Biofilm degrading composition

Abstract

The invention relates to a biofilm degrading composition comprising a DNAse, to uses thereof, and to a method of degrading biofilms comprising the application of this composition.

Description

[0001]BIOFILMS DEGRADING COMPOSITION Technical field The present invention relates to a biofilm degrading composition comprising a specific DNAse. Prior art In many fields of activity such as the agri-food sectors, communities, medical and veterinary sectors, cosmetic industries, the pharmaceutical, life science or biotechnology sectors, problematic contaminations due to the presence of microorganisms are observed very frequently. These biofilms can be treated in different ways, but it remains difficult to ensure that the treatment that will be applied will be effective. In particular, biocidal or antibiotic molecules, which are known to destroy bacteria in planktonic form, are very often ineffective against these same bacteria in biofilm form.Furthermore, in practice, and unlike in the laboratory, the composition of a biofilm contaminating a surface is generally unknown. Finally, applications in the food or medical fields, or even for GMP (Good Manufacturing Practice) production, impose constraints on the compounds that can be used, which complicates the treatment of biofilms. For example, pipes are difficult to disinfect due to reduced access. Similarly, medical instruments, such as endoscopes, are difficult to disinfect, due to (i) their surface, which has many irregularities, and (ii) the impossibility of applying many usual physical or chemical treatments, such as heat treatments, products that would damage them or be incompatible with subsequent use in a patient. Different types of enzymes have been tested and used successfully for the degradation of biofilms.However, some biofilms are not adequately deconstructed with a single enzymatic activity, while mixtures of enzymes, particularly when proteases are present, do not necessarily allow liquid compositions that are stable over time. Furthermore, as will be described below, for a biofilm of a given composition, the inventors noted that current enzymatic compositions too often exhibit significant variations in terms of effectiveness, which complicates the work of arriving at an optimal formulation. Patent application WO2022079318 describes the use of a particular nuclease (Denarase), with increased effectiveness. Patent US10954497 describes a class of HNH DNAses having a GYS motif and their effectiveness when formulated for the destruction of biofilms.Brief Summary of the Invention The present invention relates to a liquid composition for the prevention or elimination of a biofilm, comprising one or more surfactant(s), one or more sequestering agent(s) and a phosphodiesterase having deoxyribonuclease (DNAse) activity, said DNAse being one or more HNH-type DNAse(s) and / or having at least 95% identity with one of the sequences SEQ ID NO: 1 to SEQ ID NO: 13 (or SEQ ID NO: 1 to SEQ ID NO: 18). The present invention further relates to the use of this composition for the elimination of a biofilm comprising a lactic acid bacterium selected from Lacobacillus sp, Pediococcus sp, Lactococcus sp.Streptococcus sp, Teragenococcus sp, Leuconostoc sp, Oenococcus sp, Bifidobacterium sp, and / or Listeria monocytogenes, Stenotrophomonas maltophilia, Bacillus cereus, Bacillus subtilis, and / or Pseudomonas fluorescens, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Staphylococcus epidermidis or Clostridium sp. The present invention also relates to this liquid composition (for pharmaceutical use) for the removal of a biofilm present on a tissue of a patient, said tissue preferably being the oral cavity, a mucosa, a wound or in contact with an implant. The present invention also relates to a method for the removal of a biofilm on a surface comprising the application to said surface of this liquid composition.Detailed description of an embodiment of the invention The inventors sought to improve compositions against biofilms, in particular to obtain compositions whose spectrum of activity is sufficiently broad and constant, and which ensures a considerable reduction in the number of microorganisms protected by the biofilm. To do this, the inventors identified a DNAse which is particularly effective against certain types of biofilms. Furthermore, depending on the biofilms, this DNAse is very effective, either alone or in synergy with other enzymatic activities. In addition, the inventors succeeded in formulating this DNAse in such a way that it retains its activity, including over time (even in the presence of a protease in the composition or sequestering agents), which first of all allows reasonable commercial logistics between the time when the liquid (aqueous) composition is formulated and the time when it will be used.The composition according to the invention is preferably used on surfaces. In the context of the present invention, the terminology "surface" is preferably understood in the broad sense, therefore including the internal surface of a pipe, or even surfaces of reusable medical devices, in particular endoscopes and surgical instruments. Indeed, the inventors tested the effect of this DNAse alone, compared to other nucleases known for their remarkable activity, such as Denarase (as in WO2022079318), and the effect of this DNAse in synergy with other enzymes, either in a mixture or in sequential application, and noted an increased or more constant efficacy, depending on the type of biofilm to be treated.Indeed, to their surprise, the inventors compared, under well-controlled laboratory conditions, the effect on biofilms of the compositions (protease + amylase + lipase + detergent + mild sequestrant) without this DNAse and of the same compositions with the DNAse of the invention and, in the case of the compositions without this DNAse, they sometimes noted good results and sometimes mediocre results, while the compositions enriched with the DNAse of the present invention always showed very good results. Thus, it took many tests, and repeated many times, to clearly identify the effect of the DNAse of the present invention, and the results shown below are among the most spectacular. Sequence listing Sequences 1 to 11, as well as 14 to 18, include DNAses according to the invention. These are HNH type DNAses. Sequence 12 is the consensus sequence in its broadest understanding.Sequence 13 is a consensus sequence incorporating preferred amino acids at key positions, especially in the central part of the enzyme. In sequences 12 and 13, the amino acids subject to variation are indicated by the letter "X" and their structure is shown below.Certain positions are preferentially occupied by specific amino acids, which are also listed below: SEQ ID NO: 12 XPXXXPSKXXXQXQLXXLXVXXEXXMXGYSRXXFPHWXXQGXGCXTRQXVLXRDADXXSG XCPXTXGXWYSYXDGXXXXXPSXXDXDHXVPLAEAWRSGASSWXTXXRXXFANDLXGXQL IAVXASXNRXKGDQDPSTWXPXRXXAXCXYXKXWXXTKXXXXLXLQSXEKXXLXXMLXXC XY SEQ ID NO: 13 LPPGTPSKSXAQSQLNALXVXXEXSMTGYSRXXFPHWSSQGGGCDTRQVVLKRDADXXSG XCPVTSGKWYSYFDGXXFTNPSDLDIDHIVPLAEAWRSGASSWTTSKRXXFANDLNGPQL IAVXASXNRXKGDQDPSTWQPPRAAARCAYSKWWISTKYKWGLSLQSXEKXXLXXMLXXC AY 1: L, F, I, T, P; preferably L or I, preferably L; 3: P, S; preferably P; 4: G, E, V, preferably G, V, preferably G; 5: T, I; preferably T; 9 S, A, preferably S; 10. (sequences 12 and 13). A, E, Q, T, preferably E, Q; 11. A, S; preferably A; 13. S, T; preferably S; 16. N, D; preferably N; 17 A, S, G; preferably A, S; 19 (sequences 12 and 13). T, A; preferably T; 21 (sequences 12 and 13).K, Q; preferably K; 22 (sequences 12 and 13). A, P, T, S; preferably P or S; 24 (sequences 12 and 13). D, S, G; preferably D; 25. P, T, A, S; preferably P, S; 27. T, S; preferably T; 32. (sequences 12 and 13). D, N; 33. (sequences 12 and 13). L, H, K; 38. N, S, I, T; 39. S, G, preferably S; 42. S, G, N; 45. D, N; 49. I, L, V, M; 52. Q, K; 57(sequences 12 and 13), Y, S; preferably Y; 58 (sequences 12 and 13). Y, F, preferably Y at least one Y residue at positions 57 / 58, preferably positions 57 and 58 are occupied by 2 Y residues; 59. S, T; preferably S; 61 (sequences 12 and 13). N, A, T, S, D; preferably A; 64. V, T; preferably V; 66. S, T; preferably S; 68. R, K, S; preferably R, K; 73. F, Y; 76 (sequences 12 and 13). I, V; 77 (sequences 12 and 13). V, I, T, K; 78. V, F; 79. T, Y; 80. S, D, N; preferably S; 83. E, D, N; preferably E, D; 84. I, L; 86. I, V; preferably I 89.I, V; preferably I; at least 1 I at positions 86 / 89; 104. S, T; preferably T; 106. E, S, Q, A, T; 107. K, Q; preferably K; 109. (sequences 12 and 13). E, K, Q, R; preferably Q or R, preferably R 110. (sequences 12 and 13). E, S, A, N, D; preferably N or D, preferably N 116. N, T, G, S; preferably N, S; 118. P, S, preferably P; 124 (sequences 12 and 13). S, T; 127 (sequences 12 and 13). T, S, V; preferably S, V; 130 (sequences 12 and 13). S, A; preferably S; 140. Q, K; preferably Q; 142. P, T; preferably P; 144. A, V, S, Y; 145. A, G, S; preferably G; 147. R, H, K, A; preferably R; 149. G, A; 151. S, A; preferably A; 153. W, M; preferably W; 155. I, V; preferably I; 156. N, S; preferably N; 159. H, Y, S; preferably Y, H; 160. R, V, K; preferably R, K, preferably R; 161. W, Y; preferably W; 162. D, G, N, preferably G, D; 164. S, H, N; 168 (sequences 12 and 13). S, A; preferably S; 171 (sequences 12 and 13).S, T, N; preferably S; 172 (sequences 12 and 13). S, A, G, preferably S, A; 174 (sequences 12 and 13). Q, E; preferably Q; 175 (sequences 12 and 13). T, S, G; 178 (sequences 12 and 13). N, D; preferably N; 179 (sequences 12 and 13). T, G, S; preferably T, S, preferably T; 181. S, A. Thus, a first subject of the present invention relates to a liquid composition for the prevention or elimination of a biofilm, comprising one or more surfactant(s), one or more sequestering agents and a phosphodiesterase having a deoxyribonuclease activity (DNAse), said DNAse being one or more HNH type DNAse(s) (having a GYS motif) and / or having at least 95% identity with one of the sequences 1 to 13, or even with the DNAses of the sequences 1 to 18. Preferably, said liquid composition comprises at least 7% by weight of water, preferably at least 10% of water, or even at least 20% of water.Preferably, in this composition, the DNAse(s) has(have) at least 95% identity with one of the sequences 1 to 13, or even with the DNAses of the sequences 1 to 18. In the context of the present invention, the identity is preferably measured by an alignment of a sequence to be tested with one of the sequences 1 to 13 (or 1 to 18) over their entire length (182 amino acids). The default parameters of BLASTp are advantageously used, such as a Blosum62 type matrix, a "word" of size 6, a "gap" penalty of 11 and a "gap" extension penalty of 1. Thus the percentage of identity is calculated without ambiguity. Advantageously, in the above calculation, a "gap" will be counted as a single difference, regardless of its length.Similarly, when a DNAse is to be compared with sequences 12 and 13, a global alignment is carried out as described above, and an amino acid of the sequence to be compared which would be identical to the variants of sequences 12 or 13 listed at the different positions marked with the symbol "X" is counted as identical. For example, a sequence to be compared would be aligned with sequence 12 or 13. If the amino acid of this sequence to be compared at the position corresponding to position 10 of sequence 12 or 13 is an alanine, it will be considered identical. A sequence which would have an equivalent enzymatic activity, but which would be longer (e.g. addition of sequences for purification or sequences for increasing stability, or even a signal sequence), or shorter, is also covered by the present invention, provided that the enzyme in question takes up at least 95% of one of sequences 1 to 13 (or 1 to 18).Alternatively, identity can be inferred after a global alignment as above and applying a tolerance of less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3 variations or "gaps" between a target sequence and any of sequences 1 to 13 (or 1 to 18). Additional enzymes Advantageously, the composition further comprises one or more enzymatic activity(ies) chosen from one (or more) protease, a laccase, an amylase, a lipase, a cellulase, a mannanase, a β-1,6-N-acetylhexosaminidase (Dispersin B) activity and a mixture thereof, preferably one protease (or even several proteases) and / or β-1,6-N-acetylhexosaminidase (Dispersin B), preferably one protease (or even several proteases) and / or a β-1,6-N-acetylhexosaminidase (Dispersin B) and another enzyme, or several other enzymes, chosen from a laccase, an amylase, a lipase, a cellulase and a mannanase.For example, a preferred composition comprises the DNAse described above and one (or more) proteases, one (or more) amylase and one (or more) cellulase. A preferred composition may also comprise the DNAse described above and another nuclease, as well as, advantageously, a protease (or even a cellulase and / or an amylase and / or a second protease). One or more enzymes catalyzing redox reactions may advantageously be added.Indeed, even if the addition of several enzymatic activities presents difficulties, in particular at the level of stability, for example due to sequestering agents (see below) or the endogenous activity of the protease, the inventors have noticed that a presence of several different enzymatic activities offers a broader spectrum of activity, which is advantageous since, in general, the composition of the biofilms to be treated is unknown, or even the biofilms to be treated comprise several different structural elements: the already remarkable effect of the DNAse of the invention is further reinforced by the additional enzymatic activities. Other components The surfactant(s) are not particularly limited. Anionic, neutral and / or zwiterionic surfactants may be present.The inventors have noted that alkyl polyglucoside surfactants work well, as do alkyl amine oxide zwitterionic surfactants (e.g., 12 and / or 14 and / or 16 and / or 18 carbons). When the composition is to be applied to vertical surfaces, for example, a siphon, a foaming surfactant is advantageously incorporated. In addition, a foaming agent, such as betaine derivatives, and / or zwitterionic surfactants having a quaternary amine are advantageously added. Typically, these surfactants are present in a (final) content of at least 1%, weight:volume. Advantageously, their content does not exceed 15% (weight:volume; sum of the weights of the surfactants:volume).Preferably, in the composition according to the invention, the sequestering agent(s) is (are) chosen from the group consisting of citrate, carboxymethylinulin, a phosphate, a phosphonate, and amino acid derivatives (Glutamate and tetrasodium diacetate, GLDA; trisodium methylglycine diacetate, MGDA), sodium iminodisuccinate; IDS, gluconate (e.g. sodium gluconate) and mixtures thereof. Preferably, the sequestering agent(s) is (are) chosen from the group consisting of citrate, carboxymethylinulin, a phosphate, a phosphonate, gluconate and mixtures thereof. These are preferably mild sequestering agents. The inventors have noted that these sequestrants do not interfere with the activity of the DNAse of the invention, nor with that of the other enzymes listed above (when they are present). Advantageously, the composition according to the invention further comprises glycerol and / or monopropylene glycol.The inventors have noticed that this type of molecule increases the stability of the composition according to the invention. An advantageous content is approximately 5 to 30% by weight of glycerol and / or monopropylene glycol:volume of the solution, preferably 7 to 15% (weight:volume), such as 8 to 12% (weight:volume), or approximately 10%±0.5% (weight of glycerol and / or monopropylene glycol:volume). Sorbitol may also be incorporated for the same purpose. However, when the composition is intended for administration to a patient (implant, mucous membranes of the digestive or respiratory systems), it advantageously does not comprise glycerol or monopropylene glycol, or in any case less than 5%, or even less than 1%, or even less than 0.1% (weight of glycerol and / or monopropylene glycol: volume of the composition).Conversely, or in addition, the liquid composition according to the invention comprises (in addition to glycerol and / or monopropylene glycol) advantageously water, for example at least 5% by weight, preferably at least 10% by weight. Furthermore, advantageously, the liquid composition according to the invention is present in a concentrated formulation, which is diluted (e.g. 10 times, 100 times, or more) in water just before use. Some compositions are however advantageously directly formulated “ready to use”; for example compositions comprising foaming surfactants (e.g. as described above for the treatment of siphons); a more detailed description of this will be provided below. Among the foaming surfactants, there are those which have an “HLB” (Hydrophilic-lipophilic-Balance) value of between 3 and 8.Alternatively, several anionic or zwitterionic surfactants may be used, preferably in synergy with a betaine derivative, such as 1-propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-acyl (acyl being a hydrocarbon chain with an even number of carbons, for example 18). Similarly, advantageously, the composition according to the invention further comprises a preservative, preferably an isothiazolinone such as Benzisothiazolinone, or (2-)phenoxyethanol. Conversely, the compositions intended to be administered to a patient (implant, mucous membranes of the digestive or respiratory systems) advantageously do not comprise a preservative. Advantageously, the composition according to the invention comprises at least 40% (volume:volume) water. This is a composition which should not be diluted excessively before use. On the other hand, too high a water content risks harming the stability of the composition over time.Another related aspect of the present invention relates to a composition (with at least one HNH type DNAse and / or having at least 95% identity over the entire sequence with any of Sequences 1 to 13, or 1 to 18) "ready to use", comprising at least 90%, or even at least 95% of water (weight of water: weight of the composition) and from 1 to 10% (for example from 2 to 5%) of the other components (sum of the weights of the components other than water: total weight of the composition): the DNA according to the invention, the possible other enzymes (preferably one or more proteases; amylase, possibly a lipase, or even the other enzymes described above), a sequestering agent and one or more surfactants, or even one or more carrier compounds if present. Preferably such a “ready-to-use” composition comprises a foaming surfactant and / or a betaine derivative as described above.Another related aspect of the present invention relates to the use of the composition according to the invention (with at least one HNH-type DNAse and / or having at least 95% identity over the entire sequence with any of Sequences 1 to 13, or 1 to 18) for the elimination of a biofilm comprising a lactic acid bacterium selected from Lacobacillus sp, Pediococcus sp, Lactococcus sp. Streptococcus sp, Teragenococcus sp, Leuconostoc sp, Oenococcus sp, Bifidobacterium sp, and / or Listeria sp (or L. monocytogenes), Stenotrophomonas maltophilia, Bacillus sp. (B. cereus, B. subtilis) and / or Pseudomonas sp. (e.g. Pseudomonas fluorescens or Pseudomonas aeruginosa), Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Staphylococcus epidermidis, Clostridium sp.Indeed, in the case of biofilms comprising the microorganisms listed above, the inventors have noted the superiority of the DNAse according to the invention compared to other nucleases, even nucleases considered to be excellent, this superiority is measured either alone or in synergy with other enzymatic activities. Another related aspect of the present invention relates to a (pharmaceutical) composition comprising the DNAse (or several DNAses) according to the invention (with at least one HNH type DNAse and / or having at least 95% identity over the entire sequence with any of Sequences 1 to 13, or 1 to 18) for the elimination of a biofilm present on a tissue of a patient, said tissue preferably being the oral cavity, a mucosa, a wound, or in contact with an implant.In the context of the present invention, the terminology "mucosa" preferably relates to a mucosa selected from the mucosa of the respiratory system (nasal cavities, bronchi), the urogenital system, and the digestive system. Preferably, this biofilm present on the tissue of a patient comprises Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, and / or Staphylococcus epidermidis. Advantageously, this pharmaceutical composition is produced according to "GMP" standards, which means that the compounds are chosen not to cause deleterious effects to the patient (e.g., especially for implants or mucous membranes of the digestive or respiratory system, as little glycerol or propylene glycol as possible; as little preservative or other agent toxic to the patient as possible), and that the production steps follow a strict protocol validated by independent authorities (health authorities, hospital ethics committees, etc.). Another related aspect of the present invention relates to a method for removing a biofilm from a surface comprising the application to this surface of the liquid composition according to the invention, comprising the DNAse (or several DNAses) according to the invention (with at least one HNH type DNAse and / or having at least 95% identity over the entire sequence with any of Sequences 1 to 13, or 1 to 18).A variant of this method is particularly advantageous for the treatment of medical instruments, in which the medical instrument is treated with the liquid composition according to the present invention comprising the DNAse described above (often in synergy with one or more surfactants, a sequestering agent and other enzymatic activities described above, in particular, a protease, but also an amylase, or even at least a fourth enzyme), then the instrument is rinsed, then disinfected using a biocidal molecule, for example peracetic acid. Then, if the medical instrument is of delicate composition, for example, an endoscope, it is simply dried. On the contrary, if the instrument can be subjected to a sterilizing heat treatment (e.g. surgical instruments), the latter is advantageously carried out in addition.A sterilizing heat treatment is preferably a treatment in an autoclave at a temperature above 100°C and at a pressure above 1 bar, the atmosphere being advantageously saturated with water. Another advantageous sterilizing heat treatment is pasteurization. A related aspect of the present invention relates to a composition for the prevention or treatment of biofilms present on a substantially vertical surface comprising: one or more enzymatic activities selected from a DNAse (e.g. that of WO2022 / 079315 and / or of EC classes 3.1.30 and 3.1.31, or the DNAse described above of HNH type and / or having at least 95% identity over the entire sequence with any of Sequences 1 to 13, or 1 to 18), a protease, a polysaccharidase (e.g. amylase, Dispersin B, cellulase, mannanase), a lipase, a laccase and mixtures thereof; one or more surfactants comprising at least one foaming surfactant.Preferably, this composition further comprises a surfactant having a quaternary amine, said surfactant preferably being zwitterionic and / or a betaine derivative, preferably of RN structure. +(CH3)2CH2COO-. The R group is preferably a linear alkyl or an alkylamidopropylbetaine, such as cocoamidopropylbetaine. Optionally, the carboxyl group is substituted by a sulfonate group, and / or a hydroxyl group is grafted onto the alkyl chain. The (linear) alkyl chain preferably has a size between 2 and 18 carbon atoms, preferably between 12 and 16 carbon atoms. Another related aspect of the present invention relates to a method for the prevention or treatment of a biofilm on a substantially vertical surface comprising the successive steps of applying the composition for the prevention or treatment of biofilms present on a substantially vertical surface as described above to said substantially vertical surface for a predetermined time; adding a microbiocidal agent for a predetermined time.This method is particularly advantageous when the vertical surface is a siphon, preferably the emerged part of said siphon. Examples Two different biofilms were tested, so as to encounter a diversity of microorganisms: a biofilm consisting of Lactococcus lactis and a biofilm consisting of Pseudomonas fluorescens. These biofilms were generated by applying bacterial suspensions on agar at 37°C in multi-well plates (24 wells), for 48 hours (Pseudomonas) or 24 hours (Lactococcus). The quantification of the microorganisms is done by spectrometry, here at 590 nm (570 nm is also OK) following staining with “Crystal Violet”. Different conditions are provided per plate, which is shown schematically in Table 1 below:. Table 1: Overview of the different conditions for the tests. Each condition is therefore in triplicate. Bacteria are seeded in all wells, except for the “negative controls”. All solutions are sterile and are added very gently to avoid causing mechanical disturbances to the biofilms. The wells of the “Positive control” and “Negative control” conditions were incubated with 1 ml of physiological saline. The other wells were incubated for 30 minutes at 45°C with 1 ml of the following solutions (the percentages below are in weight:volume): Biorem-CIP (Realco): 1%; Denarase and DNAse: 0.01% of the enzymatic solutions, in order to ensure standardized activity; Biorem A1 +10: the solutions sold by Realco, diluted to 0.25% (A1) and 0.05% (Biorem 10).These solutions include detergents / surfactants (>1% C6 alkyl glucoside), sequestrants (a phosphonate; >15%), and several enzymatic activities, including a protease, an amylase, and a laccase. Biorem CIP also contains detergents / surfactants (>1% C6 alkyl glucoside), sequestrants (>1% sodium citrate), and a dispersing agent and several enzymatic activities, including a protease, an amylase, and a laccase. The composition was applied at 1%. Biorem compounds are also described, for example, in patent application WO2012048758A1 and corresponding patents. Then, the wells are gently emptied, taking care not to remove biofilms or their fragments. After rinsing with physiological water, the plate containing the biofilms is dried in an oven for one hour at 45°C.Once the biofilm has dried, Crystal Violet (0.1%) is added for a 15-minute incubation at room temperature, followed by three rinses with distilled water. Finally, the wells are filled with 44% acetic acid for a one-hour incubation at room temperature. The absorbance measurement at 590 nm can now be performed. If the Crystal Violet concentrations are too high and the spectrophotometer saturates, then a ten-fold dilution is performed. The results for Lactococcus lactis are shown in Table 2 below:. destruction of biofilms consisting of Lactococcus lactis and synergy with an enzymatic composition. The inventors further tested, in another series of experiments, the effect of DNAse alone or Denarase alone, and observed a very good efficiency of DNAse alone on Lactococcus lactis, which is further increased by approximately 20% by Biorem, while Denarase alone has only marginal activity. The results for Pseudomonas fluorescens are shown in Table 3 below: Table 3: Efficacy of different nucleases for the destruction of biofilms consisting of Pseudomonas fluorescens and synergy with an enzymatic composition. As for L. lactis, the inventors tested in a second series of experiments the effect of DNAse alone and observed a strong activity of DNAse alone, which is somewhat increased by Biorem. From this, the inventors conclude that the DNAse of the invention is clearly superior, especially in synergy with the less intense conditions of Biorem A1+10, compared to Biorem CIP. Advantageously, for Lactococcus lactis, where the efficacy of Biorem CIP is a little less marked, the synergy with DNAse is better demonstrated. Another series of experiments was carried out in synergy with other enzymatic compositions (Enzimed Prevent, which includes a protease, a cellulase, an amylase and a mannanase; the first 2 columns of the tables below,without or with the DNAse of the present invention), and a comparison was made with other commercial compositions: alkaline detergent compositions with protease activity (3rd column; DAP), or an enzymatic degreasing cocktail, two distinct commercial formulations (4th and 5th column; DE1 and DE2), a multi-enzymatic disinfectant detergent containing a quaternary ammonium (6th column; DMQ) or a non-enzymatic disinfection composition for endoscopes (7th column; DME). The compositions were tested at a concentration of 0,5%. The biofilm of the test species is formed in a 96-well plate. The enzymatic detergents are then diluted to their usual concentration in standard hardness water (demineralized water) and incubated on the biofilm for 1 hour at 37°C. The amount of biomass remaining after treatment and washing of the biofilm is then evaluated by crystal violet staining (2% sigma solution). This test is carried out on reference strains in 96-well plates, namely: S. aureus ATCC33591: 24h biofilm (TGN) E. coli ATCC25922: 48h biofilm (LB) P. aeruginosa ATCC27853 and PAO1: 48h biofilm (TGN) Solutions: TGN (TSB+1% glucose+2% NaCl) LB (for E.coli) “Distilled” water: sterilized by filtration (0.22 micron), finally containing 1.25 mM MgCl2, 2.5 mM CaCl2, 3.33 mM NaHCO3. 2% crystal violet (Crystal violet solution, Sigma-Aldrich, ref: HT90132-1L) 66% acetic acid (acid) 100% glacial acetic, ref: K48283163635,Millipore) Tryptic soy agar (TSA; BD benelux – ref 254086) plates The solutions were preheated to 37°C, so as to avoid thermal shocks to the biofilms. Method: D0: Start an overnight culture. 5ml TGN + 20 µl of bacteria. Incubate for 18 hours at 37°C with slow shaking, 130 rpm D1: Preparation Add 200 ml of the inoculum (OD 0.05) to each well of a 96-well plate following the plate plan (VWR). Perform a non-contamination test of the liquid preculture Streak the preculture on a TSA plate. Incubate overnight @37°C The next day,ensure there is no contamination. Biofilm growth: For S. aureus 24h @37°C Incubate the plate at 37°C for 24h in a closed box to avoid evaporation. Note the time to have a biofilm of exactly 24h. For E. coli: 48h with change of medium after 24h @37°C: - Incubate the plate at 37°C for 24h in a closed box to avoid evaporation (LB not TGN). - Carefully remove the medium by pipetting - Add 200 µl of pre-warmed LB - Incubate for another 24h @37°C in a closed box to avoid evaporation. For P. aeruginosa: 48h @37°C - Incubate the plate at 37°C for 48h in a closed box to avoid evaporation. Note the time to have a biofilm of exactly 48h. D2: After growth of the biofilm Empty the culture medium from the biofilm plates by suction,avoiding touching the biofilm Rinse the plates 1 X with pre-warmed sterile PBS (remove the PBS after 30 sec) Enzyme treatment: - Prepare the enzyme solutions at the desired concentrations according to the attached preparation protocol - Add 230 µl of the enzyme solutions into empty 96-well plates according to the plate plan - Heat these plates in a water bath at 37°C or in the oven for 15 min - Transfer 200 µl of heated enzymes into the wells of the 96-well plates containing the biofilms by pipetting horizontally - Incubate the plates at 37°C for 1 h - Empty the enzymes from the treated plates by aspiration - Rinse the plates 2X with pre-warmed sterile PBS, between each step,remove the PBS by aspiration Staining the biomass with crystal violet: - Dry the plates in the oven at 60°C overnight to fix the biofilm - Add 200 ml of 2% crystal violet solution to the plates (including the negative control) - Incubate the plates at room temperature for 15 min - Empty the plates and rinse them at least 5X with distilled water - Drain the plates for a few minutes to remove the rinsing water from the wells - Add 200 µl of 66% acetic acid to the plates (including the negative control) - Incubate the plates at room temperature for at least 1 hour - Measure the absorbance values ​​with spectramax (570nm) and make ds dilutions of the acetic acid if some values ​​on the plate are > 2, Experiment 1 Experiment 2 5 Thus, the Enzimed Prevent product alone is consistently effective against E. coli and P. aeurginosa, but its effectiveness against S. aureus is variable. However, this Enzimed composition enriched with the DNAse of the present invention shows consistently and consistently increased activity, even against S. aureus. Other cleaning or even disinfecting compositions are less effective and sometimes not even effective at all on the biofilms tested.

Claims

CLAIMS 1. A liquid composition for the prevention or elimination of a biofilm, comprising one or more surfactant(s), one or more sequestering agent(s) and a phosphodiesterase having deoxyribonuclease (DNAse) activity, said DNAse being one or more HNH-type DNAse(s) and / or having at least 95% identity with one of the sequences SEQ ID NO: 1 to SEQ ID NO:

13.

2. The composition according to claim 1 wherein the DNAse has at least 95% identity with one of the sequences SEQ ID NO: 1 to SEQ ID NO:

13.

3. The composition according to claim 1 or 2 further comprising one or more enzymatic activity(ies) chosen from a protease, a laccase, an amylase, a lipase, a cellulase, a mannanase, a β-1,6-N-acetylhexosaminidase (Dispersin B) activity and a mixture thereof, preferably a protease and / or β-1,6-N-acetylhexosaminidase (Dispersin B). 4.The composition according to any one of the preceding claims wherein the sequestering agent(s) is (are) selected from the group consisting of citrate, carboxymethylinulin, a phosphate, a phosphonate, tetrasodium glutamate diacetate (GLDA), trisodium methylglycine diacetate (MGDA), sodium iminodisuccinate (SDI), a gluconate and mixtures thereof.

5. The composition according to any one of the preceding claims further comprising glycerol and / or monopropylene glycol, preferably between 5 and 30% (weight:volume).

6. The composition according to any one of the preceding claims further comprising a preservative, of. preferably an isothiazolinone such as Benzisothiazolinone, or a phenoxyethanol.

7. The composition according to any one of the preceding claims comprising at least 7% water (weight:volume).

8. The composition according to any one of the preceding claims comprising at least 60% water (weight:volume), preferably said composition further comprising β-1,6-N-acetylhexosaminidase (Dispersin B).

9. The composition according to any one of the preceding claims comprising at least 90% water (weight:volume) and a foaming surfactant.

10. Use of the composition according to any one of the preceding claims for the removal of a biofilm comprising a lactic acid bacterium chosen from Lacobacillus sp, Pediococcus sp, Lactococcus sp.Streptococcus sp, Teragenococcus sp, Leuconostoc sp, Oenococcus sp, Bifidobacterium sp, and / or Listeria monocytogenes, Stenotrophomonas maltophilia, Bacillus cereus, Bacillus subtilis, and / or Pseudomonas fluorescens, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Staphylococcus epidermidis or Clostridium sp.

11. A liquid composition according to any one of the preceding claims 1 to 9 for the removal of a biofilm present on a tissue of a patient, said tissue preferably being the oral cavity, a mucosa, a wound or in contact with an implant.

12. A method for the removal of a biofilm on a surface comprising applying to said surface the composition. liquid according to any one of the preceding claims 1 to 9.

13. The method according to claim 12 wherein the surface is the inner surface and / or the outer surface of an endoscope and further comprising applying a disinfectant to said surfaces.

14. The method according to claim 12 wherein the surface is that of a surgical instrument and further comprising applying a disinfectant to said surface, followed by applying a sterilizing heat treatment.

15. The method according to claim 13 or 14 wherein the disinfectant is peracetic acid.

16. A composition for the prevention or treatment of biofilms present on a substantially vertical surface comprising: one or more enzymatic activities selected from a DNAse, a protease, a polysaccharidase, a lipase, a laccase and mixtures thereof; one or more surfactants comprising at least one foaming surfactant. 17.The composition according to claim 16 further comprising a surfactant having a quaternary amine, said surfactant preferably being zwitterionic.

18. A method for preventing or treating a biofilm on a substantially vertical surface comprising the successive steps of applying the composition according to claim 16 or 17 to said substantially vertical surface for a predetermined time; adding a microbiocidal agent for a predetermined time.

19. The method according to claim 18, wherein the vertical surface is a siphon, preferably the emerged part of said siphon.

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