ANTIMICROBIAL COMPOSITIONS

Abstract

Compositions for generating antimicrobial activity are described. The compositions comprise an enzyme that is able to convert a substrate to release hydrogen peroxide, a substrate for the enzyme, and a solute in the form of a sugar or sugar derivative having a solubility of at least 100 g/100 g water at 20 C. and 1 atm. The compositions may not comprise sufficient free water to allow the enzyme to convert the substrate.

Claims

1. A composition for generating antimicrobial activity, which comprises: a purified enzyme that is able to convert a substrate to release hydrogen peroxide; a purified substrate for the enzyme; and a solute in the form of a sugar or sugar derivative having a solubility of at least 100 g/100 g water at 20 C. and 1 atm, wherein the composition does not comprise sufficient free water to allow the enzyme to convert the substrate.

2. A composition according to claim 1, wherein the solute has a solubility of at least 300 g/100 g water.

3. A composition according to any preceding claim, wherein the solute is a monosaccharide.

4. A composition according to claim 4, wherein the monosaccharide is fructose.

5. A composition according to any preceding claim, wherein the solute is at least 60% by dry weight of the composition.

6. A composition according to any preceding claim, wherein the substrate is at least 30% by dry weight of the composition.

7. A composition according to any preceding claim, wherein the combined dry weight of the substrate and the solute is at least 90%, preferably at least 95% of the composition, or wherein the total amount of sugar or sugar derivative in the composition is at least 90%, by dry weight, preferably at least 95%, by dry weight.

8. A composition according to any preceding claim, comprising a buffer.

9. A composition according to any preceding claim which has a pH of 6 to 8.

10. A composition according to any preceding claim, which provides for sustained release of hydrogen peroxide at a level of less than 2 mmol/litre and/or at level of at least 0.1 mmol/litre for a period of at least twenty four hours, following dilution of the composition.

11. A composition according to any preceding claim, wherein the enzyme is a glucose oxidase and the substrate for the enzyme is D-glucose.

12. A composition according to any preceding claim which is sterile, optionally wherein the composition has been sterilised by exposure to irradiation, preferably gamma irradiation, more preferably 10-70 kGy, even more preferably 25-70 kGy, most preferably 35-70 kGy.

13. A composition according to any of claims 1 to 11 which is sterile and has been sterilised by exposure to electron beam irradiation, preferably 10 to 100 kGy, more preferably 35 to 80 kGy.

14. A composition according to any preceding claim comprising substantially no hydrogen peroxide.

15. A composition according to any preceding claim, wherein the substrate for the enzyme is the solute, or wherein the substrate for the enzyme is distinct from the solute.

16. A composition according to any preceding claim, comprising substantially no zinc oxide.

17. A composition according to any preceding claim, comprising substantially no catalase.

18. A composition according to any preceding claim comprising substantially no peroxidase.

19. A composition according to any preceding claim wherein the composition is a pharmaceutical grade composition.

20. A composition according to any preceding claim comprising 25 to 2000 ppm of the enzyme.

21. A composition according to claim 20, comprising 250 to 1500 ppm of the enzyme.

22. A composition according to any preceding claim which does not comprise honey.

23. A composition according to any of claims 1 to 22, for use as a medicament.

24. A composition according to any of claims 1 to 22, for use in prevention, treatment, or amelioration of a microbial infection.

25. A composition according to any of claims 1 to 22, for use in treatment of a wound.

26. A wound dressing comprising a dressing material for dressing a wound, and a composition according to any of claims 1 to 22.

27. A method for producing a composition for generating antimicrobial activity, which comprises: contacting a purified enzyme that is able to convert a substrate to release hydrogen peroxide with a purified substrate for the enzyme, and a solute in the form of at least one sugar or sugar derivative, the solute having a solubility greater than 100 g/100 g water at 20 C. and 1 atm, wherein the composition does not comprise sufficient free water to allow the enzyme to convert the substrate.

28. A method according to claim 27, wherein the enzyme, substrate and solute are contacted with each other in dry form, preferably in powder form.

29. A method according to claim 27, wherein the composition is in the form of a liquid or a solution, and the substrate, solute and enzyme are dissolved in water.

30. A method of sterilising a composition or wound dressing according to any of claims 1 to 26, which comprises exposing the composition or dressing to irradiation, preferably gamma irradiation or electron beam radtiation, preferably 10-70 kGy, more preferably 25-70 kGy, most preferably 35-70 kGy.

31. A method of forming an antimicrobial solution comprising diluting a composition as defined in any of claims 1 to 22 in an aqueous solution such that there is sufficient free water to allow the enzyme to convert the substrate.

32. A method according to claim 31, comprising diluting the composition to form a solution which contains 30 g/l to 150 g/l, 50 to 100 g/l or 65 to 75 g/l of the composition.

33. A solution obtained or obtainable by a method according to claim 31 or 32.

34. A solution according to claim 33 in which hydrogen peroxide is present at a concentration of at least 10 M, preferably 10 to 50 M, more preferably 20 to 30 M.

35. A solution according to claim 34, wherein the concentration of hydrogen peroxide is maintained for at least 1 hour, preferably at least 2 hours, more preferably at least 10 hours, even more preferably at least 24 hours following formation.

36. A composition comprising: a purified enzyme that is able to convert a substrate to release hydrogen peroxide; a purified substrate for the enzyme; a solute in the form of a sugar or sugar derivative having a solubility of at least 100 g/100 g water at 20 C. and 1 atm, and sufficient free water to allow the enzyme to convert the substrate, wherein hydrogen peroxide is present at a concentration of at least 10 M, preferably 10 to 50 M, more preferably 20 to 30 M.

37. A composition according to claim 36, wherein the concentration of hydrogen peroxide is maintained for at least 1 hour, preferably at least 2 hours, more preferably at least 10 hours, even more preferably at least 24 hours, following formation.

38. A solution or composition according to any of claims 33 to 37, for use in the treatment of a microbial infection that comprises a biofilm.

39. A solution or composition for use according to claim 38, wherein the microbial infection comprises Haemophilus influenza, MRSA or MSSA.

40. A solution or composition according to any of claims 33 to 37 for use in treating chronic rhinosinusitis.

41. A solution or composition as defined in any of claims 1 to 22 or 33 to 37, for use in treating an infection that comprises a biofilm, wherein the composition is administered with an antibiotic, optionally wherein the antibiotic is co-amoxiclav and/or optionally wherein the infection comprises Haemophilus influenza.

42. A solution or composition for use according to claim 41, wherein the administration is combined, concurrent, or sequential.

43. A solution or composition according to any of claims 1 to 22 or 33 to 37, comprising an antibiotic, optionally wherein the antibiotic is co-amoxiclav.

44. A kit comprising a composition or solution according to any of claims 1 to 22 or 33 to 37, and separately, an antibiotic.

45. A solution or composition according to any of claims 1 to 22 or 33 to 37, for use in the treatment of aphthous ulcers or geographic tongue.

Description

[0390] Preferred embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

[0391] FIG. 1 is a graph showing the effect of compositions of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to Surgihoney, at various concentrations.

[0392] FIG. 2 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (buffered at pH 4.03) on the growth of planktonic MRSA, at various concentrations.

[0393] FIG. 3 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (unbuffered) on the growth of planktonic MRSA, at various concentrations.

[0394] FIG. 4 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (buffered at pH 7.04) on the growth of planktonic MRSA, at various concentrations.

[0395] FIG. 5 is a table showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose, on the MIC and MBC of planktonic MRSA, at various concentrations.

[0396] FIG. 6 shows an optical microscopy images of reverse micelles in an emulsion containing Surgihoney.

[0397] FIG. 7 shows that SurgihoneyRO influences the balance of T-helper cell (Th) subsets by altering the expression of gatekeeping genes. Gene-expression profiles for Th lineage gatekeeping genes were analysed in nasal epithelial cells treated with 10 g/L or 100 g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous R-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, **** p0.0001;

[0398] FIG. 8 shows that SurgihoneyRO induces a Th.sub.17 response in nasal epithelial cells. Gene-expression profiles for Th.sub.17 related cytokines were analysed in the nasal epithelial cells treated with 10 g/L or 100 g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous R-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, **** p0.0001;

[0399] FIG. 9 shows that SurgihoneyRO can modulate the host immune response to invading pathogens in nasal epithelial cells. Gene-expression profiles for (a) matrix metalloprotease (MMP) related genes and (b) toll-like receptor (TLR) genes were analysed in nasal epithelial cells treated with 10 g/L or 100 g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous R-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, 0.0001;

[0400] FIG. 10 shows that SurgihoneyRO induces a Th.sub.17 response in mast cells. Gene-expression profiles for Th.sub.17 related cytokines were analysed in HMC-1 treated with 10 g/L or 100 g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous R-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, **** p0.0001;

[0401] FIG. 11 shows fluorimetric measurements of hydrogen peroxide. The concentration of hydrogen peroxide production was measured over a time course of 24 hours. The kinetics of hydrogen peroxide production by both SurgihoneyRO and Acacia honey were measured without the presence of cells (a), with nasal epithelial cell line (b) and with mast cell line (c). The data represents the mean with the error bars showing standard deviation of the mean for at least 3 independent experiments;

[0402] FIG. 12 shows that hydrogen peroxide treatment influences the balance of T-helper cell (Th) subsets by altering the expression of the gatekeeping genes in the same way as SurgihoneyRO. Gene-expression profiles for Th lineage gatekeeping genes were analysed by RT-PCR. (a) shows GATA3 gene expression in the nasal epithelial cells, (b) shows the expression of Th.sub.17 related cytokines for the nasal epithelial cells and (c) for the mast cells. Both cell lines were treated either with or without hydrogen peroxide (0-400 M) for 1 hour. The fold change calculated was based on normalisation to the expression of the endogenous R-actin housekeeping gene. Each point shows the mean fold change in gene expression for 5 independent experiments. The dotted line for y=0 representing the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, **** p0.0001;

[0403] FIG. 13 shows that hydrogen peroxide treatment can modulate anti-microbial and host immune response to invading pathogens in both nasal epithelial cells and mast cells. Gene-expression profiles for MMP related genes were analysed in (a) nasal epithelial cells and (b) mast cells treated with various concentrations of exogenous hydrogen peroxide (0-400 M) for 1 hour using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous R-actin housekeeping gene. For each treatment group the mean fold change in gene expression for 5 independent experiments is shown. The dotted line for y=0 representing the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, **** p0.0001;

[0404] FIG. 14 shows analysis of gene-expression of IL10 in the nasal epithelial cells treated with various concentrations of exogenous hydrogen peroxide (0-400 M) for 1 hour (a); with 10 g/L or 100 g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous R-actin housekeeping gene. Each point shows the mean fold change in gene expression for 5 independent experiments. The dotted line for y=0 representing the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p0.05, ** p0.001, *** p0.001, **** p0.0001;

[0405] FIG. 15 shows the effect of compositions of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0406] FIG. 16 shows the effect of SyntheticRO on the MIC and MBC of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

[0407] FIG. 17 shows the effect of SyntheticRO comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MSSA isolate;

[0408] FIG. 18 (a and b) compares SyntheticRO with SurgihoneyRO using planktonic MRSA and MSSA, and FIG. 18c is a table showing the MICs of a composition of the invention compared to SurgihoneyRO. Planktonic MRSA and MSSA in vitro cultures were grown in the presence of the respective compositions for 18 hours, then the absorbance (OD.sub.595) measured and compared to untreated cultures (n=6);

[0409] FIG. 19 shows 48 hour MRSA (a&c) and MSSA (b&d) in vitro biofilms treated with SyntheticRO+ (with enzyme) and SyntheticRO (without enzyme) for 24 hours (n=5), with viability measured by cfu enumeration (a&b) and biomass measured by absorbance [OD.sub.595] (c&d);

[0410] FIG. 20 shows 48 hour MRSA and MSSA biofilms treated with 71 g/l SyntheticRO+ and SyntheticRO for 24 hours and then imaged using confocal microscopy and LIVE/DEAD staining (b), with maximum biofilm height measured and compared to untreated controls (a);

[0411] FIG. 21 shows 48 hour biofilms formed by individual MRSA (a; n=7) and MSSA (b; n=5) clinical isolates treated with 71 g/l synthetic RO+ and viability measured by cfu enumeration;

[0412] FIG. 22 shows the result of Surgihoney treatment of in vitro non-typeable H. influenza biofilms. (a) NTHi in vitro planktonic cultures were grown in the presence of SurgihoneyRO or Acacia for 18 h then growth assessed by measurement of absorbance (OD.sub.595). (b) 48 hour in vitro NTHi biofilms were treated with SurgihoneyRO or Acacia for 2 h and biofilm viability measured by cfu enumeration, (c) Fluorimetric measurements of H.sub.2O.sub.2 production by different SurgihoneyRO and Acacia concentrations at 2 h. (d) 48 hour in vitro NTHi biofilms treated with HBSS adjusted to pH6.3 (equivalent pH to 71 g/L SurgihoneyRO) for 2 h and biofilm viability measured by cfu enumeration *P0.05; **P0.01; and

[0413] FIG. 23 Shows a comparison between SurgihoneyRO and co-amoxiclav in the treatment of non-typeable H. influenza biofilms. (a) 48 hour in vitro NTHi biofilms were treated with 71 g/L SurgihoneyRO and 300/60 g.Math.ml co-amoxiclav alone, and in combination for two hours with viability measured by cfu enumeration. Confocal microscopy was performed on (b) untreated 48 h NTHi biofilms, biofilms treated for 2 hours with (c) 300/60 g.Math.ml co-amoxiclav and d) 71 g/: SurgihoneyRO. Biofilms were imaged using Live/Dead staining to visualize ive cells (green fluorescence) and dead cells (red fluorescence). *P0.05.

[0414] Surgihoney may also be referred to as Surgihoney, SurgihoneyRO or SurgihoneyRO Compositions of the invention, such as compositions which comprise purified glucose, purified fructose and glucose oxidase may be referred to as SyntheticRO, synthetic honey compositions or synthetic compositions.

SPECIFIC EXAMPLES

Example 1Synthetic Honey Compositions

[0415] Samples with batch number RO contain no glucose oxidase.

[0416] Samples with batch number RO1 contain 50 ppm glucose oxidase.

[0417] Samples with batch number RO2 contain 1000 ppm glucose oxidase.

A. pH 4.03 Buffered Samples

A1. Batch No NB01p43RO

[0418] Non sterile

TABLE-US-00004 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0419] Non sterile base buffered saccharide solution.
A2. Batch No NB01 p43RO [0420] Sterile

TABLE-US-00005 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%
Description sterile base buffered saccharide solution
A3. Batch No NB01 p44RO1 [0421] Non sterile

TABLE-US-00006 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0422] Non sterile base buffered RO1 saccharide solution.

A4. Batch No NB01p44RO1

[0423] Sterile

TABLE-US-00007 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

[0424] Sterile base buffered RO1saccharide solution

A5. Batch No NB01p44RO2

[0425] Non sterile

TABLE-US-00008 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 4.03

Description

[0426] Non sterile base buffered RO2 saccharide solution.

A6. Batch No NB01p43RO2

[0427] Sterile

TABLE-US-00009 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 4.03 GOX enzyme N/A
Description Sterile base buffered RO2 saccharide solution

B. Unbuffered Samples

B1. Batch No NB01p51RO

[0428] Non sterile

TABLE-US-00010 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0429] Non sterile base buffered saccharide solution.

B2. Batch No NB01p51RO

[0430] Sterile

TABLE-US-00011 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%
Description Sterile base buffered saccharide solution

B3. Batch No NB01p51RO1

[0431] Non sterile

TABLE-US-00012 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0432] Non sterile base buffered RO1 saccharide solution.

B4. Batch No NB01p51RO1

[0433] Sterile

TABLE-US-00013 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0434] Sterile base buffered RO1saccharide solution

B5. Batch No NB01p51RO2

[0435] Non sterile

TABLE-US-00014 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

Non Sterile Base Buffered RO2 Saccharide Solution.

B6. Batch No NB01 p51 RO2

[0436] Sterile

TABLE-US-00015 Material Weight fraction Fructose 52.0% Glucose 31.0% Water 17.0%

Description

[0437] Sterile base buffered RO2 saccharide solution

C. pH 7.04 Buffered Samples

[0438] C1. Batch No NB01 p57RO [0439] Non sterile

TABLE-US-00016 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 7.04

Description

[0440] Non sterile base buffered saccharide solution.
C2. Batch No NB01 p57RO [0441] Sterile

TABLE-US-00017 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 7.04

Description

[0442] Sterile base buffered saccharide solution
C3. Batch No NB01 p57RO1 [0443] Non sterile

TABLE-US-00018 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 7.04

Description

[0444] Non sterile base buffered RO1 saccharide solution.
C4. Batch No NB01 p57RO1 [0445] Sterile

TABLE-US-00019 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 7.04

Description

[0446] Sterile base buffered RO1saccharide solution

C5. Batch No NB01p57RO2

[0447] Non sterile

TABLE-US-00020 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 7.04

Description

[0448] Non sterile base buffered RO2 saccharide solution.

C6. Batch No NB01p57RO2

[0449]

TABLE-US-00021 Material Weight fraction Fructose 52.0% Glucose 31.0% 50 mMol Citric acid/ 17.0% NaOH buffer pH 7.04

Description

[0450] Sterile base buffered RO2 saccharide solution

Example 2Efficacy of Synthetic Honey Compositions Against Planktonic MRSA

[0451] MIC and MBC were assessed for the RO1 samples (containing 50 ppm glucose oxidase) and compared to Surgihoney (also containing 50 ppm glucose oxidase). See Andrews J. M. Journal of Antimicrobial Chemotherapy (2001) 48, suppl. S1, 5-16.

[0452] The results are shown in FIGS. 1 to 5.

[0453] The results show that, like Surgihoney, synthetic compositions containing glucose, glucose oxidase and fructose are able to inhibit microbial growth.

[0454] Out of all of synthetic compositions, the synthetic composition buffered at pH7.04 had the most effective MIC. Sterilised compositions were more effective than non-sterilised compositions, and synthetic composition buffered at pH7.04 synthetic had the most effective MBC when compared to other synthetic compositions and even when compared to Surgihoney.

[0455] FIGS. 15 (a to d) and 16 show MIC and MBC results including SurgihoneyRO2 samples and synthetic RO2 samples.

[0456] pH 7.04 formulations were tested against a planktonic MSSA isolate. FIG. 17 shows the results obtained.

[0457] The synthetic RO2 composition was selected for further investigation. FIG. 18 (a, b and c) show SyntheticRO (RO2; pH7.04) compared to SurgihoneyRO using planktonic phenotype. RO indicates a product lacking enzyme activity.

Example 3Surgihoney Emulsions

Preparation

[0458] 10 g Surgihoney was dissolved in 10 ml of glycerol. 10 ml of paraffin oil was then added to a

[0459] Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5 C. After 2 minutes, 10 ml of Surgihoney-glycerol solution was added dropwise. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

Optical Microscopy

[0460] Optical microscopy revealed that the emulsion contained reverse micelles which encapsulated Surgihoney. Such micelles can be observed in FIG. 6. The average micelle diameter was found to be 178 m.

Hydrogen Peroxide Tests

[0461] Hydrogen peroxide stick tests (Purchased from Sigma Aldrich (Quantofix)) were used to detect hydrogen peroxide in the emulsion. The tests were carried out before and after addition of water, and showed that before addition of water, the emulsion produced no hydrogen peroxide, and after water was added, the emulsion tested positive for hydrogen peroxide. A positive test was indicated by a colour change to blue.

Stability Test

[0462] The emulsion maintained its capacity to generate hydrogen peroxide after storage at ambient conditions for at least four weeks.

Spray Test

[0463] The emulsion was added to a pump-action spray bottle and was found to be sprayable.

Example 4Effects of Different Parameters on Stability of Surgihoney Emulsions

[0464] The effects of changing the Surgihoney emulsion preparation method described in Example 3, one parameter at a time, were investigated. The changes and their effects are summarised Below.

[0465] i) Proportion of the oil phase to the Surgihoney-Glycerol Phase

[0466] Oil volumes greater than 10 ml, and less than 10 ml, were tested. The emulsion was found to be more stable when a lower volume of oil was used compared to the volume of the Surgihoney-glycerol phase. When the volume of the oil was less than 6 ml, the emulsion was found to separate by less than 3% in total volume over 72 hours. A volume of 4 ml allowed a separation of just 1.3% of the total volume over 72 hours. This stability is far greater than that of the method described in Example 3, which provided an emulsion with a separation of 9.4% of total volume over the same time period.

[0467] ii) Volume of PGPR

[0468] PGPR volumes up to 4 ml, and less than 1 ml, were tested. The emulsion was more stable when a higher amount of PGPR was used. At a volume of 4 ml, PGPR provided greater stability than with use of lower volumes, and far greater stability than that of the emulsion described in Example 3.

[0469] iii) Shear Rate

[0470] Shear rates from 1000 1/s to 3000 1/s were tested. The emulsion was more stable when a higher shear rate was applied. A shear rate of 3000 1/s produced the most stable emulsion. Separation of only 4.6% of the total volume over 72 hours was observed for emulsion prepared at this shear rate, compared with a separation volume of 9.4% of the total volume over the same time period for emulsion prepared as described in Example 3.

[0471] iv) Temperature

[0472] Temperatures from 20 C. to 40 C. were tested. There was no noticeable trend regard the stability of the emulsions as temperature was increased. However a temperature of 40 C. produced the most stable emulsion. Separation of only 3.1% of the total volume over 72 hours was observed for this emulsion.

[0473] v) Length of Shear

[0474] Shear times of 20 minutes and 30 minutes were tested, in addition to that used in the preparation method described in Example 3. However, there was no significant difference produced by extending the shear time.

[0475] vi) Order of Reagent Addition

[0476] The effect of changing the order in which the reagents are added to the rheometer was tested. The effect of adding all of the components before starting the rheometer was compared with the effect of adding the Surgihoney-glycerol and oil components first, then adding the PGPR after 1-2 minutes. The most stable emulsion was formed when the PGPR was added last. The resulting emulsion provided a separation volume of 2.8% of the total volume over 120 hours.

[0477] vii) Concentration of Surgihoney Dissolved in Glycerol

[0478] The following ratios of Surgihoney (g) to glycerol (ml) were tested: 1 g:1 ml; 0.5 g:1 ml; 2 g:1 ml. The ratio that produced the most stable emulsion was 1 g:1 ml, the same ratio used in the preparation method described in Example 3.

[0479] viii) Sodium Chloride

[0480] When sodium chloride is dissolved in the polar layer of the emulsion, it increases the polarity of this layer. It also forms electrostatic interactions with the lipid layer of the emulsion. The electrostatic interactions and increased polarity could improve stability and reduce coalescence. However, addition of sodium chloride (1 g, 2 g or 4 g) was not found to influence the stability of the emulsion.

[0481] The effects of the changes are summarised in the table below:

TABLE-US-00022 Stability Glycerol Paraffin PGPR Shear Temp. Order of (% total vol. Emulsion SH (g) (ml) Oil (ml) (ml) rate (1/s) ( C.) addition after 72 hrs) Ex 7 10 10 10 1 2000 37.5 Oil, then 9.4 PGPR, then SH/glycerol Ex 8 (i) 10 10 6 1 2000 37.5 Oil, then 2.8 PGPR, then SH/glycerol Ex 8 (i) 10 10 4 1 2000 37.5 Oil, then 1.3 PGPR, then SH/glycerol Ex 8 (ii) 10 10 10 4 2000 37.5 Oil, then 2.7 PGPR, then SH/glycerol Ex 8 (iii) 10 10 10 1 3000 37.5 Oil, then 4.6 PGPR, then SH/glycerol Ex 8 (iv) 10 10 10 1 2000 40.0 Oil, then 3.1 PGPR, then SH/glycerol Ex 8 (vi) 10 10 10 1 2000 37.5 SH/glycerol 2.8* and oil, then PGPR *(after 120 hrs)

Example 5Surgihoney Emulsions with High Stability

[0482] The results from the changes described above were used to design a further method of preparing Surgihoney emulsion. This method is described below.

Preparation

[0483] 10 g Surgihoney was dissolved in 10 ml of glycerol. 4, 6, 8, or 10 ml of Paraffin oil was then added to the rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 10 ml of Surgihoney-glycerol solution was then added to the rheometer. The rheometer was then started under the following conditions; Shear rate 3000 1/s, Temperature 40 C., gap 4000 m, Run time 10 minutes. After 1 minute 4 ml of PGPR (Polyglycerol polyricinoleate) was then added. Once a total of 10 minutes had elapsed the emulsion was transferred from the rheometer to a container.

TABLE-US-00023 Total Total Volume Volume Surgihoney- Separation Separation Formulation Glycerol (ratio - Paraffin oil <11 Days after 20 number 1 g:1 ml) (ml) PGPR (ml) (ml) (%) days (%) 1 10 4 10 0 0.7 2 10 4 4 0 0.9 3 10 4 4 0 1.0 4 10 4 4 0 1.6 5 10 4 6 0 1.2 6 10 4 8 0 0.9

[0484] All of the formulations were found to be highly stable, with a slight increase in stability observed as the volume of paraffin oil used was increased.

Example 6Surgihoney Cream Formulation

[0485] 1.5 g of Surgihoney was dissolved in 1.5 ml of glycerol. 1 g of sodium alginate was then dissolved in the Surgihoney-glycerol solution. Next 10 ml of Paraffin oil was added to the Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5 C., gap 4000 m, Run time 10 minutes. After 2 minutes, 1.5 ml of the Surgihoney-alginate and glycerol solution was added to the rheometer. After 3 minutes 8 ml of calcium chloride solution was added dropwise to the rheometer. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

Example 7Non-Aqueous Surgihoney Cream Formulation

[0486] The method described in Example 10 uses water to dissociate calcium chloride into its ions. This could potentially activate the Surgihoney to produce hydrogen peroxide, and limit the stability of the cream formulation. However, we have appreciated that calcium chloride can be dissociated using non-aqueous solvents, such as ethanol or acetic acid. We have also appreciated that glycerol is able to bind to free water. This property allows water to be used to dissolve the alginate, provided sufficient glycerol is present to prevent premature release of hydrogen peroxide.

[0487] The method described below uses ethanol as a solvent for calcium chloride, and glycerol to bind free water in the alginate solution.

[0488] 1 g of sodium alginate is dissolved in 15 ml water. Next 30 ml glycerol is added to the alginate solution and mixed. Then 30 g Surgihoney is then dissolved in the solution. 10 ml of Paraffin oil is then added to the rheometer (TA Instruments AR-G2) which has a Jacket Peltier and vane geometry attached. 10 ml of Surgihoney solution is then added to the rheometer. The rheometer is then started under the following conditions: Shear rate 3000 1/s, Temperature 40 C., gap 4000 m, Run time 10 minutes. After 1 minute 4 ml of PGPR (Polyglycerol polyricinoleate) is added. After 2 minutes 8 ml of non-aqueous calcium chloride solution (1M Calcium chloride in ethanol) is added dropwise to the rheometer. Once a total of 10 minutes has elapsed, the emulsion is transferred from the rheometer to a container.

Summary of Emulsion Formulations Described Above.

[0489]

TABLE-US-00024 Emulsion/ Glycerol Paraffin PGPR NaAlg(g)/ Shear Temp. Order of cream SH (g) (ml) Oil (ml) (ml) CaCl.sub.2(ml) rate (1/s) ( C.) addition Ex 7 10 10 10 1 2000 37.5 Oil, then PGPR, then SH/glycerol Ex 9 10 10 4, 6, 8, 4 3000 40.0 Oil and SH/glycerol, or 10 then PGPR Ex 10 1.5 1.5 10 1 1/8 2000 37.5 Oil and PGPR, then (aq) SH/glycerol/Na Alg, then CaCl.sub.2 Ex 11 30 30 10 4 1/8 3000 40 Oil and SH/glycerol/Na (non-aq) Alg(aq), then PGPR, then CaCl.sub.2 (non-aq)

[0490] Synthetic honey compositions of the invention could replace the Surgihoney in the Surgihoney-based emulsions and creams described herein.

Example 8Treatment of Aphthous Ulcers and Geographic Tongue with Surgihoney

[0491] Surgihoney was used to treat two patients with geographic tongue and recurrent aphthous ulcers. Surgihoney was applied topically and held over the affected areas three times a day for as long as possible before swallowing. This was continued for a week.

[0492] In both cases, the geographic tongue resolved in 48 hours. In one case, the ulcers did not progress, although this case had further aphthous ulcers 10 days after stopping treatment with Surgihoney. In the other case, the subject reported that the ulcers were not as bad as usual and no further ulcers were reported.

Example 9Treatment with Electron Beam Irradiation

[0493] Samples of activated Acacia honey were prepared. Three glass sample vials were each filled with 10 g of the activated honey. Each of the samples was tested by a Labtek PER100 peroxide test stick to determine hydrogen peroxide generation from the rate of colour generation. Samples were sterilised with electron beam irradiation at a dose of about 75 kGy and then tested for hydrogen peroxide generation.

[0494] Non Sterile Activated Acacia Honey Samples: Average measured rate of peroxide production 0.540 ppm/s standard Deviation 0.062.

[0495] Sterile Activated Acacia Honey Samples: Average measured rate of peroxide production 0.353 ppm/s standard Deviation 0.008

[0496] The honey samples maintained the ability to produce hydrogen peroxide after sterilisation with electron beam irradiation.

Example 10Immunomodulatory Effects of SurgihoneyRO Treatment on Epithelial Cells

[0497] A human nasal epithelial cell line was used to investigate the immediate effect of SurgihoneyRO (also known as Surgihoney) on the integrity and function of the epithelium. In addition, a human mast cell line was used as a representative leukocyte to investigate immunological responses to SurgihoneyRO. Mast cells were selected because of their function as sentinel cells, being located just underneath the epithelium in tissues. Mast cells have close contact to the external environment, for example being found in the nasal mucosa, where they are ideally placed to participate in the early recognition of pathogensby acting as immune effectors and modulatory cells with an essential role in linking innate and adaptive immunity in the host's defence against pathogens such as bacteria.

[0498] Another reason for using mast cells relates to findings of intracellular bacteria in mast cells (Hayes et al., J Allergy Clin Immunol 2015; 135(6):1648-51). One of the objectives of this treatment would be to develop its use to target intracellular bacteria which act as a reservoir constantly seeding bacteria into the extracellular environment and therefore promoting ongoing inflammation and development of chronicity.

[0499] One of the objectives of the study was to determine whether treatment with SurgihoneyRO could elicit an immunomodulatory effect to the host. FIG. 7 demonstrates the effects of SurgihoneyRO on the expression of four main gatekeeping genes for T cell differentiation. SurgihoneyRO treatment causes a shift in the Th balance toward Th.sub.2 and Th.sub.17 polarisation for nasal epithelial cells. The results show this as there is no observed significant increase in the expression of T-bet across the time course with either concentration of SurgihoneyRO. However there is a dose and time dependent increase in the expression of GATA3 this difference being significant at all time points (p0.05) when treated with 100 g/L. At 2 hours, there is a significant increase in expression by 2.74 fold with 100 g/L and 2.19 fold with 10 g/L of SurgihoneyRO.

[0500] FIG. 7 also shows that there is a dose and time dependent decrease in FOXP3 expression with significance at both 1 hour (0.34 fold decrease, p0.05) and 2 hours (0.60 fold decrease, p0.0001) when treated with 100 g/L SurgihoneyRO. The nasal epithelial cells display an increase in the expression of the Th.sub.17 gatekeeping gene RORC. When treating with 100 g/L SurgihoneyRO for 2 hours, the epithelial cells had a significant 1.89 fold increase in RORC expression with p0.05. Data showed no effect of cell treatment with matching concentrations (10 g/L and 100 g/L) of the non-engineered base honey (Acacia) for all genes studied (data not shown).

[0501] The polarisation of Th.sub.17 is further supported by the changes in gene expression of the Th.sub.17 related cytokines interleukin 22 (IL22) and interleukin 23 receptor (IL23R) (FIG. 8). There is a time and dose dependent increase in the expression of IL22; increasing from 0.22 to 2.05 fold (p0.05) and 1.67 (p0.01) across the time points with 100 g/L and 0.86 fold to 1.33 and 1.38 (p0.05) with 10 g/L respectively. Furthermore, there is a time and dose dependent increase in IL23R expression but only when treating with 100 g/L; increasing from 2.74 fold (p0.001) to 8.54 fold (p0.05) and finally 14.28 fold (p0.05) across the time course.

[0502] Having illustrated the ability of SurgihoneyRO to influence Th.sub.17 responses, we wanted to investigate whether the treatment had the capacity to mediate host responses to invading pathogens. Matrix metalloproteinases (MMPs) are known to be secreted by activated epithelial cells. In addition to matrix degrading and wound healing properties, MMPs also play an important role in host defence responses against infectious agents. MMP7 is involved in the activation of antimicrobial defensin, releasing mature TNF and chemokines to tackle infections. MMP9 can regulate immune responses and attract lymphocytic cell migration which facilitates tissue remodelling resulting from cleavage of extracellular matrix.

[0503] FIG. 9a demonstrates a time and dose dependent upregulation of both MMP7 and MMP9 resulting in significant fold increases following treatments with both 10 g/L and 100 g/L of SurgihoneyRO. The expression of MMP7 significantly increased to a fold change of 4.56 (p0.01) with 10 g/L and 8.04 (p0.05) with 100 g/L at 2 hours. MMP9 also significantly increased to 1.37 fold (p0.01) with 10 g/L and 3.73 fold (p0.05) with 100 g/L. There were also significant increases in the expression of both defensin 5 and TNF. There was a mean increase in defensin 5 expression of 0.9 fold (p0.01) and TNF expression increased by 4.24 fold (p0.05) following 2 hours of treatment with 100 g/L of SurgihoneyRO. This is direct evidence to support the hypothesis that SurgihoneyRO has the ability to boost the host response to invading pathogens (FIG. 9a).

[0504] An essential function of the early innate immune response to pathogens is played by Toll-Like Receptors (TLRs) whose recognition of pathogen-associated microbial patterns (PAMPs), and endogenous danger-associated molecular patterns (DAMPs) initiates downstream signalling cascades resulting in the production of pro-inflammatory and effecter cytokines that have a direct effect on the adaptive immune response. FIG. 9b shows an increase in the gene expression of two TLRs (TLR2 and TLR4) following the treatment of nasal epithelial cells with SurgihoneyRO (FIG. 9b). There is a highly significant (p0.001) mean increase of 1.85 fold in expression of TLR2 when treating with 100 g/L SurgihoneyRO at 2 hours.

[0505] We then studied the response of the mast cell line (HMC-1) to SurgihoneyRO treatment. In parallel to the effects seen with nasal epithelial cells, there is also an increase in the expression of Th.sub.17 related cytokines following SurgihoneyRO treatment. When treating with 100 g/L SurgihoneyRO for 2 hours, mast cells displayed significant increases of 1.89 fold in RORC expression (p0.01), 1.35 fold in IL22 expression (p0.05) and 0.61 fold in IL23R expression (p0.05) respectively (FIG. 10).

Example 11Hydrogen Peroxide Production by SurgihoneyRO

[0506] In addition to the potential immunomodulatory effects of SurgihoneyRO, we quantified the production of hydrogen peroxide by SurgihoneyRO for the concentrations used in this study (10 g/L and 100 g/L). The aim of this experiment was to validate whether the immunomodulatory effects observed previously were as a result of endogenous hydrogen peroxide production by SurgihoneyRO.

[0507] FIG. 11a shows that the release of hydrogen peroxide by SurgihoneyRO is significantly higher than that of the non-engineered base honey (Acacia). The production of hydrogen peroxide peaks between 2 and 6 hours at which time it is roughly 12 fold higher for 100 g/L than that for 10 g/L. Following the exponential increase in the production, the level of hydrogen peroxide remains constant until 24 hours which was the last time point measured.

[0508] The study evaluated the production of hydrogen peroxide when the SurgihoneyRO and Acacia honey were used to treat both cell lines. It showed that the maximum amount of hydrogen peroxide produced by 100 g/L SurgihoneyRO in the presence or absence of epithelial cells (FIG. 11b) or mast cells (FIG. 11c) was around 400 uM, peaking at 2 hours. There was no significant difference whether there are any cells present. The presence of cells within the culture made no significant difference suggesting that SurgihoneyRO is the main source of hydrogen peroxide in the cell culture.

Example 12Immunomodulatory Effects of Exogenous Hydrogen Peroxide Treatment

[0509] Having established the concentration of hydrogen peroxide production by SurgihoneyRO in FIG. 11, we wanted to know whether the concentration of hydrogen peroxide produced in the cell culture is able to induce immunomodulatory changes. In this case, we treated cells with an appropriate concentration range of pure hydrogen peroxide, from 0-400 uM.

[0510] The effect of the treatment with SurgihoneyRO correlated to that with a comparable concentration of pure hydrogen peroxide. FIG. 12a shows that similar to the treatment with SurgihoneyRO, exogenous hydrogen peroxide elicits a dose dependent increase in GATA3 expression in nasal epithelial cells, with a significant fold increase of 0.92 (p0.5) when treating with 400 M hydrogen peroxide for 1 hour (FIG. 12a).

[0511] A dose dependent increase in the expression of RORC was also detected following exogenous hydrogen peroxide treatment, resulting in a mean increase in expression of 14.57 fold (p0.05) for the nasal epithelial cells and 2.83 fold (p0.05) for the mast cells with 400 M hydrogen peroxide.

[0512] These changes are also associated with increases of IL22 and IL23R for both cell lines (FIGS. 12b and 12c). With the nasal epithelial cells there is a steady increase in the expression of IL22 from 0.90 fold with 40 M to 1.42 fold with 200 M and 4.55 fold with 400 M (p0.001), and with IL23R the fold change in expression increases from 0.45 to 1.20 (p0.05) and 1.87 (p0.001) across the concentration range. With the mast cells there is an increase in expression of IL22 from 0.60 fold with 40 M to 1.25 with 200 M (p0.05) and 1.81 with 400 M (p0.01), and with IL23R the fold change in expression increases from 0.47 to 0.58 and 1.05 (p0.05) across the concentration range.

[0513] The treatments with exogenous hydrogen peroxide with epithelial cells and mast cells also caused a dose dependent increase in the expression of the anti-microbial MMP7 and MMP9 (FIGS. 13a and 13b). However there is only statistical significance with the expression of MMP9 for the nasal epithelial cells (1.47 fold with 400 M, p0.01) and MMP7 for the mast cells which have an increase of 3.07 fold (p0.05) and 3.31 fold (p0.05) with 200 M and 400 M respectively.

Example 13a Protective Anti-Inflamatory Effect of SurgihoneyRO and Exogenous Hydrogen Peroxide

[0514] Exogenous hydrogen peroxide treatment can directly induce a protective anti-inflammatory effect on epithelial cells by increasing the expression of IL10. FIG. 14a shows a significant increase in expression of the anti-inflammatory cytokine IL10 to 4.63 fold (p0.05) in the nasal epithelial cells treated with 400 M hydrogen peroxide. In concordance with these effects, we also demonstrated that there is a time dependent increase in the expression of the protective and anti-inflammatory cytokine IL10; increasing from a fold change of 0.01 to 2.65 (p0.05) and 4.73 (p0.05) across the time points with 100 g/L and 0.81 to 3.01 (p0.05) and 4.85 with 10 g/L respectively (FIG. 14b). This increase in IL10 expression also correlated with the increase in GATA3 expression in the nasal epithelial cells supporting the shift towards the Th.sub.2 lineage.

[0515] These data support a protective effect of both SurgihoneyRO and hydrogen peroxide and a shift in the balance towards the Th.sub.2 lineage specifically for the nasal epithelial cells.

[0516] Taken together, these experiments clearly demonstrate an immunomodulatory effect of SurgihoneyRO treatment in both nasal epithelial and mast cells with a shift towards a Th.sub.2 and Th.sub.17 response, as well as anti-microbial and innate immunity responses. These effects could be mediated by the hydrogen peroxide production of SurgihoneyRO. It has been shown SurgihoneyRO has anti-inflammatory and wound healing effects on skin. In our study, we showed that these effects could be mediated through the upregulation of the anti-inflammatory cytokine IL10. These data shed new insights into the immunomodulatory properties of Surgihoney RO.

Example 14Evaluation of Surgihoney and Synthetic Honey Compositions on Chronic Rhinosinusitis-Related Mucosal Bacterial Strains of Staphylococcus aureus

[0517] SurgihoneyRO and synthetic RO (pH 7.04; RO2) were tested on both the in vitro planktonic phenotype and established biofilms of clinical MRSA and MSSA isolates. Biofilm viability was assessed by colony forming unit (cfu) enumeration and biomass assessed. by measurement of absorbance. Data were validated using confocal microscopy.

[0518] Materials and Methods

Bacterial Strains and Growth Conditions

[0519] S. aureus strains were sub-cultured from frozen stocks onto Colombia blood agar (CBA) plates (Oxoid, UK) and incubated for 18 h at 37 C. and 5% CO.sub.2, following which colonies were resuspended in Brain Heart Infusion (BHI) broth and grown to mid-exponential phase for experiments.

Planktonic Assays

[0520] Flat-bottomed 96-well plates (Fisher Scientific, UK) were inoculated with 1.010.sup.6 planktonic bacteria per well (grown in supplemented BHI). SurgihoneyRO and the synthetic products (RO+ and RO) were prepared in BHI and added to wells at final concentrations of 6 g/L to 383 g/L. BHI alone was added in place of treatments for untreated controls. Cultures were incubated at 37 C. and 5% CO.sub.2 for 18 hours then turbidity measured by absorbance (OD595) using an EZRead 400 spectrophotometer (Biochrom; n=6).

Biofilm Assays

[0521] Mid-exponential planktonic cultures were used to inoculate individual wells of untreated 6-well polystyrene plates (1.010.sup.8 planktonic bacteria per well; Corning Incorporated, USA). Cultures were incubated at 37 C. and 5% CO.sub.2 for 48 h, replacing spent media with fresh BHI at 24 h. Prior to treatment spent media was removed and biofilms washed twice with Hanks' balanced salt solution (HBSS; Gibco, UK). Biofilms were treated with SurgihoneyRO, RO+ or RO (all prepared in HBSS) at final concentrations of 7 to 142 g/L. HBSS alone was added in place of treatments for untreated controls. Biofilms were incubated at 37 C. and 5% CO2 for 24 h, following which the treatments were removed and biofilms washed twice with HBSS to remove unattached cells. Biofilms were resuspended in 1 ml HBSS by cell scraping and briefly vortexing, then serially diluted onto Columbia blood agar. Plates were incubated at 37 C. and 5% CO.sub.2 and colony forming units (CFUs) enumerated (n=5). To assess the total biofilm biomass 100 L of the resuspended biofilms was diluted 10-fold in BHI and the turbidity measured by absorbance (OD595) using a Jenway 6300 spectrophotometer.

Confocal Microscopy

[0522] Mid-exponential planktonic cultures were used to inoculate 35 mm untreated glass-bottom CellView cell culture dishes (1.0108 planktonic bacteria per well; Greiner Bio One, UK). Cultures were incubated at 37 C. and 5% CO.sub.2 for 48 h, replacing spent media with fresh BHI at 24 h. Media was removed, biofilms washed twice with HBSS, then treated with 71 g/L RO+, 71 g/L RO, or HBSS alone (untreated control) for 24 h at 37 C. and 5% CO.sub.2. Treatments were removed and biofilms washed twice with HBSS before staining with a LIVE/DEAD Baclight Bacterial Viability Kit (Life Technologies, UK) as per manufacturer's instructions. Biofilms were examined using an inverted Leica SP8 confocal microscope using a 63 oil immersion lens with sequential scanning of 2 m sections (Leica Microsystems, UK).

Statistical Analyses

[0523] Statistical analyses of in vitro planktonic and biofilm data were performed using one-way analysis of variance (ANOVA) and Kruskal-Wallis multiple comparisons tests. Comparative data with a P value of 0.05 were considered as statistically significant.

[0524] Results

[0525] Treatment of established 48 h MRSA and MSSA biofilms using the same isolates revealed that treatment with the RO+ product for 24 h reduced biofilm viability in both instances (FIG. 19 a&b). A log-fold reduction in viability was observed when treating MRSA biofilms with 53 and 71 g/L (p=0.0079), and a 2-log reduction when treating with 142 g/L (p=0.0159). In comparison, only a log-fold reduction was observed when treating MSSA biofilms with 36-142 g/L (p0.05). Treatment with RO had no effect on viability of biofilms formed by either of the strains tested (FIG. 19 a&b). Treatment of MRSA biofilms with either 36-71 g/L RO+ or RO for 24 h also resulted in a significant increase in overall biofilm biomass (p0.05), whilst all concentrations tested (7-142 g/L) significantly increased the biomass of MSSA biofilms (p0.05). Confocal microscopy was used to validate the biofilm viability and biomass data. Both 48 h established MRSA and MSSA biofilms demonstrated a reduction in viability and an increase in maximum biofilm thickness when treating with the 71 g/L RO and RO+ products (FIGS. 20 a&b). The MRSA biofilm maximum height increased from 25.3 m to 36.3 m (RO) and 45.3 m (RO+), whilst the MSSA biofilm increased from 19.66 m to 45.3 m (RO) and 35.99 m (RO+). Finally, treatment of 48 h established biofilms formed by several clinical MRSA (n=7) and MSSA (n=5) isolates revealed that the viability of each biofilm was reduced with an average log-fold reduction observed within each group (FIG. 21 a&b).

Example 15Activity of Surgihoney Against In Vitro Non-Typeable Haemophilus influenza Biofilms

[0526] Materials and Methods

Bacterial Strains and Growth Conditions

[0527] Bacterial strains used in this study were isolated from nasopharyngeal swabs of children aged 4 years. NTHi was sub-cultured from frozen stocks onto Colombia agar with chocolated horse blood (Oxoid, UK) and incubated for 18 h at 37 C. and 5% CO.sub.2, following which colonies were resuspended in Brain Heart Infusion (BHI) broth supplemented with 10 g/ml Hemin and 2 g/ml NAD, and grown to mid-exponential phase for experiments. Pseudomonas aeruginosa PA01 and a clinical methicillin resistant Staphylococcus aureus isolate were subcultured onto Colombia blood agar plates (Oxoid, UK) and grown in non-supplemented BHI. 65

Planktonic Assays

[0528] Flat-bottomed 96-well plates (Fisher Scientific, UK) were inoculated with 1.010.sup.6 planktonic bacteria per well (grown in supplemented BHI). SurgihoneyRO and the non-engineered base honey (Acacia) were both prepared in supplemented BHI and added to wells at final concentrations of 6 g/L to 319 g/L. Supplemented BHI alone was added in place of treatments for untreated controls. Cultures were incubated at 37 C. and 5% CO.sub.2 for 18 hours then turbidity measured by absorbance (OD595) using an EZRead 400 spectrophotometer (Biochrom; n=6). 75

Biofilm Assays

[0529] Mid-exponential planktonic cultures were used to inoculate individual wells of untreated 6-well polystyrene plates (1.010.sup.8 planktonic bacteria per well; Corning Incorporated, USA). Cultures were incubated at 37 C. and 5% CO.sub.2 for 48 h, replacing spent media with fresh supplemented BHI (NTHi) at 24 h. Prior to treatment spent media was removed and biofilms washed twice with Hanks' balanced salt solution (HBSS; Gibco, UK). Biofilms were treated with SurgihoneyRO or Acacia (both prepared in HBSS) at final concentrations of 7 to 213 g/L. To assess the effect of pH biofilms were treated with HBSS adjusted to pH6.3 (the pH of 71 g/L SurgihoneyRO in HBSS). For adjuvant assays biofilms were treated with 300 g/mL amoxicillin and 60 g/mL clavulanic acid (Co-amoxiclav). HBSS alone was added in place of treatments for untreated controls. Biofilms were incubated at 37 C. and 5% CO.sub.2 for 2 h, following which the treatments were removed and biofilms washed twice with HBSS to remove unattached cells. Biofilms were resuspended in 1 ml HBSS by cell scraping and briefly vortexing the serially diluted onto Colombia agar with chocolated horse blood (NTHi). Plates were incubated at 37 C. and 5% CO2 and colony forming units (c.f.u.) enumerated (n=4).

Confocal Microscopy

[0530] Mid-exponential planktonic cultures were used to inoculate 35 mm untreated glass-bottom CellView cell culture dishes (1.010.sup.8 planktonic bacteria per well; Greiner Bio One, UK). Cultures were incubated at 37 C. and 5% CO.sub.2 for 48 h, replacing spent media with fresh supplemented BHI at 24 h. Media was removed, biofilms washed twice with HBSS, then treated with 71 g/L SurgihoneyRO, 300/60 g.Math.ml co-amoxiclav or HBSS alone (untreated control) for 2 h at 37 C. and 5% CO.sub.2. Treatments were removed and biofilms washed twice with HBSS before staining with a LIVE/DEAD Baclight Bacterial Viability Kit (Life Technologies, UK) as per manufacturer's instructions. Biofilms were examined using an inverted Leica SP8 confocal microscope using a 63 oil immersion lens with sequential scanning of 2 m sections (Leica Microsystems, UK).

Hydrogen Peroxide Measurements

[0531] SurgihoneyRO and Acacia were prepared at a range of concentrations between 7 to 213 g/L in HBSS and incubated at 37 C. and 5% CO.sub.2 for 2 h. Hydrogen peroxide production was then measured using a Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich, UK) as per manufacturer's instructions using an untreated flat-bottomed black 96-well plate (Greiner BioOne, UK). In brief, a standard curve was generated using known concentrations of H.sub.2O.sub.2, to which SurgihoneyRO and Acacia samples were compared, including a negative HBSS control. A master mix comprised of red peroxidase, horseradish peroxidase and assay buffer was added to each standard and sample well. Samples were incubated at room temperature and protected from light for 30 minutes after which fluorescence was measured (excitation: 540 nm/emission: 590 nm; n=4).

Statistical Analyses

[0532] Statistical analyses of in vitro planktonic and biofilm data were performed using one-way analysis of variance (ANOVA) and Kruskal-Wallis multiple comparisons tests. Comparative data with a P value of 0.05 were considered as statistically significant.

[0533] Results

[0534] SurgihoneyRO Treatment Reduces In Vitro NTHi Biofilm Viability Through Increased H.sub.2O.sub.2 Generation

[0535] Treatment of established 48 h in vitro biofilms with 71 and 142 g/L SurgihoneyRO for 2 hours resulted in a 4-log and 3-log reduction in viability respectively (P0.05), whilst treatment with 213 g/L reduced viability 5-log (P0.01; FIG. 22a). In comparison, treatment with the equivalent concentrations of Acacia had no effect on biofilm viability (P=0.75; FIG. 1b). A dose-dependent increase in H.sub.2O.sub.2 levels in the surrounding media was also observed when treating with both Acacia and SurgihoneyRO (FIG. 22b). SurgihoneyRO, however, produced significantly higher levels of H.sub.2O.sub.2 at all concentrations tested, ranging from 10.7-71.2 M in comparison with 0.24-6.5 M generated when treating with the equivalent concentrations of Acacia. Furthermore, these data indicate that the minimum concentration of H.sub.2O.sub.2 effective in reducing NTHi biofilm viability, as evidenced with 71 g/l SurgihoneyRO, is approximately 14.2 to 25.7 M (FIG. 22b). To account for a pH-mediated reduction in viability NTHi biofilms were also treated with HBSS adjusted to pH6.3, revealing no effect on biofilm viability (FIG. 22c).

SurgihoneyRO is More Effective than Co-Amoxiclav in the Treatment of NTHi Biofilms

[0536] Following confirmation that 71 g/L SurgihoneyRO was effective in reducing NTHi biofilm viability the activity was compared to the conventional antibiotic co-amoxiclav, and also whether it could improve antibiotic efficacy when used as an adjuvant. Treatment of established 48 h in vitro biofilms with 71 g/L SurgihoneyRO for 2 hours resulted in a 5-log reduction in viability (P=0.029) whereas treatment with 300/60 g.Math.ml co-amoxiclav had no effect on viability (P=0.343; FIG. 2a). Combined treatment, however, did not improve co-amoxiclav efficacy (FIG. 23a). Confocal laser scanning microscopy confirmed the reduction in biofilm viability of 48 h NTHi biofilms following treatment with 71 g/L SurgihoneyRO for 2 hours, and also the ineffectiveness of co-amoxiclav (FIG. 2b-d). The confocal micrographs also demonstrated that SurgihoneyRO treatment had no obvious effect on overall biofilm biomass or ultrastructure, with all biofilms being 70 m in maximum height (FIG. 23b-d).