COMPOSITION COMPRISING AT LEAST ONE ENZYME AND AT LEAST ONE MICROBICIDAL MOLECULE FOR THE PREVENTION OR TREATMENT OF POST-IMPLANT INFECTIONS

Abstract

The present invention relates to a composition comprising at least one enzyme and at least one microbicidal molecule as combination products, for simultaneous use, separated use or use staggered over time, for use in the preventative and/or curative treatment of infections at an implant site, said infections being post-implant infections of mammalian bodies, in particular post-implant infections of the human body.

Claims

1. Composition comprising at least one enzyme and at least one microbicidal molecule as combination products.

2. Composition according to claim 1, wherein said at least one enzyme is chosen from the group composed of deoxyribonucleases (DNases), lipases, proteases and polysaccharide hydrolases including Dispersin B and cellulase.

3. Composition according to claim 1, wherein said at least one enzyme is present in a concentration ranging between 0.01 and 1000 mg/L.

4. Composition according to claim 1, wherein said at least one microbicidal molecule is present in a concentration ranging between 0.01 and 1000 mg/L.

5. Composition according to claim 1, wherein said at least one microbicidal molecule is chosen from the group composed of fluoroquinolones, glycopeptides, lipoglycopeptides, fusidic acid, penicillins, cephalosporins, carbapenems, monobactams, polymyxins, beta-lactams, macrolides, lincosamides, oxazolidinones, amphenicols, tetracyclines, aminoglycosides, rifamycins, nitrofurans, sulphonamides, nitroimidazoles antifungals (echinocandins, fluorocytosines, azoles, griseofulvins), lytic enzymes (for example endolysins or lysozyme), N-acetylcysteine, quaternary ammonium, biguanides, amines, halogenated derivatives (particularly of chlorhexidine), antimicrobial peptides, silver (Ag) derivatives, H.sub.2O.sub.2 derivatives, peroxy acids, phenolic derivatives, aldehydes, alcohols and mixtures thereof.

6. Composition according to claim 1, said composition being in the form of a sterile aqueous solution which may be injected or not, which may be diluted or not in water for injectable preparation or in the form of a soluble powder, preferably in the form of a lyophilisate.

7. Composition according to claim 1, said post-implantation infections of mammalian bodies, in particular of the human body, being post-implantation infections of Staphylococcus aureus bacteria, Staphylococcus epidermis bacteria, Escherichia coli bacteria, Escherichia faecalis bacteria, Klebsiella pneumoniae bacteria and Pseudomonas aeruginosa bacteria.

8. Composition according to claim 1, said post-implantation infections of mammalian bodies, in particular of the human body, being infections localised at an implantation site of an implantable medical device, for example a tube, a catheter or a prosthesis.

9. Composition according to claim 1, wherein the composition is effective to kill at least 1 Log 10, preferably at least 2 Log 10, preferentially at least 5 Log 10 of the microorganisms responsible for post-implantation infections of mammalian bodies, in particular in the human body.

10. A method for the treatment, at an implantation site, of post-implantation infections of mammalian bodies, in particular in the human body, comprising administering an effective amount of a composition of claim 1 to the mammalian body, wherein the at least one enzyme and least one microbicidal molecule are administered simultaneously, separately, or staggered over time.

11. The method according to claim 10, wherein the composition is administered by injection, by infiltration, by irrigation, by ingestion or by percutaneous application.

12. Method for treating, at an implantation site, post-implantation infections of mammalian bodies, in particular in the human body, with a composition comprising at least one enzyme and at least one microbicidal molecule as combination products, for simultaneous use, separated use or use staggered over time, said method comprising the following steps: administration, at an implantation site of an implantable medical device, of a composition comprising at least one enzyme and at least one microbicidal molecule, breakdown, by action of said at least one enzyme of said administered composition, of a biofilm present at said implantation site of said implantable medical device, and destruction of bacteria and/or inhibition of the growth of bacteria released from said biofilm, by action of said at least one microbicidal molecule of said administered composition.

13. Method for treating post-implantation infections of mammalian bodies, in particular in the human body, according to claim 12, wherein said administration is carried out by injection, by infiltration, by irrigation, by ingestion or by percutaneous application.

Description

[0057] Other features, details and advantages of the invention will become clear in the examples and appended figures.

[0058] FIG. 1 is a graph which illustrates the viability of the bacteria from an isolate of S. aureus (isolated from an implanted pacemaker) having developed a biofilm, following contact either with a composition according to the invention (ciprofloxacin as an antibiotic molecule at 3.2 mg/L+0.025% DNase I, 0.01% Dispersin B and 0.05% cellulase), or with a composition solely comprising ciprofloxacin as an antibiotic molecule at 3.2 mg/L.

[0059] FIG. 2 is a graph which illustrates the viability of the bacteria from an isolate of E. coli (isolated from an implanted urinary catheter) having developed a biofilm, following contact either with a composition according to the invention (ciprofloxacin as an antibiotic molecule at 3.2 mg/L+0.025% DNase I, 0.01% Dispersin B and 0.05% cellulase), or with a composition solely comprising ciprofloxacin as an antibiotic molecule at 3.2 mg/L.

[0060] FIG. 3 is a confocal microscopic image of an isolate of S. aureus (isolated from an implanted pacemaker) having developed a biofilm over 24 h.

[0061] FIG. 4 is a confocal microscopic image of an isolate of S. aureus (isolated from an implanted pacemaker) having developed a biofilm over 24 h and then treated with a composition solely comprising ciprofloxacin as an antibiotic molecule at 1 mg/L over 24 h.

[0062] FIG. 5 is a confocal microscopic image of an isolate of S. aureus (isolated from an implanted pacemaker) having developed a biofilm over 24 h and then treated with a composition according to the invention (ciprofloxacin at 1 mg/L+0.025% DNase I+0.01% Dispersin B+0.05% cellulase) over 24 h.

[0063] FIG. 6 is a graph which illustrates the cytotoxicity of DNase I, Dispersin B and cellulase alone or linked to human cell lines.

[0064] FIGS. 7a to 7d are graphs which illustrate the viability (% of viability with respect to the control) of the bacteria of an isolate of P. aeruginosa PA20 (isolated from an implanted arterial catheter) having developed a biofilm, following contact with different antibiotics of Cmin concentration in combination with different enzymes or a cocktail of enzymes.

[0065] FIGS. 8a to 8d are graphs which illustrate the viability (% of viability with respect to the control) of the bacteria of an isolate of P. aeruginosa PA500 (isolated from an implanted arterial catheter) having developed a biofilm, following contact with different antibiotics of Cmin concentration in combination with different enzymes or a cocktail of enzymes.

[0066] FIG. 9 is a graph which illustrates the viability (% of viability with respect to the control) of the bacteria of an isolate of S. aureus Sa2003/1083 (isolated from a knee prosthesis) having developed a biofilm, following contact with different antibiotics of Cmin concentration in combination with different enzymes or a cocktail of enzymes.

[0067] FIGS. 10a to 10c are graphs which illustrate the viability (% of viability with respect to the control) of the bacteria of an isolate of K. pneumoniae Kp826 (isolated from a central venous catheter) having developed a biofilm, following contact with different antibiotics of Cmin concentration in combination with different enzymes or a cocktail of enzymes.

[0068] FIGS. 11a to 11f are graphs which illustrate the cytotoxicity of compositions according to the invention on human cell lines.

EXAMPLES

Example 1

Effectiveness and Cytotoxicity of a First Composition According to the Invention Used in the Treatment of Post-Implantation Infections

A. Effectiveness

[0069] In order to test the effectiveness of a composition according to the invention comprising at least one enzyme and at least one biocidal and/or antibiotic molecule (microbicidal molecule) to treat post-implantation infections of mammalian bodies, in particular of the human body, several experiments were conducted from isolates taken from medical devices infected by biofilms in the human body following their implantation. These isolates are listed in Table 1 below.

[0070] Three different experiments, carried out according to three biofilm models, were conducted: (1) static model of in-vitro biofilm, (2) dynamic model of in-vitro biofilm and (3) dynamic model of biofilm in a bioreactor.

[0071] Furthermore, confocal microscopic images were taken for an isolate in order to visualise the dispersal of the bacteria and the effectiveness of a composition according to the invention in terms of bacteria survival.

[0072] To carry out these experiments, different compositions according to the invention were prepared under agitation (120 RPM) by dilution of at least one antibiotic and/or biocidal molecule in an aqueous solution (water buffered with 20 mM tris(hydroxymethyl)aminomethane and with a pH of 7.5) comprising at least one enzyme. For the tests presented below, ciprofloxacin was used as the antibiotic molecule.

[0073] In order to judge the effectiveness of a post-implantation treatment carried out with a composition according to the invention, the viability of the bacteria was measured by following the development of the colour of the resazurin (7-Hydroxy-3H-phenoxazin-3-on 10-oxide), this colour changing according to the redox potential which depends on microbial activity. The measurement was conventionally carried out by absorption photometry (measurement at 590 nm with the Spectramax M4 equipment). Based on the values measured for different concentrations of the antibiotic and/or biocidal molecule in the presence of an enzymatic composition fixed in the composition according to the invention, the dose-response curves, and more particularly the EC.sub.50, that is the necessary concentration of the antibiotic and/or biocidal molecules for ensuring a 50% reduction of the viability of the bacteria, which corresponds to a 50% reduction of the size of the signal measured by absorption photometry, were determined.

TABLE-US-00001 Isolate Strain Bacteria Origin 1 80124430375 S. aureus MRSA pacemaker 2 80224422456 S. aureus MRSA knee prosthesis 3 80124474762 S. aureus MRSA knee screw 4 80224420266 S. epidermis central venous catheter (CVC) 5 6081 E. coli urinary catheter 6 5701 E. coli orthopaedic implant 7 9794 E. faecalis urinary catheter 8 9781 E. faecalis urinary catheter 9 9555 E. faecalis urinary catheter 10 DIV5508 P. aeroginosa central venous catheter (CVC) 11 04/190 P. aeroginosa urinary catheter

[0074] 1. Static Model of In-Vitro Biofilm

[0075] From each of the isolates given in table 1, biofilms (n=4) were developed over 24 hours at a temperature of 37 C. in the wells of a 96-well plate containing 200 L of a TSB culture medium (Tryptic Soy Broth VWR) supplemented with 1% glucose and 2% sodium chloride.

[0076] Then, over a second 24-hour period, the biofilms developed in the wells were subjected to growing concentrations (from 0.15 to 40 mg/L) of ciprofloxacin as an antibiotic in an aqueous solution comprising 0.025% DNase I (VWR), 0.01% Dispersin B (Symbiose Biomaterials) and 0.05% cellulase (Carezyme of Novozyme).

[0077] It should be noted here that the enzyme percentages are in wt % which express the quantities by the weight of each commercial enzyme with respect to the total weight of the composition. This applies to the entirety of this document.

[0078] Before proceeding with the measurement of the viability of the bacterial cells with resazurin, the plates containing the developed biofilms were washed with a PBS buffer (pH=7.4) then incubated with resazurin (0.01 mg/L) for 1 hour in the dark.

[0079] The results obtained are listed in Table 2 below which presents, based on the established dose-response curves, the concentrations (mg/L) necessary in the antibiotic molecule in order to reduce the viability of the studied bacteria having developed a biofilm by 50%, also known as EC.sub.50 (which corresponds to a 50% reduction of the size of the signal measured by absorption photometry).

[0080] The increase in activity of the antibiotic molecule, or the decrease of its EC.sub.50, when it is linked to 0.025% DNase I, 0.01% Dispersin B and 0.05% cellulase, is also given in Table 2 (ratio of EC.sub.50: solution B/solution A).

[0081] As can be seen, for each of the isolates tested, a composition according to the invention allowed the activity of the antibiotic molecule to be systematically increased in a significant manner, also known as reducing the EC.sub.50 in a significant manner. These results indicate, in comparison with a composition only containing the antibiotic molecule, that the bacteria were reached and killed by the molecule in a much more effective manner. This is explained by the presence of enzymes which truly break the biofilm down in such a way that the bacteria are released and then come into direct contact with the antibiotic molecule.

TABLE-US-00002 TABLE 2 Concentration of the antibiotic molecule in a solution comprising the antibiotic Concentration of the antibiotic alone molecule in a solution (solution A) comprising the antibiotic in to reduce association with 0.025% DNase Increase in the viability I, 0.01% Dispersin B and 0.05% activity of of the bacteria cellulase (solution B) to reduce the by 50% the viability of the bacteria by antibiotic Isolate (EC.sub.50) 50% (EC.sub.50) molecule 1 >40.sup.a 1.5 >26.6 2 0.8 0.1 8 3 1.2 0.2 6 4 1.77 0.08 22.1 5 2.7 0.11 24.5 6 >40 0.23 >173.9 7 >40 0.25 >160 8 >40 1.4 >28.5 9 >40 2.5 >16 10 >40 1.26 >31 11 >40 0.05 >792 .sup.a>40 means that the 50% reduction of the microbial population for a concentration of 40 mg/L in the antibiotic did not occur. The EC.sub.50 is thus greater than 40 mg/L.

[0082] 2. Dynamic Model of In-Vitro Biofilm

[0083] 20 L of liquid culture from isolates 1 and 5 (n=4) was inoculated in the wells of a 96-well plate containing 180 L of a TSB culture medium (Tryptic Soy Broth VWR) supplemented with 1% glucose and 2% sodium chloride then a PEG platform was immersed so the biofilms developed on the PEG protrusions for 48 hours at a temperature of 37 C. under constant agitation at 120 RPM.

[0084] Then, over a second 24-hour period, the biofilms developed in the wells on the protrusions were subjected, still under constant agitation at 120 RPM, to growing concentrations (from 0.15 to 40 mg/L) of ciprofloxacin as an antibiotic in an aqueous solution comprising 0.025% DNase I (VWR), 0.01% Dispersin B (Symbiose Biomaterials) and 0.05% cellulase (Carezyme of Novozyme).

[0085] Before proceeding with the measurement of the viability of the bacterial cells with resazurin, the PEG plates with the protrusions containing the developed biofilms were incubated with resazurin for 2 hours in the dark.

[0086] The results obtained are listed in Table 3 below which presents, based on the established dose-response curves, the concentrations (mg/L) necessary of the antibiotic molecule in order to reduce the viability of the studied bacteria having developed a biofilm by 50% (which corresponds to a 50% reduction of the size of the signal measured by absorption photometry). The increase in activity of the antibiotic molecule, or the decrease of its EC.sub.50, when it is linked to 0.025% DNase I, 0.01% Dispersin B and 0.05% cellulase, is also given in Table 2 (ratio of EC.sub.50:solution B/solution A).

[0087] As can be seen, for isolates 1 (S. aureus isolated from a pacemaker on which a biofilm developed in the human body after implantation) and 5 (E. coli isolated from a urinary catheter on which a biofilm developed in the human body after implantation), a composition according to the invention allows the activity of the antibiotic molecule to be systematically increased, also reducing the EC.sub.50, in a significant manner.

TABLE-US-00003 Concentration of the antibiotic molecule Concentration of the in a solution antibiotic molecule in a comprising the solution comprising the antibiotic alone antibiotic in association with (solution A) to 0.025% DNase I, 0.01% Increase in reduce Dispersin B and 0.05% activity the viability of the cellulase (solution B) to of the bacteria reduce the viability of the antibiotic Isolate by 50% (EC.sub.50) bacteria by 50% (EC.sub.50) molecule 1 0.7 0.001 700 5 3.18 0.004 795

[0088] These results indicate, in comparison with a composition only containing the antibiotic molecule, that the bacteria were reached and killed by the molecule in a much more effective manner. This is explained by the presence of enzymes which truly break the biofilm down in such a way that the bacteria are released and then come into direct contact with the antibiotic molecule.

[0089] 3. Dynamic Model of Biofilm in a Bioreactor

[0090] Isolates 1 and 5 (n=3) were inoculated on polycarbonate coupons then placed in a CDC bioreactor (BioSurfaces Technologies) containing 300 ml of a TSB culture medium (Tryptic Soy Broth VWR) supplemented with 1% glucose and 2% sodium chloride in order to develop biofilms for 20 hours at a temperature of 37 C. under constant agitation at 120 RPM. The development was more particularly carried out in two successive phases, namely (1) a first incubation phase of 6 hours at a bacterial concentration of 10.sup.5 bacteria/ml and (2) a second phase of 14 hours during which continuous circulation at 10 ml/minute of the culture medium was carried out in the bioreactor through use of a peristaltic pump (Masterflex). Following this development of biofilms carried out in two phases, the latter were subjected, in the bioreactor, to a concentration of 3.2 mg/L of ciprofloxacin as an antibiotic in an aqueous solution comprising 0.025% DNase I (VWR), 0.01% Dispersin B (Symbiose Biomaterials) and 0.05% cellulase (Carezyme of Novozyme).

[0091] The coupons were then aseptically removed after 0, 4, 8, 12, 18 and 24 hours and rinsed twice in a PBS buffer before sonication of the formed biofilms. Successive dilutions in a PBS buffer (pH=7.4) were then carried out from the samples thus obtained before plating on a TSA culture medium (VWR) for counting the bacterial colonies (Log.sub.10 CFU/ml) following incubation of the culture media for 18 hours at a temperature of 37 C. and a relative humidity of 60%.

[0092] The results obtained are presented in FIGS. 1 and 2 from which it is again made clear that, for each of the isolates tested, a composition according to the invention allows the activity of the antibiotic molecule to be systematically increased in a significant manner. In fact, regarding isolate 1 (S. aureus isolated from a pacemaker on which a biofilm developed in the human body after implantation), a logarithmic reduction in the order of 5 of the number of live bacteria was observed in comparison with a logarithmic reduction in the order of 0.5 when the antibiotic molecule was used alone (see FIG. 1). The same observation was conducted for isolate 5 (E. coli isolated from an urinary catheter on which a biofilm developed in the human body after implantation): logarithmic reduction in the order of 5 of the number of live bacteria during treatment with a composition according to the invention and in the order of 0.5 for a composition containing only the antibiotic (see FIG. 2).

[0093] These results indicate, in comparison with a composition only containing the antibiotic molecule, that the bacteria were reached and killed by the molecule in a much more effective manner. This is explained by the presence of enzymes which truly break the biofilm down in such a way that the bacteria are released and then come into direct contact with the antibiotic molecule.

[0094] 4. Confocal Microscopic Images

[0095] For isolate 1 (S. aureus isolated from a pacemaker on which a biofilm developed in the human body after implantation), confocal images were taken and correspond to FIGS. 3 to 5. FIG. 3 corresponds to an image of the biofilm developed by isolate 1 over 24 h; FIG. 4 corresponds to an image of the biofilm developed by isolate 1 over 24 h after treatment (incubation) with a composition containing only the antibiotic molecule (ciprofloxacin at 1 mg/L) for 24 h; FIG. 5 corresponds to an image of the biofilm developed over 24 h by isolate 1 after treatment (incubation) with a composition according to the invention (ciprofloxacin at 1 mg/L+0.025% DNase I+0.01% Dispersin B+0.05% cellulase) for 24 h.

[0096] Before obtaining the different images, staining was carried out with the LIVE/DEAD kit (Invitrogen) for 30 minutes before rinsing with a PBS buffer.

[0097] From these confocal images it appears that, as for the tests with the biofilm models above, a composition according to the invention is much more effective than a composition not containing the antibiotic molecule. In fact, FIG. 5 (treatment with a composition according to the invention) shows that all the bacteria have been killed (destroyed) (M) whereas FIG. 4 (treatment with the antibiotic molecule alone) shows, on the contrary, that numerous bacteria are still alive (V). As before, this is explained by the presence of enzymes which will truly break the biofilm down in such a way that the bacteria are released and then come into direct contact with the antibiotic molecule.

[0098] B. Cytotoxicity

[0099] In order to judge the cytotoxicity of a composition according to the invention used in the treatment of post-implantation infections of mammalian bodies, in particular of the human body, three human cell lines, THP-1, U937 and HL-60, were used. For each line, cells (10.sup.4 cells/ml) were incubated for 4 h in the wells of a 96-well plate in the presence of an enzymatic cocktail according to the invention (0.025% DNase I+0.01% Dispersin B+0.05% cellulase) or in the presence of each of these enzymes at the same concentration as they are in the enzymatic cocktail (DNase IDNase or Dispersin BDispB or cellulaseCarezyme).

[0100] The cytotoxicity was evaluated based on the amount of lactate dehydrogenase (LDH) present in the supernatant by using the PLUS detection kit (Roche, Basel, Switzerland). The level of LDH naturally salted-out by the non-treated cells of each line was measured (negative control) as well as the maximum level of LDH released by the same cells (positive control). The cytotoxicity was calculated according to the following formula: (value measured from the samplevalue of the negative control)/(value of the positive controlvalue of the negative control)100.

[0101] The results obtained are presented in FIG. 6 where it can be seen that the enzymatic cocktail, like each of the enzymes, according to the invention shows no toxicity for human cell lines.

Example 2

Effectiveness and Cytotoxicity of Different Compositions According to the Invention Used in the Treatment of Post-Implantation Infections

[0102] Compositions other than that described in Example 1 were tested in terms of effectiveness and cytotoxicity in order to establish that different combinations of enzymes with different microbicidal molecules (antibiotics) are able to provide treatment for post-implantation infections. The object of the tests carried out in Example 2 is essentially to highlight that the present invention is not limited to a particular composition such as that of Example 1, but that a whole range of different combinations and thus different compositions according to the invention are effective in the treatment of post-implantation infections.

[0103] Different clinical strains were isolated from medical devices infected by biofilms in the human body following their implantation. These isolates, as well as their origins, are listed in Table 4 below.

TABLE-US-00004 TABLE 4 Strain Bacterial species Origin PA20 P. aeruginosa arterial catheter PA500 P. aeruginosa arterial catheter Sa2003/1083 S. aureus MRSA knee prosthesis Kp826 K. pneumoniae central venous catheter

A. Effectiveness: Static Model of In-Vitro Biofilm

[0104] a) Culturing and Formation of Biofilm from Isolates of P. aeruginosa (PA20 and PA500)

[0105] 5 L of a glycerol stock of PA20 or PA500 was added to 5 ml of LB-glucose culture medium (medium formed of a mixture of Luria Bertani (LB) and 1% glucose) and incubated for 24 h at 37 C. The bacterial suspension was then diluted with the LB-glucose medium in order to obtain an inoculum of 10.sup.6 bacteria/ml.

[0106] The biofilms were cultivated on 96-well plates following an addition of 200 L of the inoculum in each well. The plates were then incubated at 37 C. for 48 h with a replenishment of the medium after 24 h.

[0107] The biofilms obtained were then treated for 24 h with different enzymes (Dispersin B, DNase, cellulase (carezyme), savinase (protease), lipolase (lipase), amylase (stainzyme) and mannanase) in combination with different antibiotics at maximum concentration (Cmax) or minimum concentration (Cmin), Cmax being the maximum serum concentration after administration of the antibiotic and Cmin the minimum serum concentration. These concentrations Cmin and Cmax are based on those recommended by EUCAST (The European Committee on Antimicrobial Susceptibility Testing).

[0108] The treatments of the biofilm with the use of different combinations of an antibiotic with one or several enzymes were carried out in accordance with the following concentrations of antibiotics and enzymes: [0109] enzymes: 0.05%; DCC=0.025% DNase+0.01% Dispersin B (DspB)+0.05% cellulase (Ce) [0110] amikacin: Cmin=5 mg/L (Cmax=24 mg/L) [0111] tobramycin: Cmin=0.9 mg/L (Cmax=4 mg/L) [0112] moxifloxacin: Cmin=0.3 mg/L (Cmax=3.6 mg/L) [0113] meropenem: Cmin=0.1 mg/L (Cmax=20 mg/L) [0114] ciprofloxacin: Cmin=0.6 mg/L (Cmax=3.2 mg/L)

[0115] Fluorescein diacetate (FDA) was used to measure the viable P. aeruginosa (PA20 or PA500) bacteria. The plates containing the biofilms were washed twice with an MOPS buffer then incubated in FDA (100 g/ml) for 1 h in the dark. FDA is a non-fluorescent hydrolysable in a fluorescent yellow component (fluorescein) by non-specific intracellular esterases produced by the viable bacteria. The measurement was carried out by spectrophotometry (length of excitation wavelength at 494 nm and transmission at 518 nm) with a spectramax M4. The amount of fluorescein measured by fluorimetry is directly proportional to the number of viable bacteria in the medium. The results obtained are presented in FIGS. 7 and 8.

[0116] FIGS. 7a to 7d illustrate the viability (% of viability with respect to the controlnon-treated) of the bacteria of an isolate of P. aeruginosa PA20 (isolated from an implanted arterial catheter) having developed a biofilm, following contact with [0117] (A) amikacin at Cmin concentration as an antibiotic in combination with savinase (Say) as the sole enzyme or with lipolase (Li) as the sole enzyme or with savinase (Say) and lipolase (Li) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0118] (B) tobramycin at Cmin concentration as an antibiotic in combination with savinase (Say) as the sole enzyme or with amylase (Am) as the sole enzyme or with savinase (Say) and amylase (Am) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0119] (C) moxifloxacin at Cmin concentration as an antibiotic in combination with savinase (Say) as the sole enzyme or with mannanase (Ma) as the sole enzyme or with savinase (Say) and mannanase (Ma) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0120] (D) meropenem at Cmin concentration as an antibiotic in combination with DNase as the sole enzyme or with mannanase (Ma) as the sole enzyme or with DNase and mannanase (Ma) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC).

[0121] FIGS. 8a to 8d illustrate the viability (% of viability with respect to the controlnon-treated) of the bacteria of an isolate of P. aeruginosa PA500 (isolated from an implanted arterial catheter) having developed a biofilm, following contact with [0122] (A) ciprofloxacin at Cmin concentration as an antibiotic in combination with savinase (Say) as the sole enzyme or with lipolase (Li) as the sole enzyme or with savinase (Say) and lipolase (Li) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0123] (B) amikacin at Cmin concentration as an antibiotic in combination with DNase as the sole enzyme or with lipolase (Li) as the sole enzyme or with DNase and lipolase (Li) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0124] (C) tobramycin at Cmin concentration as an antibiotic in combination with DNase as the sole enzyme or with savinase (Say) as the sole enzyme or with DNase and savinase (Say) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0125] (D) moxifloxacin at Cmin concentration as an antibiotic in combination with DNase as the sole enzyme or with savinase (Say) as the sole enzyme or with DNase and savinase (Say) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC).

[0126] In the graphs of FIGS. 7a to 7d and 8a to 8d the results of a statistical test are shown (One-way ANOVA with multiple comparison Dunnett's test) carried out in order to determine if a significant difference can be observed between the Cmax of the antibiotic and the Cmin of the antibiotic associated with one or several enzymes. The characters *, ** and *** respectively mean p<0.05, p<0.01 and p<0.001.

[0127] From these graphs, it can be observed that all the combinations [antibiotic at Cmin+enzyme(s)] tested allow the viability of the bacteria of the P. aeruginosa species (both for the PA20 strain and the PA500 strain of P. aeruginosa) to be reduced in a significant manner (p value at least less than 0.05) in comparison to the sole antibiotic at a concentration of Cmax. Similarly, it can be noted that a significant difference was observed between all the combinations [antibiotic at Cmin+enzyme(s)] tested and the control (non-treated biofilm), which is not the case for a comparison between the effect of the antibiotic alone (at Cmin or Cmax) and the control (non-treated biofilm).

[0128] This shows the advantage of combining an antibiotic and at least one enzyme for the preventative and/or curative treatment of post-implantation infections at an infection site, said infections being post-implantation infections.

[0129] b) Culturing and Formation of Biofilm from Isolates of S. aureus (Sa2003/1083) and K. pneumoniae (Kp826)

[0130] 5 L of a glycerol stock of Sa2003/1083 or Kp826 was added to 5 ml of TGN medium (medium formed of a mixture of Tryptic Soy Broth, 1% glucose and 2% NaCl) and incubated for 24 h at 37 C. The bacterial suspension was then diluted with the TGN medium in order to obtain an inoculum of 10.sup.6 bacteria/ml.

[0131] The biofilms were cultivated on 96-well plates following an addition of 200 L of the inoculum in each well. The plates were then incubated at 37 C. for 24 h.

[0132] The biofilms obtained were then treated for 24 h with different enzymes in combination with different antibiotics at Cmax or Cmin.

[0133] The treatments of the biofilm with the use of different combinations of an antibiotic with one or several enzymes were carried out in accordance with the following concentrations of antibiotics and enzymes: [0134] enzymes: 0.05%; DCC=0.025% DNase+0.01% Dispersin B (DspB)+0.05% cellulase (Ce) [0135] amikacin: Cmin=5 mg/L (Cmax=24 mg/L) [0136] tobramycin: Cmin=0.9 mg/L (Cmax=4 mg/L) [0137] moxifloxacin: Cmin=0.3 mg/L (Cmax=3.6 mg/L) [0138] meropenem: Cmin=0.1 mg/L (Cmax=20 mg/L) [0139] ciprofloxacin: Cmin=0.6 mg/L (Cmax=3.2 mg/L)

[0140] The plates containing the biofilms were washed twice with a PBS buffer then incubated for 30 minutes in the dark in 200 L of resazurin (10 m/ml) in each well. Resazurin (7-Hydroxy-3H-phenoxazin-3-on 10-oxide) is a blue-coloured non-toxic dye which can spread in bacteria and then be reduced to resorufin, a fluorescent compound. The viability of the bacteria was thus measured by following the development of the fluorescence of the resorufin which is directly proportional to the number of viable bacteria in the medium.

[0141] The measurement was conventionally measured by spectrophotometry (length of excitation wavelength at 560 nm and transmission at 590 nm) with a spectramax M4. The results obtained are presented in FIGS. 9 and 10a to 10c.

[0142] FIG. 9 illustrates the viability (% of viability with respect to the controlnon-treated) of the bacteria of an isolate of S. aureus Sa2003/1083 (isolated from a knee prosthesis) having developed a biofilm, following contact with ciprofloxacin at Cmin concentration as an antibiotic in combination with DNase as the sole enzyme or with mannanase (Ma) as the sole enzyme or with DNase and mannanase (Ma) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC).

[0143] FIGS. 10a to 10c illustrate the viability (% of viability with respect to the control) of the bacteria of an isolate of K. pneumoniae Kp826 (isolated from a central venous catheter) having developed a biofilm, following contact with [0144] (A) ciprofloxacin at Cmin concentration as an antibiotic in combination with DNase as the sole enzyme or with cellulase (Ce) as the sole enzyme or with DNase and cellulase (Ce) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0145] (B) amikacin at Cmin concentration as an antibiotic in combination with Dispersin B (DspB) as the sole enzyme or with cellulase (Ce) as the sole enzyme or with Dispersin B (DspB) and cellulase (Ce) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC); [0146] (C) tobramycin at Cmin concentration as an antibiotic in combination with Dispersin B (DspB) as the sole enzyme or with cellulase (Ce) as the sole enzyme or with Dispersin B (DspB) and cellulase (Ce) as enzymes or with Dispersin B (DspB), DNase and cellulase (Ce) as enzymes (DCC).

[0147] In the graphs of FIGS. 9a to 9c the results of a statistical test are shown (One-way ANOVA with multiple comparison Dunnett's test) carried out in order to determine if a significant difference can be observed between the Cmax of the antibiotic and the Cmin of the antibiotic associated with one or several enzymes. The characters *, ** and *** respectively mean p<0.05, p<0.01 and p<0.001.

[0148] From these graphs, it can be observed that all the combinations [antibiotic at Cmin+enzyme(s)] tested allow the viability of the bacteria of the S. aureus Sa2003/1083 species and the K. pneumoniae Kp862 species to be reduced in a significant manner (p value at least less than 0.05) in comparison to the sole antibiotic at a concentration of Cmax. Similarly, it can be noted that a significant difference is observed between all the combinations [antibiotic at Cmin+enzyme(s)] tested and the control (non-treated biofilm), which is not the case for a comparison between the effect of the antibiotic alone (at Cmin or Cmax) and the control (non-treated biofilm).

[0149] This shows the advantage of combining an antibiotic and at least one enzyme for the preventative and/or curative treatment of post-implantation infections at an infection site, said infections being post-implantation infections.

[0150] B. Cytotoxicity

[0151] A protocol identical to that described under point B of Example 1 was carried out in order to judge the cytotoxicity of different combinations of an antibiotic and an enzyme on the human cell lines THP1, U937 and T24. The results obtained are presented in FIGS. 11a to 11f. In these graphs, the negative control allows the activity of the LDH released by the normal non-treated cells (spontaneous release LDH) to be determined and the positive control allows the maximum activity of the LDH released by treated cells to be determined with a cell lysis solution. To conducts these tests, the following concentrations were observed: [0152] enzymes: 0.05%; DCC=0.025% DNase+0.01% Dispersin B (DspB)+0.05% cellulase (Ce) [0153] amikacin: Cmin=5 mg/L [0154] tobramycin: Cmin=0.9 mg/L [0155] moxifloxacin: Cmin=0.3 mg/L [0156] meropenem: Cmin=0.1 mg/L [0157] ciprofloxacin: Cmin=0.6 mg/L

[0158] FIG. 11a illustrates the salting-out of the LDH (%) of the human cell lines THP1, U937 and T24 for savinase (Sav) in combination with the antibiotic ciprofloxacin (CIP) or the antibiotic tobramycin (TOB) or the antibiotic amikacin (AMI) or the antibiotic moxifloxacin (MOX). FIG. 11b illustrates the salting-out of the LDH (%) of the human cell lines THP1, U937 and T24 for DNase in combination with the antibiotic ciprofloxacin (CIP) or the antibiotic tobramycin (TOB) or the antibiotic amikacin (AMI) or the antibiotic moxifloxacin (MOX) or the antibiotic meropenem (MEROP). FIG. 11c illustrates the salting-out of the LDH (%) of the human cell lines THP1, U937 and T24 for Dispersin B (DspB) in combination with the antibiotic ciprofloxacin (CIP) or the antibiotic tobramycin (TOB) or the antibiotic amikacin (AMI). FIG. 11d illustrates the salting-out of the LDH (%) of the human cell lines THP1, U937 and T24 for lipase (Li) in combination with the antibiotic ciprofloxacin (CIP) or the antibiotic amikacin (AMI). FIG. 11e illustrates the salting-out of the LDH (%) of the human cell lines THP1, U937 and T24 for cellulase (Ce) in combination with the antibiotic tobramycin (TOB) or the antibiotic amikacin (AMI). FIG. 11f illustrates the salting-out of the LDH (%) of the human cell lines THP1, U937 and T24 for mannanase (Ma) in combination with the antibiotic ciprofloxacin (CIP) or the antibiotic meropenem (MEROP).

[0159] From these graphs in FIGS. 11a to 11f, it can be concluded that none of the combinations (antibiotic+enzyme) show any toxicity for the human cell lines tested.

[0160] C. Conclusion from Example 2

[0161] From Example 2, it is very clear that the present invention is not limited to a particular composition such as that of Example 1, but rather that a whole range of different combinations and thus different compositions according to the invention are effective in the treatment of post-implantation infections. Furthermore, it was shown that the compositions according to the invention are not cytotoxic.

[0162] It is understood that the present invention is in no way limited to the embodiments described above and that modifications may be made without departing from the scope of the appended claims.