Novel probiotic strain of Streptococcus salivarius and its uses

Abstract

The present invention relates to a newly isolated bacterial strain of the species Streptococcus salivarius (S. salivarius). More specifically, the S. salivarius strain was deposited at the BCCM under accession number LMG P-31813. Furthermore, the present invention relates to a composition comprising the isolated bacterial strain of the species S. salivarius. In another aspect, the present invention further relates to the isolated bacterial strain of the present invention or the composition of the present invention for use in immunomodulation and / or treatment and / or prevention of infection or disease.

Description

[Technical Field]

[0001] This invention relates to a newly isolated bacterial strain of the species Streptococcus salivarius (S. salivarius), which is deposited with BCCM under accession number LMGP-31813. Furthermore, this invention relates to secondary metabolites produced by the isolated bacterial strain, and compositions comprising the isolated bacterial strain and / or secondary metabolites of this invention. Furthermore, this invention relates to the use of the isolated bacterial strain, secondary metabolites, or compositions described herein. [Background technology]

[0002] Conditions or diseases of the ear, nose, and throat (ENT) are usually caused by fungal, bacterial, or viral infections in the upper tract of the respiratory system; examples of such infections include certain types of otitis media, sinusitis, and / or nasal polyposis; treatment of such forms is usually carried out with topical or oral antibiotics or anti-inflammatory drugs. Conditions of the ENT include debilitating conditions affecting the airways, voice, hearing, speech, and / or sinuses.

[0003] Among the various ENT conditions and diseases is otitis media (OM). OM refers to a group of inflammatory diseases of the middle ear caused by viral or bacterial infection. Serous otitis media (OME), in particular, is characterized by the presence of middle ear effusion (MEE) behind an intact eardrum, even without other signs or symptoms of acute inflammation (e.g., pain or fever) (Non-Patent Literature 1). Chronic OME lasting more than three months is usually treated by surgically inserting a ventilation tube into the eardrum under anesthesia. However, all surgeries carry risks, and there is both evidence supporting and opposing this surgical procedure (Non-Patent Literature 2, Non-Patent Literature 3). Therefore, the use of ventilation tubes remains a controversial issue, highlighting the need for alternatives.

[0004] Middle ear health is closely related to upper respiratory tract (URT) health. Typical ear pathogens, such as nontypeable Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis (Non-Patent Literature 4), which are normally commensal to the nasopharynx, are known to form biofilms in the middle ear of OME patients. These biofilms were not found in the healthy middle ear mucosa of patients undergoing cochlear implantation, which is an important control group with access to a non-inflammatory, non-infectious middle ear. These classic ear pathogens are also present in the URT of healthy individuals. Therefore, they are called pathogenic symbionts or commensal bacteria, which have the potential to be pathogenic in immunocompetent hosts. Recent microbiome data also point to other pathogens that may be involved in OME, such as Alloiococcus otitis and Corynebacterium otitidis (formerly known as Turicella otitidis).

[0005] In addition to immune system regulation, pathogens are controlled by beneficial bacteria present in the same habitat, either through direct interaction or immunomodulation. As a result, loss or reduction of these beneficial bacteria can lead to overgrowth of pathogens, migration to neighboring sites, and the expression of pathogenic characteristics. Long-term disruption of the microbiota has been associated with several chronic inflammatory diseases (Non-Patent Literature 5) and has also been hypothesized to be a cause of chronic OME.

[0006] Adding beneficial bacteria to prevent such disruptions or restore the disrupted microbiota is a valuable method of OME prevention and can reduce the need for surgical intervention. While such probiotic approaches (Non-Patent Literature 6) are widely used in the gastrointestinal tract, they have not been sufficiently studied for respiratory health or the prevention and treatment of otitis media (Non-Patent Literature 7). Several bacterial species have been described for use in the treatment of respiratory tract infections. For example, EP2555785 describes the use of the Streptococcus salivarius strain with access number DSM 23307 in the treatment of chronic infections of the respiratory tract, more specifically the upper respiratory tract. DSM 23307 is described as adhering to HEp-2 cells and producing bacteriocins that can inhibit the growth of S. pneumoniae and S. pyogenes. These characteristics make the strains contained in the currently marketed Rinogermina® probiotic nasal spray suitable for the treatment of bacterial and / or fungal infections of the upper respiratory tract. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Schilder AGM, Chonmaitree T, Cripps AW,Rosenfeld RM, Casselbrant ML, Haggard MP, Venekamp RP. 2016. Otitis media. NatRev Dis Primer 2:16063. [Non-Patent Document 2] Lous J, Burton MJ, Felding J, Ovesen T,Rovers M, Williamson I. 2005. Grommets (ventilation tubes) for hearing lossassociated with otitis media with effusion in children, p. CD001801.pub2. InThe Cochrane Collaboration (ed.), The Cochrane Database of Systematic Reviews. JohnWiley & Sons, Ltd., Chichester, UK.

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[0008] In light of all of the above, understanding the microorganisms that cause OME and those that defend against OME could be helpful in developing alternative therapies to antibiotics and surgery. Furthermore, there is a strong need to isolate new non-pathogenic bacterial strains that exhibit probiotic activity, for example, in the treatment of respiratory infections such as serous otitis media. [Means for solving the problem]

[0009] In this application, it has been demonstrated that a novel strain of Streptococcus salivarius has a protective effect against respiratory infections, particularly serous otitis media.

[0010] The present invention is based on the identification of a novel isolate of the Streptococcus salivarius species. Said strain was deposited with the Belgian Co-ordinated Collection of Micro-organisms (BCCM) under accession number LMG P-31813 on May 28, 2020. This strain is also further designated herein as AMBR158.

[0011] In a first aspect, the present invention relates to an isolated bacterial strain of the Streptococcus salivarius species, wherein said bacterial strain was deposited with the BCCM under accession number LMG P-31813.

[0012] In one embodiment of the present invention, the bacteria of the isolated bacterial strain are in a suspension state, lyophilized state, spray-dried state, live state or abiotic / postbiotic state, provided that the active ingredients are not destroyed.

[0013] In a further aspect, the present invention relates to a secondary metabolite herein shown as a bacteriocin produced by an isolated bacterial strain of the Streptococcus salivarius species of the present invention.

[0014] In a further aspect, the present invention relates to a composition comprising an isolated bacterial strain of the Streptococcus salivarius species of the present invention, or a secondary metabolite of a Streptococcus salivarius strain of the present invention.

[0015] In one embodiment of the present invention, the composition comprises one or more pharmaceutically acceptable excipients, aromatizing agents or carriers.

[0016] In one embodiment of the present invention, the composition contains 10 per gram of the composition 3from 10 11 including a bacterial amount within the range of CFU.

[0017] In a further aspect, the present invention relates to an isolated bacterial strain, secondary metabolite or composition according to the present invention for use as a pharmaceutical in humans or in veterinary medicine.

[0018] In a further aspect, the isolated bacterial strain, secondary metabolite or composition according to the present invention is for use in the treatment and / or prevention of infectious and / or inflammatory diseases. In a further aspect, the infectious and / or inflammatory diseases are selected from, but not limited to, upper respiratory tract infections; ear, nose and throat (ENT) infections; oral infections; dental caries; rhinosinusitis; nasal polyposis; acute otitis media; recurrent acute otitis media; otitis media with effusion; chronic suppurative otitis media; mastoiditis; halitosis; respiratory infections associated with cystic fibrosis. In an even more preferred embodiment, the isolated bacterial strain, secondary metabolite or composition of the present invention is for use in the treatment of otitis media with effusion.

[0019] In another aspect, the isolated bacterial strain, secondary metabolite or composition according to the present invention is for use in immunomodulation, and in particular, the isolated bacterial strain, the secondary metabolite or the composition is used as an adjuvant for promoting the immune response during vaccination or as an immunomodulator for preventing allergies or reducing allergic symptoms.

[0020] In a further aspect, the present invention relates to the use of an isolated bacterial strain, secondary metabolite or composition according to the present invention in the personal hygiene industry, the cleaning industry, air purification or in the manufacture of personal care / consumer / cosmetic products. In yet another embodiment of the present invention, the use of the isolated bacterial strain, secondary metabolite or composition according to the present invention is provided in the oral hygiene industry, particularly in the manufacture of oral hygiene personal care products.

[0021] In another embodiment, the present invention provides the use of isolated bacterial strains, secondary metabolites, or compositions according to the present invention as probiotics. In a further embodiment, the use of isolated bacterial strains, secondary metabolites, or compositions according to the present invention is provided in the food industry, in particular in the manufacture of fermented dairy and non-dairy products, dietary supplements, dietary food additives, and / or nutraceuticals.

[0022] In one embodiment of the present invention, the present invention relates to an isolated bacterial strain, secondary metabolite or composition according to the present invention or the use thereof, wherein the bacterial strain, secondary metabolite or composition is in any form suitable for topical, oral or respiratory administration.

[0023] Furthermore, the present invention relates to a bacterial strain, secondary metabolite, or composition according to the present invention, or the use thereof, wherein the bacterial strain, secondary metabolite, or composition is a pharmaceutically acceptable form selected from, but not limited to, a spray, cream, lotion, gel, ointment, liquid, suspension, emulsion, capsule, tablet, powder, granule, drop, inhalant, toothpaste, or mouthwash.

[0024] Furthermore, the present invention relates to a bacterial strain, secondary metabolite or composition according to the present invention or to a use according to the present invention, wherein the bacterial strain, secondary metabolite or composition is formulated to be administered through the airway by a nebulizer, with or without a propellant. [Brief explanation of the drawing]

[0025] It is emphasized that the descriptions shown herein with reference to the figures are illustrative and intended solely for illustrative purposes to illustrate different embodiments of the present invention. They are presented for the purpose of providing what is considered to be the most useful and straightforward explanation of the principles and conceptual aspects of the present invention. In this regard, no attempt has been made to show structural details of the present invention beyond what is necessary for a basic understanding of the present invention. The descriptions with drawings are intended to make it clear to those skilled in the art how some forms of the present invention are actually embodied.

[0026] [Figure 1] Amplicon sequence variants (ASVs) are significantly more abundant in the nasopharynx of chronic OME (D) and control (H), according to ANCOM analysis with strict adjustments for multiple studies. Streptococcus 5 can all be classified as members of the Streptococcus salivarius group: Streptococcus salivarius, Streptococcus vestibularis, or Streptococcus thermophilus. [Figure 2] The ability of Streptococcus species isolated from the upper respiratory tracts of healthy children to inhibit classic middle ear pathogens. Mean inhibition of (A) Haemophilus influenzae, (B) Catarrhococcus, and (C) Streptococcus pneumoniae, measured by spot assay. Each point represents the mean value of different isolated strains. [Figure 3]Inhibition of upper respiratory tract and classic and suspected middle ear pathogens (Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus catarrhalis, Streptococcus pyogenes, Staphylococcus aureus, A. otitis, and Corynebacterium otitidis) by seven S. salivarius strains isolated within the university (indicated by AMBR numbers). The isolated strains were compared to S. salivarius 24SMB and Streptococcus oralis 89a isolated with Rinogermina® probiotic nasal spray, with Hextril mouthwash (0.1% hexetidine) used as a positive control. For each pathogen, the tested isolates are classified by their mean inhibition zone diameter from minimum (left) to maximum (right). A statistical comparison of the inhibition zone was performed using 0.1% hexetidine as the baseline. [Figure 4] Survival rate in the presence of 0.03% H2O2 in PBS (simulating oxidative stress conditions relative to the initial inoculation (100%)). [Figure 5] Adhesion of intra-campus S. salivarius isolates (indicated by AMBR number) to respiratory epithelial cells (Calu-3). Adhesion rates were statistically compared to those of S. oralis 89a treated with Rinogermina® probiotic nasal spray. Rinogermina® S. salivarius 24SMB was also included in this assay. Isolates were classified from lowest to highest median adhesion rate. **: p < 0.01, ****: p < 0.0001. P values ​​were adjusted by the Holm method. [Figure 6]Immunostimulatory capacity of S. salivarius AMBR158 compared to other S. salivarius isolates and the model probiotic Lacticaseibacillus rhamnosus GG (LGG). (A) Induction of nuclear factor kappa light chain enhancer (NF-κB) in activated B cells, (B) Induction of the interferon regulator (IRF) pathway in THP1-Dual® monocytes, and (C) Induction of TLR2 / 6 receptor activation in HEK-Blue® hTLR2-TLR6 reporter cells co-cultured with bacteria are shown. Results for each condition are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test for culture conditions representing the reporter cells themselves, and immunostimulation was induced using Poly(I:C) and Pam2CSK4 controls; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. [Figure 7] Phylogenetic tree of S. salivarius strains. Light gray dots and dotted rectangles represent isolates derived from this study, while black dots represent S. salivarius strains K12 and M18, which are already commercialized as probiotics. The genome of S. salivarius 24SMB was not publicly available. AMBR158 is highlighted with an asterisk. [Modes for carrying out the invention]

[0027] The present invention will now be described further. Different embodiments of the present invention will be defined in more detail in the following sections. Each of the embodiments defined in this manner may be combined with any other embodiments unless it is explicitly shown to be contrary thereto. In particular, features that are shown to be preferred or advantageous may be combined with other features that are shown to be preferred or advantageous.

[0028] When describing the compounds of the present invention, the terms used should be interpreted according to the following definitions unless the context indicates otherwise.

[0029] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context explicitly indicates otherwise. For example, “compound” means one compound or two or more compounds.

[0030] As used herein, the terms “about” or “approximately” when referring to measurable values ​​such as parameters, quantities, and durations mean that the variation of the specified value is within ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1%, as long as such variation is appropriate for carrying out the disclosed invention. It should be understood that the values ​​themselves that the modifiers “about” or “approximately” refer to are also specifically and preferably disclosed.

[0031] This invention is based on the identification of a novel isolate of the species Streptococcus salivarius. The isolate was deposited with the Belgian Co-ordinated Collection of Microorganisms (BCCM) on May 28, 2020, under accession number LMG P-31813. In part of this specification, the isolate is also referred to as AMBR158.

[0032] As further described in the examples, the inventors of the present invention have found that the LMG P-31813 strain exhibits excellent ability to inhibit the growth of several respiratory infection-related microorganisms, including Haemophilus influenzae, Coccoccus catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Aloyococcus otitis, and Corynebacterium otitis. Such bacterial strains show potential as probiotics for the prevention and / or treatment of respiratory infections such as otitis media.

[0033] In embodiments of the present invention, the isolated bacterial strain may be provided in suspension, freeze-dried, spray-dried, live, or postbiotic form, provided that the active ingredient is not inactive. In the context of the present invention, the term “live form” means a form in which the bacteria are alive. In the context of the present invention, the term “postbiotic form” refers to a form in which the bacteria are not alive, such as in the case of an inanimate application or tyndalized version of the bacteria.

[0034] The present invention also provides secondary metabolites of the isolated bacterial strain LMG P-31813. While the S. salivarius species is well known as a producer of secondary metabolites with bacteriostatic or bactericidal activity against various other bacterial species (Reference 8), the isolated bacterial strain LMG P-31813 exhibits unique characteristics, possessing two bacteriocin loci and one lassopeptide locus.

[0035] Compositions comprising isolated bacterial strains or their secondary metabolites are also provided. The compositions of the present invention can be prepared by freeze-drying or spray-drying bacterial cultures or their secondary metabolites, and mixing both the dried bacteria or secondary metabolites in a suspension with water, or with further suitable excipients and optionally with the addition of further active ingredients.

[0036] As used herein, “composition” means any mixture of two or more products or compounds (e.g., pharmaceuticals, modifiers, etc.). It may be a solution, suspension, liquid, powder or paste, aqueous or non-aqueous formulation, or any combination thereof. In the context of the present invention, a composition is preferably a pharmaceutical composition comprising one or more pharmaceutically active ingredients, carriers, or diluents, such as a suitable sugar, copolymer PEG / PPG, or cryoprotectant.

[0037] In one embodiment, the composition comprises one or more pharmaceutically acceptable excipients, fragrances, or carriers. Examples of excipients that can be selected in such a composition include rubber, xanthan gum, carboxymethylcellulose, silicone, petrolatum, white soft paraffin, magnesium stearate, maltodextrin, mannitol, starch, glucose, trehalose, glycerin, propylene glycol, and lactose.

[0038] The composition may also include fragrances such as thyme or its extracts.

[0039] In embodiments of the present invention, the carrier provides improved bioavailability, stability, and / or durability of microorganisms.

[0040] The composition may further include one or more carriers to improve the bioavailability, stability, and durability of microorganisms or their secondary metabolites. The carriers can also improve adhesion to mucosal surfaces, such as exopolysaccharides produced by bacterial strains or their secondary metabolites, e.g., S. salivarius or Lactobacillus. Furthermore, the carrier may be a thermosensitive polymer, such as Gantrex, whose viscosity can be increased by raising the temperature, and therefore its adhesion. In another embodiment, the carrier may be hydroxypropyl methylcellulose (HPMC).

[0041] In embodiments of the present invention, the composition preferably contains 10 per gram of composition. 3 from 10 11 Contains a quantity of bacteria within the CFU range.

[0042] In another embodiment, the present invention relates to isolated bacterial strains, secondary metabolites, or compositions according to the present invention for use as pharmaceuticals in humans or veterinary medicine. In particular, the isolated bacterial strains, secondary metabolites, or compositions according to the present invention are for use in the treatment and / or prevention of infectious and / or inflammatory diseases. The infectious and / or inflammatory diseases may be selected from, but are not limited to, upper respiratory tract infections; ear, nose, and throat (ENT) infections; oral infections; dental caries; sinusitis; nasal polyposis; acute otitis media; recurrent acute otitis media; serous otitis media; chronic suppurative otitis media; mastoiditis; halitosis; respiratory infections associated with cystic fibrosis; and Covid-19. In a more preferred embodiment, the isolated bacterial strains, secondary metabolites, or compositions of the present invention are for use in the treatment of serous otitis media.

[0043] In another embodiment, the isolated bacterial strains, secondary metabolites, or compositions according to the present invention are intended for use in the treatment and / or prevention of immune-related diseases such as hay fever, allergic rhinitis, allergic sinusitis, asthma, and Covid-19.

[0044] In another embodiment, the isolated bacterial strain, secondary metabolite, or composition according to the present invention is used for immunomodulation, and in particular, the isolated bacterial strain, secondary metabolite, or composition is used as an adjuvant to enhance the immune response during vaccination. For example, we have shown that co-culturing the isolated bacterial strain AMBR158 induces nuclear factor-kappa light chain enhancer (NF-κB) in activated B cells, the interferon regulator (IRF) pathway in THP1-Dual® monocytes, and TLR2 / 6 receptor activation in HEK-Blue® hTLR2-TLR6 reporter cells. In a more preferred embodiment, vaccination is selected from vaccination against respiratory infections such as respiratory infections caused by coronaviruses, e.g., SARS-CoV virus or SARS-CoV-2 virus.

[0045] Terms such as “treatment,” “therapy,” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely stabilizes or cures the disease and / or adverse effects resulting from the disease. “Therapy” includes any treatment of disease in mammals, particularly humans, and includes: (a) preventing the development of a disease or symptom in a subject that may be predisposed to the disease or symptom but has not yet been diagnosed with it; (b) suppressing the symptoms of a disease, i.e., preventing its onset; or (c) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptom. In the context of this invention, terms such as “prevention” refer to preventing a disease or condition from occurring. In the context of this invention, the term “immunomodulation” refers to the process of altering an immune response to a desired level and / or direction.

[0046] In further embodiments, the present invention relates to the use of isolated bacterial strains, secondary metabolites, or compositions according to the present invention in the personal hygiene industry, the cleaning industry, air purification, or in the manufacture of personal care / consumer products. The personal hygiene industry includes the manufacture of personal care / consumer products for personal hygiene, such as tissues, protective masks, or sprays; in particular, the manufacture of tissues, protective masks, or sprays for the treatment and / or prevention of respiratory infections. In certain embodiments, the use of isolated bacterial strains, secondary metabolites, or compositions according to the present invention is provided in the oral hygiene industry, in particular in the manufacture of oral hygiene personal care products such as toothpaste, toothbrushes, or mouthwashes.

[0047] In another embodiment, the present invention provides the use of isolated bacterial strains, secondary metabolites, or compositions according to the present invention as probiotics, particularly as probiotics for the food industry, more preferably as probiotics in the production of dairy and non-dairy fermented products, dietary supplements, dietary food additives, and / or functional foods. Thus, the food industry may encompass fermented foods (dairy-based, worth, soy). The food industry may also include bioreactors and processing environments used in the production of food, and the bacterial strains, secondary metabolites, or compositions of the present invention may be added to food during production.

[0048] The isolated bacterial strains, secondary metabolites, or compositions according to the present invention may be in any form suitable for topical, oral, or respiratory administration. Furthermore, the isolated bacterial strains, secondary metabolites, or compositions according to the present invention may be in pharmaceutical form, but are not limited to, sprays, creams, lotions, gels, ointments, liquids, suspensions, emulsions, capsules, tablets, powders, granules, drops, inhalants, toothpastes, and mouthwashes. Finally, the isolated bacterial strains, secondary metabolites, or compositions according to the present invention can be formulated in a manner that allows them to be administered through the airway by nebulizer, with or without a propellant.

[0049] As used herein, the term “biological sample,” or ultimately simply “sample,” can encompass a variety of fluid samples, including blood and other fluid samples of biological origin, or tissue samples, or mixed fluid-cell or mixed fluid-tissue samples, obtained from an organism and usable in a diagnostic or monitoring assay. The term particularly includes clinical fluid or tissue samples, and further includes cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids, tissue biopsies, lavage fluids, aspirates, sputum, or mucus. The term also includes samples that have been manipulated in any way after procurement, such as by treatment with reagents, solubilization, or concentration of specific components. [Examples]

[0050] A case-control microbiome study of chronic serous otitis media (OME) in children identified Streptococcus salivarius as a pathogen inhibitor.

[0051] Materials and methods

[0052] Case-control microbiome studies to identify bacteria associated with health and OME.

[0053] Research plan Ethical approval was obtained from the Ethics Committee of Antwerp University Hospital to include OME and cochlear implant patients (B300201731724; clinicaltrials.gov identifier NCT03109496) for the NPcarriage study (B300201526558) and for 16S sequencing of a subset of its samples (B300201940224), and informed consent was obtained from parents or legal guardians prior to sampling. OME patients were recruited from a group of children aged 1 to 10 years who received unilateral or bilateral tympanotomy tubes, with or without concurrent adenoidectomy, to alleviate symptoms of persistent (≥3 months) OME. The first control group consisted of microbiologically healthy cochlear implant recipients aged 1 to 45 years, and the second control group consisted of children aged 6 to 30 months who were healthy enough to attend daycare (originally sampled for the NPcarriage study). Exclusion criteria included anatomical features of the URT, immune system or mucociliary system, acute or chronic URT infection, and comorbidities affecting the use of antibiotics or steroids up to one week prior to surgery. Swabs were taken from the anterior nostrils, nasopharynx, and external auditory canal, aspirated middle ear effusion, and, in the case of concurrent adenoidectomy, both adenoid tissue and swab from OME patients. Cochlear implant controls provided anterior nostril and nasopharynx swabs and middle ear irrigation solution, while daycare children provided nasopharyngeal swabs.

[0054] 16S rRNA gene V4 MiSeq sequencing

[0055] DNA from samples of cochlear implant and OME patients was extracted using the QIAamp PowerFecal DNA Kit and quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). NPcarriage study samples were received as NucliSENS® easyMAG® (BIOMERIEUX) extracted DNA. For all samples, the V4 region of the bacterial 16S rRNA gene was amplified using barcode primers 515F (5′‐TATGGTAATTGTGTGCCAGCMGCCGCGGTAA‐3′; SEQ ID No: 1) and 806R (5′‐AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT‐3′; SEQ ID No: 2), and each running sample was indexed with a unique combination of forward and reverse primer barcodes (Reference 9). The PCR mixture consisted of 200 μM deoxyribose nucleoside triphosphate (dNTPs), 3% dimethyl sulfoxide (DMSO), 1x Phusion HF buffer, 0.4 units of Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific), 0.5 μM each of forward and reverse primers, and up to 50 ng or 5 μL of DNA extract. PCR-grade water was added to this mixture to a final volume of 20 μL. The amplification conditions were 30 cycles of denaturation at 95°C for 20 seconds, followed by annealing at 55°C for 20 seconds and extension at 72°C for 1 minute. There was an initial denaturation step of 2 minutes prior to cycling, and the cycle ended with a final extension step of 10 minutes. The amplicon (amplified product) was purified by elution with 40 μL of PCR-grade water using Agentcourt AMPure XP (Beckman Coulter, A63881) according to the protocol. Following this, DNA quantification was performed using a Qubit Fluorometer 3.0 with a 2 μL amplicon, and equimolar pooling was carried out on the same day.Next, the pooled amplicons (~282 bp) were separated from other DNA fragments by gel electrophoresis (0.8% agarose at 60 V for 50 minutes) and extracted from the gel according to a protocol that involved elution to a final volume of 15 μL using a NucleoSpin® gel and PCR cleanup kit (Macherey-Nagel). The library was quantified, diluted to 2 nM, and 5 μL of the diluted solution was loaded onto a MiSeq desktop sequencer (Illumina).

[0056] Data Analysis Following sequencing of the V4 region of the 16S rRNA gene, pair readings were trimmed, error corrected, chimeras removed, and classifications were all performed using DADA2 version 1.6.0 against the EzBioCloud 16S database version from 19.01.2018. This workflow generated an ASV (amplicon sequence variant) table with single nucleotide differences resolved. Sequence extraction and PCR control served as indicators of background contamination. The tidyverse 1.2.1 package and the tidyamplicons package (https: / / github.com / SWittouck / tidyamplicons) were used for filtering contaminants and visualizing the data. ASVs of interest were further classified using the online EzBioCloud 16S-based ID Web app (updated 2020.05.13).

[0057] (Analysis of Microbiome Composition) The difference in ASV abundance between the nasopharynx of OME patients and control nasopharynx was calculated using the R tool (version 1.1.2). Permanova (vegan version 2.2.5) was used to determine the impact of metadata on microbiome composition and to compare the same anatomical locations between cases and controls. Student's t-test (ggpubr version 0.2.5) was used to compare the mean age of cases and controls. The 16S rRNA gene sequencing data generated for this study were deposited in the European Nucleotide Archive as accession number PRJEB33591.

[0058] Characterization of isolates from healthy URT

[0059] Isolation of lactic acid bacteria Samples from cochlear implant controls were plated on three different agar plates (Table 1). From each plate, one colony was selected for each morphology. Bacteria were identified by Sanger sequencing of the 16S rRNA gene (primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID No: 3) and 1492R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID No: 4)).

[0060] Table 1: Culture conditions [Table 1]

[0061] Antimicrobial screening assay The ability of isolates to inhibit the growth of URT and middle ear pathogens was tested by (1) overlaying 2 μL of 48-hour spots of the isolate onto pathogen-containing soft agar (spot assay), and (2) inoculating 30 μL of used filter-sterilized culture supernatant into perforated wells of pathogen-containing agar (radiodiffusion assay)

[17] . 0.1% hexetidine (Hextril®, Johnson & Johnson) and Todd Hewitt (TH) broth served as positive and negative controls, respectively. In some repeats, the pH of the TH broth was reduced to 5. Growth conditions are summarized in Table 1.

[0062] Antibiotic susceptibility assay The minimum inhibitory concentrations of antibiotics (ampicillin, vancomycin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol) were determined using a broth microdilution assay with 2-fold serial dilutions between 0.5 μg / mL and 128 μg / mL, along with evaluation of growth after 24-hour incubation. The cutoff values ​​for Streptococcus thermophilus were used in accordance with European Food Safety Authority (EFSA) guidelines.

[0063] Adhesion assay to airway epithelial cells Human airway epithelial cell line Calu-3 ATCC® HTB-55™ (purchased from ATCC, Molsheim Cedex, France) is used in a 75 cm³ culture medium containing 20 mL of minimal essential medium (MEM) (Life Technologies, Erembodegem, Belgium) supplemented with thermoinactivated fetal bovine serum (Thermo Fischer, Asse, Belgium) and penicillin-streptomycin (100 U / mL) (Life Technologies). 2Cultures were maintained in flasks and incubated in a humidified 5% CO2 incubator at 37 °C. The medium was changed every 3 - 4 days, and cells were passaged weekly at a split ratio of 1:2 using 0.25% trypsin-EDTA solution (Life Technologies).

[0064] To test the ability of bacterial isolates to adhere to human airway epithelium, 2x10 8 colony forming units (CFU) of bacteria were added to confluent Calu-3 cultures seeded at a density of 3x10 5 cells / cm 2 . After incubation at 37 °C, 5% CO2 for 1 h, non-adherent bacteria were removed by washing the cells once with PBS. Subsequently, the cells were trypsinized for 15 min at 37 °C, 5% CO2 to separate the Calu-3 cells and adherent bacteria. Three 10-fold serial dilutions (from 10 -2 to 10 -7 ) of the remaining cell suspension (pre) and adherent cells in PBS were plated onto TH agar and incubated overnight at 37 °C, 5% CO2. After incubation, colonies were counted and the adhesion rate was calculated by comparing the number of CFU added to the Calu-3 cells to the number of CFU recovered after the adhesion experiment.

[0065] Oxidative stress resistance test

[0066] Hydrogen peroxide assay Overnight cultures of 2x10 8 CFU were pelleted at 1400 g for 10 min, washed twice with PBS using the same centrifugation settings, and resuspended in 2 mL PBS. Three 10-fold serial dilutions in PBS were made using 200 μL, and 10 μL of each was spotted onto appropriate agar media. Next, 1350 μL was added with 0.3% (0.979 M) hydrogen peroxide (C FinalMixed with 0.03% w / w (0.0979 M) and incubated at 37°C with shaking. After 20, 40, and 60 minutes, a fixed amount was taken out, serially diluted, and spotted onto appropriate agar. Colonies were counted the following morning (Streptococci) or after 1.5 days (Lactobacilli). L. casei AMBR2, S. salivarius 24SMB, and S. oralis 89a (isolated from the probiotic nasal spray Rinogermina®, DMG Italia) were used as controls.

[0067] Induction of NF-κB and IRF pathways in human monocytes THP1-Dual® NF-κB-SEAP and IRF-Lucia luciferase human reporter monocytes were cultured according to the manufacturer's (InvivoGen) instructions. THP1-Dual® cells were cultured for 10 minutes to evaluate the induction of the NF-κB and IRF pathways. 5 Seed cells / well at a concentration of 10 in a 96-well plate. 6 UV-inactivated bacteria were combined with CFU / well. IRF was induced using a Poly(I:C) control containing 25 μg / mL lipofectamine 2000. After co-culture at 37°C and 5% CO2 for 24 hours, NF-κB induction was assessed by measuring the optical density at 405 nm in each well of a 96-well plate and by monitoring SEAP activity after the addition of p-nitrophenyl phosphate (pNPP) solution. IRF induction was assessed by monitoring luciferase activity in each well of a 96-well plate. Both readings were performed using a Synergy® HTX multimode microplate reader.

[0068] Induction of TLR2 / 6 in human cells: The HEK-Blue® The HEK-Blue® hTLR2-TLR6 reporter cells were cultured according to the manufacturer's (InvivoGen) instructions. For TLR2 / 6 activation evaluation, HEK-Blue® hTLR2-TLR6 cells were cultured in 2x10⁻¹⁶ cells. 5 Cells were seeded in a 96-well plate at a cell / well concentration and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, 10 6 CFU / well of UV-inactivated bacteria were added to HEK-Blue® hTLR2-TLR6 cells. TLR2 / 6 was induced using a 25 ng / mL Pam2CSK4 control. After co-culturing at 37°C and 5% CO2 for 24 hours, TLR2 / 6 activation was assessed by monitoring SEAP activity after addition of p-nitrophenyl phosphate (pNPP) solution by measuring the optical density at 405 nm in each well of a 96-well plate using a Synergy® HTX multimode microplate reader.

[0069] Phage induction assay S. salivarius cultured overnight was diluted 1 / 50 with TH broth and incubated at 37°C for 30 minutes, after which mitomycin C was added to a final concentration of 0, 0.1, or 0.2 μg / mL. The culture density was measured every hour for 15 hours at 600 nm (OD600). Next, the cells were pelleted at 1400 g at 4°C for 10 minutes, the supernatant was neutralized to pH 7-7.2 with 0.1 M NaOH, and then filtered to sterilize. A rectangular petri dish was prepared containing 300 μL of S. salivarius overnight culture medium in 45 mL of base TH agar and 15 mL of top TH soft agar (0.65%), and 10 μL of phage-inducing supernatant was spotted on the surface. After drying, the plate was incubated at 37°C, and the clearing zone by phage lysis was evaluated.

[0070] Bacterial whole-genome sequencing: DNA extraction and data analysis Bacterial DNA was extracted for whole-genome sequencing (WGS) as follows: Overnight cultures of 2 x 1.5 mL bacteria were incubated at 37°C for 1 hour in the presence of 1.5 μl (100 mg / mL) ampicillin. The bacteria were then pelletized (at 12000 xg for 3 minutes), washed three times with 1 mL of NaCl-EDTA, and subsequently added 100 μl of lysozyme (10 mg / mL in NaCl-EDTA) and 1 μl of RNAse (20 mg / mL). After incubation at 37°C for 1 hour with shaking, 229 μl of NaCl-EDTA, 50 μl of SDS (10%), and 20 μl of proteinase K (20 mg / mL) were added, followed by incubation at 55°C for 1 hour. Next, the protein was precipitated in a 200 μl refrigerator-chilled protein precipitation solution consisting of 6 ml of 5 M potassium acetate, 1.15 ml of glacial acetic acid, and 2.85 ml of distilled water, followed by incubation on ice for 5 minutes, and then centrifugation twice at 12000 xg and 4°C for 3 minutes, transferring the supernatant after each centrifugation step. Next, the DNA was precipitated in 600 μl of ice-cold isopropanol, pelletized at 12000 xg and 4°C for 3 minutes, and washed once with 70% ethanol. Finally, the supernatant was discarded, the DNA pellet was air-dried, incubated at 55°C for 5 minutes, and dissolved in 100 μl H2O.

[0071] DNA was sequenced on the Illumina MiSeq platform, and the resulting reads were de novo assembled using the SPAdes-based Shovill (https: / / github.com / tseemann / shovill), subsequently quality-controlled with checkM, and annotated with Prokka. The assembled contigs were screened against the ResFinder 3.2 database for the presence of transmissible antibiotic resistance genes and screened against the Virulence Factor Database (VFDB) using ABRicate (https: / / github.com / tseemann / abricate) for pathogenicity factors. Secondary metabolites were identified using antiSMASH 5.0 and BAGEL4. Genes of interest were further characterized using NCBI-BLAST.

[0072] Phylogenetic tree of S. salivarius NCBI assembly accession numbers for genomes classified as Streptococcus salivarius or Streptococcus sp001556435, with a maximum contamination rate of 5% and a minimum completeness rate of 98%, were obtained from the Genome Taxonomy Database (GTDB). The assembled genome was downloaded, and genes were predicted and translated in Prodigal. Next, progenomics was used to identify single-copy core genes based on a subset of the genome, followed by the calculation of gene occurrence rates across the entire genome, selecting over 1100 genes that existed as single copies in more than 99% of the genome. These genes were aligned, the alignments were ligated, and columns with more than 2% gaps in the sequence were discarded. Finally, phylogenetic trees were constructed using IQ-TREE with a GTR+G substitution model, and midpoint rooted and visualized in R using the phytools package.

[0073] result

[0074] Study of population and sample characteristics Samples were collected from 70 OME patients, 12 cochlear implant recipients, and 41 daycare children. Of the 523 sequences of samples, 443 were retained after quality filtering, with library sizes ranging from 2500 to 784460 and read counts of 36332 ± 49509 (mean ± standard deviation). There was no significant age difference between the OME group and the composite control group (4.38±2.42 years vs. 4.56±7.21 years, p=0.12). In the cochlear implant group, 42% of participants were female, similar to the 41% in both OME patients and daycare controls. Sixty-six OME patients had bilateral tympanotomy tubes inserted, and 28 patients underwent adenoidectomy. Sex did not significantly affect microbiome composition, and age affected only the anterior nostril microbiome (p=0.002 in the OME group, p=0.043 in the cochlear implant group). Neither lateral (unilateral and bilateral OME) nor adenoidectomy significantly affected the microbiome composition at the sampling site.

[0075] Pathological features of the middle ear during OME Of the 97 OME exudate samples, 80% were dominated by a single ASV (relative abundance of ≥50%) (Table 2). In only 33% of the exudates, the dominant ASV belonged to one of the classic otopathogenic genera Haemophilus, Moraxella, or Streptococcus, but these genera showed high prevalence, being detected in 75%, 53%, and 56% of middle ear samples, respectively. Other dominant ASVs were Alloiococcus 1 (39%), Turicella 1 (4%), Staphylococcus 1 (3%), and Corynebacterium 1 (1%). To examine the continuity of body parts, the similarity between middle ear effusion and the nasopharyngeal microbiome (1-Bray Curtis dissimilarity) was plotted against the similarity between middle ear effusion and the laterally coincidental external auditory canal microbiome. All samples predominantly containing Alloococcus, Tulicella, Staphylococcus, or Corynebacterium species were more similar to the external auditory canal (similarity score ≥ 0.574) than to the nasopharynx (similarity score ≤ 0.254). These taxa were also often dominant in the external auditory canal of all patients (data not shown), suggesting that these taxa likely originate from the external auditory canal.

[0076] Table 2: Significant prevalence of ASV (relative abundance ≥ 50%) in at least one middle ear effusion. [Table 2]

[0077] A healthy middle ear microbiome To identify bacteria associated with middle ear health, sequences were determined for 12 middle ear lavage solutions collected from microbiologically healthy cochlear implant recipients. However, after removing obvious contaminants (data not shown), the sequences had at least twice the number of reads compared to the largest negative control, and only four of these were retained after quality filtering. Furthermore, of the 107 ASVs detected in the remaining four samples, only seven were present in multiple samples, and of these, only Staphylococcus 1 and Corynebacterium 1 were not present in the negative control. ASVs detected in the negative control but expected to be found in the respiratory and ear environments were not removed from the dataset. While the impact is minimal in samples with high biomass, caution is needed in interpreting their presence in samples with very low biomass, such as healthy middle ears. Therefore, attempts to sequence the microbiome of a healthy middle ear are considered unsuccessful, as they have shown little to no bacteria to be present, which aligns with recent studies that have argued that bacterial signals detected in this body part under healthy conditions are likely due to contamination.

[0078] Comparison of nasopharyngeal microbiome in healthy individuals and those with chronic OME. To compare the URT microbiome of OME patients with that of healthy controls and identify health-related bacteria, we focused on the nasopharynx rather than the middle ear. The nasopharynx is a natural habitat for classic middle ear pathogens and is a suitable and accessible location for probiotics targeting middle ear health. The nasopharyngeal microbiome differed significantly between patients and controls (p=0.014), and was more pronounced than the difference observed between the two control groups (p=0.049). A total of 134 taxa were shared between both the control and OME groups (data not shown). Abundance variation analysis (ANCOM) with strict correction for multiple tests using the composite control dataset identified Acinetobacter 1 (A. lwoffii or A. pseudolwoffi) and Streptococcus 5 (S. salivarius, S. thermophilus or S. vestibularis) as health-related (Figure 1). The latter extends recent findings that detected a significantly higher prevalence of S. thermophilus-related taxa in the anterior nostrils of healthy controls than in patients with chronic OME (Reference 10).

[0079] Antimicrobial activity of S. salivarius against URT pathogens Next, we aimed to more closely characterize the potential beneficial properties of bacteria isolated from healthy controls, particularly their ability to control middle ear pathogens. The culturomics approach began with the isolation of 142 bacterial isolates belonging to 11 different genera (data not shown). Streptococcus strains were most frequently isolated, particularly from the nasopharynx (n=66), although A. llwoffii or A. pseudolwoffii isolates were not obtained, likely due to their low relative abundance in cochlear implant controls (0.1%). All Streptococcus isolated from healthy children belonged to one of the following groups: mitis (n=28), salivarius (n=32), or sanguinis (n=5).

[0080] The antimicrobial activity of 78 streptococcal species (53 from this study and 25 isolated from healthy adults) was screened. All species tested could inhibit the growth of Haemophilus influenzae, with S. anginosus, S. pseudopneumoniae, and S. salivarius showing the largest inhibition zone (Figure 2A). S. anginosus and S. salivarius could also inhibit Catarrococcal bacteria (M. catarrhalis), but this effect was strain-dependent (Figure 2B). S. salivarius and S. vestibularis were most effective against S. pneumoniae, with S. anginosus ranking third (Figure 2C).

[0081] Seven S. salivarius isolates (AMBR024, AMBR037, AMBR047, AMBR055, AMBR074, AMBR075, and AMBR158) were selected for WGS and more detailed in vitro characterization based on their health relevance, prevalence, and superior ability to inhibit the growth of classic middle ear pathogens. In addition to testing against Haemophilus influenzae (H. influenzae), M. catarrhalis, and Streptococcus pneumoniae, these isolates were also tested against URT pathogens isolated from OME middle ear effusion during this study: Streptococcus pyogenes and Staphylococcus aureus, as well as suspected middle ear pathogens: A. otitis and Corynebacterium otitidis (formerly Turicella otitidis). S. salivarius 24SMB and S. oralis 89a isolated from Rinogermina® (DMG ITALIA) probiotic nasal spray were used as references. These isolates were able to inhibit all pathogens tested in the spot assay (Figure 3), and AMBR158 was the most effective isolate against classic otopathogens.

[0082] Prediction of secondary metabolites with antibacterial activity The genomes of selected S. salivarius isolates were then screened for loci encoding potentially bacteriostatic or bactericidal secondary metabolites. All isolates possessed a class IIc bacteriocin-like peptide (blp) cassette containing different predicted bacteriocins, ABC transporters, and immunoproteins. AMBR074, AMBR075, and AMBR037 further encoded bacteriocins of the class IId Lactococcin 972 family, including ABC transporters and immune mechanisms. The genome of AMBR074 contained lunch peptide loci associated with Streptococcin A M49 (13) and Macedocin (14). Lasopeptides (bacteriocin class If) were detected in AMBR024 (undetermined) and AMBR158 (associated with streptomonomicin). In addition to bacteriocins, antiSMASH (15) also predicted the gramidicin NRPS (non-ribosomal peptide synthase) locus of AMBR024 (Table 3). The specific amino acid sequences of bacteriocin loci 1 and 2 and lasopeptide loci of AMBR158 are shown in Table 4.

[0083] Oxidative stress tolerance To survive URT, bacteria need to adapt to oxidative stress. Hydrogen peroxide (H2O2) is produced by neutrophils, macrophages, and some bacteria, and plays a role in host-microbe and microbe-microbe interactions.

[0084] Some respiratory bacteria, such as Streptococcus pneumoniae, can actively produce high levels of H2O2, inhibiting the growth of other bacteria in the same niche. To phenotypically characterize the ability of isolates to survive H2O2 despite the absence of catalase, isolates were exposed to 0.03% H2O2 in PBS and plated out before exposure and at 20, 40, and 60 minutes (Figure 4). All tested isolates showed some degree of resistance to H2O2, although their viability decreased over time. S. salivarius isolates were more resistant than S. oralis 89a (RGT), which did not develop colonies after 60 minutes. In previous experiments, exposure to 0.1% H2O2 for 90 minutes killed all isolates.

[0085] Safety evaluation Potential probiotics should not carry antibiotic resistance markers, as they can infect pathogens and complicate treatment, especially in mobile components. Therefore, the genomes of isolated strains were screened for antibiotic resistance markers, and susceptibility to major antibiotic classes was tested in vitro. S. salivarius AMBR055 and AMBR047 were predicted to possess the contiguous genes mefA (96% identity, 100% coverage) and mel (100% identity, 100% coverage) encoding macrolide class antibiotic efflux pumps. Phenotypic testing showed resistance only to the macrolide erythromycin (MIC 4–16 mg / L, cutoff 2 mg / L) and chloramphenicol (MIC 8 mg / L, cutoff 4 mg / L) in AMBR047. Based on this finding, AMBR055 and AMBR047 were excluded from further analysis.

[0086] As an additional safety check, we also confirmed that the potential probiotics do not contain genes encoding pathogenic factors. No pathogenic genes of concern were observed, however, two genes hit the virulence factor database (VFDB) for all isolates: psaA, which encodes a putative adheren (with 87.85%–88.92% coverage and 76.24%–76.96% identity of the pneumococcal surface adhesion gene in Streptococcus pneumoniae TIGR4) and hasC, which encodes UDP-glucose pyrophosphorylase (with 91.26%–91.85% coverage and 76.94%–77.67% identity of the gene in Streptococcus pyogenes M1 GAS), likely involved in capsular polysaccharide biosynthesis. These genes were considered adaptive factors reflecting the adaptation of non-pathogenic S. salivarius to URT, rather than actual pathogenic factors. Adhesion to host tissue is generally considered a desirable trait for most probiotic applications, as it increases the opportunities for probiotics to interact with their host and mediates the competitive elimination of pathogens that bind to the same receptors as already adhered bacteria. Regarding capsular polysaccharides, it is important to examine the molecular composition: S. salivarius produces levan or dextran capsules instead of the known virulence factor hyaluronic acid capsules found in pathogenic Streptococcus pyogenes (S. pyogenes), which mimic human connective tissue. Therefore, the hasC gene is not a concern.

[0087] Adhesion ability of S. salivarius to airway epithelium Adhesion to host mucosal and epithelial cells is known to increase the opportunities for probiotics to interact with the host, mediating the competitive elimination of pathogens that replace already adhered bacteria and bind to the same receptor (References 11, 12). Therefore, we phenotypically characterized the interaction between these S. salivarius isolates and respiratory epithelial cells Calu-3. Consistent with the fact that all isolates were able to adhere to cells and express adhesion factors, the median adhesion values ​​ranged from 1.8% (AMBR024) to 8.1% (AMBR158) (Figure 5).

[0088] Immunostimulatory capacity of S. salivarius The immunostimulatory capacity of S. salivarius AMBR158 was compared with other S. salivarius isolates and the model probiotic Lacticaseibacillus rhamnosus GG (LGG) (Figure 6). Induction of the nuclear factor-kappa light chain enhancer (NF-κB) and interferon regulator (IRF) pathways in activated B cells in THP1-Dual® monocytes (Panels A and B, respectively), and activation of the TLR2 / 6 receptor in HEK-Blue® hTLR2-TLR6 reporter cells co-cultured with bacteria (Panel C) are shown.

[0089] Phylogeny of S. salivarius The seven characterized S. salivarius isolates originated from only three children and one adult. Therefore, we investigated not only their interrelationships but also their relationship to other publicly deposited S. salivarius genomes, including the commercially available probiotic strains K12 and M18. Strain 24SMB was unavailable for analysis. The phylogenetic tree is shown in Figure 7. Two strains were obtained from Subject 1 and three from Subject 6. AMBR075 and AMBR037 isolated from Subject 6 appear to be clonal, supported by their mean nucleotide identity (ANI) value between their genomes being 99.95 and the fact that they possess the same incomplete prophage (Streptococcus virus O2105) at the same bacteriocin locus. The S. salivarius genome appeared to be branched into two clades: one clade contained isolates from subject 1 (AMBR074, AMBR075, AMBR037) and subject 6 (AMBR024, AMBR055), as well as the probiotic strain M18. The second clade contained AMBR047 and AMBR158, along with the probiotic strain K12.

[0090] Table 3: Loci that produce secondary metabolites from BAGEL4 and AntiSMASH. The names indicate the type of the most closely related bacteriocin. [Table 3]

[0091] Table 4: Amino acid sequences of bacteriocin loci 1 and 2 and laso peptide loci of the AMBR158 strain. [Table 4] JPEG0007851550000005.jpg204141JPEG0007851550000006.jpg41142

[0092] References 1. Schilder AGM, Chonmaitree T, Cripps AW, Rosenfeld RM, Casselbrant ML,Haggard MP, Venekamp RP. 2016. Otitis media. Nat Rev Dis Primer 2:16063. 2. Lous J, Burton MJ, Felding J, Ovesen T, Rovers M, Williamson I. 2005.Grommets (ventilation tubes) for hearing loss associated with otitis media witheffusion in children, p. CD001801.pub2. In The Cochrane Collaboration (ed.),Cochrane Database of Systematic Reviews. John Wiley & Sons, Ltd,Chichester, UK. 3. Schilder AGM, Manen JG, Zielhuis GA, Grievink EH, Peters SAF, Broek P. 2007.Long-term effects of otitis media with effusion on language, reading andspelling. Clin Otolaryngol Allied Sci 18:234-241. 4. Ngo CC, Massa HM, Thornton RB, Cripps AW. 2016. Predominant BacteriaDetected from the Middle Ear Fluid of Children Experiencing Otitis Media: ASystematic Review. PLoS ONE 11:e0150949. 5. Kim D, Zeng MY, N??ez G. 2017. The interplay between host immune cells andgut microbiota in chronic inflammatory diseases. Exp Mol Med 49:e339. 6. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L,Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME. 2014. The InternationalScientific Association for Probiotics and Prebiotics consensus statement on thescope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol11:506-514. 7. van den Broek MFL, De Boeck I, Kiekens F, Boudewyns A, Vanderveken OM,Lebeer S. 2019. Translating Recent Microbiome Insights in Otitis Media intoProbiotic Strategies. Clin Microbiol Rev 32:e00010-18. 8. Wescombe PA, Heng NCK, Burton JP, Chilcott CN, Tagg JR. 2009. Streptococcalbacteriocins and the case for Streptococcus salivarius as model oralprobiotics. Future Microbiol 4:819-835. 9. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. 2013.Development of a Dual-Index Sequencing Strategy and Curation Pipeline forAnalyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform.Appl Environ Microbiol 79:5112-5120. 10. Walker RE, Walker CG, Camargo CA, Bartley J, Flint D, Thompson JMD,Mitchell EA. 2019. Nasal microbial composition and chronic otitis media witheffusion: A case-control study. PLoS ONE 14:e0212473. 11. Sanders ME, Benson A, Lebeer S, Merenstein DJ, Klaenhammer TR. 2018. Sharedmechanisms among probiotic taxa: implications for general probiotic claims.Curr Opin Biotechnol 49:207-216. 12. Dunne EM, Toh ZQ, John M, Manning J, Satzke C, Licciardi P. 2014.Investigating the Effects of Probiotics on Pneumococcal Colonization Using anIn Vitro Adherence Assay. J Vis Exp 51069.

[0093] Terms in the drawings Log of Abundance Grouping Factor Acinetobacter genus Streptococcus (genus of streptococcus) Inhibition Zone Diameter Haemophilus influenzae Moraxella catarrhalis (Cataric bacterium) Streptococcus pneumoniae Alloiococcus otitidis Staphylococcus aureus Streptococcus pyogenes Turicella otitidis Survival Time Isolate Percent Adhesion Adhesion rate NF-κB induction in THP1-Dual monocytes IRF induction in THP1-Dual monocytes luminescence TLR2 / 6-mediated SEAP activity in HEK-Blue hTLR2-TLR6 reporter cells Medium culture medium

[0094] Deposit JPEG0007851550000007.jpg13383JPEG0007851550000008.jpg14080

Claims

1. An isolated bacterial strain of the species Streptococcus salivarius, which is deposited with BCCM under accession number LMG P-31813.

2. The isolated bacterial strain according to claim 1, wherein the bacteria are in a living form, in a suspension, freeze-dried state, and / or spray-dried state.

3. A composition comprising the isolated bacterial strain described in claim 1 or 2.

4. The composition according to claim 3, comprising one or more pharmaceutically acceptable excipients, fragrances, or carriers.

5. A pharmaceutical product for use as a human or veterinary medicine, comprising an isolated bacterial strain according to claim 1 or 2 or a composition according to claim 3 or 4.

6. A pharmaceutical product for use in the treatment and / or prevention of infectious diseases and / or inflammatory diseases, comprising the pharmaceutical product described in claim 5.

7. The pharmacopoeia according to claim 6, wherein the infectious and inflammatory disease is selected from upper respiratory tract infections; ear, nose and throat (ENT) infections; oral infections; pharyngeal infections, dental caries; sinusitis; nasal polyposis; acute otitis media; recurrent acute otitis media; serous otitis media; chronic suppurative otitis media; mastoiditis; halitosis; and respiratory infections associated with cystic fibrosis.

8. A pharmaceutical product for use in the treatment of serous otitis media, comprising the pharmaceutical product described in claim 7.

9. A pharmaceutical product for use as an immunomodulator, comprising the pharmaceutical product described in claim 5.

10. The pharmaceutical product according to claim 9, wherein an isolated bacterial strain or composition is used as an adjuvant to promote an immune response during vaccination.

11. The pharmaceutical product according to any one of claims 5 to 10, wherein the isolated bacterial strain or composition is in any form suitable for local, oral or respiratory administration.

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