Composition and its use

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] This invention relates to extracellular polysaccharides (EPS) derived from marine bacteria, compositions thereof, non-therapeutic uses of the EPS and compositions, and therapeutic uses of the EPS and compositions. The invention also relates to a process for obtaining the EPS. Furthermore, the invention relates to a nasal delivery system comprising at least one isolated extracellular polysaccharide, and the therapeutic use of the nasal delivery system. The invention also relates to a method for formulating nasal compositions. [Background technology]

[0002] Infectious diseases are usually caused by microorganisms that enter and spread within the body. There are many types of infectious organisms, also called pathogenic microorganisms. Here are some examples of how these microorganisms can enter the body: through the mouth, eyes, and nose; through sexual contact; through wounds or bites; and through contaminated medical devices.

[0003] People are primarily infected with these microorganisms by drinking contaminated water or eating contaminated food. They may also be infected by inhaling spores or dust, or by inhaling contaminated droplets from another person's cough or sneeze. They may also be infected by handling contaminated objects (such as doorknobs) or by having direct contact with an infected person and touching their eyes, nose, or mouth.

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

[0005] Pathogenic microorganisms can also adhere to medical devices implanted in the body (e.g., catheters, artificial joints, or artificial heart valves). They may be present on the device during implantation if it is accidentally contaminated. Infectious materials from other locations can also spread through the bloodstream and attach to already implanted devices. Because implanted devices lack natural defenses, these microorganisms can easily multiply and spread, potentially causing disease.

[0006] After entering the human body, pathogenic microorganisms must multiply or spread to cause an infection. Once multiplication or spread has begun, three scenarios can occur: 1) The microorganism continues to multiply and overwhelms the host organism's defenses. 2) A state of equilibrium is reached, causing a chronic infection. 3) The host organism destroys and eliminates the invading microorganism, with or without medical intervention.

[0007] The effectiveness of existing solutions (i.e., antibiotics, antivirals) in eliminating pathogenic infections depends on the rapid recognition of the pathogen and the application of pathogen-suppressing compositions before the microorganisms irreversibly adhere to the surface. It is highly desirable to have alternative approaches that limit the irreversible adhesion of microbial pathogens to surfaces, thereby reducing the decrease in resistance to treatment and, consequently, the impact on associated healthcare costs. [Overview of the Initiative]

[0008] According to a first embodiment, the disclosure provides isolated extracellular polysaccharides (EPS) having a weight-average molecular weight in the range of 40 to 4000 kDa, wherein the EPS is obtained or can be obtained by fermentation of marine bacteria. In one embodiment, the isolated EPS has a weight-average molecular weight (Mw) in the range of 40 to 2000 kDa, 40 to 1400 kDa, 40 to 1000 kDa, 40 to 500 kDa, 40 to 400 kDa, 40 to 300 kDa, 40 to 200 kDa, 40 to 150 kDa, or 40 to 100 kDa. In a preferred embodiment, the isolated EPS has an Mw in the range of 40 to 150 kDa. In one embodiment, the isolated EPS has a Mw in the range of 40 to 150 kDa and optionally a number-average molecular weight (Mn) in the range of 26 to 100 kDa and / or a polydispersity index (Mw / Mn) in the range of 1.2 to 1.8. In one embodiment, the isolated EPS described herein may contain 30 to 90% neutral glycosyl units, 10 to 70% aminoglycosyl units, and 0 to 15% acidic glycosyl units with respect to the total number of glycosyl units of the 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) on January 27, 2020, under depositary number NCIMB 43557. 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 isolated extracellular polysaccharides (EPS) having a weight-average molecular weight (Mw) in the range of 40 to 4000 kDa, comprising the steps of: culturing marine bacteria in a first culture medium to obtain cultured marine bacteria; 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. The isolated EPS may be any isolated EPS described herein. In one embodiment, the marine bacteria 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) on January 27, 2020, deposit number NCIMB 43557. In one embodiment, the fermentation medium may contain 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 H2O (q sp.). In one embodiment, fermentation can be carried out at pH 5-8, 40°C for 20-40 hours under 30-50% oxygenation. In one embodiment, heat treatment of the fermentation medium can be carried out by heating the fermentation medium at 85°C for 1 hour. In one embodiment, centrifugation of the fermentation medium can be carried out at 14,000 g using a disc stack centrifuge with a flow rate of 200-800 L / h. In one embodiment, filtration of the supernatant includes continuous filtration using a filtration step of 1.60-0.22 μm and / or ultrafiltration using a 10-100 kDa cutoff filter cartridge. In one embodiment, the process of the present disclosure may further include free drying of 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 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 described herein, or at least one isolated EPS obtainable or obtained by the process described herein. In one embodiment, the nasal delivery system can deliver at least one isolated EPS as a nasal drop, a liquid spray, a dry spray, a gel or an ointment. In another embodiment, at least one isolated EPS can be formulated in a composition further comprising an aqueous saline solution. In another embodiment, the nasal delivery system described herein may be for treating and / or preventing microbial pathogen infection in a subject that needs it. In another embodiment, the nasal delivery system described herein may be for use in treating and / or preventing microbial pathogen infection in a subject that needs it. In some embodiments, the nasal delivery system described herein may be for attenuating the pathogenicity of a microbial pathogen by inhibiting or reducing its colonization of the nasal cavity of a subject that needs it. In some embodiments, the nasal delivery system described herein may be for use in attenuating the pathogenicity of a microbial pathogen by inhibiting or reducing its colonization of the nasal cavity of a subject that needs it.

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

[0013] According to a sixth aspect, the present disclosure provides the isolated EPS described herein, or an isolated EPS obtained or obtainable by the processes described herein, the compositions described herein, or the nasal delivery systems described herein, for use in a method for treating and / or preventing microbial pathogen infection in a subject that needs it.

[0014] According to a seventh aspect, the present disclosure provides the use of the isolated EPS described herein, or an isolated EPS obtained or obtainable by the processes described herein, the compositions described herein, or the nasal delivery systems described herein, for the manufacture of a medicament for treating and / or preventing microbial pathogen infection.

[0015] According to an eighth aspect, the present disclosure provides the isolated EPS described herein, or an isolated EPS obtained or obtainable by the processes described herein, the compositions described herein, or a nasal delivery system, for use in a method for attenuating the pathogenicity of microbial pathogens.

[0016] According to a ninth aspect, the present disclosure provides the use of the isolated EPS described herein, or an isolated EPS obtained or obtainable by the processes described herein, the compositions described herein, or the nasal delivery systems described herein, for the manufacture of a medicament for attenuating the pathogenicity of microbial pathogens.

[0017] In a tenth aspect, the Disclosure provides a method for treating and / or preventing a microbial pathogen infection in a subject requiring such treatment, 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] In an eleventh aspect, the present disclosure provides a method for reducing the pathogenicity of a microbial pathogen infection in a subject requiring such reduction, 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 for formulating a nasal composition for reducing the pathogenicity of a microbial pathogen infection by inhibiting or reducing the colonization of microbial pathogens into the nasal cavity, the method comprising the step of mixing at least one isolated extracellular polysaccharide (EPS) derived from marine bacteria with a saline solution to obtain a nasal composition containing a salt at a concentration of 0.1% to 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 contains a salt at a concentration of 0.5% to 5% w / w. More preferably, the resulting nasal composition contains a salt at a concentration of 0.7% to 3% w / w. In one embodiment, the resulting composition contains a salt at a concentration of about 0.9% w / w. In one embodiment, the resulting composition contains a salt at a concentration of about 2.2% w / w. In one embodiment, the resulting composition contains a salt at a concentration of about 2.7% w / w.

[0020] According to any of the isolated EPS for use as disclosed herein, the compositions for use as disclosed herein, or the methods disclosed herein, 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 to prevent, inhibit, or reduce the 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 area, genitourinary 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 a subject reduces the pathogenicity of the bacterial pathogen infection by reducing or inhibiting the formation of an early bacterial biofilm and / or by disrupting an early bacterial biofilm. In another embodiment, the microbial pathogen may be a viral pathogen, and administration to an isolated EPS or composition of subject reduces the pathogenicity of viral pathogen infection by preventing or reducing the release of virions from inoculated subject cells and / or preventing or reducing infection of adjacent uninfected subject cells, thereby reducing the mortality rate of subject cells infected with the viral pathogen compared to equivalent untreated cells.

[0021] According to any of the nasal delivery systems of the Disclosure, the non-therapeutic uses of the Disclosure, the isolated EPS for use according to the Disclosure, the compositions for use according to the Disclosure, or the methods of the 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 may be a species of Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or Streptococcus mutans. In one embodiment, 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 at least one EPS in the composition of the present disclosure or in the composition used in accordance with the method and use of the present disclosure may be in the range of 0.00005 to 0.5% w / w, preferably 0.0001 to 0.1% w / w, and more preferably 0.0005 to 0.05% w / w, based on the total weight of the composition. [Brief explanation of the drawing]

[0023] [Figure 1] This represents the step-by-step process used in the manufacture of the EPS of this disclosure. [Figure 2] This shows the percentage of 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) conducted in a triple (n=3) format. *p<0.05 vs control; **p<0.01 vs control; [Figure 3]This study evaluates the antimicrobial effect of increasing EPS concentration on the culture and growth of various bacterial pathogens in two independent experiments (N=2) conducted in a triple series (n=3). OD600 values ​​and mean ± SD were compared. Cultures in the absence of EPS were used as a control. [Figure 4] The viability of HNEpC cells cultured for 2 hours and 24 hours while increasing EPS concentration is shown. The viability of TO-PRO3-negative cells cultured in complete medium for 2 hours and 24 hours was used as a control (C). Error bars represent the standard deviation of a single experiment (N=1) performed in a triple series (n=3). [Figure 5] The study demonstrates a reduction in the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus to HNEpC in the presence of gradually increasing EPS concentrations. Biofilm formation (cell adhesion) was normalized to untreated cells (control). Error bars represent the standard error of the mean values ​​from 2-4 independent experiments (N=2-4) conducted in triplicate (n=3) groups. **p<0.01 vs control; [Figure 6] The emulsion activity index is shown after mixing EPS solutions of various concentrations with vegetable oil and allowing a 24-hour resting period. The error bars represent the standard deviation of three independent experiments (N=3). [Figure 7] This report shows the survival rate of HNEpC cells infected with adenovirus or rhinovirus, or the survival rate of HNEpC cells treated with EPS (400 μg / mL) for 2 hours before or after high-dose viral infection. Cells cultured in BEBM complete medium for 48 hours were used as a positive control, and virus-inoculated cells without EPS treatment were used as a negative control. The values ​​represent the mean ± SD of TO-PRO-3 negative (viable cells) from two independent experiments (N=2) conducted in a triple series (n=3). [Figure 8]Figures 8A and 8B show the survival rates of HNEpC cells infected with adenovirus or rhinovirus, or HNEpC cells treated with EPS (200 μg / mL) for 2 hours before or after high-dose viral infection, respectively. Cells cultured in BEBM complete medium for 48 hours were used as positive controls, and virus-inoculated cells without EPS treatment were used as negative controls. The values ​​represent the mean ± SD of TO-PRO-3 negative (viable cells) from two independent experiments (N=2) conducted in a triplicate (n=3) format. [Figure 9] This shows the viability of HNEpC cells infected with adenovirus or rhinovirus, or HNEpC cells pretreated with EPS (400 μg / mL) for 2 hours before infection, or HNEpC cells post-treated with EPS for 5 days, or HNEpC cells post-treated with daily replacement of EPS for 5 days. 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) conducted in a double-chain (n=2) format. [Figure 10] Figures 10A and 10B show the viability of HNEpC cells infected with adenovirus or rhinovirus, HNEpC cells pretreated with EPS (200 μg / mL) for 2 hours before infection, HNEpC cells post-treated with EPS for 5 days, or HNEpC cells post-treated with daily replacement of EPS for 5 days, respectively. 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) conducted in a triple series (n=3); [Figure 11] This shows the viability of HNEpC cells infected with coronavirus (1 TCID50 or 0.001 TCID50), HNEpC cells pretreated with EPS (200-400 μg / mL) for 2 hours before infection, or HNEpC cells posttreated with 1 dose of EPS for 5 days. 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) conducted in a triple (n=3) configuration. [Figure 12]Figures 12A and 12B show the percentages of IL-8-producing HNEpC and IL-6-producing HNEpC, respectively, after 24 hours of stimulation with an EPS sample (400 μg / mL). Unstimulated cells and cells stimulated with IL-1β were used as negative and positive controls, respectively. The data represent the results of three independent experiments (N=3), and error bars represent standard deviation (SD). [Figure 13A] Figures 13A and 13B show: A) Expression of activated / mature molecules (CD83, CD40, and CD25) on dendritic cells (DCs) cultured for 24 hours in an EPS sample (400 μg / mL). Unstimulated cells or cells stimulated with lipopolysaccharide (LPS, 1 μg / mL) were used as negative and positive controls, respectively. The bar graphs show the mean ± SEM of the MFI for the molecules shown. DCs derived from PBMCs obtained from two different donors were used in the experiment in pairs (N=2, n=2). Error bars represent SD; B) Percentage of IL-8-producing DCs and IL-6-producing DCs after 24 hours of stimulation with an EPS sample (400 μg / mL). 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), and error bars represent SD. [Figure 13B] (As stated above.) [Modes for carrying out the invention]

[0024] This disclosure proposes a novel pathogen infection control approach based on means of reducing or preventing pathogen adhesion to surfaces. Therefore, we studied the chemical components released by marine bacteria isolated from shallow hydrothermal vents in the waters around Panarea, Italy, and investigated their ability to disrupt, control, and / or inhibit the reversible adhesion steps of pathogenic microorganisms, such as bacteria, fungi, and viruses, to biological and abiotic surfaces.

[0025] The marine-derived bacterium, specifically Bacillus licheniformis LP-T14, deposited on January 27, 2020, by Danstar Ferment AG (Poststrasse 30, Zug, 6300, Switzerland) under deposit number NCIMB 43557 with the NCIMB (The National Collection of Industrial, Food and Marine Bacteria, NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK), is found in high temperatures (50°C) and saltwater (conductivity 42.90 mS.cm). -1 These marine bacterial strains possess a unique ability to grow under extreme environmental conditions such as pH 5.4, high concentrations of hydrogen sulfide, and heavy metals, and it has been found that the extracellular polysaccharides secreted from these marine bacterial strains have unique physicochemical properties.

[0026] Therefore, this disclosure demonstrates that a novel extracellular polysaccharide (EPS) released by the marine strain Bacillus licheniformis LP-T14 has the ability to reduce the pathogenicity of pathogen infections by preventing and / or reducing the adhesion of pathogens to treated surfaces compared to untreated surfaces.

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

[0028] In the context of this disclosure, the term “pathogenicity” as used herein means the ability of a microorganism (i.e., bacteria, viruses, yeasts, fungi) to grow and / or spread within a living organism, thereby overwhelming the body’s defense mechanisms. Therefore, when used as a preventive and / or therapeutic measure, the compositions of this disclosure (indirectly) support the host’s immune system, enabling the host to effectively defend against any type of pathogenic microbial infection.

[0029] Furthermore, in the context of this disclosure, the expression “inhibits or reduces establishment” means, in relation to the compositions disclosed herein, any action having the effect of inhibiting or reducing the formation of initial biofilms, disrupting or degrading (partially or completely) initial biofilms, or disrupting or degrading (partially or completely) advanced biofilms.

[0030] In the context of this disclosure, the expression "inhibit or reduce establishment" refers to any action associated with the compositions disclosed herein that has the effect of locally disrupting the establishment of a virus in a living organism. Accordingly, the compositions have the effect of partially or completely preventing the internalization of the virus within living cells and / or limiting the spread of virions from infected cells to adjacent living cells.

[0031] Extracellular polysaccharides and their compositions Bacteria originating from the ocean possess a unique ability to grow under extreme environmental conditions such as high temperatures, saltwater, high concentrations of hydrogen sulfide, and heavy metals. Extracellular polysaccharides (EPS) produced by marine organisms have been found to have unique physical properties and molecular structures. Therefore, EPS from marine bacteria are being applied in a wide range of fields, including biotechnology, medicine, and pharmaceuticals, due to their unique properties such as water solubility, biodegradability, biocompatibility, bioadhesion, heat resistance, swelling, and gelling ability.

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

[0033] In one embodiment, at least one isolated EPS is obtained or can be obtained by fermentation of marine bacteria and an acceptable carrier. In another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or can be obtained by fermentation of Gram-positive thermophilic bacteria and an acceptable carrier thereof. In another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or can be obtained by fermentation of bacteria belonging to the genus Bacillus and an acceptable carrier thereof. In another embodiment, the composition of the present disclosure comprises at least one isolated EPS obtained or can be obtained by fermentation of bacteria belonging to the species Bacillus licheniformis and an acceptable carrier thereof. 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, extremophilic marine Bacillus licheniformis LP-T14, deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB) at NCIMB 43557, and an acceptable carrier thereof.

[0034] In one embodiment, fermentation of marine bacteria is carried out in a fermentation medium containing 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 H2O (qsp.) at pH 5-8, 40°C for 20-40 hours, under 30-50% oxygen supplementation. After fermentation is complete, and before EPS isolation, the fermentation medium is heated at 85°C for 1 hour to inactivate both the marine bacteria and the proteins derived from them.

[0035] The term “isolated” should be understood to mean that it has been materially removed from the original environment in which it is naturally produced, for example, the fermentation medium in this case. The removed material is usually purified from the environment in which it was produced. In one embodiment, the isolated form of EPS ideally does not contain a significant amount of bacterial strain, which in some embodiments may be partially or completely inactivated. In some other embodiments, the isolated form of EPS contains a negligible amount of bacterial strain. In further embodiments, the isolated form of EPS contains at most 10 1 EPS per CFU / g, at most 10 2 EPS per CFU / g, at most 10 3 EPS per CFU / g, at most 10 4 EPS of CFU / g, or at most 10 5Contains CFU / g EPS. In one embodiment, the isolated form of EPS lacks a detectable amount of protein from the bacterial strain used to produce it, which may be partially or completely inactivated when analyzed by the Lowry assay (Example 4) in some embodiments. In some embodiments, the isolated form of EPS contains a negligible amount of protein from the bacterial strain used to produce it when 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, and at most 3% w / w of protein from the bacterial strain used to produce it when analyzed by the Lowry assay (Example 4). Furthermore, after separation from the main portion of other bacterial culture medium components, the isolated EPS of this disclosure may be concentrated by filtration, ultrafiltration, evaporation, spray drying, or freeze-drying, or a combination thereof, but is not limited to these. Therefore, the isolated EPS of this disclosure may be in the form of a concentrate, suspension, powder, or lyophilized product.

[0036] In one embodiment, isolated EPS contained in the composition of the Disclosure exhibits at least one weight-average molecular weight distribution in the ranges 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 ranges 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 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, glucuronic acid, galacturonic acid, and hexuronic acid, which are uronic acids. In one embodiment, the isolated EPS of the Disclosure comprises neutral glycosyl units including glucose, rhamnose, mannose, xylose, and galactose. In one embodiment, the isolated EPS of the Disclosure comprises aminoglycosyl units including N-acetylglucosamine and N-acetylgalactosamine. In one embodiment, the isolated EPS of the Disclosure may comprise acidic glycosyl units including glucuronic acid. In one embodiment, the isolated EPS of the Disclosure contains neutral glycosyl units between 30-90%, 35-80%, and 40-75% with respect to the total number of glycosyl units of the EPS. In one embodiment, the isolated EPS of the Disclosure contains aminoglycosyl units between 10-70%, 15-65%, and 20-60% with respect to the total number of glycosyl units of the EPS. In one embodiment, the isolated EPS of the Disclosure contains acidic glycosyl units between 0-15%, 0-10%, and 0-5% with respect to the total number of glycosyl units of the EPS. In another embodiment, the isolated EPS of the Disclosure is structurally composed of glycosyl units including mannose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine. In another embodiment, the isolated EPS of the Disclosure is not structurally 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 this disclosure exhibits strong emulsifying activity even at low concentrations (i.e., 50 μg / mL, which corresponds to 0.005% EPS). -1 (Using only this method, we reached 50% E24).

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

[0040] In another embodiment, the compositions of the present disclosure contain 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, and at least 0.002% w / w of the composition. The composition comprises at least one isolated EPS having the ability to reduce the pathogenicity of microbial pathogen infection by inhibiting or reducing the colonization of the microbial pathogen on the treated surface, in amounts of %w / w, 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, and at least 0.02%w / w of the composition.

[0041] In other embodiments, the composition of the present disclosure is approximately 0.5 to 5000 μg / mL. -1, about 1 to 1000 μg / mL -1 , about 5 to 500 μg / mL -1 It may be an aqueous solution or suspension containing at least one isolated EPS having the ability to attenuate the pathogenicity of a microbial pathogen infection by inhibiting or reducing the colonization of the microbial pathogen on the treated surface.

[0042] In another embodiment, the composition of the present disclosure is at least 50 ng / mL -1 , at least 100 ng / mL -1 , at least 200 ng / mL -1 , at least 500 ng / 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 It may be an aqueous solution or suspension containing at least one isolated EPS having the ability to attenuate the pathogenicity of a microbial pathogen infection by inhibiting or reducing the colonization of the microbial pathogen on the treated surface.

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

[0044] In one embodiment, the composition disclosed herein may further comprise another EPS. In another embodiment, the composition 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 any bacterial strain tested. In another embodiment, the isolated EPS described herein, optionally provided as a composition, has no bactericidal, bacteriostatic, or antibiotic effects, independently 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 microbiome present on living tissues. In another embodiment, the isolated EPS described herein, optionally provided as a composition, does not induce cytotoxicity in the target to which they are intended, regardless of its concentration and incubation period.

[0046] In another embodiment, the isolated EPS described herein, optionally provided as a composition, substantially lacks the ability to induce an immune (cellular and / or humoral) response. In some embodiments, the isolated EPS described herein, optionally provided as a composition, lacks the ability to induce any immunological response, such as an inflammatory response and / or anergy. In another embodiment, the isolated EPS described herein, optionally provided as a composition, lacks a detectable effect on the maturation of dendritic cells to 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, cannot induce 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, cannot induce 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, cannot induce the production of IL-6 and / or IL-8.

[0047] Antibiofilm activity In the case of bacterial pathogens, pathogenic biofilms can develop on a variety of mucous membranes and epithelial cells, including the oral cavity, nasal cavity, lungs, digestive tract, skin, ophthalmology, and genitourinary tract, as well as on abiotic and hydrophobic nonpolar surfaces (e.g., Teflon, glass, and plastics) such as central veins, urinary and peritoneal dialysis catheters, endotracheal tubes, pacemakers, artificial joints, and mechanical heart valves.

[0048] Microbial pathogens embedded in such matrices can form their own ecosystems and survive, protecting themselves from metabolites or substances produced by the body's immune defenses and exogenous treatments. At the same time, they can acquire new capabilities, such as resistance genes, through intercellular communication (e.g., quorum sensing), adapting to and fighting the environment. Therefore, clinically, biofilm formation is known to be a crucial factor in the establishment and persistence of some difficult-to-treat infections. For example, extracellular polysaccharides produced by pathogenic bacteria protect them, thereby making them resistant to antimicrobial treatment.

[0049] Therefore, this disclosure, which aims to reduce the pathogenicity of bacterial infections, targets not only active or established bacterial infections but also the initial onset of pathogenicity leading to bacterial infections, i.e., the inhibition of initial biofilm formation in which bacteria reversibly adhere to surfaces.

[0050] In one embodiment, the compositions of the Disclosure impart a biofilm formation inhibitory effect to various bacterial pathogens incubated on a synthetic surface under optimal conditions compared to an untreated surface. In another embodiment, surface pretreatment with EPS according to the Disclosure inhibits initial biofilm formation, even when applied at low concentrations, compared to an untreated surface. In another embodiment, the use of the compositions of the Disclosure results in a reduction of biofilm formation from various bacterial pathogens incubated on a synthetic surface under optimal conditions. In another embodiment, surface pretreatment with the compositions reduces initial biofilm formation, even when applied at low concentrations, compared to an untreated surface. In another embodiment, the compositions of the Disclosure reduce biofilm formation of pathogenic bacteria on a synthetic surface 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%, and at least 15%, compared to an untreated synthetic surface. In another embodiment, a composition of the Disclosure having an EPS concentration of at least 200 μg / mL reduces the biofilm formation of pathogenic bacteria on a synthetic surface by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, and at least 10% compared to an untreated synthetic surface. In another embodiment, a composition of the Disclosure having an EPS concentration of 400 μg / mL reduces the biofilm formation of pathogenic bacteria on a synthetic surface by at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, and at least 20% compared to an untreated synthetic surface.

[0051] In one embodiment, the EPS of the Disclosure has the ability to reduce and / or limit the results of bacterial biofilm formation on biological surfaces, regardless of their origin. In fact, in addition to the demonstrated preventive effect of reducing and / or preventing biofilm formation, surface cleaning with the compositions of the Disclosure may also have therapeutic activity due to its ability to remove pathogenic bacterial cells from biofilms. In another embodiment, the compositions of the Disclosure promote the disruption or dissolution of biofilms on surfaces (reduction of existing colonization and pre-formed or accumulated biofilms). In some embodiments, the compositions of the Disclosure promote the disruption or dissolution of initial biofilms on surfaces. In another embodiment, surface treatment with the compositions of the Disclosure prevents, reduces, and / or inhibits bacterial re-colonization on surfaces. In another embodiment, the EPS compositions of the Disclosure reduce the biofilm formation of pathogenic bacteria 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%, and at least 10% compared to an untreated biological surface. In another embodiment, a composition of the Disclosure having an EPS concentration of at least 200 μg / mL reduces the biofilm formation of pathogenic bacteria 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%, and at least 20% compared to an untreated biological surface. In another embodiment, a composition of the Disclosure having an EPS concentration of 400 μg / mL reduces the biofilm formation of pathogenic bacteria 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%, and at least 30% compared to a biological surface.

[0052] In another embodiment, surface treatment with the composition of the present disclosure is performed no later than 8 hours, no later than 6 hours, no later than 4 hours, no later than 3 hours, no later than 2 hours, or no later than 1 hour after contact with pathogenic bacteria. Surface treatment with the composition of the present disclosure may also be applied immediately after contact with pathogenic bacteria. Surface treatment with the composition of the present disclosure may also be applied as a precautionary measure before contact with pathogenic bacteria. Thus, surface treatment with the composition of the present disclosure may 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 one or more specific sites on the body. For example, viral diseases can be contracted through direct contact with other individuals who are already ill, i.e., through contact with bodily fluids or aerosols and infected surfaces.

[0054] Once ingested, the virus enters a specific host cell, alters its cellular mechanisms to replicate, and the resulting virions then spread, infecting neighboring cells.

[0055] This disclosure also covers reducing 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 lowering the mortality rate of living cells compared to untreated cells and / or by preventing the release of virions and their spread to adjacent living cells.

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

[0057] In one embodiment, post-treatment of living cells with the composition of the Disclosure (i.e., after viral inoculation of living cells) prevents the release of virions and their spread to adjacent cells. In another embodiment, post-treatment of living cells with the composition of the Disclosure (i.e., after viral inoculation of living cells) provides a therapeutic effect that reduces the pathogenicity of viral infection compared to untreated cells for at least 1, at least 2, at least 3, at least 4, or at least 5 days after viral inoculation. In yet another embodiment, post-treatment of living cells with the composition of the Disclosure (i.e., after viral inoculation of living cells) is administered in a single dose after viral inoculation. In another embodiment, although we do not wish to be bound by theory, administration of the EPS composition of the Disclosure to human living cells forms an extracellular "barrier" that sterically prevents viral internalization, regardless of the virus considered. In one embodiment, post-treatment of living cells with the EPS composition of the Disclosure after viral inoculation increases the viability of cells compared to the viability of untreated cells. In another embodiment, post-treatment of live cells with the EPS composition according to the Disclosure after viral inoculation (1 TCID50) increases cell viability by at least 1%, at least 2%, at least 3%, and at least 4% compared to untreated cells. In some other embodiments, post-treatment of live cells with the composition according to the Disclosure after viral inoculation (0.001 TCID50) increases cell viability by at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, and at least 30% compared to untreated cells.

[0058] In another embodiment, surface treatment with the compositions of the present disclosure prevents, reduces, and / or inhibits the re-establishment of viruses on a surface.

[0059] In another embodiment, surface treatment with the composition of the Disclosure is performed no later than 8 hours, no later than 6 hours, no later than 4 hours, no later than 3 hours, no later than 2 hours, or no later than 1 hour after contact with the virus. Surface treatment with the EPS composition of the Disclosure may also be applied immediately after contact with the virus. Surface treatment with the composition of the Disclosure may also be applied as a precautionary measure before contact with the virus. Thus, surface treatment with the composition of the Disclosure may 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 this disclosure are effective in reducing the pathogenicity of microbial pathogen infections by inhibiting or reducing the colonization of microorganisms such as bacteria, fungi, yeasts, and viruses on surfaces treated with the claimed compositions.

[0061] In one embodiment, the composition according to the present disclosure is effective in reducing the pathogenicity of bacterial pathogen infections by inhibiting or reducing bacterial colonization. In some embodiments, the bacteria for which this disclosure may be relevant to specific uses include, but are not limited to, species of Staphylococcus, Streptococcus, Enterococcusae and Enterococcus, Neisseria or Branhamella, Bacillus, Propionibacterium, Corynebacterium, Listeria, Clostridium, Escherichia, Enterobacter, Proteus, Pseudomonas, and Klebsiella. This includes both Gram-negative and Gram-positive bacteria, such as those belonging to the species *Bacterium*, *Salmonella*, *Shigella*, *Campylobacter*, *Actinomyces*, *Actinobacillus*, *Acinetobacter*, *Aggregatibacter*, *Fusobacterium*, *Haemophilus*, *Mycobacterium*, *Pseudomonas*, *Porphyromonas*, *Bacteriodes*, *Treponema*, *Prevotella*, or *Eubacterium*. In some other embodiments, bacteria for which this disclosure may be relevant to specific uses include both Gram-negative and Gram-positive bacteria, for example, but not limited to, Staphylococcus aureus.Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes (Group A), Streptococcus (Viridans Group), Streptococcus agalactiae (Group B), Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Streptococcus mutans S. 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 baumanniiExamples include *Baumannii*, *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 are 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 include Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catharalis, Propionibacterium acnes, and Cutibacterium acnes.It is also known as acne, or a combination thereof.

[0062] Accordingly, the compositions relating to this disclosure may be useful for reducing the pathogenicity of bacterial pathogen infections by inhibiting and / or reducing their colonization on treated surfaces in order to prevent, reduce, and / or reduce the likelihood of microbial infections, including, but are not limited to, bacteremia, sepsis, pneumonia, meningitis, osteomyelitis, endocarditis, dental caries, periodontal disease, sinusitis, rhinitis, pink eye, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, erythema, 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 in reducing the pathogenicity of viral pathogen infections by inhibiting or reducing the colonization of viruses on surfaces or epithelium. 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 are also effective against rhinovirus, orthomyxovirus, paramyxovirus, coronavirus, adenovirus, influenza, Roussacomavirus, parainfluenza, metapneumovirus and / or Epstein-Barr virus.

[0064] Accordingly, the compositions according to this disclosure are also effective in preventing and / or treating infectious diseases such as the common cold, influenza, chills, genital herpes, and warts, and / or in preventing HIV and SARS-CoV-2 infection. 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 this disclosure are applicable to human or, more generally, animal tissues including, but not limited to, cells, epithelial cells and / or mucous membranes. Biological surfaces considered in this disclosure include, but not limited to, the oral cavity, tooth surfaces, nasal cavity, respiratory tract, pharynx, ear, ophthalmic area, urogenital tract, skin, scalp, hair and / or nails. In some embodiments, the methods of administering the compositions according to this disclosure are via the nasal cavity, respiratory tract and / or buccal route.

[0066] In one embodiment, the compositions and methods according to the Disclosure may be administered to a healthy subject having intact, healthy tissue, but may be useful when the subject has tissue that has been damaged following a wound or scratch that could lead to local and / or systemic infection. In another embodiment, the compositions and methods according to the Disclosure may be administered to a subject susceptible to infection. In yet another embodiment, the compositions and methods according to the Disclosure may be administered to an infected subject.

[0067] In one embodiment, the target administered by the composition and method according to this disclosure is an animal. In another embodiment, the target administered by the composition and method according to this disclosure is a mammal. In yet another embodiment, the target administered by the composition and method according to this disclosure is a human.

[0068] In one embodiment, for intranasal administration, the composition 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 composition according to the present disclosure may be administered via nasal drops, liquid sprays, e.g., plastic bottle atomizers, bag-on-valve aerosols and / or metered-dose inhalers. Nasal solutions are typically aqueous solutions designed to be administered into the nasal cavity as drops or sprays and are prepared to resemble nasal secretions in many respects so that normal ciliary function can be maintained. In one embodiment, the aqueous nasal solution is typically isotonic or hypertonic and exhibits a final saline concentration between 0.1% and 10% w / w based on the total weight of the nasal composition. In some embodiments, the aqueous nasal solution is typically isotonic or hypertonic and exhibits a final saline concentration between 0.5% and 5% w / w based on the total weight of the nasal composition. In some other embodiments, the aqueous nasal solution is typically isotonic or hypertonic and exhibits a final saline concentration between 0.7% and 3% w / w based on the total weight of the nasal composition.

[0069] In the case of respiratory administration, also known as inhalation, nebulization, or pneumoperitoneum, the compositions according to this disclosure are administered via a pressurized aerosol as a fine powder suspension or liquid in combination with a liquefied gas propellant. When released through a suitable valve and oral adapter, the composition is propelled into the target airway. The use of a pressurized aerosol (i.e., pneumoperitoneum, nebulizer) is considered advantageous because a fine mist is produced by the pressurized aerosol. The pressurized aerosol may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, nitrogen, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the administration unit can be determined by providing a valve for supplying a metered amount.

[0070] The compositions according to this disclosure may be applied topically to the mucosal tissue, gingival tissue, and / or tooth surface of the oral cavity by several conventional methods. For example, the gingival tissue or mucosal tissue may be rinsed with a liquid (e.g., a mouthwash, mouth spray) containing the composition according to this disclosure; or, if the composition according to this disclosure is in the form of a toothpaste (e.g., toothpaste, tooth gel, or tooth powder), the gingival / mucosal tissue or tooth is immersed in the liquid and / or foam produced by brushing the tooth. Other non-limiting examples include applying a non-abrasive gel or paste containing the claimed composition directly to the gingival / mucosal tissue or tooth, with or without the oral care devices described below; chewing gum containing the claimed composition; or chewing or sucking breath tablets, lozenges, or soluble strips 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 toothpaste. In some other embodiments, the composition according to this disclosure is applied to gingival / mucosal tissue and / or teeth via a plastic bottle atomizer or bag-on-valve aerosol. Other methods for topically applying the EPS composition according to this disclosure to the surface of gingival / mucosal tissue and teeth will be apparent to those skilled in the art.

[0071] For vaginal administration, the compositions according to this disclosure may be presented as pessaries, tampons, suppositories, creams, gels, pastes, foams, or sprays, comprising an active ingredient in addition to a carrier known to be suitable in the art.

[0072] When formulated for topical administration, the compositions according to this disclosure may contain components typical 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 tissue to which they are applied, e.g., oral cavity, tooth surface, nasal cavity, respiratory tract, pharynx, ear, ophthalmic area, genitourinary tract, skin, scalp, hair, nails, or mucous membranes. The compositions and components of this disclosure are suitable for contact with infected tissue or for general use on a target without excessive toxicity, incompatibility, instability, allergic reactions, etc. The EPS compositions according to this disclosure may optionally contain any additional components conventionally used in the art considered.

[0073] In terms of their forms, the compositions according to the Disclosure may include liquids, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solid or liquid compositions used for application to skin and other tissues where the composition may be used. Such compositions may include: antimicrobial agents, humectants and hydrates, penetrating agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, cleansing agents, gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyes, colorants, powders, viscosity modifiers, or water, and optionally include anesthetics, antipruritic agents, plant extracts, conditioning agents, darkening or lightening agents, luminescent agents, humectants, mica, minerals, polyphenols, silicones or their derivatives, sunscreens, vitamins, or phytomedicinals. In yet another embodiment, the EPS composition according to the Disclosure is formulated with the above-mentioned components that provide long-term stability to the EPS composition, which may be required for continuous or long-term treatment.

[0074] From the viewpoint of administration method, the carrier of the composition according to this 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] In the case of 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 specifically for nasal formulations is about 50 μL to about 300 μL per nostril, or about 100 μL to about 200 μL per nostril in the case of bag-on-valve aerosol delivery. In some other embodiments, the volume of the claimed composition specifically for nasal formulations is about 0.3 mL to 2 mL, or about 0.6 mL to 1.2 mL per nostril in the case of plastic bottle atomizer delivery. Other delivery media, such as soluble strips, may have dosages obtained from these ranges, taking into account adjustments of concentration and other factors known to those skilled in the art.

[0076] The daily dose of the compositions disclosed herein typically depends on the condition of the subject (healthy in the case of prophylactic treatment or ill in the case of curative treatment) and also on the surface being treated. The daily dose may be administered once a day or divided into two or more doses and administered two or more times a day.

[0077] In one embodiment, the composition according to the Disclosure is administered daily for a period of at least one day, at least three days, at least five days, or at least one week, or at least two weeks, or at least three weeks, or at least four weeks, or at least one month, or at least two months, or at least three months. In one non-limiting embodiment, it may be desirable to continue the treatment until pathogenic bacteria are no longer detected on the treated surface. In another embodiment, the EPS composition according to the Disclosure is administered to a subject as a prophylactic treatment for an undetermined period, such as once or twice daily.

[0078] The compositions according to this disclosure may be used alone or in combination with other antibiotics / antimicrobial agents / antivirals. For example, by inhibiting biofilm formation, microbial pathogens will become far more susceptible to the action of other antibiotics / antimicrobial agents / antivirals, such as those conventionally used to treat microbial pathogens. Similarly, the compositions according to this disclosure may also be used alone or in combination with other EPS.

[0079] Non-biological surface applications In one embodiment, the compositions relating to this disclosure may also be applied to non-biological surfaces, including, but not limited to, hydrophobic and nonpolar surfaces. Essentially, any medical device that is readily colonized by pathogenic microorganisms and / or at least partially coated by a biofilm is suitable for implementation of this disclosure, and this includes contact lenses, contact lens cases, ophthalmic and lens therapy solution containers, or other related products; analyte sensing devices such as electrochemical glucose sensors; drug delivery devices such as insulin pumps; hearing enhancement 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 prostheses). Medical devices that can be processed with this composition include permanent catheters (e.g., central venous catheters, dialysis catheters, long-term tunneling central venous catheters, short-term central venous catheters, peripherally inserted central venous catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters, urethral catheters, or peritoneal catheters), long-term urinary devices, tissue-adhering urinary devices, vascular grafts, vascular catheter ports, wound drainage tubes, ventricular catheters, hydrocephalus shunts, cerebral or spinal shunts, heart valves, cardiac assist devices (e.g., left ventricular assist devices), pacemaker capsules, and incontinence devices. Examples of such products include, but are not limited to, penile implants, small or temporary artificial joints, urinary dilators, cannulas, elastomers, hydrogels, surgical instruments, dental instruments, intravenous tubes, respiratory tubes, dental water lines, dental drain tubes, or feeding tubes, cloth, paper, indicator strips (paper or plastic), 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 into or implanted in a human or other animal, or placed in an insertion or implantation site such as 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 surfaces that may be desired or required to prevent biofilm-embedded microorganisms from growing or multiplying 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, patient room, clinic, or bathroom.

[0080] The compositions according to this disclosure can be administered by attaching an acceptable carrier to a decontaminated non-biological surface that will come into contact with a biological surface. In some cases, the functionality or physical stability of the compositions according to this disclosure can also be increased by adding 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 may be used. The compositions and methods according to this disclosure can be used alone or in combination with other sterilization treatments. For example, by inhibiting biofilm formation, microbial pathogens will become far more susceptible to the action of other sterilization treatments, such as those conventionally used to kill or discard microbial pathogen-specific materials from the treated surface.

[0081] In one embodiment, the composition according to the Disclosure is applied, immersed, and / or coated onto a medical device or instrument before the medical device or instrument encounters a contaminated environment and / or before the object or instrument encounters the tissue in question. In another embodiment, both the tissue in question and the medical device or instrument that comes into contact with the tissue may be treated with the composition according to the Disclosure to limit the adhesion of pathogens and / or to reduce the risk of infection (once in contact with the tissue in question). In yet another embodiment, the composition according to the Disclosure may be used to facilitate a decontamination process of reusable medical materials or instruments. In some further embodiments, the composition according to the Disclosure may be used to facilitate a decontamination process of reusable medical materials or instruments before and / or after sterilization of the medical materials or instruments.

[0082] Surfaces treated with the compositions of the Disclosure by spraying or immersion need to be partially or completely covered in order to reduce the adhesion of pathogens to the surface. The applied EPS compositions of the Disclosure may be effective in covering, 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, or a substantial proportion of the treated surface. [Examples]

[0083] [Example 1: EPS Production Process] Figure 1 shows the seven-step process involved in EPS production. A brief explanation is as follows: Reactivation and Pre-culture: The contents of the cryotube containing Bacillus licheniformis LP-T14 (deposit number NCIMB 43557) were separated into a pre-culture tube and then suspended in 9 mL of suitable liquid Zobell medium under sterile conditions. This pre-culture tube was placed under orbital stirring at 40°C and used to inoculate a liquid culture test sample prepared in 150 mL of Erlen with liquid Zobell medium under stirring at 40°C for 5 hours. Next, 150 mL of the Erlen pre-culture was inoculated into four 5 L fermentation tanks and allowed to stand at 40°C for 4–8 hours under stirring, oxygenation (30–60%), and pH (5–8) control until the optical density (OD) > 2, confirming that bacterial growth was in the initial exponential phase.

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

[0085] Heat treatment: The samples were heat-treated at 85°C for 1 hour in a 400L jacketed agitated tank to inactivate enzymes and bacteria. Centrifugation: The entire culture medium was centrifuged using a disc stack centrifuge (14,000 g, flow rate 200-800 L / h) to separate the biomass from the supernatant. Filtration and ultrafiltration: The supernatant was further treated with continuous filtration (1.60–0.22 μm) and ultrafiltration (10–100 kDa cutoff cartridge hollow fiber polysulfone) to purify and recover only high molecular weight compounds, i.e., EPS. Freeze-drying: EPS was frozen at -20°C for at least 16 hours, then freeze-dried in a freeze-dryer with a 100 kg ice volume. Packaging: The lyophilized EPS was packaged in low-density polyethylene bags sealed under vacuum and labeled before being formulated into appropriate formulations. Samples from each batch were characterized according to standard procedures.

[0086] [Example 2: EPS Characterization - Determination of Average Molecular Weight] 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.

[0087] EPS was analyzed using a size exclusion chromatography (HP-SEC) system, specifically a TDA 302 (Viscotek) equipped with a refractive index detector, a laser light scattering detector, and a viscometer detector. This system was calibrated with a certified pullulan standard (PolyCAL-PullulanSTD-105k, Malvern Panalytical) with molecular weight, polydispersity index, and intrinsic viscosity. EPS samples (100 μL, 1 mg / mL) dissolved in 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] The 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 a dn / dc parameter 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 (deposit number NCIMB 43557), as described in Example 1.

[0090] The glycosyl composition of EPS was determined using two characterization methods: the alditol acetate method (Pena et al. (2012) Methods Enzymol. 510:121-39) and the TMS-derivativeized methyl glycoside method (Santander et al. (2013) Microbiology 159:1471). A brief explanation follows:

[0091] Glycosyl composition analysis by alditol acetate (AA) method: The glycosyl composition of alditol acetate was analyzed by combined gas chromatography / mass spectrometry (GC / MS). EPS samples (250 μg) were hydrolyzed in 2 M trifluoroacetic acid (TFA) in a sealed tube at 120°C for 2 hours, reduced with NaBD4, and acetylated with acetic anhydride / TFA. The resulting alditol acetate was analyzed in electron impulse 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] GC-MS Analysis of Glycosyl Composition of TMS-Derivatived Methyl Glycoside: The glycosyl composition of per-O-trimethylsilyl (TMS) derivatives of monosaccharide methyl glycosides generated from samples by acidic methanolysis was analyzed by combined gas chromatography / mass spectrometry (GC-MS). EPS samples (250 μg) were heated with methanolic HCl in a sealed screw-cap glass test tube at 80°C for 18 hours. After cooling, the solvent was removed under a nitrogen stream, the sample was N-acetylated again, and dried again. Subsequently, the sample was derivatized with Tri-Sil® (Pierce) at 80°C for 30 minutes. GC / MS analysis of TMS methyl glycoside was performed using an Agilent 7890A GC connected to a 5975C MSD with a Supelco Equity-1 fused silica capillary column (30 mx 0.25 mm ID).

[0093] Two characterization methods indicate that mannose is the main hexose sugar detected, and N-acetylgalactosamine is the main amino sugar detected. Significant amounts of galactose, glucose, and N-acetylglucosamine are also present in the sample. Low percentages of xylose were detected, but glucuronic acid was detected only by TMS. However, as shown in Table 1, ribose, arabinose, rhamnose, fucose, and fructose were not detected using either characterization method.

[0094] [Table 1]

[0095] [Example 4: Analysis of EPS protein and nucleic acid content] 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 staining. This analysis was performed on neat EPS samples 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 minutes, the sample was analyzed by absorbance at 595 nm. The protein contamination in EPS was 0.052 ± 0.019 mg / mL. -1 It was evaluated as such, and based on the total weight of isolated EPS, it showed approximately 0.005% w / w.

[0097] Measurement of EPS nucleic acid content by 260 / 280 and 260 / 230 analysis: 2 μL of EPS sample was analyzed at 260 / 280 and 260 / 230 nm using a Nanodrop spectrophotometer. The nucleic acid content in the EPS was 58.0 ± 8.7 ng.μL. -1 It was evaluated as such, and based on the total weight of isolated EPS, it showed approximately 0.006% w / w.

[0098] [Example 5: In vitro EPS antibiofilm activity against bacterial pathogens] 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.

[0099] The following in vitro experiments describe the inhibitory effect of EPS on biofilm formation of pathogenic bacterial strains, namely: Gram-negative bacteria [Haemophilus influenzae type B ATCC9795, Klebsiella pneumoniae ATCC8047, and Pseudomonas aeruginosa ATCC27853] and Gram-positive bacteria [Staphylococcus aureus ATCC29213, Streptococcus pneumoniae sero 3 ATCC6303, and Streptococcus mutans DSM20523].

[0100] Bacterial pathogen biofilm formation was performed in 96-well polystyrene microtiter plates, and 180 μl (1.5 × 10⁶) of the overnight culture of each bacterial pathogen was collected. 8 bacteria.mL -1 Aliquots of different concentrations (50, 100, 200, and 400 μg / ml) -1The strains were inoculated with 20 μl of EPS solution in PBS, or with 20 μl of PBS as a control. Plates were incubated at 37°C for 24 hours (Streptococcus pneumoniae), 48 hours (Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus), and 96 hours (St. mutans). Shaking was not performed to allow biofilm formation, taking into account the growth rate of each strain. Incubation media were also tailored to each strain; therefore, microaerophilic or anaerobic strains were incubated under 5% CO2 or anaerobic conditions, respectively. Non-adherent bacteria were removed by washing three times with distilled sterile water, and adherent bacteria (biofilms) were stained with 0.1% crystal violet solution in 96% vol (w / v) ethanol for 25 minutes. Excess staining solution was removed by aspirating, the plates were washed with distilled sterile water (5 times), and air-dried (15 minutes). The stained biofilms were solubilized with 96% ethanol, and the 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 cultured and incubated under optimal conditions in suitable media, free from competition with other existing pathogens. Since the percentage of biofilm formation is calculated using OD (dispersive occlusion), a higher number of crystal violet-labeled cells measured after washing indicates more cells adhering to the plate and thus more biofilm formation during the incubation period. A reduction in biofilm formation was observed regardless of the pathogenic strains involved. The prophylactic antibiofilm effect of EPS is shown in Figure 2. The greatest effect was observed when EPS was added within the first two hours after inoculation of pathogenic bacteria (data not shown). When EPS was added four hours after inoculation, the effect on biofilm formation was slightly reduced (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 (deposit number NCIMB 43557), as described in Example 1.

[0104] To investigate whether the inhibitory effect of EPS on biofilm formation by bacterial pathogens may be due to direct inhibition of growth, the same experiment as in Example 5 was performed in a triple series (N=3) on two independent occasions (N=2) to evaluate antimicrobial activity. Briefly, polystyrene microtiter plates were filled with final concentrations of 50, 100, 200, and 400 μg / ml of the optimal medium (180 μl) for each test strain (OD600=0.1). -1Overnight pathogenic bacterial cultures were packed into PBS solutions containing either 20 μl of EPS or 20 μl of PBS as a control. Plates were incubated at 37°C for 24 to 96 hours (optimal growth period varied depending on the test strain) without shaking under aerobic, microaerobic, or anaerobic conditions. Antimicrobial activity was measured using a microtiter plate reader (Thermo Scientific® Multiskan® GO Microplate Spectrophotometer), with the mean OD600 of the control well compared to the mean OD600 of each test condition.

[0105] The antibacterial effect of EPS on the culture and growth of various bacterial pathogens is shown in Figure 3, comparing the OD600 values ​​(mean ± SD) of treated cells versus untreated cells after their optimal growth period. Turbidity measurement of the samples (e.g., OD600) is commonly used with a spectrophotometer to estimate bacterial concentration in the culture medium, thereby allowing for the measurement of the growth of the cell population over time. Cultures grown in the absence of EPS were used as a control.

[0106] The results shown in Figure 3 indicate that the same growth levels of pathogenic bacteria were achieved regardless of the presence or absence of EPS. This is true for various concentrations (50-400 μg / ml). -1 The presence of EPS has been shown not to adversely affect the growth of pathogenic bacteria, and more generally, the presence of EPS did not affect the growth rate of any of the bacterial strains tested. Therefore, EPS does not exhibit bactericidal, bacteriostatic, or antibiotic effects, independently of its concentration and the pathogenic bacteria considered. Thus, the presence of non-biocidal EPS demonstrates respect for the integrity of the natural microbiome 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 in vitro study was conducted to determine the putative cytotoxic effect of EPS on human nasal epithelial cells (HNEpCs). Human nasal epithelial cells (HNEpCs) were grown in BEBM complete medium until 80% confluence, as recommended by the manufacturer. The cells were washed twice with PBS and incubated in 5% CO2 medium at 37°C at concentrations of 0 (control) to 800 μg / mL. -1 Cells were cultured with EPS in the specified range for 2 or 24 hours. After culturing, cells were washed twice with PBS, harvested, and their viability was assessed by staining with the viability dye TO-PRO3. The percentage of TO-PRO3-negative cells, indicating viable cells in the culture, was assessed using a Symphony BD Sciences flow cytometer.

[0109] Figure 4 shows the time-dependent effect of increasing EPS concentration on HNEpC viability at 2 and 24 hours. HNEpC cells were selected as a model due to their high sensitivity and were incubated for 2 and 24 hours consecutively while increasing the EPS dose. As shown in Figure 4, cell viability was the same as the control. This result indicates that the presence of EPS does not induce cytotoxicity, regardless of concentration or 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 (deposit number NCIMB 43557), as described in Example 1.

[0111] Next, we evaluated the effectiveness of an EPS composition that removes two bacterial strains, namely Staphylococcus aureus and Pseudomonas aeruginosa, from a monolayer of human nasal epithelial cells.

[0112] Human nasal epithelial cells (HNEpC; PromoCell, catalog number C-12620) were cultured in BEBM complete medium until 80% confluence as recommended by the manufacturer. HNEpC cells were washed twice with PBS and cultured with pathogenic bacteria (Pseudomonas aeruginosa or Staphylococcus aureus) at 37°C, 5% CO2, and antibiotic-free medium for 2 hours. Pseudomonas aeruginosa was grown in Luria Bertani broth, and Staphylococcus aureus in tryptone soy broth. During overnight culture in broth, the bacteria were diluted in PBS at an optical density (OD600) of 0.1 for use in the experiment. Contaminated HNEpC cells were washed twice with PBS and cultured in BEBM complete medium with EPS at concentrations of 0 (control), 50, 100, 200, and 400 μg / mL. -1 The mixture was added and left for 2 hours. After washing twice with PBS, a final wash was performed with distilled H2O for 10 minutes to kill and detach the HNEpC cells and to recover all remaining bacteria. The supernatant (containing dead cells and bacteria) was then collected and, after appropriate dilution, seeded on AGAR medium (Pseudomonas aeruginosa on cetrimide agar, and Staphylococcus aureus on mannitol salt agar). Bacterial growth was analyzed by counting bacterial colonies (CFU) in the plate after 24 hours.

[0113] Figure 5 shows the in vitro efficacy of EPS for the removal of Pseudomonas aeruginosa and Staphylococcus aureus from human nasal epithelial cell (HNEpC) monolayers. These experiments were performed in triplicate (n=3) between 2–4 independent opportunities (N=2–4). Statistical significance was determined by Kruskal-Wallis one-way ANOVA. A p-value < 0.05 was considered statistically significant. Post-hoc analysis was performed using Dunn's test.

[0114] By measuring the number of pathogenic bacteria attached to nasal cells before 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 (HNEpCs). Therefore, these results demonstrated that EPS has the ability to limit the outcome of bacterial biofilm formation on biological surfaces, regardless of its origin. Indeed, in addition to the preventive effect demonstrated in Example 5, where biofilm formation was affected by the presence of EPS, this specification demonstrates that EPS washing may have therapeutic activity due to its pathogenic bacterial cell removal effect (Figure 5).

[0115] [Example 9: Emulsification / surfactant properties of EPS] 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.

[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 5-800 μg / mL was added. -1 The solutions were adjusted to reach several concentrations within a specified range. 2.0 mL of each EPS solution was added to a glass tube containing 2.0 mL of vegetable oil. The resulting heterogeneous solution was then emulsified using a vortex mixer at 3000 rpm for 2 minutes. "Vegetable oil + PBS" and "Vegetable oil + saponin (1%)" were used as negative and positive controls, respectively. Prior to the evaluation of the emulsification index (E24), the samples were allowed to stand at room temperature for 24 hours without stirring. E24 is expressed as the value obtained by dividing the height of the emulsion layer by the total liquid column height and multiplying the result by 100.

[0117] The results of three independent experiments (N=3) shown in Figure 6 demonstrate that EPS exhibits strong emulsifying activity even at low concentrations (i.e., 50 μg / mL, equivalent to 0.005% EPS). -1 (This alone resulted in an E24 of 50%). Furthermore, it was clearly shown that the greater 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 (deposit number NCIMB 43557), as described in Example 1.

[0119] We evaluated 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). A brief description follows.

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

[0121] EPS (400 μg / mL) for HNEpC infected with high doses of adenovirus or rhinovirus. -1The antiviral activity of EPS is reported in Figure 7. In the negative control group (virus inoculation only), half of the HNEpCs were affected by virus inoculation after 48 hours. However, when cells were pretreated with EPS before virus inoculation, the observed cell mortality rate was reduced to half that of the negative control group. Therefore, such pretreatment improves the viability of cells after viral infection, revealing a prophylactic property of EPS against viral infection, independent of the virus in question. EPS treatment of nasal epithelial cells pre-inoculated with a viral load was less pronounced than pretreatment (Figure 7). While we do not wish to be constrained by theory, these observations can be explained by high viral load inoculation, which allows for the internalization of a large amount of virus within HNEpCs within 2 hours, enabling rapid virion contamination of the majority of cells. These results indirectly suggest that EPS does not promote intracellular effects (i.e., induction of an immune response), but rather has an extracellular "barrier" effect that sterically inhibits viral internalization. Nevertheless, it is worth noting that inoculation with such a high viral load (i.e., 1 TCID50) does not reflect the reality of viral infections involving lower viral loads (i.e., 0.001 TCID50).

[0122] EPS concentration 200μg.mL -1 Further antiviral experiments were conducted under the same conditions, except for a reduction in the dosage. EPS (200 μg / mL) was administered to HNEpC inoculated with high doses of adenovirus or rhinovirus. -1 The antiviral activity of ) is reported in Figures 8A and 8B, respectively. These results showed prophylactic antiviral activity equivalent to that observed with high-dose EPS.

[0123] [Example 11: Ex vivo therapeutic antiviral activity of EPS against HNEpC inoculated with low doses of adenovirus or rhinovirus] 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.

[0124] The ability of EPS to prevent the spread of cell infection after virions are released from inoculated cells via viral inoculation was measured. To better reflect the reality of viral infection, these experiments were conducted 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 for viral spread after inoculation. It was also necessary to understand whether the consistent presence of EPS is required to exert a protective effect, or whether initial contact with HNEpCs is sufficient. Therefore, different cell culture conditions were implemented, either by adding EPS only as a pretreatment (similar to Example 10) or by adding it again after viral inoculation (either once at the beginning of the 5-day culture or daily for 5 days). Briefly, viable human nasal epithelial cells (HNEpCs) were grown in BEBM complete medium until 80% confluence, as recommended by the manufacturer. i) As a positive control, viable HNEpC cells were cultured in BEBM medium. As a negative control, HNEpC cells were inoculated with a low dose of viral inoculum (0.001 TCID50) for 2 hours, washed with PBS to remove uninternalized viruses, and cultured for 5 days. ii) 400 μg / mL of viable HNEpC cells were used. -1 iii) Surviving HNEpC cells were treated with EPS for 2 hours, washed with PBS, inoculated with a low dose of viral inoculum (0.001 TCID50) for 2 hours, washed with PBS, and cultured for 5 days. -1 ) Incubated for 5 days in the presence of ), and on day 1, EPS solution was added in one shot. iv) Viral HNEpC cells were inoculated with viral inoculum (low dose, 0.001 TCID50) for 2 hours, washed with PBS, and EPS (400 μg / mL) was added. -1 Incubate for 5 days in the presence of ) and daily EPS (400 μg / mL). -1The substance was added to the culture. After 5 days of culture, cells were harvested and their viability was evaluated using TO-PRO3. The percentage of TO-PRO-3 negative cells (representing viable cells) was evaluated for each condition using a Symphony BD Sciences flow cytometer.

[0125] The antiviral activity of EPS 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 more than half of the HNEpCs were affected by the virus inoculation after 5 days. The prophylactic properties of EPS pretreatment against viral infection observed in Example 10 (i.e., cells treated with the EPS composition before virus inoculation) were confirmed by cell viability close to that of the positive control after 5 days of culture. Furthermore, it was shown that when the EPS composition was applied after virus inoculation (i.e., post-treated cells), the virus could not 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 (one EPS application over 5 days, and one EPS application per day over 5 days) (Figure 9), demonstrating that EPS prevents not only virion release but also diffusion to adjacent cells. Therefore, EPS provides a therapeutic effect that reduces the pathogenicity of the virus inoculated by 50% after 5 days of incubation (Figure 9). These results reinforce the observations of Example 10, where the extracellular "barrier" effect that sterically inhibits viral internalization becomes relevant.

[0127] Therefore, the combined effects of these two effects—a preventive effect (observed in EPS pretreatment of cells before viral inoculation) and a therapeutic effect (observed in EPS posttreatment of cells after viral inoculation)—promote the elimination of viral infection. Such administration of EPS may therefore function as support for cellular mechanisms to effectively defend themselves from any type of viral infection, given that specificity for the target viral strain has not been demonstrated.

[0128] EPS concentration 200μg.mL -1 Further antiviral experiments were conducted under the same conditions except for a reduction in the dosage. EPS (200 μg / mL) was administered to HNEpC inoculated with a low dose of adenovirus or rhinovirus. -1 The antiviral activity of ) is reported in Figures 10A and 10B, respectively. These results showed both prophylactic and therapeutic antiviral activity comparable to that observed with high-dose EPS.

[0129] [Example 12: Ex vivo therapeutic antiviral activity of EPS against low-dose and high-dose coronavirus-infected HNEpC] 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.

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

[0131] EPS (200-400 μg / mL) for HNEpC vaccinated with high or low doses of coronavirus. -1 The antiviral activity of EPS is reported in Figure 11. These results demonstrate that EPS provides i) prophylactic antiviral activity as described in Example 10 of this specification, and ii) therapeutic antiviral activity as described in Example 11 of this specification, independently of the virus under consideration.

[0132] [Example 13: EPS formulation - Nasal formulation] 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.

[0133] Nasal formulation 1: Seawater and desalinated seawater were mixed at room temperature in a washed bulk tank until the desired salt concentration (0.9%, 2.2%, or 2.7%, depending on the formulation) was reached. To solubilize the lyophilized EPS of this disclosure, a certain volume of bulk solution (10 L) was taken and allowed 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 the EPS and the bulk solution were homogenized for at least 30 minutes and then filtered through 0.22 μm. This procedure was repeated once before filling nasal spray cans.

[0134] Nasal formulation 2: Sodium chloride was dissolved in water at room temperature and prepared in a washed bulk tank to a desired salt concentration of 0.9%, 2.2%, or 2.7% (depending on the desired formulation). Next, the EPS of the present disclosure was dissolved in the bulk solution to a desired EPS concentration of 0.02% or 0.04%, corresponding to 200 μg / mL or 400 μg / mL (depending on the desired formulation). Both the EPS and the bulk solution were homogenized for at least 30 minutes and then filtered through 0.22 μm. This procedure was repeated once before filling 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 (deposit number NCIMB 43557), as described in Example 1.

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

[0137] [Example 15: Pharmacological / Immunological properties of EPS disclosed herein] 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.

[0138] Since EPS produced by marine bacteria has been reported to induce selective patterns of regulatory cytokines in leukocytes and tissue cells (Okutani K. et al. Bull Jpn Soci Sci Fish 1984;50:1035-7), the objective of these assays was to determine the extent to which the EPS described herein can induce an immune response in the cells it comes into contact with. Therefore, since two different systems, namely epithelial cells and leukocytes of the innate immune system, are considered susceptible to modification when in contact with bacterial-derived substances, the assays described herein were performed on human nasal epithelial cells (HNEpCs) and human monocyte-derived dendritic cells (DCs), respectively.

[0139] More specifically, the following series of experiments were performed to assess the activation state of DCs after EPS stimulation in order to evaluate i) the production of major cytokines involved in the inflammatory pathway, such as IL-6 and IL-8, after culturing with HNEpC and EPS samples, and ii) 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) Effects of the EPS of this disclosure on HNEpC: Human nasal epithelial cells (HNEpCs) were grown in BEBM complete medium until confluence and stimulated with EPS sample (400 μg / mL) for 24 hours. Unstimulated cells and cells stimulated with IL-1β (50 ng / mL) were used as negative and positive controls, respectively. Monensin and brefeldin were used as GolgiStop (protein transport inhibitors) and added during the last 12 hours of culture. Cells were harvested, fixed with 1% paraformaldehyde, permeabilized with 0.1% saponin in PBS (Sigma-Aldrich), stained with phycoerythrin (PE) conjugate anti-IL-8 antibody and anti-IL-6 antibody (BD Bioscience), and analyzed by flow cytometry.

[0141] These two cytokines are key mediators involved in the recruitment of immune cells that cause inflammatory pathways and inflammatory phenomena. As illustrated in Figures 12A and 12B, no production of IL-6 and IL-8 was observed by HNEpCs after 24 hours of stimulation with the EPS of this disclosure, compared to unstimulated HNEpCs. Surprisingly, no cytokine production was observed upon EPS exposure, despite HNEpCs having the ability to produce these cytokines when properly stimulated with typical inflammatory molecules (i.e., IL-1β). Therefore, these results indicate that the EPS of this disclosure cannot induce an inflammatory response.

[0142] ii) EPS effect of this disclosure on DC Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors by Ficoll Hypaque density gradient centrifugation. Monocytes were isolated by adhering them to a plastic surface at 37°C and 5% CO2 for 45 minutes. Non-adherent cells were then removed by washing with phosphate-buffered saline (PBS). The resulting 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, non-adherent cells were identified as CD14. - / CD11c + / CD83 - This phenotype is characteristic of immature dendritic cells (DCs).

[0143] DCs were stimulated with EPS (400 μg / mL) as disclosed herein for 24 hours. Unstimulated cells and cells stimulated with lipopolysaccharide (LPS, 1 μg / mL) were used as negative and positive controls, respectively. For cytokine production analysis, monensin and brefeldin were added as GrogiStops and left in the DC culture for the last 12 hours. 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 at 4°C for 20 minutes. After washing the cells with PBS, the surface expression levels of the activating molecules shown by flow cytometry were analyzed. B) Cytokine production: DCs were collected, fixed with 1% paraformaldehyde, permeabilized with 0.1% saponin in PBS (Sigma-Aldrich), stained with phycoerythrin (PE) conjugate anti-IL-8 antibody and anti-IL-6 antibody (BD Bioscience), and analyzed by flow cytometry.

[0144] The effect of the EPS of this disclosure on DCs was evaluated by assessing both DC activation and CD83, CD40, and CD25 expression levels as Mean of Fluorescence Intensity (MFI), as shown in Figure 13A. Analysis of DCs after EPS stimulation revealed that the EPS of this disclosure does not affect DC maturation, as indicated by the very low expression levels of CD83, CD40, and CD25, which remained at the same level as unstimulated DCs.

[0145] As illustrated in Figure 13B, the effect of EPS on DCs and their cytokine production, specifically IL-8 and IL-6, after 24 hours of stimulation was evaluated as a percentage of cytokine-producing cells. Compared to unstimulated DCs, no production of IL-6 and IL-8 was observed in DCs stimulated with ESP for 24 hours.

[0146] It is noteworthy that the human cell types (HNEpC and DC) used in these experimental systems all possess sufficient pattern recognition receptors (PRRs) to sense the presence of pathogen-associated molecular patterns (PAMPs), including bacteria. DCs have an extremely high ability to sense potential PAMPs in order to initiate inflammatory processes. In addition, it should be noted that LPS, derived from Gram-negative bacteria and used as a positive control in the DC experiments, was effective at a concentration of 1 μg / ml, which is 400 times lower than the EPS concentration tested (Figure 13). Therefore, it has been demonstrated that the EPS described herein lacks the ability to induce an immune (cellular and / or humoral) response.

[0147] While the present invention has been described in relation to its specific embodiments, it will be understood that the claims should not be limited by the preferred embodiments described in the examples, but rather should be given the broadest interpretation consistent with the overall description.

[0148] Further aspects and embodiments of the present invention are described in the following numbered sections: 1. A composition comprising at least one isolated extracellular polysaccharide (EPS) derived from marine bacteria and a suitable carrier, wherein at least one isolated EPS has an average molecular weight in the range of 40 to 4000 kDa, and at least one isolated EPS can be obtained by fermentation of marine bacteria. 2. The composition according to item 1, each independently comprising at least two extracellular polysaccharides 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 the EPS is 40 to 2000 kDa, and optionally 100 to 1400 kDa. 4. The composition according to any one of claims 1 to 3, wherein the marine bacterium is a Gram-positive thermophilic bacterium, optionally, the Gram-positive thermophilic bacterium is Bacillus licheniformis, preferably the Gram-positive thermophilic bacterium is Bacillus licheniformis LP-T14 deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB) on January 27, 2020, under deposit number NCIMB 43557. 5. A 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 for processing medical devices, preferably the composition is a nasal composition; and / or (ii) A composition wherein the carrier is a physiological saline solution. 6. A nasal delivery system comprising at least one isolated extracellular polysaccharide (EPS) derived from marine bacteria, wherein at least one isolated EPS has an average molecular weight in the range of 40 to 4000 kDa, and at least one isolated EPS can be obtained by fermentation of marine bacteria. 7. The nasal delivery system described in item 6, wherein: (i) The average molecular weight of at least one isolated EPS is 40–2000 kDa, optionally 100–1400 kDa; and / or (ii) The marine bacterium is a Gram-positive thermophilic bacterium, and optionally, the Gram-positive thermophilic bacterium is Bacillus licheniformis, preferably Bacillus licheniformis LP-T14 deposited with the NCIMB under depositary number NCIMB 43557; (iii) A nasal delivery system delivers at least one isolated EPS as a nasal spray, liquid spray, dry spray, gel or ointment; (iv) At least one isolated EPS is formulated in a composition further comprising a physiological saline solution; (v) The nasal delivery system is for treating and / or preventing microbial pathogen infections in patients who require it; and / or (vi) Nasal delivery systems are designed to reduce the pathogenicity of microbial pathogens by inhibiting or reducing their colonization in the nasal cavity of patients who require them. Nasal delivery system. 8. Non-therapeutic use of any one of the compositions described in sub-sub 9. A composition according to any one of items 1 to 5, or a nasal delivery system according to item 6 or 7, for use in a method for treating and / or preventing microbial pathogen infections in a patient in need thereof. 10. A composition according to any one of items 1 to 5, or a nasal delivery system according to item 6 or 7, for use in a method for reducing the pathogenicity of microbial pathogens. 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 any one of the compositions described in sub-sub 12. A method for reducing the pathogenicity of a microbial pathogen infection in a patient in need thereof, comprising administering to the patient an effective amount of a composition described in any one of items 1 to 5. 13. (i) The composition is administered to a patient before, during, and / or after infection with a microbial pathogen, thereby preventing, inhibiting, or reducing the colonization and / or internalization of the microbial pathogen in at least one biological tissue of the patient, wherein the at least one biological tissue is optionally selected from the oral cavity, nasal cavity, respiratory tract, throat, ear, ophthalmic area, genitourinary tract, skin, scalp, hair, nails, and combinations thereof, preferably at least one biological tissue being the nasal cavity; and / or (ii) The microbial pathogen is a bacterial pathogen, and administration of the composition to the patient reduces or inhibits the formation of the initial bacterial biofilm and / or disrupts the initial bacterial biofilm, thereby reducing the pathogenicity of the bacterial pathogen infection; or (iii) A composition for use according to item 9 or 10, or according to item 11 or 12, wherein the microbial pathogen is a viral pathogen, and administration of the composition to a patient reduces the pathogenicity of viral pathogen infection by reducing the mortality rate of patient cells infected with the viral pathogen compared to equivalent 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. Microbial pathogens include: (i) A bacterial pathogen optionally selected from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and / or Streptococcus, preferably the bacterial pathogen being Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae It is a species of *Streptococcus pneumoniae* or *Streptococcus mutans*, or a combination thereof; (ii) Viral pathogens selected at will from the following: 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 any combination thereof; (iii) Fungal pathogens; or (iv) One or more mixtures of (i), (ii), and (iii), A nasal delivery system as described in item 6 or 7, a use as described in item 8, a composition for use as described in item 9, 10 or 13, or a method as described in any one of items 11 to 13. 15. A composition according to any one of claims 1 to 5, a use according to claim 8, a composition for use according to any one of claims 9, 10, 13 and 14, or a method according to any one of claims 11 to 14, wherein the amount of 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 for formulating a nasal composition for reducing the pathogenicity of microbial pathogen infection by inhibiting or reducing the colonization of microbial pathogens into the nasal cavity, comprising the steps of: mixing at least one isolated extracellular polysaccharide (EPS) derived from marine bacteria with physiological saline solution and optionally a suitable carrier to obtain a nasal composition containing a 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. The inventions described in the original claims of this application are listed below. [Invention 1] Isolated extracellular polysaccharides (EPS) having a weight-average molecular weight (Mw) in the range of 40 to 4000 kDa, which are obtained or can be obtained by fermentation of marine bacteria. [Invention 2] Isolated EPS according to Invention 1, having a weight-average molecular weight (Mw) in the range of 40-2000kDa, 40-1400kDa, 40-1000kDa, 40-500kDa, 40-400kDa, 40-300kDa, 40-200kDa, 40-150kDa, or 40-100kDa, preferably 40-150kDa. [Invention 3] Isolated EPS according to Invention 1 or 2, having an Mw in the range of 40 to 150 kDa, and optionally a number-average molecular weight (Mn) in the range of 26 to 100 kDa and / or a polydispersity index (Mw / Mn) in the range of 1.2 to 1.8. [Invention 4] An isolated EPS according to any one of Inventions 1 to 3, comprising 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. [Invention 5] An isolated EPS according to any one of Inventions 1 to 4, comprising mannose, galactose, glucose, N-acetylgalactosamine, and N-acetylglucosamine. [Invention 6] An isolated EPS according to any one of Inventions 1 to 5, substantially free of ribose, arabinose, rhamnose, fructose, and / or fucose. [Invention 7] The isolated EPS according to any one of Inventions 1 to 6, wherein the marine bacterium is a Gram-positive thermophilic bacterium, preferably Bacillus licheniforms, and more preferably Bacillus licheniforms LP-T14 deposited with the NCIMB (The National Collection of Industrial, Food and Marine Bacteria) on January 27, 2020, under deposit number NCIMB 43557. [Invention 8] An isolated EPS according to any one of inventions 1 to 7, substantially lacking the ability to induce an immune response. [Invention 9] The following steps: A step to obtain cultured marine bacteria by culturing marine bacteria in a first culture medium; The step of fermenting the cultured marine bacteria in a fermentation medium; A step of heat-treating the fermentation medium; The steps of obtaining the supernatant by centrifuging the fermentation medium; and The step of filtering the supernatant to obtain isolated EPS. A process for obtaining isolated extracellular polysaccharides (EPS) having a weight-average molecular weight (Mw) in the range of 40 to 4000 kDa, including [specific component]. [Invention 10] The isolated EPS is as defined in any one of inventions 2 to 6; The marine bacterium is a Gram-positive thermophilic bacterium, preferably Bacillus licheniformis, and more preferably Bacillus licheniformis LP-T14 deposited with the NCIMB (The National Collection of Industrial, Food and Marine Bacteria) on January 27, 2020, under deposit number NCIMB 43557; The fermentation medium comprises 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 H2O (qsp); The aforementioned fermentation is carried out at pH 5-8, 40°C for 20-40 hours under the addition of 30-50% oxygen; 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 performed at a rate of 200-800 L / h using a disc stack centrifuge to a volume of 14,000 g; and / or The filtration of the supernatant includes continuous filtration using a 1.60-0.22 μm filtration step and / or ultrafiltration using a 10-100 kDa cutoff filter cartridge. The process described in Invention 9. [Invention 11] The process according to invention 9 or 10, further comprising free-drying the isolated EPS to obtain freeze-dried EPS, wherein the free-drying is optionally carried out at -20°C for at least 16 hours. [Invention 12] A composition comprising at least one isolated extracellular polysaccharide (EPS) described in any one of Inventions 1 to 8, or at least one isolated EPS obtained or obtainable by a process described in any one of Inventions 9 to 11, and a suitable carrier. [Invention 13] The composition according to Invention 12, 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. [Invention 14] A nasal delivery system comprising at least one isolated extracellular polysaccharide (EPS) described in any one of Inventions 1 to 8, or at least one isolated EPS obtained or obtainable by a process described in any one of Inventions 9 to 11. [Invention 15] 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 physiological saline solution; The nasal delivery system is for treating and / or preventing microbial pathogen infections in subjects requiring it; and / or The nasal delivery system according to Invention 14, wherein the nasal delivery system is intended to reduce the pathogenicity of the microbial pathogen by inhibiting or reducing its colonization in the nasal cavity of the target that requires it. [Invention 16] Non-therapeutic use of an isolated EPS according to any one of Inventions 1 to 8, an isolated EPS obtained or obtainable by a process according to any one of Inventions 9 to 11, or a composition according to Invention 12 or 13, for application to a non-biological surface to prevent or reduce the colonization of a surface by microbial pathogens, wherein the surface is optionally the surface of a medical device. [Invention 17] Isolated extracellular polysaccharides (EPS) according to any one of Inventions 1 to 8, isolated EPS obtained or obtainable by a process according to any one of Inventions 9 to 11, a composition according to Invention 12 or 13, or a nasal delivery system according to Invention 14 or 15, for use in a method for treating and / or preventing microbial pathogen infections in a subject requiring such treatment. [Invention 18] An isolated extracellular polysaccharide (EPS) according to any one of Inventions 1 to 8, an isolated EPS obtained or obtainable by a process according to any one of Inventions 9 to 11, a composition according to Invention 12 or 13, or a nasal delivery system according to Invention 14 or 15, for use in a method for reducing the pathogenicity of a microbial pathogen. [Invention 19] A method for treating and / or preventing microbial pathogen infection in a subject requiring such treatment, comprising administering to a patient an effective amount of an isolated extracellular polysaccharide (EPS) described in any one of Inventions 1 to 8, an isolated EPS obtained or obtainable by a process described in any one of Inventions 9 to 11, or a composition described in Invention 12 or 13. [Invention 20] A method for reducing the pathogenicity of a microbial pathogen infection in a subject requiring such reduction, comprising administering to the subject an effective amount of an isolated extracellular polysaccharide (EPS) described in any one of Inventions 1 to 8, an isolated EPS obtained or obtainable by a process described in any one of Inventions 9 to 11, or a composition described in Invention 12 or 13. [Invention 21] 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 the colonization and / or internalization of the microbial pathogen on at least one biological tissue of the subject, the at least one biological tissue including epithelium such as the oral cavity, nasal cavity, respiratory tract, pharynx, ear, ophthalmic region, genitourinary 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 the formation of the initial bacterial biofilm and / or disrupts the initial bacterial biofilm, thereby attenuating the pathogenicity of the bacterial pathogen infection; and / or The microbial pathogen is a viral pathogen, and administration of the isolated EPS or the composition to the subject reduces the pathogenicity of viral pathogen infection by reducing the mortality rate of the subject cells inoculated with the viral pathogen compared to equivalent untreated cells, preventing or reducing the release of virions from the inoculated subject cells, and / or preventing or reducing infection of adjacent uninoculated subject cells. An isolated EPS or composition for use as described in Invention 17 or 18, or the method according to Invention 19 or 20. [Invention 22] The aforementioned microbial pathogens are as follows: The bacterial pathogen is 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 Bacterial pathogens optionally selected from the pneumoniae and / or Streptococcus mutans species; Viral pathogens selected at will 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 A nasal delivery system according to Invention 15, a use according to Invention 16, an isolated EPS or composition for use according to any one of Inventions 17, 18, or 21, or a method according to any one of Inventions 19 to 21. [Invention 23] The nasal delivery system, use, isolated EPS for use, composition for use, or method according to Invention 22, wherein the microbial pathogen is a bacterial pathogen selected from Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and / or Streptococcus mutans species. [Invention 24] The nasal delivery system, use, isolated EPS for use, composition for use, or method according to Invention 22, wherein the microbial pathogen is a viral pathogen selected from coronavirus OC43, adenovirus, and / or rhinovirus / enterovirus (picornavirus). [Invention 25] The composition according to Invention 12 or 13, the use according to Invention 16, the composition for use according to any one of Inventions 17, 18 and 21-24, or the method according to any one of Inventions 19-24, 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 0.00005-0.5% w / w, preferably 0.0001-0.1% w / w, or more preferably 0.0005-0.05% w / w. [Invention 26] A method for formulating a nasal composition for reducing the pathogenicity of microbial pathogen infection by inhibiting or reducing the colonization of microbial pathogens into the nasal cavity, comprising the steps of: mixing at least one isolated extracellular polysaccharide (EPS) derived from marine bacteria with physiological saline solution and optionally a suitable carrier to obtain a nasal composition containing a 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. [Discussion 27] The method according to invention 26, wherein the at least one isolated EPS is as defined in any one of inventions 1 to 8. [Invention 28] The method according to invention 27 or 28, wherein the salt concentration is approximately 0.9% w / w, approximately 2.2% w / w, or approximately 2.7% w / w.

Claims

1. An isolated extracellular polysaccharide (EPS) having a weight-average molecular weight (Mw) in the range of 40 to 4000 kDa, obtained by fermentation of marine bacteria, wherein the marine bacteria is Bacillus licheniformis. The aforementioned EPS is (i) When the biological surface is treated with the EPS, the colonization of microbial pathogens (where the microbial pathogens are bacteria and / or viruses) on the biological surface treated with the EPS is prevented and / or reduced, (ii) Those that cannot induce an inflammatory response, The aforementioned Bacillus licheniforms is Bacillus licheniforms LP-T14, which was deposited with the NCIMB (The National Collection of Industrial, Food and Marine Bacteria) on January 27, 2020, under deposit number NCIMB 43557. The isolated extracellular polysaccharide (EPS) mentioned above.

2. The isolated EPS according to claim 1, wherein the isolated EPS has a weight-average molecular weight (Mw) in the range of 40 to 2000 kDa.

3. The isolated EPS according to claim 1 or 2, wherein the isolated EPS has an Mw in the range of 40 to 150 kDa.

4. An isolated EPS according to any one of claims 1 to 3, having a number-average molecular weight (Mn) in the range of 26 to 100 kDa and / or a polydispersity index (Mw / Mn) in the range of 1.2 to 1.

8.

5. The isolated EPS according to any one of claims 1 to 4, wherein the isolated EPS comprises 30 to 90% neutral glycosyl units, 10 to 70% aminoglycosyl units, and 0 to 15% acidic glycosyl units, relative to the total number of glycosyl units of the EPS.

6. The isolated EPS is This includes mannose, galactose, glucose, N-acetylgalactosamine and N-acetylglucosamine; and / or Free from ribose, arabinose, rhamnose, fructose and / or fucose. Isolated EPS according to any one of claims 1 to 5.

7. A composition comprising at least one isolated extracellular polysaccharide (EPS) according to any one of claims 1 to 6, and a suitable carrier.

8. The composition according to claim 7, 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.

9. The composition according to claim 7 or 8, wherein the amount of at least one isolated extracellular polysaccharide (EPS) in the composition, based on the total weight of the composition, is in the range of 0.00005 to 0.5% w / w.

10. A nasal delivery system comprising at least one isolated extracellular polysaccharide (EPS) according to any one of claims 1 to 6, for delivering at least one of the isolated EPS as a nasal spray, liquid spray, dry spray, gel or ointment.

11. The at least one isolated EPS is formulated in a composition further comprising a physiological saline solution; The nasal delivery system is for treating and / or preventing microbial pathogen infections in subjects requiring it; and / or The nasal delivery system according to claim 10, wherein the nasal delivery system is for reducing the pathogenicity of microbial pathogens by inhibiting or reducing their colonization in the nasal cavity of a target requiring such delivery.

12. Non-therapeutic use of isolated EPS according to any one of claims 1 to 6, for application to a non-biological surface to prevent or reduce the colonization of the surface by microbial pathogens.

13. Non-therapeutic use of the composition according to any one of claims 7 to 9 for application to a non-biological surface to prevent or reduce the colonization of the surface by microbial pathogens.

14. The non-therapeutic use according to claim 12 or 13, wherein the surface is the surface of a medical device.

15. (i) Methods for treating and / or preventing microbial pathogen infections in subjects requiring such treatment, (ii) Methods for reducing the pathogenicity of microbial pathogens An isolated extracellular polysaccharide (EPS) according to any one of claims 1 to 6, for use in the following.

16. (i) Methods for treating and / or preventing microbial pathogen infections in subjects requiring such treatment, (ii) Methods for reducing the pathogenicity of microbial pathogens A composition according to any one of claims 7 to 9 for use in the following.

17. (i) Methods for treating and / or preventing microbial pathogen infections in subjects requiring such treatment, (ii) Methods for reducing the pathogenicity of microbial pathogens A nasal delivery system according to claim 10 or 11 for use in the following:

18. The isolated EPS is administered to the subject before, during, and / or after infection with the microbial pathogen, thereby preventing, inhibiting, or reducing the colonization and / or internalization of the microbial pathogen on at least one biological tissue of the subject, the at least one biological tissue including epithelium such as the oral cavity, nasal cavity, respiratory tract, pharynx, ear, ophthalmic region, genitourinary tract, skin, scalp, hair, nails, and combinations thereof; The microbial pathogen is a bacterial pathogen, and the administration of the isolated EPS to the subject reduces or inhibits the formation of the initial bacterial biofilm and / or disrupts the initial bacterial biofilm, thereby attenuating the pathogenicity of the bacterial pathogen infection; and / or The microbial pathogen is a viral pathogen, and the administration of the isolated EPS to the subject reduces the pathogenicity of viral pathogen infection by reducing the mortality rate of the inoculated target cells compared to equivalent untreated cells, preventing or reducing the release of virions from the inoculated target cells, and / or preventing or reducing infection of uninoculated adjacent target cells. Isolated EPS for use according to claim 15.

19. The composition is administered to the subject before, during, and / or after infection with the microbial pathogen, thereby preventing, inhibiting, or reducing the colonization and / or internalization of the microbial pathogen on at least one biological tissue of the subject, the at least one biological tissue including epithelium such as the oral cavity, nasal cavity, respiratory tract, pharynx, ear, ophthalmic region, genitourinary tract, skin, scalp, hair, nails, and combinations thereof; The microbial pathogen is a bacterial pathogen, and administration of the composition to the subject reduces or inhibits the formation of an initial bacterial biofilm and / or destroys the initial bacterial biofilm, thereby attenuating the pathogenicity of the bacterial pathogen infection; and / or The microbial pathogen is a viral pathogen, and the administration of the composition to the subject reduces the pathogenicity of viral pathogen infection by reducing the mortality rate of the subject cells inoculated with the viral pathogen compared to equivalent untreated cells, preventing or reducing the release of virions from the inoculated subject cells, and / or preventing or reducing infection of adjacent uninoculated subject cells. The composition for use according to claim 16.

20. The aforementioned microbial pathogens are as follows: bacterial pathogens; Bacterial pathogens selected at will from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and Streptococcus; Viral pathogens; Viral pathogens selected at will 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 rhinovirus / enterovirus (picornavirus); or fungal pathogen The nasal delivery system according to claim 10 or 11, wherein at least one of the above.

21. The aforementioned microbial pathogens are as follows: bacterial pathogens; Bacterial pathogens selected at will from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and Streptococcus; Viral pathogens; Viral pathogens selected at will 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 rhinovirus / enterovirus (picornavirus); or fungal pathogen A non-therapeutic use according to any one of claims 12 to 14, which is at least one of the above.

22. The aforementioned microbial pathogens are as follows: bacterial pathogens; Bacterial pathogens selected at will from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and Streptococcus; Viral pathogens; Viral pathogens selected at will 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 rhinovirus / enterovirus (picornavirus); or fungal pathogen An isolated EPS for use according to claim 15 or 18, which is at least one of the following.

23. The aforementioned microbial pathogens are as follows: bacterial pathogens; Bacterial pathogens selected at will from the genera Cutibacterium, Haemophilus, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, and Streptococcus; Viral pathogens; Viral pathogens selected at will 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 rhinovirus / enterovirus (picornavirus); or fungal pathogen A composition for use according to claim 16 or 19, which is at least one of the following.

24. The nasal delivery system according to claim 20, wherein the bacterial pathogen is a bacterial pathogen optionally selected from the following species: Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus mutans.

25. The non-therapeutic use according to claim 21, wherein the bacterial pathogen is a bacterial pathogen optionally selected from the species Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus mutans.

26. The isolated EPS for use according to claim 22, wherein the bacterial pathogen is a bacterial pathogen optionally selected from the species Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus mutans.

27. ​​The composition for use according to claim 23, wherein the bacterial pathogen is a bacterial pathogen optionally selected from the species Cutibacterium acnes, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catharalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus mutans.

Images