Osteocrin, Lebetin or ANP for Destroying Bacterial Biofilms

Abstract

The present invention relates to a natriuretic peptide selected from the group made up of (i) osteocrin, an osteocrin propeptide of osteocrin derivative, (ii) a lebetin, a lebetin fragment, or a lebetin or lebetin fragment derivative, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative, for the use thereof in a method for therapeutically treating a bacterial infection associated with a bacterial biofilm in a subject, wherein said natriuretic peptide disperses the bacterial biofilm. In a particular embodiment, the natriuretic peptide is used in combination with an antibiotic.

Claims

1-10. (canceled)

11. A method of therapeutically treating a bacterial infection associated with a bacterial biofilm in a subject in need thereof, the method comprising: administering a therapeutically effective amount of a natriuretic peptide to the subject to disperse the bacterial biofilm, wherein the natriuretic peptide is selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment, or a lebetin or lebetin fragment derivative, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative; thereby therapeutically treating the bacterial infection associated with the bacterial biofilm in the subject.

12. The method according to claim 11, wherein the bacterial infection is an infection with Pseudomonas aeruginosa.

13. The method according to claim 11, wherein the subject suffers from a chronic respiratory pathology.

14. The method according to claim 11, wherein the subject suffers from cystic fibrosis.

15. The method according to claim 11, wherein the subject has an implant or prosthesis, or suffers from major burns or a surface infection.

16. The method according to claim 11 further comprising administering a therapeutically effective amount of an antibiotic in combination with the natriuretic peptide.

17. The method according to claim 16, wherein the antibiotic is an aminoglycoside antibiotic or a quinolone.

18. The method according to claim 11, further comprising administering a therapeutically effective amount of at least one other additional natriuretic peptide in combination with the first natriuretic peptide.

19. A pharmaceutical composition comprising: (a) a natriuretic peptide selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment, or a lebetin or lebetin fragment derivative, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative; and (b) an antibiotic.

20. A pharmaceutical antibacterial combination for therapeutically treating a bacterial infection associated with a bacterial biofilm in a subject in need thereof, the pharmaceutical antibacterial combination comprising: (a) a natriuretic peptide selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment, or a lebetin or lebetin fragment derivative, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative; and (b) an antibiotic for simultaneous, separate, or sequential use with the natriuretic peptide.

21. An in vitro or ex vivo method to disperse a bacterial biofilm, the method comprising: applying to the bacterial biofilm a natriuretic peptide selected from the group consisting of (i) osteocrin, an osteocrin propeptide or osteocrin derivative, (ii) a lebetin, a lebetin fragment, or a lebetin or lebetin fragment derivative, and (iii) an ANP peptide, ANP propeptide or ANP peptide derivative; thereby dispersing the bacterial biofilm.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0175] FIG. 1 gives graphs representing the growth of P. aeruginosa in the presence of different concentrations of ANP.

EXAMPLES

Example 1: Effect of ANP Alone (0.1 μM) on Biofilms in Formation, on Preformed Biofilms of P. aeruginosa and on the Growth of P. aeruginosa

[0176] This example shows the action of ANP on pre-formed biofilms of P. aeruginosa.

Material and Methods

Tested Substances, Bacterial Strains and Bacterial Cultures

[0177] The P. aeruginosa wild-type strain PA14 used was supplied by the Harvard Medical School (Boston, Mass.) (Liberati et al. (2006) Proc. Natl. Acad. Sci. USA 103: 2833-2838).

[0178] The bacterial strains were cultured at 37° C. in Luria Bertani medium (LB) under agitation.

Formation of the P. aeruginosa Biofilm Under Dynamic Conditions

[0179] After 3 h pre-culture in LB medium at 37° C., P. aeruginosa was inoculated at OD.sub.600=0.08 in LB medium and sub-cultured for 2 h.

[0180] The ANP (Calbiochem Merck) was added 2 h after the start of culture, a time corresponding to the middle of the exponential growth phase of the bacteria. Final bacterial density and absence of contamination were controlled by seeding.

[0181] The biofilms were formed under dynamic conditions at 37° C. in a 3-channel flow chamber as described in Bazire et al. (2010) J. Bacteriol. 192:3001-3010. Briefly, from an 18 h pre-culture, a bacterial suspension at OD.sub.600=0.08 prepared in sterile physiological saline solution was injected into each channel of the flow chamber. The bacteria were left for 2 h at 37° C. under static conditions (no flow) so that they adhered to the glass slide. Study of biofilm formation was then conducted under a flow of LB medium (2.5 ml/h) for 24 h at 37° C.

Destruction of the P. aeruginosa Biofilm Under Dynamic Conditions.

[0182] To study the impact of ANP on 24 h pre-formed biofilms, 300 μl of ANP (0.1 μM) or 300 μl of sterile ultra-pure water (control conditions) were injected into different channels of the flow chamber. Treatment of the preformed biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0183] The biofilms were observed under a confocal laser scanning microscope (Zeiss LSM710 (Zeiss) after being stained for 15 min with 5 μM of Syto9 Green fluorochrome (Invitrogen). Images were taken in the different layers of the biofilm allowing three-dimensional reconstitution; at least 3 images were taken at different points of one same channel of the flow chamber. The files of each image were then analysed with COMSTAT software (Heydorn et al. (2000) Microbiology 146:2395-2407); 3 images of at least 3 independent experiments were analysed allowing determination of the average and maximum thicknesses of the biofilms and of bacterial biovolume.

Cell Culture

[0184] The line of human pulmonary epithelial cells of type II A549 (ATCC-CCL185TM, ATCC Manassas, Va.) was placed in culture at 37° C. under 5% CO.sub.2 atmosphere in DMEM medium (Lonza) supplemented with 10% foetal calf serum (Lonza) and 1% penicillin and streptomycin (Penistrep, Lonza). Under routine cell culture, the cells were seeded in a 25 ml flask and used at 80% confluence.

[0185] For cytotoxicity tests, the cells were seeded in 24-well plates to a final density of 3×10.sup.5 cells per well and cultured 48 h before use. For a minimum time of 24 h before the infection tests, the cells were deprived of antibiotics and foetal calf serum through the addition of a medium without fresh serum.

Measurement of the Release of Cytosolic Lactate Dehydrogenase (LDH) by A549 Cells

[0186] LDH is a stable cytosolic enzyme released into the culture medium after cell lysis and hence a marker of cell death. The amount of LDH released by the eukaryote cells in the presence of bacteria, whether or not exposed to ANP (concentration of 1 μM at 10 nM), was determined using the Cytotox 96 enzymatic test (Promega, Charbonnières, France). The A549 cells were incubated for 6 h on a control (without treatment) or with P. aeruginosa PA14 (pre-treated with ANP) with a multiplicity of infection of 10. A lysis buffer consisting of Triton X-100 solution (9% in water), was employed to determine the maximum LDH potentially released by the A549 cells under the experimental conditions (100% LDH release). A level of background noise was determined using the culture medium alone and defined as 0% LDH release, to exclude contribution by the culture medium. Percentage LDH release in the cell population was then calculated with the equation:

[00001]$\%LDH=\frac{\mathrm{OD}\mathrm{sample}}{\mathrm{OD}100\%}\times 100$

The test was sufficiently sensitive to measure an LDH concentration equivalent to lysis of 1 of the cell population.

Results

[0187] Effect of ANP on a Preformed Biofilm of P. aeruginosa

[0188] The inventors examined the potential anti-biofilm activity of ANP on an established biofilm of P. aeruginosa. From this perspective, 24 h after the formation of a P. aeruginosa biofilm in a dynamic flow system, the inventors exposed the bacteria to ANP for 2 h at 0.1 μM. Under these conditions, the inventors observed dispersion of the biofilm which had been perturbed after exposure to ANP and absence of the mushroom-shaped structures seen under the control conditions and characteristic of a P. aeruginosa biofilm. The bacterial biomass was reduced after exposure to ANP for 2 hours at 0.1 μM by 81.4±4.1% (P<0.001) compared with the biomass present under the control conditions. In parallel, the inventors observed a major reduction in the thickness of the biofilm after 2 h exposure to ANP.

[0189] Similar results were obtained when the preformed biofilm was exposed to ANP for 30 minutes. Under these conditions, the inventors observed that the biofilm is strongly dispersed by ANP, and the mushroom-shaped structures observed under the control conditions and characteristic of a P. aeruginosa biofilm, were absent after exposure to ANP. The remaining bacterial biomass was reduced after exposure to ANP (30 minutes) by 80.2±2.0% (P<0.05) compared with the biomass present under the control conditions.

Effect of ANP on the Growth of P. aeruginosa

[0190] To determine a potential direct effect of ANP on the growth of P. aeruginosa, the inventors studied the impact of ANP at 1 μM and 0.1 μM on the growth of P. aeruginosa P14 in a liquid medium. None of the tested ANP concentrations affected bacterial growth as shown in FIG. 1.

[0191] Therefore, the effect of ANP on preformed biofilms cannot be attributable to action on growth of the bacterium.

Effect of ANP on Bacterial Cytotoxicity in Cultured Human Pulmonary Cells.

[0192] The cytotoxic activity of P. aeruginosa exposed to different concentrations of ANP (1 μM to 10 nM) was studied on A549 pulmonary cells. The inventors observed that ANP, at any concentration, had no impact on the cytotoxic activity of P. aeruginosa in pulmonary cells, as shown in the Table below.

TABLE-US-00002 % LDH Experimental conditions (dead cells) PA14 non-treated 56.4 ± 5.5 PA14 + ANP (10.sup.−6 M) 58.9 ± 5.8 PA14 + ANP (10.sup.−7 M) 53.5 ± 5.3 PA14 + ANP (10.sup.−8 M) 54.5 ± 5.5

Conclusion

[0193] The inventors have therefore shown in this example that ANP, used at a concentration of 0.1 μM, fully prevents the formation of a P. aeruginosa biofilm and strongly destroys a preformed biofilm of P. aeruginosa.

[0194] For a long time, studies to prevent the formation of biofilms focused on the initial steps of biofilm formation, including the adhering and maturation of the biofilm. The concentrations of drugs needed to inhibit the formation of a biofilm are generally much lower than those required to destroy or perturb a preformed biofilm.

[0195] The main advantage of ANP evidenced herein by the inventors, is that use thereof at 0.1 μM for 2 h, even 30 minutes, is sufficient to disperse about 80% of the biofilm structure. This is of particular interest especially with a view to use in synergy with antibiotics.

[0196] This effect of ANP on a P. aeruginosa biofilm is greater than the inhibitory effect on formation observed previously with CNP, and exhibits an additional advantage since CNP slightly increased bacterial virulence and cytotoxicity by activating bacterial quorum-sensing systems.

[0197] The inventors have shown here that ANP, despite its strong anti-biofilm action, does not kill bacteria and does not increase the cytotoxicity of P. aeruginosa in cultured human pulmonary cells. The fact that the bacteria are not killed is of particular interest since this prevents the emergence of resistant strains.

Example 2: Dose-Dependent Effect of ANP on Preformed Biofilms of P. aeruginosa

[0198] This example shows that the action of ANP alone on preformed biofilms of P. aeruginosa is dependent upon the dose used and can be seen on and after 0.3 ng/ml.

Material and Methods

[0199] The material and methods used in this example are the same as those described above in Example 1 (Destruction of the P. aeruginosa biofilm under dynamic conditions).

Results

[0200] The inventors studied the potential anti-biofilm activity of ANP on an established biofilm of P. aeruginosa at different ANP concentrations: 0.3 ng/ml, 3 ng/ml and 30 ng/ml, comparing with the activity given in Example 1 ([ANP]=300 ng/ml or 0.1 μM), after exposure for 2 h or 30 min.

[0201] The inventors observed that the biofilm is strongly dispersed by ANP on and after the lowest concentration of 0.3 ng/ml and as soon as after exposure for 30 min.

[0202] The results obtained are given in the following Table.

TABLE-US-00003 Biovolume Biovolume Experimental (2 h) % Inhibition (30 min) % Inhibition conditions (μm.sup.3/μm.sup.2) (2 h) (μm.sup.3/μm.sup.2) (30 min) PA14 non-treated  21.1 ¶ 22.19 ANP (0.3 ng/ml) 10.6  45.1 (**)  9.5 57.5 (*) ANP (3 ng/ml) 6.3 65.9 (***) 8.8 59.1 (*) ANP (30 ng/ml) 4.4 72.5 (***) 6.7 69.0 (*) ANP (300 ng/ml) 4.9 81.4 (***) 4.4 80.2 (*) ¶: Mean of all controls; (*): p < 0.05 vs non-treated biofilms; (**): p < 0.01 vs non-treated biofilms; (***): p < 0.001 vs non-treated biofilms.

[0203] This example therefore confirms the advantage of ANP which is active on and after a very low concentration and starting from an exposure time of 30 min.

Example 3: Effect of Osteocrin on Preformed Biofilms of P. aeruginosa

[0204] This example shows the action of osteocrin alone on preformed biofilms of P. aeruginosa at a dose of 10.sup.−8 M.

Material and Methods

[0205] The material and methods used in this example are the same as those described above in Example 1 (Destruction of the P. aeruginosa biofilm under dynamic conditions).

Results

[0206] The inventors studied the potential anti-biofilm activity of osteocrin on an established P. aeruginosa biofilm at a concentration of 10.sup.−8 M and after exposure for 2 h.

[0207] The inventors observed that the biofilm was strongly and significantly dispersed by osteocrin.

TABLE-US-00004 Biovolume (2 h) % Inhibition Experimental conditions (μm.sup.3/μm.sup.2) (2 h) PA14 non-treated (control) 29.8 Osteocrin (10.sup.−8 M) 12.1 59.4 (***)

Example 4: Effect of the Lebetin 2α Peptide on Preformed Biofilms of P. aeruginosa

[0208] This example shows the action of the lebetin L2 alpha peptide alone on preformed biofilms of P. aeruginosa, at a dose of 10.sup.−8 M.

Material and Methods

[0209] The material and methods used in this example are the same as those described above in Example 1 (Destruction of the P. aeruginosa biofilm under dynamic conditions).

Results

[0210] The inventors studied the potential anti-biofilm activity of the lebetin L2 alpha peptide on an established biofilm of P. aeruginosa at a concentration of 10.sup.−8 M and after exposure for 2 h.

[0211] The inventors observed that the biofilm was strongly and significantly dispersed by the lebetin L2 alpha peptide.

TABLE-US-00005 Biovolume (2 h) % Inhibition Experimental conditions (μm.sup.3/μm.sup.2) (2 h) PA14 non-treated (control) 29.7 Lebetin L2 alpha (10.sup.−8 M) 8.9 70.2 (***)

Example 5: Effect of the Lebetin 1β Peptide on Preformed Biofilms of P. aeruginosa

[0212] This example shows the action of the lebetin L1 beta peptide alone on preformed biofilms of P. aeruginosa at a dose of 10.sup.−8 M.

Material and Methods

[0213] The material and methods used in this example are the same as those described above in Example 1 (Destruction of the P. aeruginosa biofilm under dynamic conditions).

Results

[0214] The inventors studied the potential anti-biofilm activity of the lebetin L1 beta peptide on an established biofilm of P. aeruginosa at a concentration of 10.sup.−8 M and after 2 h exposure.

[0215] The inventors observed that the biofilm was strongly dispersed by the lebetin L1 beta peptide.

TABLE-US-00006 Biovolume (2 h) % Inhibition Experimental conditions (μm.sup.3/μm.sup.2) (2 h) PA14 non-treated (control) 21.0 Lebetin L1 beta (10.sup.−8 M) 6.9 67.0

Example 6: Effect of a Combination of ANP and Tobramycin on Preformed Biofilms of P. aeruginosa

[0216] This example shows the synergic action of the ANP+tobramycin combination on preformed biofilms of P. aeruginosa.

Material and Methods

[0217] The material and methods used in this example are the same as those described above in Example 1, with the following specifications:

[0218] To study the impact of ANP in combination with tobramycin on 24 h preformed biofilms (as described in Example 1), 300 μl of a mixture of ANP (1 nM) and tobramycin (50 μg/ml) or 300 μl of tobramycin alone (control conditions) were injected into different channels of the flow chamber. Treatment of the preformed biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0219] The biofilms were observed under a confocal laser scanning microscope as described in Example 1. To test the membrane integrity of the bacteria, a mixture of 5 μM SYTO 9 Green and 0.3 μM propidium iodide (PI) was used (Live/Dead BacLight Bacterial Viability Kit, Invitrogen).

Results

[0220] The inventors studied the potential anti-biofilm activity of the combination ANP (10.sup.−9 M; 3 ng/ml)+tobramycin (50 μg/ml or 10 μg/ml) on an established biofilm of P. aeruginosa.

[0221] The biovolume of the biofilm was determined and the results are given in the Tables below.

TABLE-US-00007 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 13.5 Tobramycin (50 μg/ml) 3.26 75.9 ANP (10.sup.−9 M) 4.53 66.4 Tobramycin (50 μg/ml) + ANP (10.sup.−9 M) 0.5 96.3

TABLE-US-00008 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 13.5 Tobramycin (10 μg/ml) 2.9 78.5 ANP (10.sup.−9 M) 4.53 66.4 Tobramycin (10 μg/ml) + ANP (10.sup.−9 M) 0.7 94.8

[0222] Under these conditions, the inventors observed that the biofilm was strongly dispersed by the combination ANP (1 nM)+tobramycin (50 μg/ml), which acts in synergy to obtain 96.3% destruction of the biofilm. Similarly, the combination ANP (1 nM)+tobramycin (10 μg/ml), acts in synergy to obtain 94.8% destruction of the biofilm.

Example 7: Effect of an ANP and Ciprofloxacin Combination on Preformed Biofilms of P. aeruginosa

[0223] This example shows the synergic action of the ANP+ciprofloxacin combination on preformed biofilms of P. aeruginosa.

Material and Methods

[0224] To study the impact of ANP in combination with ciprofloxacin on 24 h preformed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and ciprofloxacin (0.01 μg/ml or 0.04 μg/ml) or 300 μl of ciprofloxacin alone (control conditions) were injected into different channels of the flow chamber. Treatment of the preformed biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0225] As described in Example 1, the biofilms were observed under a confocal laser scanning microscope after being stained for 15 min with 5 μM of Syto9 Green fluorochrome (Invitrogen).

Results

[0226] The inventors studied the potential anti-biofilm activity of the combination ANP (10.sup.−8 M; 30 ng/ml)+ciprofloxacin (0.04 μg/ml or 0.01 μg/ml) on an established biofilm of P. aeruginosa.

[0227] The biovolume of the biofilm was determined and the results are given in the Tables below.

TABLE-US-00009 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 12.6 Ciprofloxacin (0.04 μg/ml) 0.7 99.3 Ciprofloxacin (0.04 μg/ml) + ANP (10.sup.−8 M) 0.5 99.5

TABLE-US-00010 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 15.5 Ciprofloxacin (0.01 μg/ml) 1.55 90.0 Ciprofloxacin (0.01 μg/ml) + ANP (10.sup.−8 M) 0.47 97.0

[0228] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the combination ANP+ciprofloxacin, compared to treatment with the ciprofloxacin antibiotic alone.

[0229] Therefore, the combination with ANP allows 97% destruction of the biofilm to be obtained in combination with a ciprofloxacin concentration of 0.01 μg/ml.

Example 8: Effect of a Combination of ANP and Colistin on Preformed Biofilms of P. aeruginosa

[0230] This example shows the synergic action of the ANP+colistin combination on preformed biofilms of P. aeruginosa.

Material and Methods

[0231] To study the impact of ANP in combination with colistin on 24 h preformed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and colistin (1 μg/ml) or 300 μl of colistin alone (control conditions) were injected into different channels of the flow chamber. Treatment of the preformed biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0232] As described in Example 1, the biofilms were observed under a confocal laser scanning microscope, after being stained for 15 min with 5 μM of Syto9 Green fluorochrome (Invitrogen).

Results

[0233] The inventors studied the potential anti-biofilm activity of the combination ANP (10.sup.−8 M; 30 ng/ml)+colistin (1 μg/ml) on an established biofilm of P. aeruginosa.

[0234] The biovolume of the biofilm was determined and the results are given in the Tables below.

TABLE-US-00011 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 18.5 ANP (10.sup.−8 M) 4.9 73.5 Colistin (1 μg/ml) 2.1 88.6 Colistin (1 μg/ml) + ANP (10.sup.−8 M) 0.5 97.3

[0235] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the combination ANP+colistin (1 μg/ml), compared to treatment with the colistin antibiotic alone or treatment with ANP alone.

[0236] Therefore, the combination with ANP allows 97.3% destruction of the biofilm to be obtained, in combination with a colistin concentration of 1 μg/ml.

Example 9: Effect of a Combination of ANP and Imipenem on Preformed Biofilms of P. aeruginosa

[0237] This example shows the synergic action of the ANP+Imipenem combination on preformed biofilms of P. aeruginosa.

Material and Methods

[0238] To study the impact of ANP in combination with Imipenem on 24 h preformed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and Imipenem (0.5 μg/ml) or 300 μl of Imipenem alone (control conditions) were injected into different channels of the flow chamber. Treatment of the preformed biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0239] As described in Example 1, the biofilms were observed under a confocal laser scanning microscope after being stained for 15 min with 5 μM of Syto9 Green fluorochrome (Invitrogen).

Results

[0240] The inventors studied the potential anti-biofilm activity of the combination ANP (10.sup.−8 M; 30 ng/ml)+Imipenem (0.5 μg/ml) on an established biofilm of P. aeruginosa.

[0241] The biovolume of the biofilm was determined and the results are given in the Tables below.

TABLE-US-00012 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 28.0 ANP (10.sup.−8 M) 6.9 75.4 Imipenem (0.5 μg/ml) 7.0 75.0 Imipenem (0.5 μg/ml) + ANP (10.sup.−8 M) 0.6 97.9

[0242] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the ANP+Imipenem (0.5 μg/ml) combination, compared to treatment with the Imipenem antibiotic alone or with ANP treatment alone.

[0243] Therefore, the combination with ANP allows 97.9% destruction of the biofilm to be obtained in combination with an Imipenem concentration of 0.5 μg/ml.

Example 10: Effect of a Combination of ANP and Polymyxin B on Preformed Biofilms of P. aeruginosa

[0244] This example shows the synergic action of the ANP+Polymyxin B combination on preformed biofilms of P. aeruginosa.

Material and Methods

[0245] To study the impact of ANP in combination with polymyxin B on 24 h preformed biofilms (as described in Example 1), 300 μl of a mixture of ANP (10 nM) and Polymyxin B (4 μg/ml) or 300 μl of Polymyxin B alone (control conditions) were injected into different channels of the flow chamber. Treatment of the preformed biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0246] As described in Example 1, the biofilms were observed under a confocal laser scanning microscope after being stained for 15 min with 5 μM of Syto9 Green fluorochrome (Invitrogen).

Results

[0247] The inventors studied the potential anti-biofilm activity of the combination ANP (10.sup.−8 M; 30 ng/ml)+Polymyxin B (4 μg/ml) on an established biofilm of P. aeruginosa.

[0248] The biovolume of the biofilm was determined and the results are given in the Tables below.

TABLE-US-00013 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 18.2 ANP (10.sup.−8 M) 4.5 75.3 Polymyxin B (4 μg/ml) 7.4 59.3 Polymyxin B (4 μg/ml) + ANP (10.sup.−8 M) 3.0 83.5

[0249] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by the ANP+Polymyxin B (4 μg/ml) combination, compared to treatment with the Polymyxin B antibiotic alone or with ANP treatment alone.

[0250] Therefore, the combination with ANP allows 83.5% destruction of the biofilm to be obtained in combination with a Polymyxin B concentration of 4 μg/ml.

Example 11: Effect of Sequential Treatment with ANP Followed by Tobramycin on Preformed Biofilms of P. aeruginosa

[0251] This example shows the sequential action of treatment with ANP followed by treatment with tobramycin on preformed biofilms of P. aeruginosa.

Material and Methods

[0252] The material and methods used in this example are the same as those described above in Example 1, with the following specifications:

[0253] To study the impact of sequential exposure to ANP and then to tobramycin on 24 h preformed biofilms (as described in Example 1), 300 μl of ANP (1 nM) (control conditions) were injected into two channels of the flow chamber. Treatment of the biofilm was conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment. Next, 300 μl of tobramycin (50 μg/ml) or 300 μl of milliQ sterile water (control conditions) were injected into the two channels of the flow chamber pre-exposed to ANP. The second treatment of the preformed biofilm was again conducted for 2 h at 37° C. under static conditions. After the 2 h, the flow of medium was again applied for 15 min to remove any cells which may have detached from the biofilm under action of the treatment.

[0254] As described in Example 1, the biofilms were observed under a confocal laser scanning microscope after being stained for 15 min with 5 μM of Syto9 Green fluorochrome (Invitrogen).

Results

[0255] The inventors studied the potential anti-biofilm activity of sequential exposure to ANP (10.sup.−9 M; 3 ng/ml) followed by tobramycin (50 μg/ml) on an established biofilm of P. aeruginosa.

[0256] The biovolume of the biofilm was determined and the results are given in the Tables below.

TABLE-US-00014 Biovolume Experimental conditions (μm.sup.3/μm.sup.2) % Inhibition PA14 non-treated 13.1 ANP (10.sup.−9 M) 4.1 68.7 ANP (10.sup.−9 M) (2 h) then Tobramycin (50 μg/ml) (2 h) 0.7 94.7

[0257] Under these conditions, the inventors observed that the biofilm was even more strongly dispersed by twofold sequential treatment with ANP (10.sup.−9 M) (2 h) then tobramycin (50 μg/ml) (2 h) compared to treatment with ANP alone.

[0258] Therefore, sequential treatment with ANP (2 h) then tobramycin (2 h) allows 94.7 destruction of the biofilm to be obtained.