Compositions and uses thereof

Abstract

The present disclosure relates to exopolysaccharides produced by marine bacteria, compositions thereof, uses of said compositions, and methods for attenuating the pathogenicity of a microbial pathogen infection, either bacterial or viral, by inhibiting or reducing the colonization of said microbial pathogens on biological and / or non-biological surfaces.

Description

[Technical field]

[0001] The present invention relates to exopolysaccharides (EPS) derived from marine bacteria, compositions thereof, non-therapeutic uses of said EPS and compositions, and therapeutic uses of said EPS and compositions. The present invention also relates to a process for obtaining said EPS. The present invention further relates to a nasal delivery system comprising at least one isolated exopolysaccharide, and therapeutic uses of said nasal delivery system. The present invention also relates to a method of formulating a nasal composition. [Background technology]

[0002] Infectious diseases are usually caused by microorganisms that invade and spread in the body. There are many types of infectious organisms, also called pathogenic microorganisms. Here are some examples of how these microorganisms can get into the body: through the mouth, eyes, or nose, through sexual contact, through a cut or bite, or through a contaminated medical device.

[0003] People become infected with these organisms primarily by drinking contaminated water or eating contaminated food, by inhaling spores or dust, or by breathing in contaminated droplets from someone else's cough or sneeze, by handling contaminated objects (e.g. door handles), or by having direct contact with a contaminated person and then touching their eyes, nose, or mouth.

[0004] Some microorganisms are spread in bodily fluids such as blood, semen, and faeces. They can also enter the body, for example, through sexual contact with an infected partner. Pathogenic microorganisms can enter the body through human and animal bites and other wounds that puncture the skin.

[0005] Pathogenic microorganisms can also attach to medical devices that are implanted in the body (e.g., catheters, artificial joints, or artificial heart valves). They may be present on the device at the time of implantation if the device is accidentally contaminated. Infectious material from elsewhere may also spread through the bloodstream and attach to an already implanted device. Because implanted devices lack natural defenses, such microorganisms can easily grow and spread, causing disease.

[0006] After entering the human body, pathogenic microorganisms must multiply or spread to cause an infection. After growth or spread begins, three scenarios can occur: 1) the microorganism continues to grow and overwhelms the host organism's defenses, 2) an equilibrium is reached, causing a chronic infection, or 3) the host organism destroys and eliminates the invading microorganism, with or without medical treatment.

[0007] The effectiveness of existing solutions (i.e., antibiotics, antivirals) in clearing pathogenic infections depends on rapid recognition of the pathogen followed by application of a pathogen-inhibiting composition before the microorganisms become irreversibly attached to the surface. It would be highly desirable to provide a different approach that would limit the irreversible attachment of microbial pathogens to surfaces, allowing for a reduction in the phenomenon of resistance to treatment and therefore the associated impact on medical costs. Summary of the Invention

[0008] According to a first aspect, the present disclosure provides an isolated exopolysaccharide (EPS) having a weight average molecular weight in the range of 40-4000 kDa, wherein the EPS is obtained or obtainable by fermentation of a marine bacterium. In one embodiment, the isolated EPS has a weight average molecular weight (Mw) in the range of 40-2000 kDa, 40-1400 kDa, 40-1000 kDa, 40-500 kDa, 40-400 kDa, 40-300 kDa, 40-200 kDa, 40-150 kDa, or 40-100 kDa. In a preferred embodiment, the isolated EPS has a Mw in the range of 40-150 kDa. In one embodiment, the isolated EPS has a Mw in the range of 40-150 kDa, and optionally a number average molecular weight (Mn) in the range of 26-100 kDa and / or a polydispersity index (Mw / Mn) in the range of 1.2-1.8. In one embodiment, the isolated EPS described herein may contain 30-90% neutral glycosyl units, 10-70% aminoglycosyl units, and 0-15% acidic glycosyl units, with respect to the total number of glycosyl units of said EPS. In another embodiment, the isolated EPS described herein may contain mannose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine. In some further embodiments, the isolated EPS described herein may be substantially free of ribose, arabinose, rhamnose, fructose, and / or fucose. In another embodiment, the marine bacterium may be a gram-positive thermophilic bacterium, preferably Bacillus licheniformis, more preferably Bacillus licheniformis LP-T14, deposited with The National Collection of Industrial, Food and Marine Bacteria (NCIMB) under deposit number NCIMB 43557 on January 27, 2020. In another embodiment, the isolated EPS described herein may substantially lack the ability to induce an immune response.

[0009] According to a second aspect, the present disclosure provides a process for obtaining an isolated exopolysaccharide (EPS) having a weight average molecular weight (Mw) in the range of 40 to 4000 kDa, the process comprising the steps of: culturing a marine bacterium in a first culture medium to obtain a cultured marine bacterium; fermenting the cultured marine bacterium in a fermentation medium; heat treating the fermentation medium; centrifuging the fermentation medium to obtain a supernatant; and filtering the supernatant to obtain an isolated EPS. The isolated EPS may be an isolated EPS described herein. In one embodiment, the marine bacterium may be a Gram-positive thermophilic bacterium, preferably Bacillus licheniformis, more preferably Bacillus licheniformis LP-T14, deposited in The National Collection of Industrial, Food and Marine Bacteria (NCIMB) on January 27, 2020 under deposit number NCIMB 43557. In one embodiment, the fermentation medium may include sea salt (40 g / L), tryptone (6 g / L), yeast extract (6 g / L), antifoaming agent (0.33 mL / L), dextrose (12 g / L) and deionized HO (qsp.). In one embodiment, the fermentation may be carried out at pH 5-8, 40° C. for 20-40 hours, under 30-50% oxygenation. In one embodiment, the heat treatment of the fermentation medium may be carried out by heating the fermentation medium at 85° C. for 1 hour. In one embodiment, the centrifugation of the fermentation medium may be carried out at 14000 g using a disc stack centrifuge with a flow rate of 200-800 L / h. In one embodiment, the filtration of the supernatant includes sequential filtration using 1.60-0.22 μm filtration steps, and / or ultrafiltration using 10-100 kDa cutoff filter cartridges. In one embodiment, the process of the present disclosure may further include free drying the isolated EPS to obtain free-dried EPS. In one embodiment, free drying is carried out at -20°C for at least 16 hours.

[0010] According to a third aspect, the present disclosure provides a composition comprising at least one isolated EPS as described herein and a suitable carrier.The composition of the present invention can be an oral composition, a nasal composition, a topical composition, a transdermal composition, an ophthalmic composition, or a composition formulated for application to a medical device.

[0011] According to a fourth aspect, the present disclosure provides a nasal delivery system comprising at least one isolated EPS as described herein or at least one isolated EPS obtained or obtainable by the process described herein. In one embodiment, the nasal delivery system may deliver at least one isolated EPS as a nasal drop, liquid spray, dry spray, gel or ointment. In another embodiment, the at least one isolated EPS may be formulated in a composition further comprising a saline solution. In another embodiment, the nasal delivery system described herein may be for treating and / or preventing a microbial pathogen infection in a subject in need thereof. In another embodiment, the nasal delivery system described herein may be for use in treating and / or preventing a microbial pathogen infection in a subject in need thereof. In some embodiments, the nasal delivery system described herein may be for attenuating the pathogenicity of a microbial pathogen by inhibiting or reducing colonization by the microbial pathogen of the nasal cavity of a subject in need thereof. In some embodiments, the nasal delivery systems described herein may be for use to attenuate the virulence of a microbial pathogen by inhibiting or reducing colonization by the microbial pathogen of the nasal cavity of a subject in need thereof.

[0012] According to a fifth aspect, the present disclosure provides a non-therapeutic use of an isolated EPS as described herein, or an isolated EPS obtained or obtainable by a process as described herein, or a composition as described herein, for treating a non-biological surface to prevent or reduce colonization of the surface by a microbial pathogen. In one embodiment, the surface may be a surface of a medical device.

[0013] According to a sixth aspect, the present disclosure provides an isolated EPS as described herein, or an isolated EPS obtained or obtainable by a process as described herein, a composition as described herein, or a nasal delivery system as described herein, for use in a method for treating and / or preventing a microbial pathogen infection in a subject in need thereof.

[0014] According to a seventh aspect, the present disclosure provides the use of an isolated EPS as described herein, or an isolated EPS obtained or obtainable by a process as described herein, a composition as described herein, or a nasal delivery system as described herein, for the manufacture of a medicament for the treatment and / or prevention of a microbial pathogen infection.

[0015] According to an eighth aspect, the present disclosure provides an isolated EPS as described herein, or obtained or obtainable by a process as described herein, a composition as described herein, or a nasal delivery system, for use in a method for attenuating the virulence of a microbial pathogen.

[0016] According to a ninth aspect, the present disclosure provides the use of an isolated EPS as described herein, or an isolated EPS obtained or obtainable by a process as described herein, a composition as described herein, or a nasal delivery system as described herein, for the manufacture of a medicament for attenuating the pathogenicity of a microbial pathogen.

[0017] According to a tenth aspect, the present disclosure provides a method of treating and / or preventing a microbial pathogen infection in a subject in need thereof, comprising administering to the subject an effective amount of an isolated EPS as described herein, or an isolated EPS obtained or obtainable by a process as described herein, or a composition as described herein.

[0018] According to an eleventh aspect, the present disclosure provides a method for attenuating the pathogenicity of a microbial pathogen infection in a subject in need thereof, comprising administering to the subject an effective amount of an isolated EPS as described herein, or an isolated EPS obtained or obtainable by a process as described herein, or a composition as described herein.

[0019] According to a twelfth aspect, the present disclosure provides a method of formulating a nasal composition for attenuating the virulence of a microbial pathogen infection by inhibiting or reducing colonization of the nasal cavity by the microbial pathogen, comprising the step of mixing at least one isolated exopolysaccharide (EPS) derived from a marine bacterium with a saline solution to obtain a nasal composition comprising a salt concentration of 0.1%-10% w / w. In one embodiment, the at least one isolated EPS may be an isolated EPS according to the present disclosure. In a preferred embodiment, the resulting nasal composition comprises a salt concentration of 0.5%-5% w / w. More preferably, the resulting nasal composition comprises a salt concentration of 0.7%-3% w / w. In one embodiment, the resulting composition comprises a salt concentration of about 0.9% w / w. In one embodiment, the resulting composition comprises a salt concentration of about 2.2% w / w. In one embodiment, the resulting composition comprises a salt concentration of about 2.7% w / w.

[0020] According to any of the isolated EPS for use according to the present disclosure, compositions for use according to the present disclosure, or methods of the present disclosure, at least one isolated EPS or composition described herein may be administered to a subject before, during, and / or after infection with a microbial pathogen, thereby preventing, inhibiting, or reducing colonization and / or internalization of the microbial pathogen in at least one biological tissue of the subject. In one embodiment, the at least one biological tissue may be selected from the oral cavity, nasal cavity, respiratory tract, pharynx, ear, ophthalmic region, urogenital tract, skin, scalp, hair, nails, and combinations thereof. In another embodiment, the microbial pathogen may be a bacterial pathogen, and administration of the isolated EPS or composition to the subject reduces or inhibits initial bacterial biofilm formation and / or disrupts the initial bacterial biofilm, thereby attenuating the virulence of the bacterial pathogen infection. In another embodiment, the microbial pathogen may be a viral pathogen, and administration of the isolated EPS or composition to a subject attenuates the pathogenicity of the viral pathogen infection by reducing the mortality of subject cells infected with the viral pathogen compared to comparable untreated cells, by preventing or reducing the release of virions from inoculated subject cells, and / or by preventing or reducing infection of adjacent uninfected subject cells.

[0021] According to any of the nasal delivery system of the present disclosure, the non-therapeutic use of the present disclosure, the isolated EPS for use according to the present disclosure, the composition for use according to the present disclosure, or the method of the present disclosure, the microbial pathogen may be at least one of a bacterial pathogen, a viral pathogen, or a fungal pathogen. In one embodiment, the at least one bacterial pathogen may be selected from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and Streptococcus. In some embodiments, at least one of the bacterial pathogens can be of the species Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or Streptococcus mutans. In one embodiment, the at least one bacterial pathogen may be selected from Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and / or Streptococcus mutans species.In another embodiment, the at least one viral pathogen may be selected from influenza A, influenza A subtype H1N1 / 2009 / pdm09, influenza A subtype H1, influenza A subtype H3, influenza B (orthomyxovirus), coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, SARS-CoV-2 (coronavirus), parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus A / B, human metapneumovirus A / B (paramyxovirus), adenovirus, or rhinovirus / enterovirus (picornavirus). In a further embodiment, the at least one viral pathogen may be selected from coronavirus OC43, adenovirus, and / or rhinovirus / enterovirus (picornavirus).

[0022] The amount of the at least one EPS in the compositions of the present disclosure or in compositions used in accordance with the methods and uses of the present disclosure may be in the range of 0.00005-0.5% w / w, preferably 0.0001-0.1% w / w, and more preferably 0.0005-0.05% w / w, based on the total weight of the composition. [Brief description of the drawings]

[0023] [Figure 1] 1 depicts the step-by-step process used to manufacture the EPS of the present disclosure. [Diagram 2] Figure 1 shows the biofilm formation (%) of various bacterial pathogens at different EPS concentrations. Biofilm formation was normalized to untreated bacterial cells (control). Error bars represent the standard deviation of two independent experiments (N=2) performed in triplicate (n=3). *p<0.05 vs control; **p<0.01 vs control; [Diagram 3]Antibacterial effect evaluation of increasing concentrations of EPS on culture growth of various bacterial pathogens in two independent experiments (N = 2) performed in triplicate (n = 3) is shown, comparing OD600 values, mean ± SD. Cultures grown in the absence of EPS were used as control; [Figure 4] Viability of HNEpCs cultured with increasing concentrations of EPS for 2 and 24 h is shown. Viability of TO-PRO3-negative cells cultured in complete medium for 2 and 24 h was used as a control (C). Error bars represent the standard deviation of one experiment (N=1) performed in triplicate (n=3); [Diagram 5] Figure 1 shows the reduction in adhesion of P. aeruginosa and S. aureus to HNEpC in the presence of increasing concentrations of EPS. Biofilm formation (cell attachment) was normalized to untreated cells (control). Error bars represent the standard error of the mean of 2–4 independent experiments (N=2–4) performed in triplicate (n=3). **p<0.01 vs control; [Figure 6] Figure 1 shows the emulsifying activity index after mixing various concentrations of EPS solutions with vegetable oil and then allowing a 24-h rest period. Error bars represent the standard deviation of three independent experiments (N=3); [Figure 7] The viability of HNEpCs infected with adenovirus or rhinovirus, or treated with EPS (400 μg / mL) for 2 h before or after high-dose virus infection is shown. Cells cultured in BEBM complete medium for 48 h were used as positive controls, and virus-inoculated cells without EPS treatment were used as negative controls. Values ​​represent the mean ± SD of TO-PRO-3 negative (viable cells) from two independent experiments (N = 2) performed in triplicate (n = 3); [Figure 8]Figure 8A and Figure 8B show the viability of HNEpCs infected with adenovirus or rhinovirus, or treated with EPS (200 μg / mL) for 2 h before or after high-dose virus infection, respectively. Cells cultured in BEBM complete medium for 48 h were used as positive controls, and virus-inoculated cells without EPS treatment were used as negative controls. Values ​​represent the mean ± SD of TO-PRO-3 negative (viable cells) from two independent experiments (N=2) performed in triplicate (n=3); [Figure 9] The viability of HNEpCs infected with adenovirus or rhinovirus, or pretreated with EPS (400 μg / mL) for 2 h before infection, or posttreated with EPS addition for 5 days, or with EPS replacement daily for 5 days, is shown. The viability of TO-PRO-3 negative cells after 5 days of culture in BEBM complete medium was used as a control. Error bars represent the standard deviation of two independent experiments (N=2) performed in duplicate (n=2); [Figure 10] Figure 10A and Figure 10B show the viability of HNEpCs infected with adenovirus or rhinovirus, respectively, or pretreated with EPS (200 μg / mL) for 2 h before infection, or posttreated with EPS addition for 5 days, or with EPS replacement every day for 5 days. Viability of TO-PRO-3 negative cells after 5 days of culture in BEBM complete medium was used as a control. Error bars represent the standard deviation of two independent experiments (N=2) performed in triplicate (n=3); [Figure 11] The viability of HNEpCs infected with coronavirus (1 TCID50 or 0.001 TCID50), or pretreated with EPS (200–400 μg / mL) for 2 h before infection, or posttreated for 5 days with one dose of EPS added, is shown. The viability of TO-PRO-3 negative cells after 5 days of culture in BEBM complete medium was used as a control. Error bars represent the standard deviation of two independent experiments (N=2) performed in triplicate (n=3); [Figure 12]Figures 12A and 12B show the percentages of IL-8-producing HNEpCs and IL-6-producing HNEpCs, respectively, upon stimulation with EPS samples (400 μg / mL) for 24 h. Unstimulated cells or cells stimulated with IL-1β were used as negative and positive controls, respectively. Data represent the results of three independent experiments (N=3), and error bars represent SD; [Figure 13A] Figures 13A and 13B show: A) expression of activation / maturation molecules (CD83, CD40 and CD25) on dendritic cells (DCs) cultured with EPS samples (400 μg / mL) for 24 h. Unstimulated cells or cells stimulated with lipopolysaccharide (LPS, 1 μg / mL) were used as negative and positive controls, respectively. Bar graphs show mean ± SEM of MFI of indicated molecules. DCs derived from PBMCs obtained from two different donors were used in duplicate experiments (N=2, n=2). Error bars represent SD; B) percentage of IL-8- and IL-6-producing DCs stimulated with EPS samples (400 μg / mL) for 24 h. Unstimulated cells or cells stimulated with LPS were used as negative and positive controls, respectively. Data represent the results of three independent experiments (N=2, n=2), error bars represent SD. [Figure 13B] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] This disclosure proposes a novel approach to pathogen infection control based on measures consisting of reducing or preventing the attachment of pathogens to surfaces. Thus, chemical components released by marine bacteria isolated from shallow hydrothermal vents in the waters around the island of Panarea (Italy) were studied for their ability to disrupt, control, and / or inhibit the reversible attachment step of pathogenic microorganisms such as bacteria, fungi, and viruses to biological and non-biological surfaces.

[0025] A bacterium from a marine source, namely Bacillus licheniformis LP-T14 deposited at NCIMB (The National Collection of Industrial, Food and Marine Bacteria, NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK) on January 27, 2020 by Danstar Ferment AG, Poststrasse 30, Zug, 6300, Switzerland, under the deposit number NCIMB 43557, showed a high bioactivity in high-temperature (50 °C), saline water (electrical conductivity 42.90 mS.cm -1 ), have the unique ability to grow under extreme environmental conditions such as low temperature, pH 5.4, and high concentrations of hydrogen sulfide and heavy metals, and the exopolysaccharides secreted by these marine strains have been found to have unique physicochemical properties.

[0026] Thus, the present disclosure demonstrates that a novel exopolysaccharide (EPS) released by the marine strain Bacillus licheniformis LP-T14 has the ability to attenuate the virulence of pathogen infection by preventing and / or reducing pathogen attachment to treated surfaces compared to untreated surfaces.

[0027] The present disclosure provides EPS, compositions thereof, uses of the compositions and methods for attenuating the pathogenicity of microbial pathogen infections, where the pathogenicity of microbial pathogen infections is attenuated by inhibiting or reducing the colonization of the microbial pathogens responsible for the infection on biological and / or non-biological surfaces.

[0028] In the context of the present disclosure, the term "pathogenic" as used herein refers to the ability of a microorganism (i.e., bacteria, virus, yeast, fungus) to grow and / or spread within an organism, thereby overwhelming the body's defense mechanisms. Thus, when used as a prophylactic and / or therapeutic treatment, the compositions of the present disclosure (indirectly) support the host's immune system, allowing the host to effectively defend against any type of pathogenic microbial infection.

[0029] Still in the context of the present disclosure, the phrase "inhibit or reduce colonization" in relation to the compositions disclosed herein refers to any action that has the effect of interfering with the placement of pathogenic bacteria on a surface and thus their proliferation: i.e., including but not limited to, inhibiting or reducing the formation of an initial biofilm, disrupting or degrading (partially or completely) an initial biofilm, disrupting or degrading (partially or completely) an advanced biofilm.

[0030] In the context of the present disclosure, the phrase "inhibit or reduce establishment" refers to any action associated with the compositions disclosed herein that has the effect of locally disrupting the establishment of the virus in the organism. Thus, the compositions have the effect of, but are not limited to, partially or completely preventing the internalization of the virus in living cells and / or limiting the spread of virions from infected cells to adjacent living cells.

[0031] Exopolysaccharides and compositions thereof Bacteria from marine sources have the unique ability to grow under extreme environmental conditions such as high temperature, salt water, and high concentrations of hydrogen sulfide and heavy metals, and the exopolysaccharides (EPS) produced by marine species have been found to have unique physical properties and molecular structures. Therefore, marine bacterial EPS has been applied in a wide range of fields such as biotechnology, medicine, and pharmaceuticals due to its unique properties such as water solubility, biodegradability, biocompatibility, bioadhesiveness, heat resistance, swelling ability, and gelling ability.

[0032] EPS is a polysaccharide, produced by bacterial fermentation and then released into the culture medium. It is composed of monosaccharides linked together via glycosidic bonds. EPS is composed of one or more types of monosaccharides, and repeating units of ordered or randomly distributed monosaccharide blocks can be observed. The EPS backbone can be linear or branched, in which case the branch arms can have different lengths and contain either the same or different monosaccharide units.

[0033] In one embodiment, the at least one isolated EPS is obtained or obtainable by fermentation of a marine bacterium and its acceptable carrier. In another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or obtainable by fermentation of a gram-positive thermophilic bacterium and its acceptable carrier. In another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or obtainable by fermentation of a bacterium belonging to the genus Bacillus and its acceptable carrier. In another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or obtainable by fermentation of a bacterium belonging to the species Bacillus licheniformis and its acceptable carrier. In yet another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or obtainable by fermentation of a specific bacterial strain, the extreme thermophilic marine Bacillus licheniformis LP-T14 deposited in the National Collection of Industrial, Food and Marine Bacteria (NCIMB) under NCIMB 43557, and an acceptable carrier thereof.

[0034] In one embodiment, fermentation of the marine bacteria is carried out in a fermentation medium containing sea salt (40 g / L), tryptone (6 g / L), yeast extract (6 g / L), antifoam (0.33 mL / L), dextrose (12 g / L) and deionized HO (qsp.) at pH 5-8, 40° C. for 20-40 hours under 30-50% oxygen addition. After fermentation is complete, and prior to EPS isolation, the fermentation medium can be heated to 85° C. for 1 hour to inactivate both the marine bacteria and proteins derived from said marine bacteria.

[0035] The term "isolated" should be taken to mean that the substance is removed from the original environment in which it is naturally produced, such as the fermentation medium in this case. The removed substance is usually purified from the environment in which it was produced. In one embodiment, the isolated form of EPS ideally does not contain significant amounts of bacterial strains, which in some embodiments may be partially or completely inactivated. In some other embodiments, the isolated form of EPS contains negligible amounts of bacterial strains. In further embodiments, the isolated form of EPS contains at most 10 1 CFU / g EPS, at most 10 2 CFU / g EPS, at most 10 3 CFU / g EPS, at most 10 4 CFU / g of EPS, or at most 10 5CFU / g of EPS. In one embodiment, the isolated form of EPS lacks detectable amounts of proteins from the bacterial strain used to produce it, which may be partially or completely inactivated in some embodiments, as analyzed by the Lowry assay (Example 4). In some embodiments, the isolated form of EPS contains negligible amounts of proteins from the bacterial strain used to produce it, as analyzed by the Lowry assay (Example 4). In some further embodiments, the isolated form of EPS contains at most 0.1% w / w, at most 0.5% w / w, at most 1% w / w, at most 2% w / w, at most 3% w / w of proteins from the bacterial strain used to produce it, as analyzed by the Lowry assay (Example 4). Furthermore, after separation from the majority of other bacterial culture medium components, the isolated EPS of the present disclosure may be concentrated by, but is not limited to, filtration, ultrafiltration, evaporation, spray drying or freeze drying, or a combination thereof. Thus, the isolated EPS of the present disclosure is in the form of a concentrate, suspension, powder, or lyophilizate.

[0036] In one embodiment, the isolated EPS contained in the composition of the present disclosure exhibits at least one weight average molecular weight distribution in the range of 40-4000 kDa, 40-2000 kDa, 40-1000 kDa, 40-500 kDa, 40-400 kDa, 40-300 kDa, and 40-200 kDa as determined by size exclusion chromatography (HP-SEC). In one embodiment, the isolated EPS exhibits at least two weight average molecular weight distributions in the range of 40-4000 kDa, 40-2000 kDa, 40-1000 kDa, 40-500 kDa, 40-400 kDa, 40-300 kDa, and 40-200 kDa as determined by size exclusion chromatography (HP-SEC).

[0037] In one embodiment, the isolated EPS of the present disclosure comprises neutral glycosyl units, aminoglycosyl units, and acidic glycosyl units. Examples of neutral glycosyl units include, but are not limited to, glucose, rhamnose, mannose, xylose, and galactose. Examples of aminoglycosyl units include, but are not limited to, galactosamine, glucosamine, N-acetylglucosamine, and N-acetylgalactosamine. Examples of acidic glycosyl units include, but are not limited to, uronic acids, including glucuronic acid, galacturonic acid, and hexuronic acid. In one embodiment, the isolated EPS of the present disclosure comprises neutral glycosyl units including glucose, rhamnose, mannose, xylose, and galactose. In one embodiment, the isolated EPS of the present disclosure comprises aminoglycosyl units including N-acetylglucosamine and N-acetylgalactosamine. In one embodiment, the isolated EPS of the present disclosure may comprise acidic glycosyl units including glucuronic acid. In one embodiment, the isolated EPS of the present disclosure comprises between 30-90%, 35-80%, 40-75% neutral glycosyl units, based on the total number of glycosyl units of the EPS. In one embodiment, the isolated EPS of the present disclosure comprises between 10-70%, 15-65%, 20-60% aminoglycosyl units, based on the total number of glycosyl units of the EPS. In one embodiment, the isolated EPS of the present disclosure comprises between 0-15%, 0-10%, 0-5% acidic glycosyl units, based on the total number of glycosyl units of the EPS. In another embodiment, the isolated EPS of the present disclosure is structurally composed of glycosyl units including mannose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine. In another embodiment, the isolated EPS of the present disclosure is structurally not composed of ribose, arabinose, rhamnose, fructose, and / or fucose. In another embodiment, the isolated EPS of the present disclosure is substantially free of ribose, arabinose, rhamnose, fructose and / or fucose.

[0038] The isolated EPS of the present disclosure exhibits strong emulsifying activity even at low concentrations (i.e., 50 μg.mL, which corresponds to 0.005% EPS). -1 (50% of E24 was reached using only

[0039] In one embodiment, the method of the present disclosure comprises the administration of an effective amount of at least one EPS, optionally provided as a composition. The term "effective amount" refers to a concentration of isolated EPS sufficient to ensure attenuation of the pathogenicity of a microbial pathogen infection by inhibiting or reducing colonization by the microbial pathogen on a treated surface as compared to an untreated surface. In some embodiments, the concentration of the isolated EPS ranges from 0.00005% to 0.5% w / w based on the total weight of the composition. In some other embodiments, the concentration of the isolated EPS ranges from 0.0001% to about 0.1% w / w based on the total weight of the composition. In yet another embodiment, the concentration of the isolated EPS ranges from 0.0005% to about 0.08% w / w based on the total weight of the composition.

[0040] In another embodiment, the composition of the present disclosure comprises at least 0.000005% w / w of the composition, at least 0.00001% w / w of the composition, at least 0.00002% w / w of the composition, at least 0.00005% w / w of the composition, at least 0.0001% w / w of the composition, at least 0.0002% w / w of the composition, at least 0.0005% w / w of the composition, at least 0.001% w / w of the composition, at least 0.002% w / w of the composition, at least 0.0001% w / w of the composition, at least 0.002% w / w of the composition. % w / w of the composition, at least 0.004% w / w of the composition, at least 0.005% w / w of the composition, at least 0.0075% w / w of the composition, at least 0.01% w / w of the composition, at least 0.015% w / w of the composition, at least 0.02% w / w of the composition of at least one isolated EPS capable of reducing the pathogenicity of a microbial pathogen infection by inhibiting or reducing colonization of the treated surface by said microbial pathogen.

[0041] In other embodiments, the compositions of the present disclosure comprise a concentration of about 0.5 to 5000 μg.mL -1, about 1~1000μg.mL -1 , about 5~500μg.mL -1 The treatment may be an aqueous solution or suspension comprising at least one isolated EPS having the ability to attenuate the pathogenicity of a microbial pathogen infection by inhibiting or reducing colonization of a treated surface by said microbial pathogen.

[0042] In another embodiment, the composition of the present disclosure has a concentration of at least 50 ng.mL -1 , at least 100ng.mL -1 , at least 200 ng.mL -1 , at least 500ng.mL -1 , at least 1 μg.mL -1 , at least 2 μg.mL -1 , at least 5 μg.mL -1 , at least 10 μg.mL -1 , at least 20 μg.mL -1 , at least 40 μg.mL -1 , at least 50 μg.mL -1 , at least 75 μg.mL -1 , at least 100 μg.mL -1 , at least 150 μg.mL -1 , at least 200 μg.mL -1 The treatment may be an aqueous solution or suspension comprising at least one isolated EPS having the ability to attenuate the pathogenicity of a microbial pathogen infection by inhibiting or reducing colonization by said microbial pathogen on a treated surface.

[0043] The compositions disclosed herein comprise at least one isolated exopolysaccharide derived from marine bacteria and a suitable carrier. The selection of the carrier(s) is made according to the type of final formulation. In one embodiment, the carrier of the compositions herein can increase but not decrease the biological activity (i.e., anti-biofilm activity and / or antiviral activity) of the composition. In another embodiment, the carrier of the compositions herein is immunologically inactive. In another embodiment, the carrier of the compositions herein can be a combination of one or more carriers.

[0044] In one embodiment, the compositions disclosed herein may further comprise another EPS. In another embodiment, the compositions disclosed herein may further comprise polyglutamic acid.

[0045] In one embodiment, apart from its antibiofilm activity (e.g., in vitro), the isolated EPS described herein, optionally provided as a composition, does not adversely affect the growth of pathogenic bacteria, and more generally, the presence of EPS does not affect the growth rate of all bacterial strains tested. In another embodiment, the isolated EPS described herein, optionally provided as a composition, does not have bactericidal, bacteriostatic, and antibiotic effects, independent of its concentration and the pathogenic bacteria considered. In another embodiment, the isolated EPS described herein, optionally provided as a composition, also respects the integrity of the natural microflora present on living tissue. In another embodiment, the isolated EPS described herein, optionally provided as a composition, does not induce cytotoxicity in the subject intended to receive them, regardless of its concentration and its incubation period.

[0046] In another embodiment, the isolated EPS described herein, optionally provided as a composition, is substantially devoid of the ability to induce an immune (cellular and / or humoral) response. In some embodiments, the isolated EPS described herein, optionally provided as a composition, is devoid of the ability to induce any immunological response, such as inflammatory response and / or anergy. In another embodiment, the isolated EPS described herein, optionally provided as a composition, is devoid of a detectable effect on the maturation of dendritic cells into immunostimulatory antigen-presenting cells. In yet another embodiment, the isolated EPS described herein, optionally provided as a composition, does not induce CD83, CD40 and / or CD25 expression from dendritic cells. In another embodiment, the isolated EPS described herein, optionally provided as a composition, is not capable of inducing the proliferation of cells expressing IL-6 and / or IL-8. In some other embodiments, the isolated EPS described herein, optionally provided as a composition, is not capable of inducing the production of inflammatory mediators, such as cytokines, chemokines and / or prostaglandins. In yet another embodiment, the isolated EPS described herein, optionally provided as a composition, is incapable of inducing the production of IL-6 and / or IL-8.

[0047] Anti-biofilm activity In the case of bacterial pathogens, pathogenic biofilms can develop not only on various mucosal and epithelial cells, oral, nasal, pulmonary, gastrointestinal, cutaneous, ophthalmic, and genitourinary tracts, but also on non-biological and hydrophobic non-polar surfaces (e.g., Teflon, glass and plastic), such as central venous, urinary and peritoneal dialysis catheters, endotracheal tubes, pacemakers, artificial joints and mechanical heart valves.

[0048] Microbial pathogens embedded in such matrices can survive by forming their own ecosystem, where they can defend themselves against the body's immune defenses and metabolites or substances produced by exogenous treatments, while at the same time acquiring new capabilities, such as resistance genes, through cell-cell communication (e.g., quorum sensing) to adapt and confront the environment. Clinically, therefore, biofilm formation is known to be a key factor in the establishment and persistence of some difficult-to-treat infectious diseases. For example, exopolysaccharides produced by pathogenic bacteria protect the pathogenic bacteria, thereby making them refractory to antimicrobial treatments.

[0049] Thus, the present disclosure, which aims to attenuate the pathogenicity of bacterial infections, is directed not only to active or established bacterial infections, but also to the inhibition of the initial onset of pathogenicity that leads to bacterial infection, i.e., the initial biofilm formation in which bacteria reversibly attach onto a surface.

[0050] In one embodiment, the composition of the present disclosure imparts a biofilm formation inhibitory effect on various bacterial pathogens incubated under optimal conditions on synthetic surfaces compared to untreated surfaces. In another embodiment, surface pretreatment with EPS according to the present disclosure inhibits initial biofilm formation even when applied at low concentrations compared to untreated surfaces. In another embodiment, the use of the composition of the present disclosure results in a reduction in biofilm formation from various bacterial pathogens incubated under optimal conditions on synthetic surfaces. In another embodiment, surface pretreatment with the composition reduces initial biofilm formation even when applied at low concentrations compared to untreated surfaces. In another embodiment, the composition of the present disclosure reduces pathogenic bacterial biofilm formation on synthetic surfaces by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15% compared to untreated synthetic surfaces. In another embodiment, a composition of the present disclosure having an EPS concentration of at least 200 μg / mL reduces pathogenic bacterial biofilm formation on a synthetic surface by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% compared to an untreated synthetic surface. In another embodiment, a composition of the present disclosure having an EPS concentration of 400 μg / mL reduces pathogenic bacterial biofilm formation on a synthetic surface by at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to an untreated synthetic surface.

[0051] In one embodiment, the EPS of the present disclosure has the ability to reduce and / or limit the consequences of bacterial biofilm establishment on biological surfaces, regardless of their origin. Indeed, in addition to the demonstrated preventative effect of reducing and / or preventing biofilm formation, surface cleaning with a composition according to the present disclosure may also have therapeutic activity due to its ability to remove pathogenic bacterial cells from biofilms. In another embodiment, the composition according to the present disclosure promotes the destruction or dissolution of biofilms on surfaces (reduction of existing colonization and pre-formed or accumulated biofilms). In some embodiments, the composition according to the present disclosure promotes the destruction or dissolution of initial biofilms on surfaces. In another embodiment, surface treatment with a composition according to the present disclosure prevents, reduces and / or inhibits bacterial recolonization on the surface. In another embodiment, the EPS composition of the present disclosure reduces pathogenic bacterial biofilm formation on biological surfaces by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% compared to untreated biological surfaces. In another embodiment, a composition of the present disclosure having an EPS concentration of at least 200 μg / mL reduces pathogenic bacterial biofilm formation on a biological surface by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% as compared to an untreated biological surface. In another embodiment, a composition of the present disclosure having an EPS concentration of 400 μg / mL reduces pathogenic bacterial biofilm formation on a biological surface by at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% as compared to an untreated biological surface.

[0052] In another embodiment, the surface treatment with the composition according to the present disclosure is performed within 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour after contact with pathogenic bacteria. The surface treatment with the composition according to the present disclosure can also be applied immediately after contact with pathogenic bacteria. The surface treatment with the composition according to the present disclosure can also be applied as a preventative measure before contact with pathogenic bacteria. Thus, the surface treatment with the composition according to the present disclosure can be applied at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, or at least 8 hours before potential contact with pathogenic bacteria.

[0053] Antiviral activity Viral diseases or infections are transmitted by viruses that attack the body at one or more specific locations. For example, a person may contract a viral disease through direct contact with another individual who is already sick, i.e., through bodily fluids or aerosols, as well as contact with infected surfaces.

[0054] Once inoculated into the body, the virus enters specific host cells and converts the cellular machinery to multiply, and the virions thus synthesized then spread and infect neighboring cells.

[0055] The present disclosure is also directed to attenuating the pathogenicity of viral infections by inhibiting and / or reducing the colonization of viral pathogens on treated surfaces. In other words, the reduction and / or inhibition of surface colonization of viral pathogens is achieved by reducing the mortality rate of viable cells compared to untreated cells and / or by preventing the release of virions and their spread to adjacent viable cells.

[0056] In one embodiment, pretreatment of live cells with a composition according to the present disclosure prior to virus inoculation emphasizes the preventative properties of the EPS composition against virus infection, regardless of the virus considered. In one embodiment, pretreatment of live cells with a composition according to the present disclosure prior to virus inoculation reduces cell mortality compared to untreated cells. In yet another embodiment, pretreatment of live cells with a composition according to the present disclosure (i.e., prior to virus inoculation of live cells) is administered in a single dose prior to virus inoculation. In another embodiment, pretreatment of live cells with a composition according to the present disclosure prior to virus inoculation increases cell survival after virus infection (regardless of the virus considered) compared to untreated cell survival. In another embodiment, pretreatment of live cells with a composition according to the present disclosure prior to virus inoculation (1 TCID50) increases cell survival by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15% compared to untreated cell survival. In another embodiment, pretreatment of live cells with a composition according to the present disclosure prior to virus inoculation (0.001 TCID50) increases cell viability by at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to untreated cell viability.

[0057] In one embodiment, post-treatment of live cells with a composition according to the present disclosure (i.e., after inoculation of live cells with a virus) prevents the release of virions as well as their spread to neighboring cells. In another embodiment, post-treatment of live cells with a composition according to the present disclosure (i.e., after inoculation of live cells with a virus) provides a therapeutic effect that reduces the pathogenicity of the virus infection compared to untreated cells for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days after inoculation of the virus. In yet another embodiment, post-treatment of live cells with a composition according to the present disclosure (i.e., after inoculation of live cells with a virus) is administered in a single dose after inoculation of the virus. In another embodiment, without wishing to be bound by theory, administration of an EPS composition according to the present disclosure to live human cells forms an extracellular "barrier" that sterically impedes internalization of the virus, regardless of the virus considered. In one embodiment, post-treatment of live cells with an EPS composition according to the present disclosure after inoculation of the virus increases the viability of the cells compared to the viability of untreated cells. In another embodiment, post-treatment of live cells with an EPS composition according to the present disclosure after virus inoculation (1 TCID50) increases cell viability by at least 1%, at least 2%, at least 3%, at least 4% compared to untreated cell viability. In some other embodiments, post-treatment of live cells with a composition according to the present disclosure after virus inoculation (0.001 TCID50) increases cell viability by at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30% compared to untreated cell viability.

[0058] In another embodiment, surface treatment with a composition according to the present disclosure prevents, reduces and / or inhibits viral recolonization on the surface.

[0059] In another embodiment, the surface treatment with the composition according to the present disclosure is performed within 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour after contact with the virus. The surface treatment with the EPS composition according to the present disclosure can also be applied immediately after contact with the virus. The surface treatment with the composition according to the present disclosure can also be applied as a preventative measure before contact with the virus. Thus, the surface treatment with the composition according to the present disclosure can be applied at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, or at least 8 hours before potential contact with the virus.

[0060] pathogen The compositions according to the present disclosure are effective to attenuate the virulence of microbial pathogen infections by inhibiting or reducing the establishment of microorganisms such as bacteria, fungi, yeasts and viruses on surfaces treated with the claimed compositions.

[0061] In one embodiment, a composition according to the present disclosure is effective to attenuate the virulence of a bacterial pathogen infection by inhibiting or reducing bacterial colonization. In some embodiments, bacteria to which the present disclosure may be of particular use include, but are not limited to, Staphylococcus species, Streptococcus species, Enterococcus spp., Enterococcus spp., Neisseria or Branhamella species, Bacillus species, Propionibacterium species, Corynebacterium species, Listeria species, Clostridium species, Escherichia species, Enterobacter species, Proteus species, Pseudomonas species, Klebsiella species, and the like. ella species, Salmonella species, Shigella species, Campylobacter species, Actinomyces species, Actinobacillus species, Acinetobacter species, Aggregatibacter species, Fusobacterium species, Haemophilus species, Mycobacterium species, Pseudomonas species, Porphyromonas species, Bacteriodes species, Treponema species, Prevotella species, or Eubacterium species, both gram-negative and gram-positive bacteria. In some alternative embodiments, bacteria to which the present disclosure may be of particular use include both gram-negative and gram-positive bacteria, such as, but not limited to, Staphylococcus aureus.aureus, Methicilin Resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes (group A), Streptococcus spp. (viridans group), Streptococcus agalactiae (group B), Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Streptococcus mutans mutans, S. sanguis, S. oralis, S. mitis, S. salivarius, S. gordonii, Neisseria gonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis, Bacillus anthracis, Bacillus subtilis, Propionibacterium acnes, Corynebacterium diphtheriae, Listeria monocytogenes, Clostridium tetani, Clostridium difficile difficile, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Campylobacter jejuni, Actinobacillus actinomycetumcomitans, Acinetobacter baumanniibaumannii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Haemophilus influenzae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Porphyromonas gingivalis, Bacteriodes forcythus, Treponema denticola, Prevotella intermedia, or Eubacterium nodatum. In one embodiment, the bacterial species is Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus mutans, Moraxella catarrhalis, Propionibacterium acnes (also known as Cutibacterium acnes), or a combination thereof. In yet another embodiment, the bacterial species is selected from the group consisting of Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catharalis, Propionibacterium acnes, Cutibacterium acnes, and the like.acnes), or a combination thereof.

[0062] Thus, compositions according to the present disclosure may be useful for attenuating the pathogenicity of bacterial pathogen infections by inhibiting and / or reducing colonization on treated surfaces to prevent, attenuate, and / or reduce the likelihood of microbial infection(s) including, but not limited to, bacteremia, septicemia, pneumonia, meningitis, osteomyelitis, endocarditis, caries, periodontal disease, sinusitis, rhinitis, pink eye, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne, redness, atopic dermatitis, psoriasis, wound infections, burn infections, fasciitis, bronchitis, or various abscesses, hospital-acquired infections, and / or opportunistic infections.

[0063] In another embodiment, the compositions according to the present disclosure are effective for attenuating the virulence of viral pathogen infections by inhibiting or reducing viral colonization on surfaces or epithelia. The EPS compositions according to the present disclosure are effective against certain viruses, such as HIV, herpes simplex virus, cytomegalovirus and / or human papillomavirus, but also against rhinovirus, orthomyxovirus, paramyxovirus, coronavirus, adenovirus, influenza, Rous-Sacoma virus, parainfluenza, metapneumovirus and / or Epstein-Barr virus.

[0064] Thus, compositions according to the present disclosure are also effective for preventing and / or treating infectious diseases such as colds, influenza, intolerance to colds, genital herpes, warts, and / or for preventing HIV and SARS-CoV-2 infections. In some embodiments, the virus is influenza, coronavirus, SARS-CoV-2, parainfluenza, respiratory syncytial virus, metapneumovirus, or a combination thereof.

[0065] biological surface applications In one embodiment, the compositions and methods according to the present disclosure are applicable to humans, or more generally to animal tissues, including, but not limited to, cells, epithelial cells and / or mucous membranes. Biological surfaces considered in the present disclosure include, but are not limited to: oral cavity, dental surfaces, nasal cavity, respiratory tract, pharynx, ear, ophthalmic area, urogenital tract, skin, scalp, hair and / or nails. In some embodiments, the method of administration of the compositions according to the present disclosure is nasal, respiratory and / or buccal route.

[0066] In one embodiment, the compositions and methods according to the present disclosure can be administered to healthy subjects with intact healthy tissue, but are useful when the subject has tissue that has been destroyed after a wound or scratch that may lead to local and / or systemic infection.In another embodiment, the compositions and methods according to the present disclosure can be administered to subjects susceptible to infection.In another embodiment, the compositions and methods according to the present disclosure can be administered to subjects with infection.

[0067] In one embodiment, the subject to which the compositions and methods according to the present disclosure are administered is an animal. In another embodiment, the subject to which the compositions and methods according to the present disclosure are administered is a mammal. In yet another embodiment, the subject to which the compositions and methods according to the present disclosure are administered is a human.

[0068] In one embodiment, for intranasal administration, the compositions according to the present disclosure may be administered in the form of an ointment or gel applied directly to the nasal mucosa. In another embodiment, the compositions according to the present disclosure may be administered via nasal drops, liquid sprays, such as plastic bottle atomizers, bag-on-valve aerosols, and / or metered dose inhalers. Nasal solutions are typically aqueous solutions designed to be administered to the nasal cavity in drops or sprays, and are prepared to resemble nasal secretions in many respects so that normal ciliary action can be maintained. In one embodiment, the aqueous nasal solutions are typically isotonic or may be hypertonic, and have a final saline concentration of between 0.1% and 10% w / w based on the total weight of the nasal composition. In some embodiments, the aqueous nasal solutions are typically isotonic or may be hypertonic, and have a final saline concentration of between 0.5% and 5% w / w based on the total weight of the nasal composition. In some alternative embodiments, the aqueous nasal solutions may be generally isotonic or hypertonic, and provide a final saline concentration of between 0.7% and 3% w / w based on the total weight of the nasal composition.

[0069] For respiratory administration, also known as inhalation, nebulization or pneumoperitoneum, the compositions according to the present disclosure are administered via pressurized aerosol as a fine powder suspension or solution in conjunction with a liquefied gas propellant. Upon release through a suitable valve and oral adaptor, the composition is propelled into the subject's airways. A fine mist is generated by pressurized aerosol (i.e., pneumoperitoneum, nebulizer), so its use is considered advantageous. Pressurized aerosols may contain suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, nitrogen, carbon dioxide or other suitable gases. For pressurized aerosols, dosage units can be determined by providing a valve to deliver a metered amount.

[0070] The composition according to the present disclosure may be applied topically to oral mucosal tissue, oral gingival tissue, and / or tooth surface in some conventional manner. For example, the gingival or mucosal tissue may be rinsed with a liquid (e.g., mouthwash, mouth spray) containing the composition according to the present disclosure; or, if the composition according to the present disclosure is in the form of a dentifrice (e.g., toothpaste, tooth gel, or tooth powder), the gingival / mucosal tissue or tooth is bathed in the liquid and / or foam generated by brushing the teeth. Other non-limiting examples include directly applying a non-abrasive gel or paste containing the claimed composition to the gingival / mucosal tissue or tooth with or without an oral care device as described below; chewing a gum containing the claimed composition; chewing or sucking a breath tablet, lozenge, or dissolving strip containing the claimed composition. In some embodiments, the method of applying the composition to the gingival / mucosal tissue and / or tooth is via rinsing with a mouthwash or via brushing with a dentifrice. In some alternative embodiments, the method of applying the compositions according to the present disclosure to the gingival / mucosal tissue and / or teeth is via a plastic bottle atomizer or a bag-on-valve aerosol. Other methods of topically applying the EPS compositions according to the present disclosure to the gingival / mucosal tissue and tooth surfaces will be apparent to those skilled in the art.

[0071] For vaginal administration, compositions according to the present disclosure may be presented as pessaries, tampons, suppositories, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0072] When formulated for topical administration, the compositions according to the present disclosure may contain typical components in topical pharmaceutical compositions, medical devices such as wound dressings, or cosmetic compositions, such as carriers, vehicles, or media. Specifically, the carriers, vehicles, or media are compatible with the tissues to which they are applied, such as the oral cavity, dental surfaces, nasal cavity, respiratory tract, pharynx, ears, ophthalmic areas, urogenital tract, skin, scalp, hair, nails, or mucous membranes. The compositions and components of the present disclosure are suitable for contacting infected tissues or for general use on subjects without undue toxicity, incompatibility, instability, allergic response, etc. The EPS compositions according to the present disclosure may optionally contain any additional components conventionally used in the field under consideration.

[0073] In terms of their form, the compositions according to the present disclosure may include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solid or liquid compositions used for application to the skin and other tissues where the compositions may be used. Such compositions may include the following: antimicrobial agents, moisturizing and hydrating agents, penetrating agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyes, colorants, powders, viscosity modifiers, or water, and may optionally include anesthetics, anti-itch actives, plant extracts, conditioning agents, darkening or lightening agents, illuminating agents, moisturizing agents, mica, minerals, polyphenols, silicones or derivatives thereof, sunscreens, vitamins, or phytomedicinals. In yet another embodiment, the EPS compositions according to the present disclosure are formulated with the above-mentioned ingredients that provide the EPS compositions with long-term stability, which may be required for continuous or long-term treatment.

[0074] In terms of the method of administration, the carrier of the composition according to the present disclosure may be selected to provide a residence time of the composition on the treated surface of at least 1 minute, or at least 5 minutes, or at least 10 minutes, or at least 15 minutes, or at least 20 minutes, or at least 25 minutes, or at least 30 minutes, or at least 1 hour, or at least 2 hours, or at least 4 hours, or at least 8 hours after application.

[0075] For liquid formulations, the volume of the claimed composition may be adjusted according to an estimate of the total surface area of ​​the tissue to be treated for a given human or animal. For example, for mouthwash formulations, about 1 mL to about 30 mL, e.g., about 3 mL to about 25 mL, or about 5 mL to about 20 mL; or for nasal spray formulations, about 0.05 mL to about 2 mL, e.g., about 0.1 mL to about 1.5 mL. In some embodiments, the volume of the claimed composition dedicated to nasal formulations is about 50 μL to about 300 μL per nostril, or about 100 μL to about 200 μL per nostril for bag-on-valve aerosol delivery. In some alternative embodiments, the volume of the claimed composition dedicated to nasal formulations is about 0.3 mL to 2 mL, or about 0.6 mL to 1.2 mL per nostril for plastic bottle atomizer delivery. Other delivery vehicles, such as dissolving strips, may have dosages obtained from these ranges, taking into account adjustments for concentration and other factors known to those skilled in the art.

[0076] The daily dosage of the compositions according to the present disclosure typically depends on the condition of the subject (healthy for preventive treatment or diseased for curative treatment) and also on the surface to be treated. The daily dosage may be administered once a day or in two or more doses administered two or more times a day.

[0077] In one embodiment, the composition according to the present disclosure is administered daily for a period of at least 1 day, at least 3 days, at least 5 days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 1 month, or at least 2 months, or at least 3 months.In a non-limiting embodiment, it may be desirable to continue treatment until no pathogenic bacteria are detected on the treated surface.In another embodiment, the EPS composition according to the present disclosure is administered to a subject as a preventative treatment for an undetermined period of time, such as once a day or twice a day.

[0078] The composition according to the present disclosure can be used alone or in combination with another antibiotic / antibacterial / antiviral agent.For example, the inhibition of biofilm production will make microbial pathogens much more sensitive to the action of another antibiotic / antibacterial / antiviral agent, such as those that are traditionally used to treat microbial pathogens specific.Similarly, the composition according to the present disclosure can also be used alone or in combination with another EPS.

[0079] Non-biological surface applications In one embodiment, the composition according to the present disclosure can also be applied to non-biological surfaces, including, but not limited to, hydrophobic and non-polar surfaces. Essentially any medical device that is susceptible to being colonized by pathogenic microorganisms and / or is at least partially covered by biofilm is suitable for implementing the present disclosure, including contact lenses, contact lens cases, ophthalmic and lens treatment solution containers, or other related products, analyte sensing devices such as electrochemical glucose sensors, drug delivery devices such as insulin pumps, hearing enhancing devices such as cochlear implants, urine contact devices (e.g., urethral stents, urethral catheters), blood contact devices (including cardiovascular stents, venous access devices, valves, vascular grafts, hemodialysis and biliary stents), or body tissue and tissue fluid contact devices (including biosensors, implants and artificial organs). Medical devices that may be treated with the compositions include permanent catheters (e.g., central venous catheters, dialysis catheters, long-term tunneled central venous catheters, short-term central venous catheters, peripherally inserted central venous catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters, urinary catheters, or peritoneal catheters), long-term urinary devices, tissue-adhered urinary devices, vascular grafts, vascular catheter ports, wound drain tubes, ventricular catheters, hydrocephalus shunts, cerebral or spinal shunts, heart valves, cardiac assist devices (e.g., left ventricular assist devices), pacemaker capsules, incontinence devices. , penile implants, miniature or temporary artificial joints, urinary dilators, cannulas, elastomers, hydrogels, surgical instruments, dental instruments, tubing such as intravenous tubes, breathing tubes, dental water lines, dental drainage tubes, or feeding tubes, fabric, paper, indicator strips (paper indicator strips or plastic indicator strips), adhesives (e.g., hydrogel adhesives, hot melt adhesives, or solvent-based adhesives), bandages, wound dressings, orthopedic implants, or other devices used in the medical field.Further medical devices include, but are not limited to, devices that may be inserted or implanted into a human or other animal, or that may be placed at an insertion or implantation site, such as the skin near the insertion or implantation site, and that include at least one surface that is susceptible to colonization by biofilm-embedded microorganisms. Medical devices also include any other surface that may be desired or required to prevent the proliferation or growth of biofilm-embedded microorganisms on at least one surface of the medical device, or to remove or clean biofilm-embedded microorganisms from at least one surface of the medical device, such as the surface of equipment in an operating room, emergency room, hospital room, clinic, or bathroom.

[0080] The composition according to the present disclosure can be administered with the addition of an acceptable carrier onto a decontaminated non-biological surface that will come into contact with a biological surface. In some cases, the functionality or physical stability of the composition according to the present disclosure can also be increased by the addition of various additives to the aqueous solution or suspension. Additives such as, but not limited to, polyols (including sugars), amino acids, surfactants, polymers, other proteins or certain salts can be used. The composition and method according to the present disclosure can be used alone or in combination with another sterilization treatment. For example, the inhibition of biofilm production will make microbial pathogens much more susceptible to the action of another sterilization treatment that is traditionally used to kill or discard microbial pathogen-specific from the treated surface.

[0081] In one embodiment, the composition according to the present disclosure is applied, soaked and / or coated on the medical device or instrument before the medical device or instrument encounters a contaminated environment and / or before the object or instrument encounters the tissue of the subject. In another embodiment, both the tissue of the subject and the medical device or instrument that contacts said tissue can be treated with the composition according to the present disclosure to limit pathogen attachment and / or reduce the risk of infection (once in contact with the tissue of the subject). In another embodiment, the composition according to the present disclosure can be used to facilitate the decontamination process of reusable medical materials or instruments. In some further embodiments, the composition according to the present disclosure can be used to facilitate the decontamination process of reusable medical materials or instruments before and / or after sterilization of the medical materials or instruments.

[0082] Surfaces treated with a composition according to the present disclosure, whether by spraying or dipping, should be partially or completely covered to reduce pathogen adhesion to said surface. The applied EPS composition according to the present disclosure may be effective to cover a substantial percentage of the treated surface, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the total surface. EXAMPLES

[0083] [Example 1: EPS production process] The seven-step process carried out to produce EPS is shown in Figure 1. Briefly, it is as follows: Reactivation and preculture: The contents of a cryotube containing Bacillus licheniformis LP-T14 (deposit number NCIMB 43557) were separated into a preculture tube and then suspended in 9 mL of adapted liquid Zobell medium, adhering to sterile conditions. This preculture tube was placed under orbital agitation at 40 °C and used to inoculate the liquid medium test specimens made in 150 mL Erlen in liquid Zobell medium under agitation at 40 °C for 5 h. Four 5 L fermenters were then inoculated with 150 mL of Erlen preculture and placed at 40 °C for 4–8 h under agitation, oxygenation (30–60%), and pH (5–8) control until the optical density (OD) was >2, ensuring that the growth of the bacteria was in the early exponential phase.

[0084] Fermentation: A 500 L fermenter was filled with 150 L of sterile fermentation medium consisting of sea salt (40 g / L), tryptone (6 g / L), yeast extract (6 g / L), antifoam (0.33 mL / L), dextrose (12 g / L), and deionized H2O (qsp.). The fermentation medium was inoculated with 5 L of pre-fermentation culture and kept under stirring, oxygenation 30-60%, and pH control of 5-8 for 20-40 h at 40 °C. Both the growth (OD) and glucose consumption of the strain were monitored until the end of the fermentation.

[0085] Heat treatment: Heat treatment was carried out at 85°C for 1 hour in a 400L jacketed stirred tank to inactivate enzymes and bacteria. Centrifugation: The entire culture medium was centrifuged in a disc stack centrifuge (14000g, flow rate 200-800L / h) to separate the biomass and the supernatant. Filtration and ultrafiltration: The supernatant was further treated with successive filtration (1.60–0.22 μm) and ultrafiltration steps (10–100 kDa cutoff cartridge hollow fiber polysulfone) to purify and recover only the high molecular weight compounds, i.e., EPS. Freeze-drying: EPS was frozen at -20°C for at least 16 h and then freeze-dried in a freeze-dryer with an ice capacity of 100 kg. Packaging: The lyophilized EPS was packaged in vacuum sealed low density polyethylene bags and labeled before being formulated into the appropriate formulation. Samples from each batch were characterized according to standard procedures.

[0086] [Example 2: EPS Characterization-Average Molecular Weight Determination] The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0087] EPS was analyzed by a size-exclusion chromatography (HP-SEC) instrument, i.e., TDA 302 (Viscotek) equipped with a refractive index detector, and laser light scattering and viscometer detectors. The system was calibrated with pullulan standards (PolyCAL-PullulanSTD-105k, Malvern Panalytical) certified for molecular weight, polydispersity index, and intrinsic viscosity. EPS samples (100 μL, 1 mg / mL) dissolved in the mobile phase (0.2 M NaNO3 + 0.05% NaN3, pH 6.81) were injected into a GMPWX column (7.8 mm x 30 cm, Tosoh Bioscience) at a flow rate of 0.6 mL / min at 40 °C.

[0088] Analytical results (average of two analyses per EPS sample) are reported in terms of weight average molecular weight (Mw; 40–150 kDa), number average molecular weight (Mn; 26–100 kDa), and polydispersity index (Mw / Mn; 1.2–1.8), all using dn / dc parameters of 0.135–0.147, and in terms of % recovery (94–99%), which represents the ratio between the concentration calculated from the refractive index detector and the experimental concentration.

[0089] Example 3: EPS characterization - Determination of glycosyl composition The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0090] The glycosyl composition of EPS was characterized according to two characterization methods: the alditol acetate method (Pena et al. (2012) Methods Enzymol. 510:121-39) and the TMS-derivatized methyl glycoside method (Santander et al. (2013) Microbiology 159:1471). Briefly:

[0091] Glycosyl composition analysis by alditol acetate (AA) method: Glycosyl composition analysis was performed by combined gas chromatography / mass spectrometry (GC / MS) of alditol acetate. EPS samples (250 μg) were hydrolyzed in 2 M trifluoroacetic acid (TFA) for 2 h at 120 °C in sealed tubes, reduced with NaBD4, and acetylated with acetic anhydride / TFA. The resulting alditol acetates were analyzed in electron impact ionization mode on an Agilent 7890A GC connected to a 5975C MSD. Separation was performed on a 30 m Supelco SP-2331 bonded-phase fused silica capillary column. Separation of amino sugars was achieved using a separate EC-1 column.

[0092] Glycosyl composition analysis of TMS-derivatized methyl glycosides by GC-MS: Glycosyl composition analysis was performed by combined gas chromatography / mass spectrometry (GC-MS) of per-O-trimethylsilyl (TMS) derivatives of monosaccharide methyl glycosides generated from samples by acidic methanolysis. EPS samples (250 μg) were heated with methanolic HCl at 80 °C for 18 h in a closed screw-cap glass test tube. After cooling, the solvent was removed under a stream of nitrogen, and the samples were re-N-acetylated and dried again. The samples were then derivatized with Tri-Sil® (Pierce) at 80 °C for 30 min. GC / MS analysis of TMS methyl glycosides was performed on an Agilent 7890A GC connected to a 5975C MSD using a Supelco Equity-1 fused silica capillary column (30 mx0.25 mm ID).

[0093] The two characterization methods indicate that mannose is the predominant hexose sugar detected, and N-acetylgalactosamine is the predominant amino sugar detected. Significant amounts of galactose, glucose, and N-acetylglucosamine are also present in the samples. A low percentage of xylose was also detected, while glucuronic acid was only detected with the TMS method. However, ribose, arabinose, rhamnose, fucose, and fructose were not detected using either characterization method, as shown in Table 1.

[0094] [Table 1]

[0095] Example 4: EPS protein and nucleic acid content analysis The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposit number NCIMB 43557) as described in Example 1.

[0096] EPS protein content analysis: Protein content was determined using a modified Lowry assay, i.e. BioRad total protein stain. The analysis was performed on neat samples of EPS according to the manufacturer's instructions and quantified using a BSA standard curve. Briefly, 10 μL of sample was mixed with 200 μL of a 1:4 dilution of the reagent. After 10 min, samples were analyzed by absorbance at 595 nm. Protein contamination in EPS was 0.052 ± 0.019 mg.mL -1 was estimated to be approximately 0.005% w / w based on the total weight of the isolated EPS.

[0097] Determination of EPS nucleic acid content by 260 / 280 and 260 / 230 analysis: 2 μL of EPS sample was analyzed on a Nanodrop spectrophotometer at 260 / 280 and 260 / 230 nm. Nucleic acid contamination in EPS was 58.0 ± 8.7 ng.μL -1 was estimated to be approximately 0.006% w / w based on the total weight of the isolated EPS.

[0098] Example 5: In vitro EPS anti-biofilm activity against bacterial pathogens The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0099] The following in vitro experiments describe the inhibitory effect of EPS on biofilm formation of pathogenic strains, namely: Gram-negative bacteria [Haemophilus influenzae type B ATCC 9795, Klebsiella pneumoniae ATCC 8047 and Pseudomonas aeruginosa ATCC 27853] and Gram-positive bacteria [Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae serotype 3 ATCC 6303 and Streptococcus mutans DSM 20523].

[0100] Biofilm formation of bacterial pathogens was performed in 96-well polystyrene microtiter plates, with 180 μl (1.5 × 10) of overnight culture of each bacterial pathogen. 8 bacteria.mL -1 ) were diluted with aliquots of different concentrations (50, 100, 200 and 400 μg.ml -1Plates were inoculated with 20 μl of EPS solution in PBS (100 μl) or with 20 μl of PBS as a control. Plates were incubated at 37°C for 24 h (St. pneumoniae), 48 h (H. influenzae, P. aeruginosa, K. pneumoniae, S. aureus) or 96 h (St. mutans) without shaking to allow for the growth rate of each strain and for biofilm production. The incubation medium was also adapted to each strain, therefore incubation of microaerophilic or anaerobic strains was performed under 5% CO2 or anaerobic conditions, respectively. Non-adherent bacteria were removed by washing three times with distilled sterile water and attached bacteria (biofilm) were stained with 0.1% crystal violet solution in 96% vol (w / v) ethanol for 25 min. Excess staining solution was removed by aspiration, and plates were washed (5 times) with distilled sterile water and air-dried (15 min). Stained biofilms were solubilized with 96% ethanol, and optical density (OD) was measured at 585 nm using a microtiter plate reader (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer). Biofilm formation (%) was calculated according to the following formula:

[0101]

number

[0102] In these experiments, pathogenic bacterial strains were cultivated and incubated under optimal conditions, in adapted media, and in the absence of competition from other pre-existing pathogens. The percentage of biofilm formation was calculated by OD, so the more crystal violet-labeled cells measured after washing, the more cells were attached to the plate and the more biofilm formed during the incubation period. The reduction in biofilm formation was observed regardless of the pathogenic strain involved. The preventive anti-biofilm effect of EPS is shown in Figure 2. The greatest effect was observed when EPS was added within the first 2 hours after inoculation with pathogenic bacteria (data not shown). The effect on biofilm formation is slightly reduced when EPS was added 4 hours after inoculation (data not shown).

[0103] [Example 6: In vitro EPS antibacterial activity experiment] The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0104] To investigate whether the inhibitory effect of EPS on biofilm formation by bacterial pathogens could be due to direct growth inhibition, the antibacterial activity was evaluated in triplicate (N=3) experiments similar to those in Example 5 on two independent occasions (N=2). Briefly, in polystyrene microtiter plates, EPS was added at final concentrations of 50, 100, 200 and 400 μg.ml in optimal medium (180 μl) for each test strain (OD600=0.1). -1Plates were filled with overnight pathogenic bacterial cultures containing 20 μl of EPS in PBS solution or 20 μl of PBS as a control. Plates were incubated at 37°C without shaking under aerobic, microaerobic or anaerobic conditions for periods ranging from 24 h to 96 h (optimal growth period varied depending on the test strain). Antimicrobial activity was assessed by measuring OD600 values ​​using a microtiter plate reader (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer) and comparing the average OD600 of control wells to the average OD600 of each test condition.

[0105] The antimicrobial effect of EPS on culture growth of various bacterial pathogens is shown in Figure 3, comparing the OD600 values, mean ± SD, of treated vs. untreated cells after the respective optimal growth periods. Measurement of the turbidity of a sample (e.g., OD600) is commonly used in a spectrophotometer to estimate the bacterial concentration in the medium, which allows the growth of a cell population over time to be measured. Cultures grown in the absence of EPS were used as controls.

[0106] The results shown in Figure 3 show that the same pathogenic bacterial growth levels were achieved with or without EPS. This was achieved at various concentrations (50–400 μg.ml -1 ) show that the presence of EPS does not have a negative effect on the growth of pathogenic bacteria and, more generally, the presence of EPS did not affect the growth rate of all the bacterial strains tested. Thus, EPS does not exhibit a bactericidal, bacteriostatic or antibiotic effect, independent of its concentration and the pathogenic bacteria considered. It is therefore demonstrated that the presence of EPS, without a biocidal effect, respects the integrity of the natural microflora that should be present on living tissues.

[0107] [Example 7: EPS cytotoxicity assay] The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposit number NCIMB 43557) as described in Example 1.

[0108] A preliminary test of the putative cytotoxic effect of EPS on human nasal epithelial cells (HNEpC) was performed in vitro. Human nasal epithelial cells (HNEpC) were grown in BEBM complete medium as recommended by the manufacturer until 80% confluent. Cells were washed twice with PBS and incubated at 37 °C, 5% CO2 in medium with 0 (control)–800 μg.mL -1 Cells were cultured for 2 or 24 hours with EPS ranging from 0.1 to 0.2 mM. After incubation, cells were washed twice with PBS, harvested, and viability assessed by staining with the viability dye TO-PRO3. The percentage of TO-PRO3-negative cells, indicating viable cells in the cultures, was assessed on a Symphony BD Sciences flow cytometer.

[0109] The time-dependent effect of increasing concentrations of EPS on the viability of HNEpC after 2 and 24 hours is shown in Figure 4. HNEpC cells were chosen as a model because of their high sensitivity and were successively incubated with increasing doses of EPS for 2 and 24 hours. As shown in Figure 4, cell viability was the same as in the control. This result indicates that the presence of EPS does not induce cytotoxicity, regardless of the concentration and incubation time.

[0110] [Example 8: Ex Vivo EPS Assay Using HNEpC Cells] The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0111] Next, the effectiveness of the EPS composition to remove two bacterial strains, Staphylococcus aureus and Pseudomonas aeruginosa, from monolayers of human nasal epithelial cells was evaluated.

[0112] Human nasal epithelial cells (HNEpC; PromoCell, catalog number C-12620) were cultured in BEBM complete medium to 80% confluence as recommended by the manufacturer. HNEpC cells were washed twice with PBS and incubated with pathogenic bacteria (P. aeruginosa or S. aureus) at 37°C, 5% CO2, in antibiotic-free medium for 2 h. P. aeruginosa was grown in Luria Bertani broth and S. aureus in tryptone soy broth. Upon overnight incubation in broth, bacteria were diluted in PBS at an optical density (OD600) of 0.1 and used for experiments. Contaminated HNEpC cells were washed twice with PBS and cultured in BEBM complete medium with EPS at 0 (control), 50, 100, 200, and 400 μg.mL. -1 The plates were then washed twice with PBS, followed by a final wash in distilled HO for 10 min to kill and detach HNEpC cells and recover any remaining bacteria. The supernatants (containing dead cells and bacteria) were then collected and plated on AGAR medium (cetrimide agar for P. aeruginosa and mannitol salt agar for S. aureus) after appropriate dilution. Bacterial growth was analyzed by counting bacterial colonies (CFU) in the plates after 24 h.

[0113] The in vitro efficacy of EPS for the elimination of P. auroginosa and S. aureus from human nasal epithelial cell (HNEpC) monolayers is shown in Figure 5. These experiments were performed in triplicate (n = 3) on two to four independent occasions (N = 2 to 4). Statistical significance was determined by Kruskal Wallis one-way analysis of variance. A p-value < 0.05 was considered statistically significant. Post-hoc analysis was performed using the Dunn test.

[0114] By measuring the number of pathogenic bacteria attached to nasal cells before the osmotic shock (washing with deionized water), these experiments indirectly measured the effect of EPS on the detachment of pathogenic bacteria from their support, i.e., nasal epithelial cells (HNEpC). Thus, these results showed that EPS has the ability to limit the consequences of bacterial biofilm installation on biological surfaces, regardless of their origin. Indeed, in addition to the preventive effect demonstrated in Example 5, where the formation of biofilm was influenced by the presence of EPS, it was demonstrated herein that EPS washing may have therapeutic activity due to its pathogenic bacterial cell removal effect (Figure 5).

[0115] Example 9: Emulsifying / surface active properties of EPS The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0116] The emulsifying / surfactant activity of EPS was determined by the method described by Song et al. (Preparation of Calcipotriol Emulsion using bacterial exopolysaccharides as emulsifier for percoutaneous treatment of Psoriasis Vulgaris. Int J Mol Sci. 2019, 20;21(1) 2019). Briefly, lyophilized EPS was resuspended in PBS and diluted at 5–800 μg.mL -1 The EPS solutions were mixed in a glass tube containing 2.0 mL of vegetable oil. The resulting heterogeneous solutions were then mixed for 2 min at 3000 rpm using a vortex mixer for emulsification. "Vegetable oil + PBS" and "Vegetable oil + saponin (1%)" were used as negative and positive controls, respectively. Prior to emulsification index evaluation (E24), the samples were allowed to stand at room temperature for 24 h without stirring. E24 is expressed as the height of the emulsified layer divided by the total liquid column height multiplied by 100.

[0117] The results of three independent experiments (N = 3) shown in Figure 6 demonstrated that EPS exhibits strong emulsifying activity even at low concentrations (i.e., 50 μg.mL, which corresponds to 0.005% EPS). -1 (E24 alone reached 50%.) Furthermore, it was clearly demonstrated that the higher the amount of EPS in the environment, the greater the emulsifying effect.

[0118] [Example 10: Ex vivo prophylactic antiviral activity of EPS against HNEpC inoculated with high doses of adenovirus or rhinovirus] The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0119] The ability of EPS to prevent viral infection of viable human nasal epithelial cells inoculated with human adenovirus 2 (ATCC VR-846) and human rhinovirus 16 (ATCC VR-283) was evaluated.

[0120] Human nasal epithelial cells (HNEpCs) were cultured in BEBM complete medium as recommended by the manufacturer until 80% confluent. Cells were cultured at 400 μg.mL -1After being treated with 100 μg.mL-1 EPS and incubated for 2 h at 37 °C and 5% CO2 and washed with PBS, a high load of rhinovirus or adenovirus (1 TCID50, representing the amount of virus that causes a cytopathic effect in 50% of the inoculated cells after 48 h) was inoculated and incubated in the same conditions. Alternatively, live HNEpCs were infected with the same inoculum for 2 h at 37 °C and 5% CO2, washed, and then treated with EPS 400 μg.mL-1 for another 2 h at 37 °C and 5% CO2. Untreated live cells (only BEBM complete medium) served as a positive control, and virus-inoculated live cells without EPS treatment served as a negative control. After washing with PBS, pre-treated cells (i.e., cells treated with EPS composition before virus inoculation), post-treated cells (i.e., cells treated with EPS composition after virus inoculation) and controls were cultured in BEBM medium for 48 h at 37 °C and 5% CO2. After 48 hours of culture, cells were harvested and viability was assessed by staining with the viability dye TO-PRO-3. The percentage of TO-PRO-3 negative cells, indicating viable cells in the culture, was assessed using a Symphony BD Sciences flow cytometer.

[0121] EPS against HNEpCs infected with high doses of adenovirus or rhinovirus (400 μg.mL -1) is reported in Figure 7. In the negative control (virus inoculation only), half of the HNEpCs were affected by the virus inoculation after 48 h. However, when the cells were pretreated with EPS before virus inoculation, the observed cell mortality was reduced to half of that of the negative control. Such pretreatment therefore improves the survival rate of the cells after virus infection, revealing the preventive properties of EPS against virus infection, independent of the virus considered. EPS treatment of nasal epithelial cells inoculated with a virus load in advance is less pronounced than pretreatment (Figure 7). Without wishing to be bound by theory, such observations can be explained by a high virus load inoculation, which allows a large amount of virus to be internalized within the HNEpCs in 2 h, allowing rapid virion contamination of the majority of the cells. These results indirectly showed that EPS does not promote an intracellular effect (i.e. the induction of an immune response), but rather has an extracellular "barrier" effect that sterically impedes virus internalization. Nevertheless, it is worth mentioning that inoculation with such a high viral load (i.e., 1 TCID50) does not reflect the real-life viral infection involving inoculation with a lower viral load (i.e., 0.001 TCID50).

[0122] EPS concentration 200μg.mL -1 Further antiviral experiments were performed under the same conditions, except that the concentration of EPS (200 μg.mL ) against HNEpC inoculated with high doses of adenovirus or rhinovirus was reduced to 100 μg.mL . -1 ) are reported in Figures 8A and 8B, respectively. These results demonstrated preventative antiviral activity comparable to that observed with high doses of EPS.

[0123] [Example 11: Ex vivo therapeutic antiviral activity of EPS against HNEpCs inoculated with low doses of adenovirus or rhinovirus] The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0124] The ability of EPS to prevent the spread of cell infection after virions are released from inoculated cells by viral inoculation was measured. To better match the reality of viral infection, these experiments were performed using lower viral loads of both human adenovirus 2 (ATCC VR-846) and human rhinovirus 16 (ATCC VR-283) than those shown in Example 10 (i.e., 0.001 TCID50), while extending the cell culture period to 5 days to allow viral spread after inoculation. We also sought to understand whether the consistent presence of EPS was required to exert a protective effect, or whether the first contact with HNEpC was sufficient. Thus, different cell culture conditions were performed, where EPS was added only as a pretreatment (as in Example 10) or further added after viral inoculation (either once at the beginning of the 5-day culture or daily for 5 days). Briefly, viable human nasal epithelial cells (HNEpC) were grown as recommended by the manufacturer in BEBM complete medium until 80% confluent. i) As a positive control, viable HNEpCs were cultured in BEBM medium, and as a negative control, HNEpCs were inoculated with a virus inoculum (low dose, 0.001 TCID50) for 2 h, washed with PBS to remove uninternalized virus, and cultured for 5 days. ii) Viable HNEpCs were cultured in BEBM medium for 400 μg.mL -1 100 μg.mL EPS for 2 h, washed with PBS, inoculated with virus inoculum (low dose, 0.001 TCID50) for 2 h, washed with PBS and cultured for 5 days. iii) Viable HNEpCs were inoculated with virus inoculum (low dose, 0.001 TCID50) for 2 h, washed with PBS and cultured for 5 days. -1 ) for 5 days, with one shot of EPS solution added on day 1. iv) Viable HNEpCs were inoculated with virus inoculum (low dose, 0.001 TCID50) for 2 h, washed with PBS, and incubated with EPS (400 μg.mL -1 ) for 5 days and then cultured daily in the presence of EPS (400 μg.mL -1) was added to the cultures. After 5 days of culture, cells were harvested and viability was assessed by TO-PRO3. The percentage of TO-PRO-3 negative cells (representing viable cells) was assessed for each condition by a Symphony BD Sciences flow cytometer.

[0125] The EPS antiviral activity against HNEpCs inoculated with low doses of adenovirus or rhinovirus is reported in Figure 9: the negative control (low dose virus inoculation only) showed that after 5 days more than half of the HNEpCs were affected by the virus inoculation. The preventive properties of EPS pretreatment (i.e., cells treated with the EPS composition before inoculation with the virus) against virus infection observed in Example 10 were confirmed by cell viability close to that of the positive control after 5 days of culture. Furthermore, it is shown that when the EPS composition was applied after virus inoculation (i.e., post-treated cells), the virus was unable to induce the same cell mortality as the negative control (i.e., virus without EPS treatment).

[0126] Similar results were obtained in two post-treatment experiments (EPS application once for 5 days, and EPS application once a day for 5 days) (Figure 9), demonstrating that EPS prevents not only the release of virions but also their spread to neighboring cells. Thus, EPS provides a therapeutic effect that reduces the pathogenicity of the virus inoculum by 50% after 5 days of incubation (Figure 9). These results reinforce the observations in Example 10, where an extracellular "barrier" effect that sterically impedes virus internalization would be an issue here.

[0127] Thus, these two combined effects, i.e., preventive effect (observed by EPS pretreatment of cells before virus inoculation) and therapeutic effect (observed by EPS posttreatment of cells after virus inoculation), promote the disappearance of viral infection. Such administration of EPS may therefore function as a support for the cellular machinery to effectively defend itself against any kind of viral infection, although specificity for the targeted viral strain has not been proven.

[0128] EPS concentration 200μg.mL -1 Further antiviral experiments were performed under the same conditions, except that the effect of EPS (200 μg.mL ) on HNEpC inoculated with low doses of adenovirus or rhinovirus was reduced to 10 μg.mL . -1 ) are reported in Figures 10A and 10B, respectively. These results demonstrated both preventative and therapeutic antiviral activity comparable to that observed with high doses of EPS.

[0129] Example 12: Ex vivo therapeutic antiviral activity of EPS against HNEpCs infected with low and high doses of coronavirus. The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0130] The ability of EPS to prevent viral infection of viable human nasal epithelial cells inoculated with human coronavirus OC43 (HCoV-OC43) at high (1 TCID50) and low (0.001 TCID50) viral loads was measured. Human coronavirus OC43 (HCoV-OC43), used in the studies herein, is the virus most commonly associated with human infections, accounting for 5–30% of human respiratory infections (Lau SKP et al., 2011, Journal of Virology). Briefly, human nasal epithelial cells (HNEpC) were cultured in BEBM complete medium as recommended by the manufacturer until 80% confluent. Cells were cultured at 200–400 μg.mL -1 100 μg.mL of EPS, incubated at 37 °C and 5% CO2 for 2 h, washed with PBS, and then inoculated with a coronavirus dose (1 TCID50 or 0.001 TCID50) and incubated under the same conditions. Alternatively, live HNEpCs were inoculated with the same inoculum at 37 °C and 5% CO2 for 2 h, washed, and then incubated with 200–400 μg.mL of EPS. -1The cells were then treated for an additional 2 hours at 37°C and 5% CO2. Untreated live cells (BEBM complete medium only) were used as a positive control, and virus-inoculated live cells without EPS treatment were used as a negative control. After washing with PBS, pre-treated cells (i.e., cells treated with EPS composition before virus infection), post-treated cells (i.e., cells treated with EPS composition after virus infection) and controls were cultured in BEBM medium for 48 hours at 37°C and 5% CO2. After 48 hours of culture, cells were harvested and viability was assessed by staining with the viability dye TO-PRO-3. The percentage of TO-PRO-3 negative cells, representing live cells in the cultures, was assessed on a Symphony BD Sciences flow cytometer.

[0131] EPS against HNEpC inoculated with high or low doses of coronavirus (200–400 μg.mL -1 ) is reported in Figure 11. These results demonstrated that EPS provides: i) preventative antiviral activity as described in Example 10 herein, and ii) therapeutic antiviral activity as described in Example 11 herein, independently of the virus considered.

[0132] Example 13: EPS formulation - nasal formulation The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0133] Nasal formulation 1: Seawater and desalted seawater were mixed at room temperature in a washed bulk tank until the desired salt concentration (0.9% or 2.2% or 2.7% according to the formulation) was reached. To solubilize the lyophilized EPS of the present disclosure, a volume of bulk solution (10 L) was taken to reach the desired EPS concentration of 0.02% or 0.04%, corresponding to 200 μg / mL or 400 μg / mL (depending on the desired formulation). Both EPS and bulk solution were homogenized for at least 30 minutes and then filtered at 0.22 μm. This operation was repeated once before filling into nasal spray cans.

[0134] Nasal formulation 2: Sodium chloride was dissolved in water at room temperature to reach the desired salt concentration of 0.9%, 2.2% or 2.7% (depending on the desired formulation) in a washed bulk tank. EPS of the present disclosure was then dissolved in the bulk solution to reach the desired EPS concentration of 0.02% or 0.04%, which corresponds to 200 μg / mL or 400 μg / mL (depending on the desired formulation). Both EPS and bulk solution were homogenized for at least 30 minutes and then filtered through 0.22 μm. This operation was repeated once before filling into nasal spray cans.

[0135] Example 14: EPS formulation - topical formulation (cream) The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0136] Polyacrylate crosspolymer-6 (1.5% w / w) and water (63.0% w / w) were mixed thoroughly before the addition of xanthan gum (4.0% w / w) and glycerin (2.0). The resulting mixture (Phase A) was warmed to 70-75°C under continuous mixing. A mixture of sucrose polystearate (3.0% w / w), glyceryl dibehenate (1.7% w / w), hydrogenated jojoba oil (21.0% w / w) was warmed to 70-75°C and then added to Phase A under continuous mixing to obtain Phase B. Phase B was mixed for emulsification at 70-75°C for 20 minutes and then the emulsified Phase B was cooled to 30°C. EPS (0.02-0.04% w / w), Vitamin E acetate and fragrance were added to the emulsified Phase B at 30°C under constant agitation. The pH of the resulting mixture was adjusted to pH 4.5-5.0 with a solution of citric acid in water (qsp.).

[0137] Example 15: Pharmacological / immunological properties of the EPS disclosed herein The EPS used in the examples herein was obtained by fermentation of Bacillus licheniformis LP-T14 (deposition number NCIMB 43557) as described in Example 1.

[0138] Since it has been reported that EPS produced by marine bacteria can induce selected patterns of regulatory cytokines in white blood cells and tissue cells (Okutani K. et al. Bull Jpn Soci Sci Fish 1984;50:1035-7), the purpose of these assays was to determine to what extent the EPS of the present disclosure can induce immune responses in cells with which the EPS will come into contact.Therefore, since two different systems, namely epithelial cells and white blood cells of the innate immune system, are likely to be modified when in contact with bacteria-derived substances, the assays herein were carried out on human nasal epithelial cells (HNEpC) and human monocyte-derived dendritic cells (DC), respectively.

[0139] More specifically, the following series of experiments were performed to assess i) the production of key cytokines involved in inflammatory pathways, such as IL-6 and IL-8, after culture of EPS samples with HNEpCs, and ii) the activation state of DCs after EPS stimulation to evaluate the expression of the most important activation / maturation markers, such as CD83, CD40 and CD25, and the production of inflammatory cytokines (IL-6 and IL-8).

[0140] i) Effect of EPS of the present disclosure on HNEpC: Human nasal epithelial cells (HNEpC) were grown to confluence in BEBM complete medium and stimulated with EPS samples (400 μg / mL) for 24 h. Cells left unstimulated or stimulated with IL-1β (50 ng / mL) served as negative and positive controls, respectively. Monensin and brefeldin were used as GolgiStop (protein transport inhibitors) and were added for the last 12 h of culture. Cells were harvested, fixed with 1% paraformaldehyde, permeabilized with saponin 0.1% (Sigma-Aldrich) in PBS, stained with phycoerythrin (PE)-conjugated anti-IL-8 and anti-IL-6 antibodies (BD Bioscience), and then analyzed by flow cytometry.

[0141] These two cytokines are the main mediators involved in the recruitment of immune cells to initiate inflammatory pathways and inflammatory events. As shown in Figures 12A and 12B, compared to unstimulated HNEpCs, no IL-6 and IL-8 production was observed by HNEpCs after 24 hours of stimulation with EPS of the present disclosure. Surprisingly, no cytokine production was observed upon EPS exposure, even though HNEpCs have the ability to produce these cytokines when appropriately stimulated with typical inflammatory molecules (i.e., IL-1β). Thus, these results indicate that EPS of the present disclosure cannot induce inflammatory responses.

[0142] ii) The effect of the disclosed EPS on DC Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors by Ficoll Hypaque density gradient centrifugation. Monocytes were isolated by attachment to plastic surfaces for 45 min at 37 °C and 5% CO2. Non-adherent cells were then removed by washing with phosphate-buffered saline (PBS). The obtained cells were cultured in complete RPMI medium in the presence of IL-4 (20 ng / mL, Miltenyi Biotec, Germany) and GM-CSF (25 ng / mL, Sargramostim). After 6 days of culture, the non-adherent cells were CD14 - / CD11c + / CD83 - These cells display a phenotype that is characteristic of immature dendritic cells (DCs).

[0143] DCs were stimulated with EPS (400 μg / mL) as disclosed herein for 24 hours. Cells left unstimulated or stimulated with lipopolysaccharide (LPS, 1 μg / mL) served as negative and positive controls, respectively. For cytokine production analysis, monensin and brefeldin were used as GlogiStops and were added and left for the last 12 hours of DC culture. A) DC activation: After 24 hours, DCs were harvested and stained with anti-CD83 (anti-CD83-PE), anti-CD40 (anti-CD40-AlexaFluor 647) and anti-CD25 (anti-CD25-PacificBlue) monoclonal antibodies for 20 minutes at 4°C. Cells were washed with PBS and then analyzed for surface expression levels of the indicated activation molecules by flow cytometry. B) Cytokine production: DCs were harvested, fixed with 1% paraformaldehyde, permeabilized with saponin 0.1% (Sigma-Aldrich) in PBS, stained with phycoerythrin (PE)-conjugated anti-IL-8 and anti-IL-6 antibodies (BD Bioscience), and analyzed by flow cytometry.

[0144] The effect of the EPS of the present disclosure on DCs was evaluated by assessing both DC activation by analyzing the expression levels of CD83, CD40 and CD25 as Mean of Fluorescence Intensity (MFI), as depicted in Figure 13 A. Analysis of DCs following EPS stimulation revealed that the EPS of the present disclosure did not affect DC maturation, as shown by the expression levels of CD83, CD40 and CD25, which remained very low and comparable to unstimulated DCs.

[0145] As shown in Figure 13B, the effect of the EPS of the present disclosure on DCs and their cytokine production, i.e., IL-8 and IL-6, after 24 hours of stimulation was evaluated as the percentage of cytokine-producing cells. Compared to unstimulated DCs, no IL-6 or IL-8 production was observed by DCs after 24 hours of stimulation with ESP.

[0146] It is noteworthy that both human cell types used in these experimental systems (HNEpC and DC) are well endowed with pattern recognition receptors (PRRs) that sense the presence of pathogen-associated molecular patterns (PAMPs), including bacteria. DCs are highly capable of sensing potential PAMPs to initiate inflammatory processes. In addition, it is noted that LPS, derived from Gram-negative bacteria and used in DC experiments as a positive control, was effective at 1 μg / ml, i.e., 400-fold lower than the EPS concentrations tested ( FIG. 13 ). Thus, it was demonstrated that the EPS of the present disclosure has no ability to induce immune (cellular and / or humoral) responses.

[0147] While the present invention has been described in connection with specific embodiments thereof, it will be understood that the claims should not be limited by the preferred embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0148] Further aspects and embodiments of the invention are described in the following numbered paragraphs: 1. A composition comprising at least one isolated exopolysaccharide (EPS) derived from a marine bacterium and a suitable carrier, wherein the at least one isolated EPS has an average molecular weight in the range of 40 to 4000 kDa, and the at least one isolated EPS is obtainable by fermentation of the marine bacterium. 2. The composition according to item 1, comprising at least two exopolysaccharides each independently having an average molecular weight in the range of 40 to 4000 kDa. 3. The composition according to item 1 or 2, wherein the average molecular weight of EPS is 40 to 2000 kDa, optionally 100 to 1400 kDa. 4. The marine bacterium is a gram-positive thermophilic bacterium, and optionally, the gram-positive thermophilic bacterium is Bacillus licheniformis, preferably, the gram-positive thermophilic bacterium is Bacillus licheniformis LP-T14 deposited in the National Collection of Industrial, Food and Marine Bacteria (NCIMB) on January 27, 2020 under the deposit number NCIMB 43557. The composition according to any one of claims 1 to 3. 5. The composition according to any one of items 1 to 4, (i) the composition is an oral composition, a nasal composition, a topical composition, a transdermal composition, an ophthalmic composition, or a composition formulated to treat a medical device, preferably, the composition is a nasal composition; and / or (ii) A composition, wherein the carrier is a saline solution. 6. A nasal delivery system comprising at least one isolated exopolysaccharide (EPS) derived from a marine bacterium, wherein the at least one isolated EPS has an average molecular weight in the range of 40 to 4000 kDa, and wherein the at least one isolated EPS is obtainable by fermentation of the marine bacterium. 7. The nasal delivery system according to paragraph 6, wherein: (i) the average molecular weight of at least one isolated EPS is between 40 and 2000 kDa, optionally between 100 and 1400 kDa; and / or (ii) the marine bacterium is a Gram-positive thermophilic bacterium, optionally wherein the Gram-positive thermophilic bacterium is Bacillus licheniformis, preferably wherein the Gram-positive thermophilic bacterium is Bacillus licheniformis LP-T14 deposited at NCIMB under deposit number NCIMB 43557; (iii) the nasal delivery system delivers the at least one isolated EPS as a nasal drop, liquid spray, dry spray, gel, or ointment; (iv) the at least one isolated EPS is formulated in a composition further comprising a saline solution; (v) the nasal delivery system is for treating and / or preventing a microbial pathogen infection in a patient in need thereof; and / or (vi) the nasal delivery system is for attenuating the virulence of a microbial pathogen by inhibiting or reducing colonization by the nasal passages of a patient in need thereof; Nasal delivery system. 8. Non-therapeutic use of a composition according to any one of paragraphs 1 to 5 for treating a non-biological surface to prevent or reduce colonisation of the surface by microbial pathogens, optionally wherein the surface is a surface of a medical device. 9. The composition of any one of paragraphs 1 to 5, or the nasal delivery system of paragraph 6 or 7, for use in a method for treating and / or preventing a microbial pathogen infection in a patient in need thereof. 10. The composition of any one of paragraphs 1 to 5, or the nasal delivery system of paragraph 6 or 7, for use in a method for attenuating the pathogenicity of a microbial pathogen. 11. A method for treating and / or preventing a microbial pathogen infection in a patient in need thereof, comprising administering to the patient an effective amount of the composition described in any one of paragraphs 1 to 5. 12. A method for attenuating the pathogenicity of a microbial pathogen infection in a patient in need thereof, comprising administering to the patient an effective amount of the composition described in any one of items 1 to 5. 13. (i) a composition is administered to a patient before, during, and / or after infection with a microbial pathogen, thereby preventing, inhibiting, or reducing microbial pathogen colonization and / or internalization of a microbial pathogen in at least one biological tissue of the patient, optionally wherein the at least one biological tissue is selected from the oral cavity, nasal cavity, respiratory tract, throat, ear, ophthalmic area, urogenital tract, skin, scalp, hair, nails, and combinations thereof, preferably wherein the at least one biological tissue is the nasal cavity; and / or (ii) the microbial pathogen is a bacterial pathogen, and administration of the composition to the patient attenuates the virulence of the bacterial pathogen infection by reducing or inhibiting initial bacterial biofilm formation and / or disrupting the initial bacterial biofilm; or (iii) The composition for use according to clause 9 or 10, or the method according to clause 11 or 12, wherein the microbial pathogen is a viral pathogen, and administration of the composition to a patient attenuates the pathogenicity of the viral pathogen infection by reducing the mortality of patient cells infected with the viral pathogen compared to comparable untreated cells, preventing or reducing the release of virions from infected patient cells, and / or preventing or reducing infection of adjacent uninfected patient cells. 14. A microbial pathogen is: (i) a bacterial pathogen optionally selected from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and / or Streptococcus, preferably the bacterial pathogen is Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae or Streptococcus mutans species, or a combination thereof; (ii) a viral pathogen optionally selected from influenza A, influenza A subtype H1N1 / 2009 / pdm09, influenza A subtype H1, influenza A subtype H3, influenza B (orthomyxovirus), coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, SARS-CoV-2 (coronavirus), parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus A / B, human metapneumovirus A / B (paramyxovirus), adenovirus or rhinovirus / enterovirus (picornavirus), or a combination thereof; (iii) a fungal pathogen; or (iv) a mixture of one or more of (i), (ii) and (iii); The nasal delivery system according to item 6 or 7, the use according to item 8, the composition for use according to item 9, 10 or 13, or the method according to any one of items 11 to 13. 15. The composition according to any one of items 1 to 5, the use according to item 8, the composition for use according to any one of items 9, 10, 13 and 14, or the method according to any one of items 11 to 14, wherein the amount of the at least one EPS in the composition based on the total weight of the composition is in the range of 0.00005 to 0.5% w / w, preferably 0.0001 to 0.1% w / w, more preferably 0.0005 to 0.05% w / w. 16. A method of formulating a nasal composition for attenuating the pathogenicity of a microbial pathogen infection by inhibiting or reducing colonization of the nasal cavity by the microbial pathogen, comprising the step of mixing at least one isolated exopolysaccharide (EPS) derived from a marine bacterium with a saline solution and optionally a suitable carrier to obtain a nasal composition comprising salt at a concentration of 0.1%-10% w / w, preferably 0.5%-5% w / w, more preferably 0.7%-3% w / w.

Claims

1. 1. An isolated exopolysaccharide (EPS) having a weight average molecular weight (Mw) in the range of 40 to 4000 kDa, the EPS being obtained or obtainable by fermentation of a marine bacterium.

2. The isolated EPS has a weight average molecular weight (Mw) in the range of 40-2000 kDa, 40-1400 kDa, 40-1000 kDa, 40-500 kDa, 40-400 kDa, 40-300 kDa, 40-200 kDa, 40-150 kDa, or 40-100 kDa, preferably 40-150 kDa; the isolated EPS has a Mw in the range of 40-150 kDa, optionally a number average molecular weight (Mn) in the range of 26-100 kDa and / or a polydispersity index (Mw / Mn) in the range of 1.2-1.8; the isolated EPS comprises 30-90% neutral glycosyl units, 10-70% aminoglycosyl units, and 0-15% acidic glycosyl units relative to the total number of glycosyl units of the EPS; the isolated EPS comprises mannose, galactose, glucose, N-acetylgalactosamine and N-acetylglucosamine; the isolated EPS is substantially free of ribose, arabinose, rhamnose, fructose and / or fucose; The marine bacterium is a Gram-positive thermophilic bacterium, preferably the Gram-positive thermophilic bacterium is Bacillus licheniformis, more preferably the Gram-positive thermophilic bacterium is Bacillus licheniformis LP-T14 deposited at the National Collection of Industrial, Food and Marine Bacteria (NCIMB) on January 27, 2020 under the deposit number NCIMB 43557; and / or The isolated EPS substantially lacks the ability to induce an immune response; 2. The isolated EPS of claim 1.

3. Steps below: culturing the marine bacterium in a first culture medium to obtain a cultured marine bacterium; fermenting the cultured marine bacteria in a fermentation medium; heat treating the fermentation medium; Centrifuging the fermentation medium to obtain a supernatant; and filtering the supernatant to obtain isolated EPS.

1. A process for obtaining an isolated exopolysaccharide (EPS) having a weight average molecular weight (Mw) in the range of 40 to 4000 kDa, comprising:

4. The isolated EPS is as defined in claim 2; The marine bacterium is a gram-positive thermophilic bacterium, preferably the gram-positive thermophilic bacterium is Bacillus licheniformis, more preferably the gram-positive thermophilic bacterium is Bacillus licheniformis LP-T14 deposited at the National Collection of Industrial, Food and Marine Bacteria (NCIMB) on January 27, 2020 under the deposit number NCIMB 43557; The fermentation medium contained sea salt (40 g / L), tryptone (6 g / L), yeast extract (6 g / L), antifoam (0.33 mL / L), dextrose (12 g / L) and deionized H ２ O(qsp); The fermentation is carried out at pH 5-8, 40° C. for 20-40 hours under 30-50% oxygen addition; The heat treatment of the fermentation medium is carried out by heating the fermentation medium at 85° C. for 1 hour; The centrifugation of the fermentation medium is carried out at 14000 g using a disc stack centrifuge with a flow rate of 200-800 L / h; said filtration of the supernatant comprises sequential filtration using 1.60-0.22 μm filtration steps and / or ultrafiltration using a 10-100 kDa cutoff filter cartridge; and / or The process further comprises free-drying the isolated EPS to obtain freeze-dried EPS, and optionally, the free-drying is performed at −20° C. for at least 16 hours.

4. The process of claim 3.

5. 10. A composition comprising at least one isolated exopolysaccharide (EPS) according to claim 1 or 2, or at least one isolated EPS obtained or obtainable by the process according to claim 3 or 4, and a suitable carrier.

6. 6. The composition of claim 5, which is an oral composition, a nasal composition, a topical composition, a transdermal composition, an ophthalmic composition, or a composition formulated for application to a medical device.

7. 5. A nasal delivery system comprising at least one isolated exopolysaccharide (EPS) according to claim 1 or 2, or at least one isolated EPS obtained or obtainable by the process according to claim 3 or 4.

8. The nasal delivery system delivers at least one isolated EPS as a nasal drop, liquid spray, dry spray, gel, or ointment; The at least one isolated EPS is formulated in a composition further comprising a saline solution; said nasal delivery system is for treating and / or preventing a microbial pathogen infection in a subject in need thereof; and / or 8. The nasal delivery system of claim 7, wherein the nasal delivery system is for attenuating the pathogenicity of a microbial pathogen by inhibiting or reducing colonization of the nasal cavity of a subject in need thereof.

9. 7. Non-therapeutic use of the isolated EPS of claim 1 or 2, the isolated EPS obtained or obtainable by the process of claim 3 or 4, or the composition of claim 5 or 6, for application to a non-biological surface to prevent or reduce colonization of the surface by microbial pathogens, optionally the surface being a surface of a medical device.

10. 10. The isolated exopolysaccharide (EPS) of claim 1 or 2, the isolated EPS obtained or obtainable by the process of claim 3 or 4, the composition of claim 5 or 6, or the nasal delivery system of claim 7 or 8, for use in a method for treating and / or preventing a microbial pathogen infection in a subject in need thereof.

11. 10. An isolated exopolysaccharide (EPS) as described in claim 1 or 2, an isolated EPS obtained or obtainable by a process as described in claim 3 or 4, a composition as described in claim 5 or 6, or a nasal delivery system as described in claim 7 or 8, for use in a method for attenuating the pathogenicity of a microbial pathogen.

12. The isolated EPS or the composition is administered to the subject before, during, and / or after infection with the microbial pathogen, thereby preventing, inhibiting, or reducing colonization of and / or internalization of the microbial pathogen on at least one biological tissue of the subject, the at least one biological tissue comprising epithelium such as the oral cavity, nasal cavity, respiratory tract, pharynx, ear, ophthalmic region, urogenital tract, skin, scalp, hair, nails, and combinations thereof; the microbial pathogen is a bacterial pathogen, and administration of the isolated EPS or the composition to the subject reduces or inhibits initial bacterial biofilm formation and / or disrupts initial bacterial biofilms, thereby attenuating the pathogenicity of the bacterial pathogen infection; and / or wherein the microbial pathogen is a viral pathogen, and administration of the isolated EPS or composition to the subject reduces the pathogenicity of viral pathogen infection by reducing mortality of cells of the subject inoculated with the viral pathogen compared to comparable untreated cells, preventing or reducing release of virions from inoculated cells of the subject, and / or preventing or reducing infection of adjacent uninoculated cells of the subject.

12. An isolated EPS or composition for use according to claim 10 or 11.

13. The microbial pathogen is: A bacterial pathogen is optionally selected from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and / or Streptococcus; preferably, said bacterial pathogen is Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus a bacterial pathogen optionally selected from the species P. pneumoniae and / or Streptococcus mutans; a viral pathogen optionally selected from influenza A, influenza A subtype H1N1 / 2009 / pdm09, influenza A subtype H1, influenza A subtype H3, influenza B (orthomyxovirus), coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, SARS-CoV-2 (coronavirus), parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus A / B, human metapneumovirus A / B (paramyxovirus), adenovirus and / or rhinovirus / enterovirus (picornavirus); or fungal pathogen The nasal delivery system according to claim 8, the use according to claim 9, or the isolated EPS or composition for use according to any one of claims 10 to 12, which is at least one of:

14. the microbial pathogen is a bacterial pathogen selected from Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and / or Streptococcus mutans species; or The microbial pathogen is a viral pathogen selected from coronavirus OC43, adenovirus, and / or rhinovirus / enterovirus (picornavirus); 14. A nasal delivery system, use, isolated EPS for use, or composition for use as described in claim 13.

15. The composition according to claim 5 or 6, the use according to claim 9, the composition for use according to any one of claims 10 to 14, wherein the amount of the at least one isolated EPS in the composition based on the total weight of the composition is in the range of 0.00005 to 0.5% w / w, preferably 0.0001 to 0.1% w / w, or more preferably 0.0005 to 0.05% w / w.

16. 1. A method of formulating a nasal composition for attenuating the pathogenicity of a microbial pathogen infection by inhibiting or reducing colonization of the nasal cavity by the microbial pathogen, comprising the step of mixing at least one isolated exopolysaccharide (EPS) derived from a marine bacterium with a saline solution and optionally a suitable carrier to obtain a nasal composition comprising salt at a concentration of 0.1% to 10% w / w, preferably 0.5% to 5% w / w, more preferably 0.7% to 3% w / w.

17. The at least one isolated EPS is as defined in claim 1 or 2; and / or the salt concentration is about 0.9% w / w, about 2.2% w / w or about 2.7% w / w; 17. The method of claim 16.

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