10 day treatment with Surgihoney;

[0339] FIG. 22 shows: (A) a photograph of a CA MRSA superficial infection; (B) a photograph of the same infection after a 5 day treatment with Surgihoney; and (C) a photograph of the same infection after a 10 day treatment with Surgihoney;

[0340] FIG. 23 shows the results of an assay for the cytotoxic activity of Surgihoney;

[0341] FIG. 24 shows hydrogen peroxide production by dried Surgihoney granules, and dried Surgihoney that has been dissolved in water to form a solution; and

[0342] FIG. 25 shows hydrogen peroxide production by the solution of Surgihoney of FIG. 24 after various time periods, up to four days.

[0343] The Examples below describe determination of the in vitro antibacterial activity of a composition for use according to the invention (Surgihoney) against important biofilm-forming burn wound pathogens.

[0344] Surgihoney (SH) has previously demonstrated highly potent inhibitory and cidal activity against a wide range of planktonic Gram-positive and Gram-negative bacteria in both laboratory tests and clinical practice. Prophylactic application to caesarean wounds has demonstrated eradication of resistant organisms and reduced rates of colonisation and infection.

[0345] Given the global concerns surrounding antimicrobial resistance, and the challenges posed by biofilms, the in vitro antibacterial activity of three different formulations of SH (SH1, SH2 and SH3) against biofilm-forming isolates of Pseudomonas aeruginosa and Acinetobacter baumannii was investigated to assess whether SH can i) prevent the formation of a biofilm and ii) eradicate or prevent seeding of a pre-formed biofilm.

[0346] The anti-biofilming properties of Surgihoney formulations SH1, SH2 and SH3 were investigated and compared against standard Manuka honey using two biofilm assays. These enabled in vitro measurement of the ‘Minimum Biofilm Inhibition Concentration’ (MBIC), and the ‘Minimum Biofilm Eradication Concentration’ (MBEC) of SH. Further experiments were performed to compare the anti-biofilming activity of SH to a range of commercial dressings, including one that contains 20% honey (L-Mesitran net).

EXAMPLE 1

[0347] Prevention of Biofilm Formation by Surgihoney Compared with Manuka Honey

[0348] This example describes a first experiment (Experiment 1), which compares the effect of Surgihoney (SH1), and Manuka honey (MH), and a second experiment (Experiment 2), which compares the effect of three different strength preparations of Surgihoney (SH1, SH2, and SH3) on biofilm formation by biofilm-producing isolates of Pseudomonas aeruginosa (control strain PA01, and clinical burn wound isolate 1054) and Acinetobacter baumannii (control strain AYE, and UK ACI clone NCTC_13420 (C59)).

Methods

[0349] For each neat honey sample, one loopful of honey was placed into 100 μl water in a well of a 96-well microtitre plate. For the diluted honey samples, approximately 2.5 ml honey was placed into a universal with 6 ml of water. 3 ml of this mixture was then serially diluted down to 1:2048. 100 μl of each serial dilution was added to a different well of the 96-well plate.

[0350] Overnight cultures of the isolates were diluted in Muller-hinton broth to an OD600 of 0.1. 100 μl of the diluted overnight culture was added to each neat or diluted honey sample well. Each positive control well contained 100 μl diluted overnight culture, and 100 μl water. Each negative control well contained 200 μl broth or 200 μl neat honey sample.

[0351] The plate was incubated for 72 hours at 33° C. (wound temperature) to encourage biofilm formation. The plate was then developed using crystal violet dye (which binds to dead and living biofilms), and visualised following solubilisation of the dye using 70% ethanol.

[0352] A spectrophotometer was then used to assess the optical density (OD) of the solubilised dye. The OD reading corresponds to the amount of incorporated crystal violet. Higher OD readings represent dark wells, and greater mass of biofilm. The OD readings were then plotted to make a graph.

Results

[0353] The results are shown in FIGS. 1-3, and summarised in the table below, which records the Minimum Biofilm Inhibitory Concentration (MBIC) for each strain/isolate (using an OD of 0.3 as a cut-off):

TABLE-US-00010 MBIC Experiment 1 Experiment 2 Strain/Isolate SH1 MH SH1 SH2 SH3 PA01 1:2 Neat.sup.1 1:4* 1:16* 1:32* 1054 1:2 Neat.sup.1 1:8* 1:32* 1:16* AYE 1:4 1:2 1:8* 1:64* 1:64* C59 1:4 1:2 1:8* 1:64* 1:32* .sup.1The activity of Manuka honey against Pseudomonas was sporadic *= Statistically significant (P < 0.005) when student t-test performed to compare growth with honey and positive control.

[0354] The results show that Surgihoney SH1 prevented Pseudomonas biofilm formation when used neat or at 1:2 dilution, and prevented Acinetobacter biofilm formation when used neat or at 1:2 or 1:4 dilution.

[0355] Manuka honey had a sporadic effect on Pseudomonas biofilm production when used neat, but prevented Acinetobacter biofilm formation when used neat or at 1:2 dilution.

[0356] Surgihoney SH2 and SH3 also prevented Pseudomonas and Acinetobacter biofilm formation, but were able to do so at lower concentrations than SH1 Surgihoney.

[0357] There appeared to be little significant difference in the ability of Surgihoney SH2 and SH3 to inhibit Pseudomonas and Acinetobacter biofilm formation.

[0358] Biofilm formation by Acinetobacter appeared to be more sensitive to Surgihoney and Manuka honey treatment than Pseudomonas biofilm formation.

Conclusions

[0359] It was concluded from these results that Surgihoney is able to prevent biofilm formation of biofilms, and that Surgihoney SH2 and SH3 were able to prevent biofilm formation at lower concentrations than Surgihoney SH1. Each Surgihoney preparation was able to prevent biofilm formation at a lower concentration than Manuka honey.

EXAMPLE 2

[0360] Prevention of Biofilm Formation by Different Strength Preparations of Surgihoney Compared with Manuka Honey

[0361] This example describes the effect of Surgihoneyl (SH1), Surgihoney 2 (SH2), Surgihoney 3 (SH3) and Manuka honey (MH), on biofilm formation by biofilm-producing isolates of Pseudomonas aeruginosa (PS_1586] and PS_6749) and Acinetobacter baumannii (ACI_C60 and ACI_19606).

Methods

[0362] Each honey was placed in a 37° C. incubator for 30 minutes. Approximately 3 ml each honey was then placed into a universal, and 3 ml sterile water was added. This mixture was then vortexed vigorously and serially diluted down to 1:4096.

[0363] 100 μl of a diluted overnight culture of each isolate was added to 100 μl of diluted honey in a 96-well microtitre plate, and incubated for 72 hours at 33° C. (wound temperature) to encourage biofilm formation.

[0364] The plate was then developed using crystal violet dye, and visualised following solubilisation of the dye using 70% ethanol.

[0365] A spectrophotometer was then used to assess the optical density (OD) of the solubilised dye. The OD reading corresponds to the amount of incorporated crystal violet. Higher OD readings represent dark wells, and greater mass of biofilm. The OD readings were then plotted to make a graph.

Results

[0366] The results are shown in FIGS. 4-7, and summarised in the table below, which records the Minimum Biofilm Inhibitory Concentration (MBIC) for each isolate (using an OD of 0.3 as a cut-off):

TABLE-US-00011 MBIC Isolate SH1 SH2 SH3 MH PS_1586 1:8 1:64 1:32.sup.1 1:2.sup.2 PS_6749 1:8 1:512 1:512 1:16.sup.3 ACI_C60 1:16.sup.4 1:128.sup.4 1:16.sup.1 1:4 ACI_19606 1:16 1:64 1:128 1:2 .sup.11:64 dilution not tested .sup.2Enhanced biofilming observed at 1:4 dilution compared to lower dilutions (see below) .sup.3Enhanced biofilming observed at 1:32 dilution compared to lower dilutions (see below) .sup.4Enhanced biofilming observed at 1:8 to 1:32 dilutions compared to lower dilutions (see below) The positive controls (POS) show that all the isolates were able to form a biofilm, and the negative controls (NEG) show that there was no contamination.

[0367] For some of the isolates, enhanced biofilming is observed at certain concentrations, compared with biofilming at higher and lower concentrations, of the Surgihoney or Manuka honey. This effect has been observed occasionally with other biocides. It is believed to be due to the ‘stress’ of the biocide causing enhanced biofilming.

Conclusions

[0368] It was concluded from these results that each Surgihoney preparation (SH1, SH2, and SH3) was able to prevent each of the isolates tested from forming a biofilm. The SH2 and SH3 Surgihoney preparations were able to do so at lower concentrations than S1 Surgihoney.

[0369] The optimum Surgihoney preparation for preventing biofilm formation of the isolates tested was SH2. A dilution of 1:64 SH2 was able to prevent biofilm formation of each isolate tested.

[0370] Each strength preparation of Surgihoney tested (SH1, SH2, and SH3) was generally able to prevent biofilm formation at lower concentrations than Manuka honey.

EXAMPLE 3

Prevention of Pre-Formed Biofilm Seeding by Surgihoney

[0371] This example describes the effect of Surgihoney SH1, SH2, and SH3 preparations, and Manuka honey (MH), on the prevention or reduction of the seeding of pre-formed biofilms produced by two isolates of Acinetobacter baumannii (ACI_AYE and ACI_C59).

Methods

[0372] 200 μl of a serially diluted overnight culture of Acinetobacter baumannii (ACI_AYE or ACI_C59) was added to each well of a 96-well microtitre plate. A PCR peg plate was placed on top of the microtitre plate so that each well contains a ‘peg’ on which a biofilm can form. The 96-well plate was then incubated for 72 hours at 33° C. to encourage biofilms to grow.

[0373] After 72 hours, the pegs were washed and then placed into a further 96-well plate with wells containing the test agent (Surgihoney or Manuka honey), or broth alone (for the controls). After 24 hours, the pegs were washed and then placed into another 96-well plate containing sterile broth for overnight incubation. The OD of the broth was assessed the following day using the spectrophotometer (as described in Examples 1 and 2 above). Turbid broth has a high OD and represents successful seeding of the biofilm.

[0374] Finally, the pegs were subjected to a crystal violet assay (as described in Examples 1 and 2) to prove that biofilms were present on the pegs in the first place.

Results

[0375] The results are shown in FIGS. 8 and 9.

[0376] In the Figures, the y-axis represents the OD of the broth and, therefore, the amount of seeding. As expected, a large amount of biofilm seeding was observed with each positive control, and minimal background turbidity was observed with each negative control. Biofilms were present on all of the pegs. The results allow determination of the Minimum Biofilm Eradication Concentration (MBEC) of Surgihoney.

SH1:

[0377] Reduced seeding was observed as low as the 1:16 dilution of SH1 for both isolates. Some turbidity was observed at the 1:2 dilution. It is believed that this may have been an artefact due to some of these test wells being in the corner of the plate and, therefore, being more difficult to wash. Some turbidity was also observed at the 1:8 dilution. Again, this is believed to have been an artefact, possibly because these test wells were next to the positive control on the plate.

SH2:

[0378] Reduced seeding was observed for the 1:2 to 1:32/1:64 dilutions for both isolates. As for SH1, some turbidity was observed at the 1:2 dilution. This may have been due to some of these test wells being in the corner of the plate and, therefore, being more difficult to wash.

SH3:

[0379] Reduced seeding was observed for the 1:2 to 1:32/1:64 dilutions for both isolates. As for

[0380] SH1 and SH2, some turbidity was observed at the 1:2 dilution. This may have been due to some of these test wells being in the corner of the plate and, therefore, being more difficult to wash.

MH:

[0381] Reduced seeding was observed for the 1:2 to 1:16/1:32 dilutions.

Conclusions

[0382] The results confirm that Surgihoney prevented or reduced the seeding of pre-formed biofilms. The SH2 and SH3 preparations of Surgihoney were able to prevent or reduce biofilm seeding at lower concentrations than the SH1 preparation, although there appeared to be little difference in potency between the SH2 and SH3 preparations. The SH2 and SH3 preparations appeared to be slightly more potent than Manuka honey in reducing biofilm seeding.

EXAMPLE 4

[0383] Prevention of Biofilm Formation by Surgihoney Compared with Commercially Available Wound Dressings

[0384] This example describes the effect of Surgihoney SH1, SH2, and SH3 preparations on prevention of biofilm formation by Pseudomonas aeruginosa (control strain PA01), and Acinetobacter baumannii (control strain AYE), in comparison with deactivated Surgihoney (DE), Manuka honey (MH: ‘Comvita Manukacare 18+’), acetic acid (AA), and several commercially available wound dressings, and wound creams.

Methods

[0385] Overnight cultures of the isolates were diluted in Muller-hinton broth to an OD600 of 0.1. Surgihoney SH1, SH2, and SH3, deactivated Surgihoney (DE), and Manuka honey (MH: ‘Comvita Manukacare 18+’), were tested at serial dilutions of 1:2-1:256 (using water as diluent). 1 ml honey was placed in each well with 1 ml diluted isolate culture.

[0386] 1 cm.sup.2 of each of the following commercially available dressings was added to 1 ml water and 1 ml diluted isolate culture: Mepilex Ag; Urgotul Ag; Acticoat (Ag); Urgotul (no AM agent); Mesitran Net (honey-based dressing); Polymem (no AM agent).

[0387] Commercially available creams Trimovate and Flamazine (used for treatment of skin infections) were also tested. Acetic acid was also tested from 5% down to 0.04%.

[0388] The plate was incubated for 72 hours at 33° C. (wound temperature) to encourage biofilm formation. The plate was then developed using crystal violet dye, and visualised following solubilisation of the dye using 70% ethanol.

[0389] A spectrophotometer was then used to assess the optical density (OD) of the solubilised dye. The OD reading corresponds to the amount of incorporated crystal violet. Higher OD readings represent dark wells, and greater mass of biofilm. The OD readings were then plotted to make a graph.

Results

[0390] The results are shown in FIGS. 10 and 11.

[0391] The results show that Acticoat and Mepilex Ag dressings (and to a lesser extent, Urgotel silver) were effective at preventing biofilm formation of both strains, as was the Flamazine cream. The Urgotul and Polymem dressings, and Mesitran net (a commercial honey-based dressing), and the Trimovate cream did not appear to be effective in preventing biofilm formation.

[0392] Acetic acid was effective at preventing biofilm formation down to 0.31% (AYE) and 0.1% (PA01).

[0393] All of the Surgihoneys tested were effective at preventing biofilm formation of both strains at 1:2 and 1:4 dilution, at least, and some at lower concentrations (for example, SH2 was effective against AYE at 1:64 dilution).

[0394] The SH2 and SH3 Surgihoneys were more effective in preventing biofilm formation at lower concentrations than SH1 Surgihoney, and each Surgihoney preparation was more effective at lower concentrations than Manuka honey (see, for example, the effect of the 1:8 dilution for each). Manuka honey was effective in preventing biofilm formation down to 1:2 to 1:4 dilution.

[0395] The results also show that the deactivated Surgihoney (DE) was effective in preventing biofilm formation. However, when the DE honey was tested for hydrogen peroxide activity, it was found to retain some hydrogen peroxide activity, and so did not appear to have been fully inactivated.

Conclusions

[0396] It was concluded from these results that Surgihoney was comparable in preventing biofilm formation to several commercially available wound dressings, and more effective than Mesitran net (a commercial honey-based dressing).

[0397] In the above examples, four isolates of A. baumanii, and three of P. aeruginosa were tested and Surgihoney was able to prevent biofilm formation of all isolates in a dose-dependent manner. Pre-formed biofilms of A. baumannii were additionally exposed to all SH formulations for 24 hours. Reduced seeding of the biofilms was observed for both strains tested and was again dose-dependent.

[0398] The dressings experiment (Example 4) revealed SH to be equally or more effective in biofilm prevention than three of the eight commercial creams and dressings tested. Furthermore, in this in vitro test, SH was more effective than L-Mesitran net at preventing biofilm formation of single isolates of A. baumanii and P. aeruginosa.

[0399] These results show that Surgihoney has potent anti-biofilming activity against key Gram-negative pathogens of burn wounds, and superior activity to the majority of commercial dressings tested. This composition can be used in place of antibiotics, and in an era of increasing antibiotic resistance, help to reduce inappropriate antibiotic use.

EXAMPLE 5

[0400] Inhibition of Biofilm Formation by Surgihoney Compared with Manuka Honey

[0401] This example describes an experiment to test the effect of Surgihoneyl (SH1) and Manuka Honey (MH) on biofilm formation by multi-drug resistant (MDR) biofilm-producing isolates of Pseudomonas aeruginosa (VIM positive) and CRE Klebsiella pneumoniae, and two different biofilm-producing isolates of Acinetobacter baumanii.

Methods

[0402] The isolates were cultured with different concentrations of SH1 or MH, and MBIC calculations were made, by similar methods to those described in Example 2.

Results

[0403] The results are shown in FIG. 12. The results show that SH1 was able to prevent each of the isolates from forming a biofilm. SH1 was able to prevent biofilm formation of MDR biofilm-producing isolates of Pseudomonas aeruginosa (VIM positive) and CRE Klebsiella pneumonia at lower concentrations than MH.

EXAMPLE 6

[0404] Surgihoney Treatment of CRS-Associated S. aureus Planktonic Populations

[0405] The minimum inhibitory concentration (MIC) of Surgihoney for different isolates of CRS-associated S. aureus planktonic populations was determined using a 96-well plate-based absorbance assay. The isolates tested were one Methicillin-resistant Staphylococcus aureus (MRSA) isolate, and four Methicillin-sensitive Staphylococcus aureus (MSSA) isolates—3 polyp isolates, and 1 mucosal isolate. The Surgihoney concentrations tested were 180 g/L, 225, 270, 315, 360, 405, 450, 495, 540, 720, and 900 g/L Surgihoney. Absorbance at OD.sub.595 was measured after 18 hours.

[0406] The results are shown in FIG. 13. The results show that the MIC of Surgihoney for planktonic MRSA was 540 g/L, and the MIC of Surgihoney for planktonic MSSA was 720 g/L.

[0407] MRSA and MSSA isolates were subcultured onto chocolate blood agar plates with effective concentrations of Surgihoney (360, 405, 450, 495, 540, 720, 900 g/L) to determine the minimum bactericidal concentration (MBC) of Surgihoney for MRSA and MSSA. Photographs of the plates are shown in FIG. 14. The photographs show that the MBC of Surgihoney for planktonic MRSA was 720 g/L, and the MBC of Surgihoney for planktonic MSSA was 900 g/L.

EXAMPLE 7

[0408] Surgihoney Treatment of CRS-Associated S. aureus Biofilms

[0409] An MRSA isolate was grown to mid-exponential phase by static culture in a polystyrene 6-well plate. The 6-well plate was inoculated and supplemented with nutritionally weaker media, and cultured for 48 hours (with fresh media exchange after 24 hours). Biofilms were washed to remove planktonic population, and treated with 500 g/L and 1,000 g/L Surgihoney for 18 hours. The treatment and planktonic population was removed, and the biofilms were re-suspended. The absorbance at OD.sub.600 was measured to determine the overall biomass (comprising cellular and extracellular material). The biofilms were cultured onto chocolate blood agar plates for colony forming unit enumeration to determine the percentage of viable cells remaining in the biofilm after treatment.

[0410] The results are shown in FIG. 15. The results show that treatment of a CRS-associated MRSA biofilm with Surgihoney reduced biofilm biomass in a dose-dependent manner, with 500 g/L Surgihoney reducing biomass by approximately one-half, and 100 g/L Surgihoney reducing biomass by approximately two-thirds, compared with the biofilm biomass without treatment. The number of viable cells remaining in the biofilm was also dramatically reduced, with 500 g/L Surgihoney reducing the percentage of viable cells to 0.5%, and 100 g/L Surgihoney reducing the percentage of viable cells to 0.13%.

EXAMPLE 8

Sprayable Compositions

[0411] A composition containing 1% by weight polyethylene oxide (PEO), 79% by weight deionised water and 20% by weight Surgihoney was added to a pump action spray device.

[0412] The spray device was used to spray the composition on to a hydrogen peroxide test strip. The test strip indicated the presence of hydrogen peroxide in the composition.

[0413] After five months, the spray was re-tested for its ability to generate hydrogen peroxide, by spraying on to a hydrogen peroxide test strip. The test strip indicated the presence of hydrogen peroxide in the composition.

EXAMPLE 9

Antimicrobial Activity of Surgihoney

[0414] The antimicrobial activity of Surgihoney (SH) and two prototype modified honeys made by Apis mellifera (honeybee) against Staphylococcus aureus (NCIMB 9518) was tested. We also examined a number of modified types of Surgihoney for the ability to change the level of production of hydrogen peroxide from the samples.

[0415] Methods: Surgihoney (SH) was compared with two modified honeys, Prototype 1 (PT1) and Prototype 2 (PT2) using a bioassay method against a standard strain of Staphylococcus aureus. Further work studied the rate of generation of hydrogen peroxide from these preparations.

[0416] Results: Surgihoney antimicrobial activity was shown to be largely due to hydrogen peroxide production. By modification of Surgihoney, two more potent honey prototypes were shown to generate between a two- and three-fold greater antibacterial activity and up to ten times greater peroxide activity.

[0417] Conclusions: Surgihoney is a wound antiseptic dressing that shows good antimicrobial activity. Two further honey prototypes have been shown to have antimicrobial activity that is possible to be enhanced due to demonstrated increases in peroxide activity.

Methods

1. Determination of Honey Activity by Bioassay Method

[0418] The antibacterial activity of Surgihoney (S) and two modified honeys, Prototype 1 (PT1) and Prototype 2 (PT2) was measured using Staphylococcus aureus (NCIMB 9518) and expressed as the equivalent % phenol. Values were calculated of the mean from three sample replicates tested, repeated on three days.

Assay Method.

[0419] The agar well diffusion method used was adapted from the punch plate assay for inhibitory substances described in the Microbiology Standard Methods Manual for the New Zealand Dairy Industry (1982) [Bee Products Standards Council: Proposed standard for measuring the non peroxide activity of honey. In. New Zealand: Bee Products Standards Council; 1982.].

Inoculum Preparation.

[0420] Overnight culture was adjusted to an absorbance of 0.5 measured at 540 nm using sterile nutrient broth as a blank and a diluents and a cuvette with a 1 cm pathway.

Assay Plate Preparation.

[0421] A volume of 100 μl of the culture adjusted to 0.5 absorbance was used to seed 150 ml nutrient agar to make the assay plates. The agar was swirled to mix thoroughly and poured into large petri dishes which had been placed on a level surface. As soon as the agar was set the plates were placed upside down overnight before using the next day. For assay these seeded plates were removed from 4° C. and allowed to stand at room temperature for 15 min before cutting 7.0 mm diameter wells into the surface of the agar. 250 μl of test material (sample or standard) was placed into each well.

Catalase Solution.

[0422] A 200 mg/ml solution of catalase from bovine liver (Sigma C9322, 2900 units/mg) in distilled water was prepared fresh each day.

Sample Preparation.

[0423] Primary sample solutions were prepared by adding 4 g of sample to 4 ml of distilled water in universals and placed at 37° C. for 30 minutes to aid mixing. To prepare secondary solutions, 2 ml of the primary sample solution was added to 2 ml of distilled water in universals and mixed for total activity testing and 2 ml of the primary sample solution was added to 2 ml of catalase solution and mixed for non-peroxide activity.

Preparation of Phenol Standards.

[0424] Standards (w/v) 10%, 30%, 50% phenol were prepared by dissolving phenol in water. Phenol standards were brought to room temperature in the dark before use and were mixed thoroughly before addition to test wells. Each standard was placed in three wells to test in triplicate. Standards were kept at 4° C. with an expiry date of one month.

Sample and Standard Application.

[0425] All samples and standards were tested in triplicate by adding 250 μl to each of 3 wells.

Plate Incubation.

[0426] After application of samples the plates were incubated for approximately 18 hours at 37° C. The diameter of inhibition zones, including the diameter of the well (7.0 mm), was recorded.

Calculation of Antibacterial Activity of Samples.

[0427] The mean diameter of the clear zone around each phenol standard was calculated and squared. A standard graph was plotted of % phenol against the square of the mean diameter of the clear zone. A best-fit straight line was obtained using linear regression and the equation of this line was used to calculate the activity of each diluted honey sample from the square of the mean measurement of the diameter of the clear zone. To allow for the dilution (assuming the density of the Surgihoney to be 1.35 g/ml) this figure was multiplied by a factor of 4.69 and the activity of the samples was then expressed as the equivalent phenol concentration (% w/v).

[0428] Total Activity: all the activity, including activity due to hydrogen peroxide (H.sub.2O.sub.2). Non-Peroxide Activity: H.sub.2O.sub.2 is removed by treating samples with catalase enzyme.

2. Determination of Honey Activity by H.sub.2O.sub.2 Method

[0429] The activity was measured using the Merckoquant® 1.10011. & 1.10081.

Peroxide Test Kits.

[0430] Concentrations expressed as the equivalent mg/L H.sub.2O.sub.2.

[0431] Samples were diluted 1:10 with purified water. Following 5 min incubation, all samples were measured for H.sub.2O.sub.2 production each hour over a 12 hour period followed by 24 and 48 hour time points.

Method of Determination.

[0432] Peroxidase transfers oxygen from the peroxide to an organic redox indicator, which is then converted to a blue coloured oxidation product. The peroxide concentration is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. The reaction zone of the test strip is immersed into the Surgihoney sample for 1 sec, allowing excess liquid to run off the strip onto an absorbent paper towel and after 15 seconds (Cat. No. 110011), 5 seconds (Cat. No. 110081), after which a determination of the colour formed in the reaction zone more precisely coincided with the colour fields scale.

Results

1. Activity Rating

[0433] The antimicrobial activity produced by the modification of the honey samples resulted in a two-fold and almost three-fold respectively increase in phenol activity with PT1 and PT2 compared with Surgihoney alone. The results for the three samples of Surgihoney (SH) and two modified prototypes, PT1 and PT2 are shown in the Table below.

[0434] Table showing the peroxide and non-peroxide antibacterial activities of Surgihoney (SH) and two modified prototypes, PT1 and PT2 against Staphylococcus aureus (NCIMB 9518).

TABLE-US-00012 Total Activity Non-Peroxide Sample Name Batch No. (% phenol) Activity (% phenol) Surgihoney 2015-06-018B 32 0 Surgihoney PT1 HHI4110311 65 7 Surgihoney PT2 HHI14110312 83 10
2. Determination of Honey Activity by H.sub.2O.sub.2 Method

[0435] The prototype modifications are observed to generate up to seven and ten times the hydrogen peroxide activity of Surgihoney. The results for the three samples are shown in FIG. 16A. By taking the maximum level of hydrogen peroxide output for each of the three honey prototypes and plotting this against the total phenol activity a linear relationship is observed (FIG. 16B).

Discussion

[0436] The results from this work show that the main antimicrobial activity of Surgihoney and two modified prototypes, PT1 and PT2 are due to hydrogen peroxide. This is a similar finding to certain other honeys from a variety of floral sources. However, unlike previous work the availability of hydrogen peroxide from the samples is able to be enhanced and at 12 hours is seven and ten times respectively the value for Surgihoney alone. There is a striking linear relationship between the antimicrobial activity and the maximum output of hydrogen peroxide from the three honey prototypes.

[0437] This peroxide activity offers potent antimicrobial activity that is ideally suited for a wound dressing that is applied to acute or chronic wounds to treat or prevent wound infections. Whilst a small amount of catalase is present in wounds and serum level of catalase in males has been reported as 50 kU/I it has been shown that catalase activity in healing wounds actually decrease during the first week post-wounding and activity levels of catalase recover to its original level at two weeks post-wounding. Such concentrations of catalase are thus extremely unlikely to influence the antimicrobial activity observed with exogenously applied Surgihoney or the two modified prototypes, PT1 and PT2.

[0438] The ideal characteristics for an antimicrobial wound dressing are: effectiveness, lack of toxicity, ease of use, patient and clinician acceptability and value for money. Hydrogen peroxide is an effective antimicrobial and is already used as a biocide for its potent activity against vegetative bacteria, yeasts and spores. It produces its antimicrobial effect through chemical oxidation of cellular components.

[0439] The human toxicity of hydrogen peroxide is concentration dependent and one study has claimed that the differential concentrations for antimicrobial and human toxicity might overlap. By contrast, certain preparations of honey have been shown to be an effective antimicrobial agent by supplying low concentrations of hydrogen peroxide to wounds continuously over time rather than as a large amount at the time of dressing and without such toxicity. Indeed there is compelling evidence that where physiological levels of hydrogen peroxide are applied to mammalian cells there is a stimulation of biological responses and activation of specific biochemical pathways in these cells.

[0440] Clearly Surgihoney and the two modified prototypes, PT1 and PT2 are antimicrobial dressings that offer effective hydrogen peroxide release over at least 24 hours.

Conclusions

[0441] Surgihoney and the two modified prototypes, PT1 and PT2 have been shown to have potent antimicrobial activity against a standard strain of Staphylococcus aureus. These antimicrobial activities have been shown to be due to hydrogen peroxide. The activity is scalable and can be described in terms of hydrogen peroxide activity. These modified honeys offer a dressing that is effective, non-toxic and easy to administer.

EXAMPLE 10

Nebulised Surgihoney

[0442] This example describes nebulisation of Surgihoney, and the antimicrobial effect of nebulised Surgihoney.

[0443] One 10 g sachet of SH1 Surgihoney was dissolved in 4 ml sterile 0.9% saline solution and warmed in a water bath to liquefy the honey. Thus, the liquefied solution contained 250% SH1 Surgihoney. The liquefied honey was placed in the medicine reservoir of a nebuliser face mask tub. This was connected to a standard Salter respiratory nebuliser and switched on. Nebulised vapour smelling and tasting of honey was produced by the nebuliser (see FIG. 17A). A volunteer inhaled the nebulised Surgihoney, with no ill effects.

[0444] The antimicrobial effect of the nebulised Surgihoney was assessed against a strain of Staphylococcus aureus. Absorbent paper discs were inoculated with S. aureus prepared to a concentration of 10.sup.3-10.sup.4 cfu/ml. The discs were placed in a tube (simulating the bronchus). Nebulised Surgihoney was passed over the paper discs for 5, 15, 20, or 30 minutes. The discs were then cultured on blood agar plates to determine the number of surviving colony forming units. A control disc inoculated with S. aureus that was not contacted with nebulised Surgihoney was also cultured.

[0445] Photographs of the blood agar plates are shown in FIG. 18. The number of colony forming units (cfu/ml) for each plate is shown in the plot in FIG. 17B, and the approximate number of cfu/ml after each exposure time is recorded in the Table below.

TABLE-US-00013 Time (minutes) Approximate number of cfu/ml 0 8000 5 600 15 100 20 5 30 3

[0446] It was concluded that Surgihoney can readily be nebulised. Nebulised Surgihoney appears to be safe to inhale, with no observed adverse effects in one volunteer. The nebulised Surgihoney has significant antimicrobial activity, reducing the bacterial load by 1000 fold in a simulated respiratory tract model.

Effectiveness of Nebulised Surgihoney In Vivo, for the Treatment of Respiratory Disease

[0447] A patient with severe bronchiectasis was colonised in the respiratory tract with Mycobacterium avium and Mycobacterium abscessus. The patient was treated by administering nebulised SH1 Surgihoney (prepared as described above), daily, for two months. The treatment cleared the Mycobacterium in the patient's respiratory tract as indicated by analysing the patient's sputum samples.

EXAMPLE 11

[0448] Surgihoney Treatment of CRS-Associated S. aureus Biofilms

[0449] MSSA static biofilms were grown in vitro, and then treated with Surgihoney, as described in Example 7 above. The results are shown in FIG. 19. The results show that, although treatment of a CRS-associated MSSA biofilm with 500 g/L Surgihoney did not appear to reduce biofilm biomass, treatment with 1000 g/L Surgihoney reduced biofilm biomass by approximately one-half, compared with the biofilm biomass without treatment. The number of viable cells remaining in the biofilm was also dramatically reduced, with 500 g/L Surgihoney reducing the percentage of viable cells by 78% to 22%, and 100 g/L Surgihoney reducing the percentage of viable cells by 94% to 6%.

[0450] The results of Examples 6, 7, and 11 demonstrate that Surgihoney has potent bactericidal and inhibitory effects on MRSA and MSSA in both planktonic and biofilm forms.

EXAMPLE 12

[0451] Surgihoney Treatment of Ischaemic Ulcer Chronically Infected with Pseudomonas Aeruginosa

[0452] A 77 year-old male with peripheral vascular disease had developed long-standing ischaemic ulcers chronically infected with Pseudomonas aeruginosa (see FIG. 20(A)). After a 7 day treatment with Surgihoney, a significant clinical improvement was seen (FIG. 20 (B)).

EXAMPLE 13

Use of Surgihoney as a Topical Anti-MRSA Treatment

[0453] The World Health Organisation's (WHO) 2014 report on global surveillance of antimicrobial resistance has revealed that the world has reached a critical point. 1 in 5 of hospital-acquired infections are now attributed to Methicillin-resistant Staphylococcus aureus (MRSA). The UK Department of Health's annual report (2014 to 2015) of MRSA bacteremia reported 874 community-acquired cases, and 349 hospital-acquired cases. The effect on the national health service (NHS) is significant because it causes a delay of elective surgery, prolonged hospital stay, and long-term antibiotic treatment.

[0454] Mupirocin (Bactroban) is a topical antibacterial treatment effective against Gram-positive bacteria, including MRSA, currently used as part of the decolonisation regime. However, recent evidence indicates increasing resistance of MRSA to Mupirocin.

Aims

[0455] 1. To compare the efficacy of Surgihoney versus Mupirocin on MRSA isolates in the in vitro setting.
2. To conduct a small scale proof-of-principle clinical study examining the feasibility of using Surgihoney as a novel MRSA decolonisation therapy in MRSA-positive clinical subjects.

Methodology

Recruitment and Phenotyping of Clinical Subjects:

[0456] Patients identified as MRSA carriers as a result of pre-assessment screening will be recruited into the study.

Specimen Acquisition, Isolation of MRSA and In Vitro Bacterial Analysis:

[0457] The antimicrobial activity of Surgihoney will be assessed in vitro on both planktonic and biofilm MRSA phenotypes. The bactericidal and inhibitory function of Surgihoney will be compared to current standard therapy (Mupirocin).

Proof of Principle Clinical Study:

[0458] A small scale proof of principle clinical study will be conducted to assess the feasibility of using Surgihoney as a novel MRSA decolonisation therapy in the nasal cavity.

[0459] The results from this study will allow use of Surgihoney as a topical therapy in MRSA nasal carriers and surgical patients with MRSA-infected wounds.

EXAMPLE 14

[0460] Clinical Response of 114 Chronic Wounds to Treatment with Surgihoney

TABLE-US-00014 Mean Number wound Mean of duration number Wound type patients Average age (months) comorbidity Leg ulcers 37 76 (32-91) 8 4 Pressure ulcers 19 76 (45-97) 5.4 3.8 Surgical wounds 14 54 (0-76)  1.9 4.7 Diabetic ulcers 9 67 (53-87) 4.2 4 Central Catheter 2 44 n/a 3 Site Infections Suprapubic catheter 1 61 1 2 site Traumatic wounds 12 72.8 (21-90)   2 3.2 Other topical 3 63 (22-95) n/a n/a infections Developing world 17 40.5 (23-82)   3.6 2.2 % Improvement Reduction >20% in healing in Reduction criteria (Dx, Wound bacterial in wound slough, Mean duration of type* load % size % inflammation) treatment (days) Leg ulcers 88 68 92   24 (8-130) Pressure 100 63 89  27.4 (14-80) ulcers Surgical 87 71 86  34.5 (14-62) wounds Diabetic 100 100 100  35.5 (10-131) ulcers Central 100 n/a 100  9 Catheter Site Infections Suprapubic 100 n/a 100 12 catheter site Traumatic 100 58 100 32.3 (7-90) wounds Other n/a n/a n/a 37.3 )8-94) topical infections Developing n/a 88 94 19.6 (8-64) world

EXAMPLE 15

Paediatric MRSA Wound Infection

[0461] FIG. 21 shows the effects of treating a MRSA wound infection with Surgihoney over 10 days.

EXAMPLE 16

CA MRSA Superficial Infection

[0462] FIG. 22 shows the effect of treating a CA MRSA infection with Surgihoney, over 5 days and over 10 days.

EXAMPLE 17

Anti-Viral Activity of Surgihoney

[0463] SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus (HSV) (50□g honey and 50□l virus) and incubated for 1 hour at 37° C. A dilution series (10.sup.−2, 10.sup.−3, 10.sup.−4, 10.sup.−5) was then made from the mixture, and the dilutions were used in a plaque reduction assay. Controls with no honey, or with control honey were also performed. The number of viral plaques formed for each dilution was recorded. The results are shown in the Table below.

TABLE-US-00015 Dilu- Experiment 1 Experiment 2 Honey tion well 1 well 2 well 3 well 1 well 2 well 3 SH1 −2 * * * * * * −3 1 1 5 0 0 0 −4 0 1 1 0 0 0 −5 0 0 0 0 0 0 SH2 −2 * * * * * * −3 0 0 0 0 0 0 −4 0 0 0 0 0 0 −5 0 0 0 0 0 0 Control −2 100 95 88 108 128 106 Honey −3 13 15 11 14 12 15 −4 2 1 2 3 2 2 −5 0 0 0 0 0 1 No −2 160 158 164 Honey −3 28 22 18 −4 6 4 1 −5 1 0 1

[0464] The results show that SH1 and SH2 Surgihoney was strongly virucidal against HSV in both experiments.

EXAMPLE 18

Cytotoxic Activity of Surgihoney

[0465] SH1 or SH2 Surgihoney (50 μg honey diluted 10.sup.−2, 10.sup.−3, 10.sup.−4, 10.sup.−5) was incubated on cells for 2 days. The number of live cells, and the total number of cells was counted (percentage viability=live/total×100). The results are shown in the table below, and in FIG. 23.

[0466] The results show that SH1 Surgihoney was cytotoxic at the 10.sup.−2 dilution, and cytostatic at the 10.sup.−3 and 10.sup.−4 dilutions, and that SH2 Surgihoney was cytostatic at the 10.sup.−2, 10.sup.−3 and 10.sup.−4 dilutions. SH1 and SH2 Surgihoney were not cytotoxic or cytostatic at the 10.sup.−5 dilution.

[0467] It is concluded from the results in Examples 17 and 18 that Surgihoney can be administered at doses which are virucidal but not cytotoxic or cytostatic.

TABLE-US-00016 Number of live cells Total number of cells Percentage viability Dilu- standard standard standard Condition tion rep1 rep2 Ave deviation rep1 rep2 Ave deviation rep1 rep2 Ave deviation DMEM — 1300000 2100000 1700000 565685.4 1400000 2600000 2000000 848528.1 92.9 80.8 86.8 8.5 Control −2 1200000 880000 1040000 226274.2 1300000 1000000 1150000 212132 92.3 88.0 90.2 3.0 honey −3 2700000 2400000 2550000 212132 2800000 2600000 2700000 141421.4 96.4 92.3 94.4 2.9 −4 3400000 2800000 3100000 424264.1 3600000 3000000 3300000 424264.1 94.4 93.3 93.9 0.8 −5 2100000 1300000 1700000 565685.4 2200000 1500000 1850000 494974.7 95.5 86.7 91.1 6.2 SH1 −2 120000 70000 95000 35355.34 370000 350000 360000 14142.14 32.4 20.0 26.2 8.8 −3 380000 380000 380000 0 400000 600000 500000 141421.4 95.0 63.3 79.2 22.4 −4 430000 780000 605000 247487.4 470000 850000 660000 268700.6 91.5 91.8 91.6 0.2 −5 1800000 2200000 2000000 282842.7 2000000 2400000 2200000 282842.7 90.0 91.7 90.8 1.2 SH2 −2 320000 360000 340000 28284.27 390000 400000 395000 7071.068 82.1 90.0 86.0 5.6 −3 450000 570000 510000 84852.81 760000 730000 745000 21213.2 59.2 78.1 68.6 13.3 −4 460000 690000 575000 162634.6 660000 790000 725000 91923.88 69.7 87.3 78.5 12.5 −5 1600000 1700000 1650000 70710.68 1800000 2000000 1900000 141421.4 88.9 85.0 86.9 2.7

EXAMPLE 19

Dried Surgihoney

[0468] Dried honey granules (K24289) were supplied by Kanegrade Limited (Stevenage, UK). The dried honey granules were produced by vacuum drying and contained honey solids with skimmed milk powder. The water content was less than 3%.

[0469] The dried honey granules were activated by adding glucose oxidase at SH1 and SH2 levels (see above).

[0470] FIG. 24 (left) demonstrates that adding water to the activated dried honey granules led to the immediate production of hydrogen peroxide at between 50 and 100 ppm, as detected by a hydrogen peroxide indicator strip.

[0471] FIG. 24 (right) demonstrates the result of adding 2 g of the activated dried honey granules to 30 g of sterile warm water (at approximately 35° C.). The activated honey granules dissolved easily with no scum formation. Even after four days, there was no settling out, as the granules remained fully dissolved.

[0472] FIG. 25 demonstrates that the activity of the dissolved active honey was retained over an extended period of time. Top row (left to right): 0 hours; 30 mins; 60 mins; 90 mins. Bottom row (left to right): 6 hours, 16.5 hours; 4 days. After four days, the level of hydrogen peroxide remained constant at between 50 and 100 ppm. This shows that the glucose oxidase remains active and there was sufficient glucose present to produce hydrogen peroxide over an extended period.

EXAMPLE 20

Stability of Aqueous Surgihoney Compositions

[0473] A sample was prepared with the following composition, on 9 Nov. 2014:

[0474] SH2 Surgihoney: 20% by weight

[0475] PVA: 5% by weight

[0476] Water: 75% by weight

[0477] The sample was tested on 16 Oct. 2015 using a hydrogen peroxide test strip and was found to still be producing hydrogen peroxide at a level of between 30-50 ppm.

[0478] A sample was prepared by mixing SH2 Surgihoney with water in a ratio of 1:15 (Surgihoney:water, by weight), on 14 Sep. 2015. The sample was tested on 16 Oct. 2015 using a hydrogen peroxide test strip and was found to still be producing hydrogen peroxide at a level of 30-50 ppm.

EXAMPLE 21

Use of Surgihoney for the Topical Treatment of Lower Genital Tract Infections

[0479] Surgihoney has been used to treat persistent vaginal discharge that had not been responsive to standard therapy. The indications were vaginal discharge with various aetiologies e.g. bacterial vaginosis and general bacterial vaginal discharge.

[0480] Tampons coated with Surgihoney were inserted into the vagina and were replaced every 24 hours. The therapeutic outcome was good and there were no reported adverse effects.

EXAMPLE 22

Use of Surgihoney to Treat CPE

[0481] A patient developed a soft tissue infection of the foot whilst in India and was diagnosed with necrotising fasciitis. This required extensive debridement of dead tissue as well as antibiotics. Post-operatively, the patient was found to be colonised in the debrided wounds with CPE (Carbapenemase-producing Enterobacteriaceae). The patient required isolation and was treated with Surgihoney, which cleared the organisms.

EXAMPLE 23

Compositions Comprising Non-Aqueous Solvent

Sample 1

[0482] Lightweight hydro entangled fabric (40 g per square meter)

[0483] Coated with 25% w/w honey/glycerol solution

[0484] Weight of coating on fabric 100 g per square meter

[0485] Suggested application: Absorbent fabric in a wound dressing.

Sample 2

[0486] Lightweight hydro entangled fabric (40 g per square meter)

[0487] Coated with 75% w/w honey/glycerol solution

[0488] Weight of coating on fabric 100 g per square meter

[0489] Suggested application: Absorbent fabric in a wound dressing.

Sample 3

[0490] Lightweight hydro entangled fabric (40 g per square meter)

[0491] Coated with 25% w/w honey/glycerol solution

[0492] Weight of coating on fabric 300 g per square meter

[0493] Suggested application: Anti bacterial wipe

Sample 4

[0494] Lightweight hydro entangled fabric (40 g per square meter)

[0495] coated with 75% w/w honey/glycerol solution

[0496] Weight of coating on fabric 300 g per square meter

[0497] Suggested application: Anti bacterial wipe

Sample 5

[0498] Paper like fabric (42 g per square meter)

[0499] Coated with 25% w/w honey/glycerol solution

[0500] Weight of coating on fabric 100 g per square meter

[0501] Suggested application: Anti bacterial wipe

Sample 6

[0502] Boots Wound Dressing coated with 25% w/w honey/glycerol solution

[0503] Weight of coating on fabric 400 g per square meter

Sample 7

[0504] Boots Adhesive Wound Dressing with absorbent pad coated with 25% w/w honey/glycerol solution

[0505] Weight of coating on fabric 150 g per square meter

Sample 8

[0506] Lightweight hydro entangled fabric (40 g per square meter)

[0507] Coated with 25% w/w Surgihoney/glycerol solution

[0508] Weight of coating on fabric 100 g per square meter

[0509] Suggested application: Absorbent fabric in a wound dressing.

Sample 9

[0510] Light weight hydro entangled fabric (40 g per square meter)

[0511] Coated with 25% w/w Surgihoney/glycerol solution

[0512] Weight of coating on fabric 300 g per square meter

[0513] Suggested application: Absorbent fabric in a wound dressing.