PARAPHARMACEUTICAL OR PHARMACEUTICAL COMPOSITION ADMINISTRABLE TO A LIVING BEING, PREFERABLY A HUMAN BEING, COMPRISING AT LEAST ONE ENZYME FOR THE TREATMENT AND/OR PREVENTION OF BACTERIAL INFECTIONS INVOLVING BIOFILM FORMATION

Abstract

A parapharmaceutical or pharmaceutical composition is administrable to living beings, and in particular to human beings. The composition includes at least one endoribonuclease enzyme chosen from the EC 3.1.30 and EC 3.1.31 classes, preferably in a content of from 10 to 1000 U/ml, preferably 50 to 500 U/ml, preferably from 100 to 500 U/ml. The composition is for curative or preventive treatment for dermatological infections or for infections which develop on superficial or deep burns and wounds.

Claims

1. A method of treatment with a parapharmaceutical or pharmaceutical composition, the parapharmaceutical or pharmaceutical composition comprising at least one endoribonuclease enzyme selected from the group consisting of enzymes belonging to the enzyme classes EC 3.1.30 and EC 3.1.31 and mixtures thereof, to potentiate a microbicidal agent, comprising an antibiotic, an antifungal, or a disinfectant, the method comprising administering a therapeutically effective dose of the parapharmaceutical or pharmaceutical composition to human beings in the treatment and/or the prevention of bacterial infections involving biofilm formation.

2. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation is a curative or preventive treatment for dermatological infections or infections developing on burns and superficial or deep wounds.

3. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation is a curative and/or preventive treatment for post-implantation infections associated with infection of tissues around a medical device implanted in the body or of a medical device implanted in the body.

4. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, in a personal hygiene or cosmetic treatment, comprising nail care, an oral solution, a mouthwash, a toothpaste, an eye bath, an eye lens cleaning solution, a cleaning solution for dental appliances/prostheses, toothbrushes, skin care for acne.

5. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein said composition further comprises a series of additional enzymes comprising one, two, three, four, five, six, seven, or eight enzyme(s).

6. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 5, wherein an enzyme from said series of additional enzymes of the said composition is selected from the group consisting of glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1.), carboxylic ester hydrolases (EC 3.1.1), proteases and peptidases (EC 3.4.), and mixtures thereof.

7. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein said composition comprises at least a second enzyme selected from the group of glycosidases (EC 3.2.1), preferably a glucanase as comprising endo-β-1 3-glucanase, and/or an endoglucanase from the group EC.3.2.1.39.

8. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 7, wherein said composition comprises at least one third enzyme selected from the group consisting of glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC 3.1.1), peptidases and proteases (EC 3.4), and mixtures thereof, selected from the group consisting of glycosidases (EC 3.2.1) and peptidases (EC 3.4), the composition comprises at least dispersin B, cellulase and endoglucanase (β 1,3).

9. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial Infections involving biofilm formation is a curative or preventive treatment for a bacterial infection of tissues surrounding an implanted orthopaedic implant or of the implanted orthopaedic implant.

10. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation is a curative or preventive treatment for a bacterial infection of tissues surrounding an implanted dental implant or of the implanted dental implant.

11. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation is a curative or preventive treatment for a bacterial infection of tissues surrounding a catheter, a cannula, a probe, a prosthesis, an endosseous implant, a zygomatic implant, an orthodontic implant, a dental prosthesis, a retainer, a valve, a drain, a stent, a tube for artificial respiration or an implanted screw or of a catheter, a cannula, a probe, a prosthesis, an endosseous implant, a zygomatic implant, an orthodontic implant, a dental prosthesis, a retainer, a valve, a drain, a stent, an artificial respiration tube or an implanted screw.

12. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation comprises an application of said composition to a wound, an infection, tissues or a medical device, comprising an application of a woven or non-woven support impregnated with said parapharmaceutical or pharmaceutical composition.

13. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation comprises an application of said composition to a wound, an infection, tissues, comprising an application of a dressing in the form of a gel comprising said parapharmaceutical or pharmaceutical composition.

14. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation comprises an application of said composition to a wound, infection, tissues or a medical device, comprising an application of an aqueous, buffered solution of said parapharmaceutical or pharmaceutical composition, by infiltration, by irrigation, by injection, by percutaneous application, by inhalation, by mouthwash or eyewash, or by dabbing.

15. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein the treatment and/or prevention of bacterial infections involving biofilm formation comprises an application of said composition to a wound, infection, tissue or medical device, by an application of a paste, cream, ointment, or a viscous solution comprising said parapharmaceutical or pharmaceutical composition.

16. The method of treatment with a parapharmaceutical or pharmaceutical composition according to claim 1, wherein said said antibiotic and/or antifungal, phage, or said disinfectant and said composition form a combination product for simultaneous, separate or staggered use over time.

17. Parapharmaceutical or pharmaceutical composition for administration to human beings, comprising at least one endoribonuclease enzyme selected from classes EC 3.1.30 and EC 3.1.31, at a concentration of 10 to 1000 U/ml.

18. The parapharmaceutical or pharmaceutical composition according to claim 17, wherein said at least one endoribonuclease enzyme is of bacterial origin and/or belongs to class EC 3.1.30.1 or EC 3.1.30.2.

19. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising a series of additional enzymes, comprising one, two, three, four, five, six, seven, or eight enzyme(s).

20. The parapharmaceutical or pharmaceutical composition according to claim 19, wherein an enzyme from said series of additional enzymes of said composition is selected from the group consisting of glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC3.1.1) and peptidases (EC 3.4), and mixtures thereof, the additional enzyme is glucanase comprising endo-β-1 3-glucanase, and/or an endoglucanase from the group EC.3.2.1.39.

21. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising at least a second enzyme selected from the group of glycosidases (EC 3.2.1).

22. The parapharmaceutical or pharmaceutical composition according to claim 21, further comprising at least a third enzyme selected from the group comprising glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC 3.1.1), proteases and peptidases (EC 3.4).

23. The parapharmaceutical or pharmaceutical composition according to claim 22, comprising at least a fourth enzyme selected from the group comprising glycosidases EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC 3.1.1), proteases and peptidases (EC 3.4), comprising as additional enzymes a glucanase comprising endo-β-1 3-glucanase and/or an endoglucanase from the group EC.3.2.1.39, Dispersin B and cellulase.

24. The parapharmaceutical or pharmaceutical composition according to claim 23, comprising at least a fifth enzyme selected from the group comprising glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC 3.1.1), proteases and peptidases (EC 3.4).

25. The parapharmaceutical or pharmaceutical composition according to claim 24, comprising at least a sixth enzyme selected from the group comprising glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC 3.1.1), proteases and peptidases (EC 3.4).

26. The parapharmaceutical or pharmaceutical composition according to claim 25, comprising at least a seventh enzyme selected from the group comprising glycosidases (EC 3.2.1), deoxyribonucleases (EC 3.1.21), oxidoreductases (EC 1), carboxylic ester hydrolases (EC 3.1.1), proteases and peptidases (EC 3.4).

27. The parapharmaceutical or pharmaceutical composition according to claim 26, comprising at least an eighth enzyme selected from the group comprising glycosidases (class EC 3.2.1), deoxyribonucleases (class EC 3.1.21), oxidoreductases (EC class 1.), carboxylic ester hydrolases (EC class 3.1.1), proteases and peptidases (EC class 3.4.), and mixtures thereof.

28. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising at least one microbicidal molecule comprising an antibiotic, an antiseptic, antifungal, a phage, or one or more microbicidal peptides conditioned separately or together with said endoribonuclease.

29. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising a quorum sensing inhibitor.

30. The parapharmaceutical or pharmaceutical composition according to claim 17, in the form of a solution, further comprising an enzyme buffer selected from the group consisting of Tris-HCl, TGN, TBS, PBS, HEPES, MES, PIPES, MOPS, BES, TES, phosphate buffer and citrate buffer, containing 0 to 2 mM MgCl2, containing 0 to 2 mM CaCl2), and 0 to 500 mM NaCl.

31. The parapharmaceutical or pharmaceutical composition to claim 17, in the form of a solution, further comprising an enzyme buffer comprising 0 to 50% of stabilising agent comprising polyol, arginine, calcium formate, or glucose.

32. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising at least one surfactant.

33. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising at least one preservative.

34. The parapharmaceutical or pharmaceutical composition according to claim 17, further comprising at least one sequestrant.

35. The parapharmaceutical or pharmaceutical composition according to claim 17, in the form of a solution, for example a mouthwash, an eye bath, a lotion, an irrigation solution, a solution for injection.

36. The parapharmaceutical or pharmaceutical composition according to claim 17, in the form of a hydrophilic dressing, for example a hydrogel.

37. The parapharmaceutical or pharmaceutical composition according to claim 17, in immobilised form on a woven or non-woven, dry support or on a medical device or in impregnated form on a woven or non-woven support.

38. The parapharmaceutical or pharmaceutical composition according to claim 17, in the form of a topical composition.

39. The parapharmaceutical or pharmaceutical composition according to claim 17, in the form of a sterile solution which for administration by infiltration, by irrigation, by injection, by percutaneous application and by inhalation.

40. The parapharmaceutical or pharmaceutical composition according to claim 17, adapted as a potentiator for a microbicidal agent, comprising an antibiotic and/or an antifungal, a phage, or a disinfectant, in the treatment and/or the prevention of bacterial infections involving biofilm formation.

41. The parapharmaceutical or pharmaceutical composition according to claim 17, for a curative or preventive treatment for dermatological infections or infections developing on burns and superficial or deep wounds.

42. The parapharmaceutical or pharmaceutical composition according to any one of claims 17 to 38 claim 17, for curative and/or preventive treatment of post-implantation infections associated with the infection of tissues around a medical device implanted in the body or of a medical device implanted in the body.

43. The parapharmaceutical or pharmaceutical composition according to claim 17, for a personal hygiene or cosmetic treatment, comprising nail care, an oral solution, a mouthwash, a toothpaste, eye bath, eye lens cleaning solution, dental appliance cleaning solution, toothbrush cleaning solution, skin care for acne.

44. The parapharmaceutical or pharmaceutical composition according to claim 17, for a curative or preventive treatment for a bacterial infection of tissues surrounding an implanted orthopaedic implant or of the implanted orthopaedic implant.

45. The parapharmaceutical or pharmaceutical composition according to claim 17, for a curative or preventive treatment for a bacterial infection of tissues surrounding an implanted dental implant or of the implanted dental implant.

46. The parapharmaceutical or pharmaceutical composition according to claim 17, for a curative or preventive treatment of a bacterial infection of tissues surrounding a catheter, a cannula, a probe, a prosthesis, an endosseous implant, a zygomatic implant, an orthodontic implant, a dental prosthesis, a retainer, a valve, a drain, a stent, a tube for artificial respiration or an implanted screw or of a catheter, a cannula, a probe, a prosthesis, an endosseous implant, a zygomatic implant, an orthodontic implant, a dental prosthesis, a retainer, a valve, a drain, a stent, a tube for artificial respiration or an implanted screw.

47. The parapharmaceutical or pharmaceutical composition according to claim 17, for treatment and/or prevention of bacterial infections involving biofilm formation which comprises an application of said composition to a wound, infection, tissues or a medical device, by an application of a woven or non-woven support impregnated with said parapharmaceutical or pharmaceutical composition or on which said parapharmaceutical or pharmaceutical composition is immobilised.

48. The parapharmaceutical or pharmaceutical composition according to claim 17, for treatment and/or prevention of bacterial infections involving biofilm formation which comprises an application of said composition to a wound, an infection, tissues, by an application of a dressing in gel form, comprising a hydrogel comprising said parapharmaceutical or pharmaceutical composition.

49. The parapharmaceutical or pharmaceutical composition according to claim 17, for treatment and/or prevention of bacterial infections involving biofilm formation which comprises an application of said composition to a wound, infection, tissues or a medical device, by an application of an aqueous buffered solution of said parapharmaceutical or pharmaceutical composition, by infiltration, by irrigation, by injection, by percutaneous application, by inhalation, by mouthwash or eyewash, by dabbing, or by soaking.

50. The parapharmaceutical or pharmaceutical composition according to claim 17, for treatment and/or prevention of bacterial infections involving biofilm formation which comprises an application of said composition to a wound, an infection, tissues or a medical device, by an application of a paste or of a viscous solution comprising said parapharmaceutical or pharmaceutical composition.

51. The parapharmaceutical or pharmaceutical composition according to claim 28, for a combination product for simultaneous, separate or staggered use over time.

52. The parapharmaceutical or pharmaceutical composition according to claim 17, for surgical use.

53. A non-therapeutic method of using subtilisin (EC.3.4.21.62) comprising potentiating one or more anti-microbiological molecules for a biofilm comprising Candida albicans.

54. The method according to claim 53, wherein the anti-microbiological molecules are an antifungal and/or an antibiotic, wherein said antibiotic is an antibiotic effective against Gram-negative bacteria in planktonic form and Gram-positive bacteria in planktonic form.

55. Pharmaceutical composition comprising subtilisin (EC.3.4.21.62) for the treatment of a biofilm comprising Candida albicans, said biofilm affecting a patient.

56. The pharmaceutical composition according to claim 55 for sequential use with an antifungal agent and/or an antibiotic, preferably wherein the antifungal is active against Candida albicans (in planktonic form) and the antibiotic is active against Gram-negative and/or Gram-positive bacteria (in planktonic forms).

57. The pharmaceutical composition according to claim 55 for curative treatment of tissues surrounding an orthopaedic implant, a catheter, a cannula, a probe, a prosthesis, an endosseous implant, a zygomatic implant, an orthodontic implant, a dental prosthesis, a retainer plate, a valve, a drain, a stent, a tube for artificial respiration or an implanted screw, of an epidermis, a wound or a mucosa inside the body being infected with a biofilm comprising Candida albicans.

Description

[0120] Other characteristics, details and advantages of the invention will be included in the description given below, on a non-limiting basis and with reference to the drawings and the examples.

[0121] FIG. 1 is a graph which illustrates the reduction in the biomass percentage of the biofilm of several strains of Staphylococcus aureus, after contact with a composition according to the invention with a concentration of 500 U/ml for denarase.

[0122] FIG. 2 is a graph which illustrates the reduction in the biomass percentage of the biofilm formed by different strains of S. aureus and Staphylococcus epidermidis, after contact with a composition according to the invention with a concentration of 100 U/ml for denarase compared with a composition comprising 100 U/ml of RNase I, with a composition comprising 100 U/ml for each enzyme in a mixture of RNase 1, RNase A and DNase 1.

[0123] FIG. 3 shows a series of four graphs which illustrates the decrease in the biomass percentage of the biofilm of different strains of S. aureus and S. epidermidis, after contact with a composition according to the invention with a concentration of 500 U/ml for denarase compared with a composition comprising a CDD enzyme cocktail comprising 500 U/ml of denarase, 0.06 U/ml of dispersin B and 7 U/ml of cellulase, compared with a composition comprising 0.06 U/ml of dispersin B, compared with a composition comprising 7 U/ml of cellulase.

[0124] FIG. 4 shows a series of three graphs which illustrates the decrease in the biomass percentage of the biofilm of multiple strains of S. aureus and S. epidermidis, after contact with a composition comprising only 500 U/ml of denarase, compared with a composition comprising an enzyme cocktail comprising 500 U/ml of denarase and 0.06 U/ml of dispersin B, compared with a composition comprising an enzyme cocktail comprising 500 U/ml of denarase and 7 U/ml of cellulase.

[0125] FIG. 5 shows a series of two graphs which illustrates the decrease in the biomass percentage of the biofilm of different strains of P. aeruginosa, after contact with a composition comprising 500 U/ml of denarase, compared with a composition comprising a CDD enzyme cocktail comprising 500 U/ml of denarase, 0.06 U/ml of dispersin B and 7 U/ml of cellulase.

[0126] FIG. 6 shows a series of four graphs which illustrates the reduction in viability (expressed in log.sub.10 (CFU/ml) of the S. aureus ATCC33591 biofilm after curative treatment of the biofilm with a composition according to the invention comprising denarase at 500 U/ml followed by 24 hours of incubation with different vancomycin concentrations of 0, 10, and 20 mg/L, as well as the reduction in the biomass (expressed in absorbance at 570 nm) of the S. aureus ATCC33591 biofilm after curative treatment of the biofilm with a composition according to the invention comprising denarase at 500 U/ml followed by 24 hours of incubation with different vancomycin concentrations of 0, 10 and 20 mg/L.

[0127] FIG. 7 shows a series of three graphs which illustrates the viability in the S. aureus ATCC33591 biofilm expressed in log.sub.10 (CFU/ml) after curative treatment of the biofilm with a composition comprising a CDD enzyme cocktail comprising 500 U/ml of denarase, 7 U/ml of cellulase and 0.06 U/ml of dispersin B followed by 24 hours of incubation with 0 mg/L or 20 mg/L of vancomycin, compared with a composition comprising a BDD enzyme cocktail comprising 500 U/ml of denarase, 1% v/v of BlazePro and 0.06 U/ml of Dispersin B followed by 24 hours of incubation with 0 mg/L or 20 mg/L of vancomycin, compared with a composition comprising an FDD enzyme cocktail comprising 500 U/ml of denarase, 1% v/v Flavourzyme and 0.06 U/ml Dispersin B followed by 24 hours of incubation with 0 mg/L or 20 mg/L of vancomycin.

[0128] FIG. 8 shows the effect of different concentrations of vancomycin (0 and 20 mg/L) on the viability expressed in log.sub.10 (CFU/ml) on the biofilm of different strains of S. aureus and S. epidermidis after curative treatment with different tri-enzyme compositions. The CDD tri-enzyme composition comprises an enzyme cocktail composed of 7 U/ml of cellulase. 0.06 U/ml of dispersin B and 500 U/ml of denarase, compared with a CDD2 enzyme composition comprising a tri-enzyme cocktail of 70 U/ml of cellulase, 0.32 U/ml of dispersin B and 500 U/ml of denarase, compared with an ADD enzyme composition comprising a tri-enzyme cocktail composed of 70 U/ml of cellulase, 2000 U/ml alpha-amylase and 500 U/ml denarase.

[0129] FIG. 9 shows the effect of 20 mg/L of vancomycin on the biomass expressed in % and the viability expressed in log.sub.10 (CFU/ml) of the biofilm of different strains of S. aureus and S. epidermidis formed in the presence of compositions according to the invention. The CDD2 tri-enzyme composition includes a tri-enzyme cocktail composed of 70 u/ml of cellulase, 0.32 U/ml of dispersin B and 500 U/ml of denarase: compared with an ADD enzyme composition comprising a tri-enzyme cocktail consisting of 70 U/ml cellulase, 2000 U/ml alpha-amylase and 700 U/ml denarase.

[0130] FIG. 10 shows the effect of different concentrations of vancomycin (0 and 20 mg/L) on the viability expressed in log.sub.10 (CFU/ml) of the S. aureus ATCC33591 and S. epidermidis ATCC35984 biofilm formed in the presence of compositions according to invention. The CDD enzyme composition comprising a tri-enzyme cocktail composed of 7 U/ml of cellulase. 0.06 U/ml of dispersin B and 500 U/ml of denarase: The CDD2 enzyme composition comprising a ti-enzyme cocktail composed of 70 U/ml of cellulase, 0.32 U/ml of dispersin B and 500 U/ml of denarase: compared with an ADD enzyme composition comprising a tri-enzyme cocktail consisting of 70 U/ml cellulase, 2000 U/ml alpha-amylase and 500 U/ml denarase.

[0131] In the figures, identical or similar elements have the same references.

[0132] Other characteristics and advantages of the present invention will be drawn from the non-limiting description which follows, and with reference to the drawings and the examples.

Efficacy of a Composition According to the Invention for Reducing the Biomass of Biofilms Involved in Infections of the Human Body

[0133] In order to test the efficacy of a composition according to the invention, comprising at least one endoribonuclease enzyme, several experiments were carried out on different bacterial strains (clinical isolates and ATCC collection strains) (Table 1).

TABLE-US-00001 TABLE 1 Isolate/strain species origin ATCC29213 S. aureus MSSA Wound (ATCC collection) ATCC33591 S. aureus MRSA Clinical isolate (ATCC collection) 7832 S. aureus MRSA Deep wound 7841 S. aureus MRSA Superficial wound 1142-004 S. aureus MSSA Wound 1144-20 S. aureus MSSA Wound ATCC35984 S. epidermidis MRSE Venous catheter (ATCC collection) PAO1 P. aeruginosa Wound ATCC27853 P. aeruginosa Infected blood (ATCC collection) 618 P. aeruginosa Superficial wound

[0134] The effect of a treatment with the composition according to the invention on the biomass of biofilms was studied in a static model in 96-well plates. The curative enzyme treatment was carried out on a preformed 24-hour biofilm. To test its efficacy in reducing the biomass of biofilms, the composition according to the invention was brought into contact with the biofilm for 1 hour at 37° C. The enzyme solution was then removed, and the detached biomass was removed via rinsing steps. Crystal violet (CV) dye was then used to quantify residual biomass. The latter interacts with dead and living cells, and the macromolecules of the extracellular matrix of the biofilm (such as DNA and exopolysaccharides). CV staining was quantified via spectrophotometry by measuring the absorbance at 570 nm.

1.1 Culture Method, Enzyme Treatment, Quantification of Biomass and Analysis of Results

[0135] The different strains of S. aureus and P. aeruginosa (Table 1) were inoculated in 5 ml of TGN culture medium (Tryptic Soy Broth VWR+1% glucose+2% NaCl) from 20 μl of a stored glycerol stock at −80° C. The strains were then aerobically incubated at 130 rpm at 37° C. After 18 hours of growth, the precultures were diluted in TGN to reach an optical density at 620 nm (OD.sub.620) of 0.05, which corresponds to +/−5*10.sup.6 CFU/ml. Two hundred microlitres of each culture (n=4 for each condition tested) were placed in the wells of a 96-well plate and incubated at 37° C. without agitation for 24 hours to allow the biofilm to form. The biofilms were then washed 2× with 200 μl PBS pH 7.5 (Sigma) in order to eliminate planktonic cells (i.e. cells that do not adhere to the biofilm). The composition according to the invention or the control compositions CT− (negative control) and CT+ (positive control) (200 μl) were added to the washed biofilm and the biofilm was incubated for 1 hour at 37° C. (n=4 for each condition). The plates were then emptied, the biofilm was washed 2× with 200 μl PBS pH 7.5 (Sigma) and dried at 60° C. for 18 h before the biomass was analysed.

[0136] To quantify the residual biomass, a 1% CV solution (Sigma) was added to the biofilms at room temperature for 15 min. The unfixed dye was then washed off with distilled water. The dye attached to the biofilm was then detached and dissolved with 66% peracetic acid and absorbance at 570 nm (A570) was measured in a SpectraMax M3 multimode reader. This absorbance value corresponds to the biomass of the biofilm.

[0137] To analyse the effect of the compositions according to the invention, a statistical test (one-way ANOVA and Tukey's post-hoc test) was carried out by comparing the absorbance values of the untreated biofilms (negative control, CT−=treatment with buffer without enzyme) and biofilms treated with the enzyme compositions including a positive control, CT+. This positive control includes a dye, two non-anionic surfactants, a sequestrant, a preservative, an enzyme stabiliser, a solvent, seven enzymes including two proteases (EC 3.4), one laccase (EC 1.10.3.2), one mannanase (EC 3.2 0.1.78 and EC 3.2.1.101), one amylase (EC 3.2.1.1) and one lipase (EC 3.1.1.3). The difference is significant when the pvalue of the statistical test is less than or equal to 0.05 (* used on graphs). When this is specified in the comparative examples, the activity of the various enzyme compositions is compared (one-way ANOVA and post-hoc Tukey test).

1.2 Results

Example 1.—Composition According to the Invention Containing Denarase in TGN

[0138] First, a composition comprising denarase (c-Lecta), which is an endoribonuclease from S. morcescens belonging to the EC 3.1.30 enzyme class, was tested at a concentration of 500 U/ml in TGN. The negative control (CT−) was composed of TGN only. The A.sub.570 absorbance of the CV measured on the CT− corresponds to 100% biomass. As shown in FIG. 1, the biomass percentage of an S. aureus ATCC 29213, ATCC 33591, 7832, 7841, 1142-004 and 1144-20 biofilm decreases by 75%, 76%, 72%, 71%, 47% and 52% respectively. Statistical analysis (ANOVA and Tukey's post-hoc test) indicates that in all cases, this difference is significant (pvalue s 0.05) compared with CT−. This decrease suggests that the effect of a microbicidal agent will be potentiated after biofilm is treated with denarase.

Comparison of the Efficacy of the Composition from Example 1 with Other Endoribonucleases

[0139] The effect on the biomass of denarase was compared to the effects of a negative control (TGN), a positive control (EnziQure® 1% in TGN: OneLife sa), and compositions containing RNase I only (Escherichia coli endoribonuclease: class EC4.6.1: ThermoFisher Scientific), and a mixture of RNase 1, RNase A (bovine endoribonuclease class EC 4.6.1: ThermoFisher Scientific) and DNase I (bovine endoribonuclease class EC 3.1.21: ThermoFisher Scientific).

[0140] The compositions are listed in Table 2.

TABLE-US-00002 TABLE 2 Composition - Example Enzyme Concentration Buffer Example 1 (Ex. 1) Denarase 100 U/ml TGN Comparative Example 1 RNase I 100 U/ml TGN (CE. 1) Comparative Example 2 RNase I, 100 U/ml of TGN (CE. 2) RNase A and each enzyme DNase I Negative control CT− (NA) (NA) TGN Positive control CT+ EnziQure ® 1% TGN

[0141] These tests were carried out with the aim of comparing the disruptive effect on biofilms of the different nucleases at 100 U/ml in TGN. These compositions were tested on a 24-hour S. aureus ATCC33591 and 7832, and S. epidermidis ATCC35984 biofilm (n=4) by following the protocol described in point 1.1.1.

[0142] FIG. 2 shows that RNase I (endoribonuclease) according to comparative example CE1 has little or no significant effect on the decrease in the biomass percentage of the biofilms (1 biofilm/3 is significantly reduced compared to CT−). The addition of RNase A (endoribonuclease) and DNase I (endodeoxyribonuclease) in addition to RNase I according to comparative example CE2 slightly improves the disruptive activity of the composition of comparative example CE1A (2 biofilms/3 are significantly reduced). Interestingly, denarase (example 1) (endoribonuclease) significantly reduces the biomass of ⅔ biofilms, like the CE2 nuclease mixture, but more effectively: 46% vs. 30% for the ATCC33591 strain and 37% vs 13% for the ATCC35984 strain. Since the non-specific endoribonuclease activity of denarase makes it possible to digest both DNA and RNA, it is unexpected to find that an endoribonuclease specifically selected from the EC 3.1.30 or EC 3.1.31 class allows a more effective reduction of biofilm biomass than a DNase I-RNase I-RNase A mixture. The scientific reasons for this observation are not yet known.

1.2.3 Comparison of the Efficacy of Denarase in Curative Treatment with Other Enzyme Compositions

[0143] The anti-biomass activity in the curative treatment of denarase (500 U/ml) was then compared with that of cellulase (Sigma: 7 U/ml) and dispersin B (GIGA: 0.06 U/ml), and to that of a tri-enzyme cocktail composed of denarase, cellulase and dispersin B at the same concentrations. These four compositions (Ex1, CE3, CE4 and Ex 2) were tested on S. aureus ATCC33591, ATCC29213 and S. epidermidis ATCC35984 biofilms following the protocol detailed in point 1.1.1, with the only difference that the mixtures were made in a 20 mM Tris-HCl buffer pH 7. In this experiment, the CT− is 20 mM Tris-HCl pH 7 without enzymes and its biomass corresponds to 100%. The CT+ is composed of 1% EnziQure® in this same buffer. The compositions are listed in Table 3.

TABLE-US-00003 TABLE 3 Composition - Example Enzyme Concentration Buffer Example 2 (Ex. 2) Denarase 500 U/ml Tris-HCl 20 mM Comparative Example 3 Cellulase 7 U/ml Tris-HCl 20 mM (CE. 3) Comparative Example 4 Dispersin B 0.06 U/ml Tris-HCl 20 mM (CE. 4) Example 3 (Ex. 3) Denarase, 500 U/ml Tris-HCl 20 mM cellulase and 7 U/ml Dispersin B 0.06 U/ml Negative control CT− (NA) (NA) Tris-HCl 20 mM Positive control CT+ EnziQure ® 1% Tris-HCl 20 mM

[0144] It can be seen in FIG. 3 that the composition comprising denarase (Ex.2) is the composition (FIG. 3A) leading to the greatest reduction in the biomass of each of the biofilms with a significant reduction and greater than 80% of the biomass of the three biofilms tested (FIG. 3A). Indeed, cellulase (CE. 3) (FIG. 3B) has no significant effect and dispersin B (CE. 4) (FIG. 3C) decreases biofilm from 70% (ATCC29213) to 27% (ATCC33591). The mixture of the 3 enzymes (Ex. 3) (FIG. 3D) is as effective as denarase alone (Ex.2) on 2 biofilms/3. However, the composition from Example 3 is less effective than the denarase from Example 2 on the biofilm of the strain ATCC29213. These results suggest that, under the experimental conditions tested, there is no synergistic effect between denarase, cellulase and dispersin B.

1.2.4 Comparison of the Disruptive Activity of the Biomass

[0145] To confirm the absence of synergy between denarase, cellulase and dispersin B, the disruptive activity of the biomass of denarase ([Example 2 (Ex.2)]—(FIG. 4A) was compared with that of enzyme duos denarase+dispersin B ([Example 4 (Ex.4)]—(FIG. 4B) and denarase+cellulase—([Example 5 (Ex.5)](FIG. 4C). The strains, experimental conditions and enzyme concentrations used are the same as in the experiment described above. In this experiment, the CT− is 20 mM Tris-HCl pH 7 without enzymes and its biomass corresponds to 100%. The CT+ is composed of 1% EnziQure® in this same buffer. The compositions tested are listed in Table 4.

TABLE-US-00004 TABLE 4 Composition - Example Enzyme Concentration Buffer Example 2 (Ex. 2) Denarase 500 U/ml Tris-HCl 20 mM Example 4 (Ex. 4) Denarase and 500 U/ml and Tris-HCl 20 mM Dispersin B 0.06 U/ml Example 5 (Ex. 5) Denarase and 500 U/ml and Tris-HCl 20 mM cellulase 7 U/ml Negative control CT− (NA) (NA) Tris-HCl 20 mM Positive control CT+ EnziQure ® 1% Tris-HCl 20 mM

[0146] Similarly to what was observed previously, the results obtained indicate that the addition of dispersin B (Example 4—FIG. 4B) or cellulase (Example 3—FIG. 4C) does not significantly increase the activity of denarase from Example 2 on the strains of staphylococci tested (FIG. 4A). We therefore do not observe any synergistic effect associated with the combination of denarase with cellulase or dispersin B.

1.2.5 Comparison of Disruptive Activity on P. aeruginosa

[0147] The disruptive activity on the biofilm of denarase according to Example 1 (500 U/ml) and of a tri-enzymatic mixture according to Example 6 composed of denarase (500 U/ml), cellulase (7 U/ml) and dispersin B (0.06 U/ml) was tested on three strains of P. aeruginosa (PAO1, ATCC27853 and 618) as a curative treatment (TGN buffer). In this experiment, the CT− is the TGN without enzymes and its biomass corresponds to 100%. The CT+ is composed of 1% EnziQure® in this same buffer.

[0148] The compositions tested are listed in Table 5.

TABLE-US-00005 TABLE 5 Composition - Example Enzyme Concentration Buffer Example 1 (Ex. 1) Denarase 500 U/ml TGN Example 6 (Ex. 6) Denarase and 500 U/ml and TGN Dispersin B 0.06 U/ml and cellulase 7 U/ml Negative control CT− (NA) (NA) TGN Positive control CT+ EnziQure ® 1% TGN

[0149] The results presented in FIG. 5 show that denarase (Ex.1: FIG. 5B) and the tri-enzymatic composition from Example 6 (Ex. 6: FIG. 5A) are equally effective in reducing the biomass of the three biofilms tested with the respective reductions of 94%, 85%, and 44% in the biomass of P. aeruginosa PAO1, ATCC27853, and 618 biofilms, in the presence of denarase. No significant difference is observed between the two compositions. These results indicate that there is no synergy between denarase, cellulase and dispersin B to reduce the biomass of P. aeruginosa biofilms. Also, interestingly, the denarase and composition from Example 6 is more effective than the multi-enzyme detergent enziQure (CT+: contains seven enzymes) in decreasing the biomass of the P. aeruginosa 618 biofilm.

Efficacy of a Composition According to the Invention in Combination with a Microbicide to Reduce the Biomass and the Viability of Biofilms Involved in Infections of the Human Body

[0150] For the tests described below, vancomycin was used as the antibiotic molecule. The antibiotic was added at different concentrations in TGN for 24 hours at 37° C. on the biofilms pretreated with the enzyme composition according to the invention (see the below sections). The biofilm was then washed before carrying out the biomass and viability measurements.

[0151] Two different biofilm models were used: static biofilm in 96-well plate (as described previously) and biofilm on titanium coupons. Titanium was chosen because it is a material widely used in the manufacture of implants (e.g. orthopaedic prostheses, dental implants, heart valves, pacemakers, etc.). The techniques used to measure viability differ between the two models and are detailed below.

2.1 Biofilm in 96-Well Plates

[0152] In this model, cell viability was measured indirectly, by measuring the metabolism of cells in the biofilm. Indeed, viability was quantified by tracking changes in the colour of resazurin (7-Hydroxy-3H-phenoxazin-3-one 10 oxide) (prestoBlue, ThermoFisher). This blue coloured compound turns pink when reduced to resorufin by the redox activity of the biofilm, activity which is directly proportional to the number of metabolically active cells in the biofilm. The quantification of resorufin was carried out by measuring fluorescence. To correlate the number of cells present in the biofilm with the fluorescent signal, a calibration curve was produced in each experiment by measuring the fluorescent signal emitted by various known cell concentrations.

2.1.1 Method of Culture, of Enzyme Treatment, of Quantifying Biomass and Viability and Analysing the Results

[0153] The experimental method for cultivating the strains and forming the biofilm is described in point 1.1 of this document. After 24 hours of biofilm formation at 37° C. in the wells of the 96-well plate, the biofilm was washed 2× with PBS (200 μl) and treated for 1 hour at 37° C. with the composition according to the invention or the control compositions (CT− and CT+) (n=4 for each condition tested). The biofilm was then washed 2× with PBS (200 μl). Two hundred microlitres of different concentrations of vancomycin prepared in TGN were then added to the biofilm and the plate was incubated for 24 hours at 37° C. Biofilms that were not treated with vancomycin were incubated in the presence of TGN only. The biofilms were then washed 2× with PBS (200 μl) before biomass and viability were analysed. Biomass was quantified via the CV staining technique following the procedure described in point 1.1. For the analysis of biomass, prestoBlue reagent (ThermoFisher) was diluted at a ratio of 1:10 in the CA-MHB (VWR) culture medium and 200 μl were added to the wells. To calculate the calibration curve, the biofilm of two CT− samples not treated with enzymes or with vancomycin was detached and homogenised in 100 μl of CA-MHB and different dilutions of this sample were carried out in CA-MHB (from 10 to 10). Ten microlitres of each dilution were then spread out on a TSA dish to perform a bacterial count. One hundred microlitres of a diluted 5×-CaMHB prestoBlue mixture were then added to 90 μl of the different dilutions. The plate containing all the samples was then incubated for 24 hours at 37° C. in the spectraMax M3 plate reader. Fluorescence was measured every 10 minutes for 18 hours (excitation 560 nm, emission 590 nm).

[0154] To analyse the effect of the compositions according to the invention, a statistical test (one-way ANOVA and Tukey's post-hoc test) was carried out. This test compares the values of fluorescence (viability) or absorbance at 570 nm (biomass) of the negative control CT− (=treatment with buffer without enzymes) and of biofilms treated with the enzyme compositions, and does so at each vancomycin concentration. The difference is significant when the pvalue of the statistical test is less than or equal to 0.05 (* used on graphs). When this is specified in the comparative examples, the activity of the various enzyme compositions is compared (one-way ANOVA and post-hoc Tukey test).

2.1.2 Results

[0155] FIG. 6 illustrates the potentiated effect of a microbicidal agent (vancomycin) as a result of the composition from Example 1 or Example 2 comprising denarase on the S. aureus ATCC33591 biofilm. The composition was tested in TGN (Ex. 1—FIGS. 6A and 6C) and in 20 mM TrisHCl pH7 (Ex. 2—FIGS. 6B and 6D). The CT− and CT+ controls were composed of buffer without enzymes. The CT+ controls were composed of 1% enziQure® (v/v) in buffer (TGN or 20 mM TrisHCl). After enzyme treatment for 1 hour at 37° C., the biofilms were incubated for 24 hours at 37° C. in the presence of the following concentrations of vancomycin: 0 mg/L, 10 and 20 mg/L.

[0156] The viability results obtained (FIGS. 6A and 6B) show that in the absence of enzyme treatment, vancomycin (10 mg and 20 mg/L) does not reduce (FIG. 5A) or only very slightly reduces (FIG. 5B) the viability of the S. aureus ATCC33591 biofilm. On the other hand, when the biofilm is pretreated with denarase from Example 1 (in TGN) and Example 2 (in 20 mM TrisHCl pH7), a significant reduction compared to the CT− of 1.35 logs and 0.5 logs, respectively, is observed after treatment with 20 mg/L of vancomycin. These decreases correspond to a decrease in viability of 96.5 and 80%, respectively, compared to the respective CT− controls without enzymes at 20 mg/L of vancomycin. The potentiating effect of denarase on vancomycin 20 mg/L is also observed on biomass with a reduction in biomass of 84% and 70% in TGN and Tris, respectively (FIGS. 6C and 6D). A similar but less significant effect on viability and biomass was measured in the presence of 10 mg/L vancomycin: 50% reduction in viability and 80% reduction in biomass in TGN, compared to CT−.

2.2 Biofilm on Titanium Coupons

[0157] In this model, viability was quantified after detachment and homogenisation of the biofilm via the technique of spreading and counting cells on a TSA culture media (VWR).

2.2 Method of Culture, of Enzyme Treatment, of Quantifying Biomass and Viability and Analysing the Results

[0158] Titanium coupons (n=3 for each condition) were placed in the wells of a 12-well plate and incubated at 37° C. with agitation (50 rpm) for 24 hours in 3 ml of bacterial inoculum at an OD of 0.005 (106 CFU/ml) in order to allow biofilm to form. The coupons were then washed 2× with PBS (2 ml) and treated for 1 hour at 37° C. with the composition according to the invention or the solution without enzymes (CT−=20 mM TrisHCl pH7). The coupons were then washed twice with PBS (2 ml) and reincubated in 750 μl of TGN containing 0 or 20 mg/L of vancomycin for 24 hours at 37° C. with agitation (50 rpm). After two washes with PBS (2 ml), the biofilm was detached and homogenised in 2 ml of PBS. Different dilutions were carried out (from 10 to 10) and 10 μl of each dilution was spread on a TSA dish. Colonies (CFU) were counted after 18 hours of incubation at 37° C.

[0159] To analyse the effect of the compositions according to the invention, a statistical test (one-way ANOVA and Tukey's post-hoc test) was carried out. This test compares the fluorescence (viability) or absorbance (biomass) values of the CT− negative control (=treatment with buffer without enzymes) and of the biofilms treated with the enzyme compositions, at each vancomycin concentration. The difference is significant when the pvalue of the statistical test is less than or equal to 0.05 (* used on graphs). When this is specified in the comparative examples, the activity of the various enzyme compositions is compared (one-way ANOVA and post-hoc Tukey test).

2.1.2 Results

Potentiating Effect of Three Ti-Enzymatic Compostions on the Efficacy of Vancomycin

[0160] FIG. 7 illustrates the potentiating effect of three tri-enzyme compositions containing denarase on the microbicidal effect of vancomycin at 20 mg/L: (i) Example 8 (Ex. 8): cellulase (7 U/ml), denarase (500 U/ml) and dispersin B (0.06 U/ml), (ii) Example 9 (Ex. 9): flavourzyme (1% v/v), denarase (500 U/ml) and dispersin B (0.06 U/ml) and (iii) BDD: Blaze® Pro (1% v/v), denarase (500 U/ml) and dispersin B (0.06 U/ml) on S. aureus ATCC33591. The buffer used is 20 mM Tris-HCl pH7.

[0161] The compositions are listed in Table 6.

TABLE-US-00006 TABLE 6 Composition - Example Enzyme Concentration Buffer Example 3 (Ex. 3) Denarase and 500 U/ml and Tris-HCl 20 mM Dispersin B and 0.06 U/ml cellulase 7 U/ml Example 8 (Ex. 8) Denarase and 500 U/ml Tris-HCl 20 mM Flavourzyme 1% v/v And Dispersin B 0.06 U/ml Example 9 (Ex. 9) Denarase and 500 U/ml Tris-HCl 20 mM Blaze ® Pro 1% v/v And Dispersin B 0.06 U/ml Negative control CT− (NA) (NA) Tris-HCl 20 mM Positive control CT+ EnziQure ® 1% Tris-HCl 20 mM

[0162] The results show that in the absence of enzyme treatment (CT−) vancomycin 20 mg/L does not decrease viability in biofilm. On the other hand, the pretreatment of biofilms with the three compositions significantly potentiate the effect of vancomycin. Indeed, with the three cocktails a similar reduction of 2.5 logs is observed in the presence of 20 mg/L of vancomycin (=99.8% reduction in viability).

Potentiating Effect of 3 Tri-Enzymatic Compostions on Vancomycin

[0163] FIG. 8 illustrates the potentiating effect of three tri-enzyme compositions containing denarase on the microbicidal effect of vancomycin at 20 mg/L: (i) Example 6: cellulase (7 U/ml), denarase (500 U/ml) and dispersin B (0.06 U/ml), (ii) Example 10: cellulase (70 U/ml), denarase (500 U/ml) and dispersin B (0.32 U/ml) and (iii) Example 11: alpha-amylase (2000 U/ml): cellulase (70 U/ml), denarase (500 U/ml) and dispersin B (0.32 U/ml). These cocktails were tested as a curative treatment on S. aureus ATCC33591 (FIG. 8A), S. epidermidis ATCC35984 (FIG. 8B) and S. aureus 144-20 (FIG. 8C) biofilms. The buffer used is TGN.

[0164] The compositions are listed in Table 7.

TABLE-US-00007 TABLE 7 Composition - Example Enzyme Concentration Buffer Example 6 (Ex. 6) Denarase and 500 U/ml and TGN Dispersin B and 0.06 U/ml cellulase 7 U/ml Example 10 (Ex. 10) Denarase and 500 U/ml TGN Dispersin B and 0.32 U/ml cellulase 70 U/ml Example 11 (Ex. 11) Denarase and 500 U/ml TGN alpha-amylase 2000 U/ml And Dispersin B 0.32 U/ml Negative control CT− (NA) (NA) TGN Positive control CT+ EnziQure ® 1% TGN

[0165] The results show that in the absence of enzyme treatment (CT−) vancomycin 20 mg/L does not significantly reduce viability in biofilm. On the other hand, the pretreatment of biofilms with the three compositions significantly potentiates the effect of vancomycin on the viability of the three strains tested. Under these conditions, a reduction in viability of 2.5 to 3 logs was observed in the presence of 20 mg/L vancomycin compared with the CT−'s not treated with the cocktails.

Efficacy of a Composition According to the Invention in Combination with a Microbicide to Prevent the Formation of Biofilm Involved in Infections of the Human Body

3.1 Model of Biofilm in 96-Well Plates

[0166] The capacity of the composition according to the invention to prevent the formation of biofilm was tested on a model of biofilm in a 96-well plate. In this experimental set-up, the enzyme compositions were added to the initial bacterial inoculum (TGN medium containing 5*106 CFU/ml), i.e. before biofilm formation. After 24 hours of growth at 37° C., the biofilms were washed and incubated in the presence of vancomycin (20 mg/L) for 24 hours at 37° C., as described in point 2.1.1. The biomass value (CV staining) and the viability (measurement of metabolism) of the biofilms were then measured and compared with the CT− condition where the starting inoculum did not contain enzymes (n=4 in each condition).

[0167] The compositions tested are listed in Table 8.

TABLE-US-00008 TABLE 8 Composition - Example Enzyme Concentration Buffer Example 10 (Ex. 10) Denarase and 500 U/ml TGN Dispersin B and 0.32 U/ml cellulase 70 U/ml Example 11 (Ex. 11) Denarase and 500 U/ml TGN alpha-amylase 2000 U/ml And Dispersin B 0.32 U/ml Negative control CT− (NA) (NA) TGN Positive control CT+ EnziQure ® 1% TGN

[0168] FIG. 9 shows the preventive effect of two compositions according to the invention containing denarase (examples 10 & 11), on different strains of S. aureus and S. epidermidis. In the presence of 20 mg/L vancomycin, the two cocktails significantly reduce the quantity of biomass formed (FIG. 9A) and the viability of the biofilm (FIG. 9B), in varying degrees between the strains.

3.2 Titanium Coupon Biofilm Model

[0169] The preventive effect on biofilm formation of the compositions according to the invention was tested using a titanium coupon model. In this experiment, the coupons were incubated for 24 hours at 37° C. with a bacterial inoculum (106 CFU/ml) in TGN containing different enzyme compositions, or not (=CT−). After biofilm formation, coupons were incubated in the presence (Van Omg/L) or the presence of 20 mg/L of vancomycin for 24 hours before analysing viability using the technique of spreading on TSA and counting.

[0170] The compositions tested are listed in Table 7.

TABLE-US-00009 TABLE 7 Composition - Example Enzyme Concentration Buffer Example 6 (Ex. 6) Denarase and 500 U/ml and TGN Dispersin B and 0.06 U/ml cellulase 7 U/ml Example 10 (Ex. 10) Denarase and 500 U/ml TGN Dispersin B and 0.32 U/ml cellulase 70 U/ml Example 11 (Ex. 11) Denarase and 500 U/ml TGN alpha-amylase 2000 U/ml And Dispersin B 0.32 U/ml Negative control CT− (NA) (NA) TGN Positive control CT+ EnziQure ® 1% TGN

[0171] FIG. 10 illustrates the preventive effect of three tri-enzyme compositions containing denarase on the microbicidal effect of vancomycin at 20 mg/L: (i) Example 6: cellulase (7 U/ml), denarase (500 U/ml) and dispersin B (0.06 U/ml), (ii) Example 10: cellulase (70 U/ml), denarase (500 U/ml) and dispersin B (0.32 U/ml) and (iii) Example 11: alpha-amylase (2000 U/ml): cellulase (70 U/ml), denarase (500 U/ml) and dispersin B (0.32 U/ml). These cocktails were tested as preventive treatment on the S. aureus ATCC33591 biofilm (FIG. 10A) and S. epidermidis ATCC35984 biofilm (FIG. 10B).

[0172] The results indicate that in the presence of vancomycin 20 mg/L the three cocktails significantly reduce viability in the biofilm formed by S. aureus ATCC33591 (decrease of 2.5 to 3 logs) (FIG. 10A) and S. epidermidis ATCC35984 (decrease of 2 to 3 logs) (FIG. 10B) after 24 hours of incubation. These observations show that the compositions according to the invention containing denarase prevent biofilm formation, and consequently potentiate the effect of an antibiotic on its capacity to reduce viability within biofilm.

[0173] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made thereto without departing from the scope of the attached claims.

[0174] For example, denarase was used in the examples, but it goes without saying that the examples could have illustrated the activity of the Benzonase® Endonuclease provided by the company Millipore Sigma

4.1 Biofilms Comprising Candida

[0175] The inventors then attempted to treat biofilms comprising Candida albicans, either in monoculture or in complex biofilm models comprising Candida albicans and prokaryotes. Different strains of Candida albicans were used: the ATCC24433 strain, as well as strains isolated from orthopaedic prosthesis infections. Subtilisin (protease from Bacillus licheniformis) and lyticase (from Arthrobacter luteus or Bacillus subtilis) were obtained at Sigma. The other enzymes are those used above.

[0176] The antifungal drug Caspofungin (Merck Sharp & Dohme) and, for complex biofilms additionally comprising one or more prokaryotes, the antibiotics Moxifloxacin HCl (Bayer) or Meropenem (Mylan) were used (24 hours of treatment, after the application of enzymes).

[0177] Cytotoxicity tests were performed on MG63 osteoblasts and other cell types.

[0178] Both subtilisin (already at concentrations of about 0.5 U/mL) and Lyticase (already at 10 U/mL) are toxic to osteoblasts, but not necessarily toxic to other cells. These are the concentrations that will be used on biofilms.

[0179] Surprisingly, the inventors observed that subtilisin (two treatment durations were tested: 30 minutes and 1 hour) sensitised biofilms comprising Candida albicans (associations with Gram-positive bacteria, here, S. aureus and/or Gram-negative bacteria: here. E. coli, were tested), or consisting solely of Candida albicans. A combination of Denarase® with Lyticase (combination applied for 30 minutes or 1 hour) also showed a synergistic effect on these biofilms. The combination of subtilisin with Denarase® did not show any advantage over the effect obtained by subtilisin alone on biofilms comprising Candida albicans.

[0180] The inventors tested a sequential application of a composition comprising Denarase, then subtilisin, with increased efficacy.