Abstract:
A new microbial strain of the species  Streptococcus salivarius  for use in the treatment of inflammatory processes with or without infectious etiology. A further object of the present invention compositions including the strain and uses thereof.

Description:
[0001]    The present invention provides a new microbial strain of the species  Streptococcus salivarius  for use in the treatment of inflammatory processes with or without infectious etiology. A further object of the present invention are compositions comprising said strain and uses thereof. 
       STATE OF THE ART 
       [0002]    Many of the ENT (Ear, Nose and Throat) diseases may originate from a fungal or bacterial infection in the upper tracts of the respiratory system; examples of such infections are some forms of otitis, sinusitis and/or nasal poliposis: usually the treatment of such forms is performed by using topical or oral antibiotics or anti-inflammatory agents. 
         [0003]    Recently clinical studies have demonstrated that the administration of streptococci such as  Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis  in the form of spray to patients affected by Acute Otitis Media (AOM) interferes and/or inhibits the growth of pathogenic microorganisms responsible of the disease. However, these species of microorganisms have the serious disadvantage to be classified as potentially pathogenic species. 
         [0004]    Recently the use of bacteria as a probiotic agent is continuously developing thanks to their capacity to maintain or restore the host&#39;s natural microbiome by interference with and/or inhibition of other microrganisms, mediated by antimicrobial peptide production such as bacteriocines. 
         [0005]    More than 700 bacterial species are present in the oral cavity and, maintaining the bacterial communities unaltered, has a significant impact on general health, by either preventing or causing infections. Changes in the structure of this complex community could contribute to a shift in the balance of the resident microflora to a disease-associated species compositions (Aas et al., 2005; Caglar et al., 2005; Marsh et al., 1991). 
         [0006]    Bacterial interference, such as antagonism, has a fundamental role in keeping the balance of the microbial ecology associated with the ability of bacterial species to interfere during surface colonization. This phenomenon represents an interesting mechanism of defence due to the capability of endogenous microflora to interfere or inhibit the growth of potential pathogens (Falagas et al., 2008). 
         [0007]    In the oral cavity,  Streptococcus salivarius , a non pathogenic and predominant colonizer in the oral microbiome, is one of the major bacteriocin producers which is able to coexist in the same environment and reduce the frequency of colonization of the main pathogens involved in the upper respiratory tract infections (Wescombe et al., 2009). For this reason  S. salivarius  is a good candidate for oral probiotic in humans. Probiotics are traditionally associated with gut health, many probiotics are used to prevent and treat several diseases mainly in the intestinal tract, and recently many studies have been involved in the development of oral probiotic applications. Clinical studies have demonstrated that the administration of streptococci such as  Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis  in the form of spray to patients affected by Acute Otitis Media (AOM) interferes and/or inhibits the growth of pathogenic microorganisms responsible of the disease. However, this species of microorganisms have the serious disadvantage to be classified as potentially pathogenic species. 
         [0008]    The international patent application WO2004/072272 describes the use of a biologically pure culture of  S. salivarius , isolated from patients oral cavity, in antibacterial compositions for the treatment of otitis media. 
         [0009]    Walls et al. disclose that  Streptococcus salivarius  produces bacteriocin-like inhibitory substances (BLIS) which inhibit the activity of acute otitis media pathogens in the nasopharingeal floraof children and suggest that due to their low pathogenicity  S. salivarius  should be incorporated into trial of bacteriotherapy of recurrent AOM (Walls et al., 2003). 
         [0010]    Recently Power et al. (Power et al. Eur J Clin Microbiol Infect Dis. 2008, 1261-3) have carried out preliminary studies on a group of children affected by AOM by administrating orally a pediatric composition comprising the strain  S. salivarius  K12. This strain has been previously used as a probiotic for oral hygiene and anti-halitosis. 
         [0011]    The study carried out by Power and colleagues has revealed that only in a small percentage of the treated patients, the strain  S. salivarius  K12 has colonized the upper respiratory tract causing an improvement in symptoms of the disease being treated. The low capacity of the so far isolated strains of  S. salivarius  to colonize the upper respiratory tract makes less efficient their use in the adjuvant therapy against infections of the respiratory tracts. 
         [0012]    So it was so strongly felt the need to isolate new non pathogenic strains that in addition to bactericide activity provide high capacity to colonize the respiratory tract. 
       SUMMARY OF THE INVENTION 
       [0013]    The inventors have succeeded in isolating from the nasopharynx of a healthy voluntary, a new bacterial strain belonging to the species  S. salivarius  deposited at the Institute Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) under the filing number DSM 23307 in date 4 Feb. 2010. 
         [0014]    The inventors, by in vitro experiments, show that this specific strain of  Streptococcus salivarius  is characterized by:
       i) high inhibitory activity towards  S. pneumoniae , stable in various culture conditions (BAC and TSYE);   ii) inhibitory activity towards particularly virulent and antibiotics multi-resistant serotypes responsible of invasive infections such as strain  S. pneumoniae  19A;   iii) inhibitory activity towards  S. pyogenes  M-type 1;   iv) high adhesion capacity to the cells HEp-2 (epithelial cells of human carcinoma of the larynx) up to 57%;   v) absence of virulence genes;   vi) complete sensitivity to antibiotics.       
 
         [0021]    Adhesion capacity of this strain to cells HEp-2, together with the properties not belonging to a pathogenic or potentially pathogenic species and producing bacteriocins able to inhibit the growth of  S. pneumoniae  and  S. pyogenes , makes the strain of  Streptococcus salivarius  selected by the inventors and any other strain of  Streptococcus salivarius  with such features particularly suitable for treating bacterial and/or fungal infections of the upper respiratory tract. The utility of such organisms, that can be administered by pharmaceutical compositions, lies in their ability to colonize the respiratory tracts competing pathogenic species. It is therefore clear that adhesion ability of the administered strains to the HEp-2 type cells plays a key role for the efficacy of the same. The pattern of adhesion in vitro on cells derived from upper respiratory tract provides the adhesion and the retention of the bacteriocins producing strains. 
         [0022]    Therefore, object of the present invention is a bacterial strain belonging to the  Streptococcus salivarius  species characterized by the ability to adhere to HEp-2 cells. 
         [0023]    A further object of the invention is said bacterial strain as above defined and compositions comprising it for treating infections and/or inflammations of the upper respiratory tract. 
         [0024]    Compositions comprising said bacterial strain and one or more carriers and/or diluents and/or excipients are object of the invention as well. 
         [0025]    The advantages, features and the use modes of the present invention are evident from the following detailed description in some embodiments, presented as an example and without limitation. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0026]      FIG. 1  is showing bacterial adhesion to HEP-2 cell layer. Cell layers were observed after Giemsa staining using light microscopy. (1)  Streptococcus salivarius  K12, (2),  Streptococcus salivarius  24SMB, (3)  Streptococcus salivarius  4SMB, (4) negative control. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    The present invention provides a new bacterial strain belonging to the species  Streptococcus salivarius  isolated by the inventors from the nosepharynx of a human voluntary subject; the strain has been identified by phenotypic and genotypic analysis. 
         [0028]    The inventors have analyzed several nasal and pharyngeal swabs and several bacterial species have been isolated therefrom, but in one case only it has been isolated and selected a strain with the desired characteristics. The strain has a typical morphology of the  S. salivarius  species with a round shape of the colony and size of 1-2 mm in diameter, with entire and smooth margins. The bacterial strain can be grown on culture medium “ Mitis salivarius ” at 35° C., preferably in presence of 5% CO 2 . The strain is able to adhere to HEp-2 cells and to inhibit the growth of the pathogen  S. pneumoniae  by bacteriocins production. 
         [0029]    The strain has been called  Streptococcus salivarius  24 SMBc and submitted in date 4 Feb. 2010 at the Institute Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) GmbH, Braunschweig, Germany, under the filing number DSM 23307. 
         [0030]    As already previously described the ability to adhere to HEp-2 cells makes this strain and even other strains belonging to the species  Streptococcus salivarius  having such feature particularly suitable for treating infections and/or inflammations of the upper respiratory tract, preferably for treating infections causing diseases such as acute otitis media, recurrence otitis media, nasal polyposis, sinusitis. 
         [0031]    In the present description are defined as upper respiratory tracts the nasal and paranasal cavities, the pharynx, the larynx. 
         [0032]    Object of the present invention are also compositions comprising strains of  Streptococcus salivarius  as above defined and one or more carriers, diluents and/or excipients. 
         [0033]    Said compositions preferably comprise the bacterial strain  Streptococcus salivarius  filing number DSM 23307. 
         [0034]    Bacteria can be in suspension, freeze-dryed or inactivated, provided they are not killed. The preparation of the compositions of the invention can then be implemented by freeze-drying of bacterial cultures, mixing freeze-dryed both in suspension with water or with further suitable excipients and optionally with addition of further active principles. 
         [0035]    The amount of bacteria in said composition is preferably in the range between 10 3  and 10 10  CFU for each gram of composition. 
         [0036]    Examples of excipients that can be used in such compositions are: rubber, xanthan, carboxylmethyl cellulose, silicone, Vaseline, white soft, magnesium stearate, maltodextrin, mannitol, starch, glucose, glycerine, propylene glycol, and similar. 
         [0037]    Said compositions may include also carriers idoneous to improve the bioavailability, the stability and the endurance of the microrganism. 
         [0038]    Said composition may comprise carriers to further improve the adhesion of the microorganism adhesion on the mucosal surface such as the EG56 polymer (Bis-Methoxy PEG-13 PEG-438/PPG-110 SMDI Copolymer), a heat-sensitive polymer able to increase the viscosity and thus the adhesiveness by increasing the temperature or Gantrex (PVM/MA Copolimer). 
         [0039]    Said compositions may be in any form considered by the expert of the technical field suitable to be administered topically, orally, or through the respiratory tract. 
         [0040]    For administration through the respiratory tract in the present description it has to be intended nasal or by inhalation administration. 
         [0000]    Examples of suitable pharmaceutical forms are cream, lotion, gel, ointment, solution, suspension, emulsion, capsule, tablet, powder, granules, sprays, drops. 
         [0041]    Preferably compositions may be formulated to be administered through the respiratory tract in a nebulizer, with or without propellants. 
         [0000]    Such compositions can be prepared according to techniques and protocols known to the expert of the technical field. Said composition may even contain anti-inflammatory agents such as 18-beta glycyrrhetinic acid. 
         [0042]    Object of the present invention are the compositions above described useful for treating infections of the upper respiratory tract, preferably for treating infections causing diseases such as acute otitis media, recurrence otitis media, nasal polyposis, sinusitis. 
       EXAMPLES 
     Collection of Nasal and Pharyngeal Swabs from Patients 
       [0043]    Thirty one children aged between 10 and 12 years have been involved in this study. Children who had one, few or any AOM episode have been selected. Patients who received antibiotics in the previous two weeks, had an operation on the upper respiratory tract or with anatomic abnormalities of the respiratory tract have been excluded. 
         [0044]    A nasal and pharyngeal swab has been collected respectively from the nostrils and the mouth of each patient with a cotton wool soaked in sterile calcium alginate. 
       Microbiological Test 
       [0045]    In order to highlight the presence of bacterial flora of nasal and pharyngeal swab samples were collected as above described, all samples have been plated onto Mitis Salivarius agar (Difco), a selective medium for streptococci, and onto “chocolate agar” (Columbia Agar Base, OXOID) containing 5% horse blood in order to determine bacterial microflora. 
         [0046]    Cultures have been incubated for 18 hours at 37° C. in presence of 5% CO 2  and atmospheric pressure. All strains have been frozen at −70° C. in “Brain heart infusion broth” (OXOID) with 20% glycerol. 
       BLIS (Bacteriocin-Like Inhibitory Substance) Test 
       [0047]    Each colony morphologically distinct and isolated, obtained from the growth of bacteria as described above has been assayed for the ability to inhibit the most representative strains causing otitis:  S. pyogenes  2812A,  S. pneumoniae  11ATN,  H. influenzae  3ATF,  S. aureus, E. coli, P. aeruginosa, S. salivarius  ATCC13419 , M. catarrhalis . The ability to inhibit pathogen strains has been assayed by the “BLIS test” as originally developed by Walls et al. (Med microbial 52 (2003)). Assays have been performed by using two different media: Trypticase Soy Yeast Extract Calcium Agar (TSYCa)+2% Yeast Extract and Blood agar+calcium carbonate (BACa). Results have shown that the strain of  Streptococcus salivarius  identified by filing number DSM 23307 is able to inhibit the growth of  S. pneumoniae  both in TSYCa and BACa medium. Furthermore, it has been evaluated the ability of strain  S. salivarius  DSM 23307 to inhibit particularly virulent and multi-resistant strains of  S. pneumoniae  19A and  S. pyogenes  M-type 1. 
       Analysis of Virulence Genes 
       [0048]    In  S. salivarius  DSM 23307 the presence of virulence genes particularly diffuse in streptococci such as sag A, smez-2 and speB, respectively responsible of the production of the toxin streptolisin S, the mitogenic exotoxin and the eritrogenic exotoxin. The assays have been performed by PCR and hybridization with specific probes. 
         [0049]    In particular, total bacterial DNA was extracted in agarose plugs as previously described (Santagati et al., 2009). Following restriction with SacII enzyme (TaKaRa BIO), macro-restriction fragments were resolved in 1% agarose gel using 0.5× tris-borate-ethylene diamine tetra-acetic acid buffer (BioRad) at 14° C. The CHEF DR PFGE (BioRad) system was used, switch and run times were 1″ to 15″ for 20 hrs, with a voltage gradient of 6V/cm 2 . The macro-restriction fragments were visualized by a blue-light trans-illuminator (Safe Imager Invitrogen) after staining with 1×SYBR Green (SYBR Safe DNA gel staining Invitrogen) in TBE 0.5×. The macro-restriction fragments were transferred from the gel to a nylon Hybond N+membrane (Amersham) using a Vacuum blotter 785 (BioRad) and denaturing solutions (NaOH 0.5M/NaCl 1.5M). DNA fragments were immobilized by UV irradiation (Ultraviolet Crosslinker, Amersham). The hybridization assay with sagA, smeZ-2, speB probes and further probes specific for spec, speJ, speG, prtF and sof were performed using the “ECL Direct Nucleic Acid Labelling and detection System” (RPN 3000 Amersham), following the protocol provided with the kit. The probes were obtained by PCR from the  S. pyogenes  SF370 and 2812A genome and purified with the QIAquick purification kit (Quiagen) using the primers listed in table 1. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Primers used in this study 
               
             
          
           
               
                   
                   
                   
                 Amplicon 
                   
               
               
                   
                 Primer 
                   
                 size 
                 Accession 
               
               
                 Gene 
                 name 
                 Primer sequence (5′-3′) 
                 (bp) 
                 number 
               
               
                   
               
               
                 sivA 
                 MS386 
                 TGATTTAACGCAACATCATCATCAACCTT 
                 640 
                 GQ857551 
               
               
                   
                   (*) MS387 
                 CATACATCAAATATCCATTCCTTG (SEQ 
                   
                   
               
               
                   
                   
                 ID NO: 1) 
                   
                   
               
               
                   
               
               
                 nisA 
                 MS389 
                 CAGGTGCATCACCACGCATTACAACCACT 
                 600 
                 DQ146939 
               
               
                   
                   (* )MS390 
                 GAGTATCCAATCTTATACCC (SEQ ID 
                   
                   
               
               
                   
                   
                 NO: 2) 
                   
                   
               
               
                   
               
               
                 nisF 
                 MS391 
                 TGGAACAGTCTGTGGTTTATTAGGAGTCA 
                 560 
                 U17255 
               
               
                   
                   (* )MS392 
                 CATTCCTCCATGCACAATCTTAA (SEQ 
                   
                   
               
               
                   
                   
                 ID NO: 3) 
                   
                   
               
               
                   
               
               
                 NsaB 
                 MS394 
                 ATCCTATGGTGAAGAGTATCGAGATCTAG 
                 774 
                 DQ146939 
               
               
                   
                   (*) MS395 
                 CCCCTAGATACTTAGAACAACC (SEQ 
                   
                   
               
               
                   
                   
                 ID NO: 4) 
                   
                   
               
               
                   
               
               
                 mutII 
                 MS396 
                 GGGTGGTAATCGTTGGTGGCAAGGCATAT 
                 560 
                 U40620 
               
               
                   
                   (* )MS397 
                 CGCCTACTACTTGAAGGCTG (SEQ ID 
                   
                   
               
               
                   
                   
                 NO: 5) 
                   
                   
               
               
                   
               
               
                 mutIiI 
                 MS398 
                 TTGTGTCGCATGTCAGCGGCGGAGTTGAT 
                 664 
                 AF207710 
               
               
                   
                   (*) MS399 
                 CTCGTGTTATAACGGGC (SEQ ID NO: 
                   
                   
               
               
                   
                   
                 6) 
                   
                   
               
               
                   
               
               
                 sagA 
                 MS380 
                 ATGTTAAAATTTACTTCAAATATTTTATT 
                 300 
                 CP000829 
               
               
                   
                   (* )MS381 
                 ATTTACCTGGCGTATAACTTCCG (SEQ 
                   
                   
               
               
                   
                   
                 ID NO: 7) 
                   
                   
               
               
                   
               
               
                 smeZ-2 
                 MS382 
                 GTAATTGATTTTAAAACCAGTCATAACGA 
                 400 
                 AF086626 
               
               
                   
                   (*) MS383 
                 TACTTTCTTTATCTCTATATCCTAC 
                   
                   
               
               
                   
                   
                 (SEQ ID NO: 8) 
                   
                   
               
               
                   
               
               
                 speB 
                 MS384 
                 GGCATGTCCGCCTACTTTACCGACAGGTG 
                 800 
                 NC_002797 
               
               
                   
                   (*) MS385 
                 CACGAAGCGCAGAAG (SEQ ID NO: 
                   
                   
               
               
                   
                   
                 9) 
                   
                   
               
               
                   
               
               
                 speC 
                 MS410 
                 TACTGATTTCTACTATTTCACCTATCATC 
                 447 
                 NC_002737 
               
               
                   
                   (*) MS411 
                 TCTGATTTTAAAGTCAATTTCCTGG 
                   
                   
               
               
                   
                   
                 (SEQ ID NO: 10) 
                   
                   
               
               
                   
               
               
                 speG 
                 MS412 
                 GCTATGGAAGTCAATTAGCTTATGCAGCC 
                 488 
                 NC_007297 
               
               
                   
                   (*) MS413 
                 GATGTATAACGCGATTCCGA (SEQ ID 
                   
                   
               
               
                   
                   
                 NO: 11) 
                   
                   
               
               
                   
               
               
                 speJ 
                 MS414 
                 CACTCCTTGTACTAGATGAGGTTGCACGC 
                 508 
                 NC_007297 
               
               
                   
                   (*) MS415 
                 ATACGAAATCATACCAGTAG (SEQ ID 
                   
                   
               
               
                   
                   
                 NO: 12) 
                   
                   
               
               
                   
               
               
                 prtF 
                 MS400 
                 CGGAGTATCAGTAGGACATGCGGACTCCC 
                 882 
                 NC_011375 
               
               
                   
                   (* )MS401 
                 ACCAACATTGCTTAATCCA (SEQ ID 
                   
                   
               
               
                   
                   
                 NO: 13) 
                   
                   
               
               
                   
               
               
                 sof 
                 MS402 
                 ATGCCTGGTTGGGTATCTTCGGTAGAGAA 
                 406 
                 NC_011375 
               
               
                   
                   (*) MS403 
                 CAAAACGTTCTGCGCCTA (SEQ ID 
                   
                   
               
               
                   
                   
                 NO: 14) 
               
               
                   
               
               
                   (*) reverse primers 
               
               
                 Results have shown the absence of such virulence genes. 
               
             
          
         
       
     
       Adhesion Test 
       [0050]    To perform the test cells HEp-2 (ATCC CCL 23) have been cultured in essential minimal Eagle media (EMEM) (Invitrogen, Carlsbad, Calif.). The media was added with 10% bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml).  Streptococcus salivarius  bacteria DSM23307 before being used in the adhesion assay have been grown for 16-18 hours in a 5 ml Todd Hewitt media. Bacteria density has been adjusted according to spectrophotometer readings in order to have a range of density between 10 5  and 10 6  CFU/ml before the test. The adhesion test has been performed in HEp-2 cells as described in Benga L. et al. In particular, the resulting number of adherent bacteria was obtained by subtraction from the total number of CFU and expressed as percentage of adherence. All experiments were performed in duplicate wells and performed at least three times; wells containing only cells were used as controls. Bacterial adhesion to the HEp-2 cell layer was performed on microscope cover glasses according to the following protocol. Briefly, 2×10 8  cells resuspended in PBS were incubated with a monolayer of HEp-2 cell for 1 h at 37° C. After washes with PBS, the cells were fixed with 3 ml of methanol and incubated for 8 min at room temperature. After removal of methanol, cells were stained with 3 ml of Giemsa stain solution (1:29, Carlo Erba, Milan Italy) for 30 min at room temperature. Wells were washed and then dried at 30° C. for 1 h. Adherent bacteria were examined microscopically (100× magnification) in 20 random microscopic fields obtaining count and average. 
         [0000]    Adhesion indexes (ADI; number of bacteria/100 HEp-2 cells); strong adhesion: ADI&gt;2500; good adhesion: 500&lt;ADI&lt;2500; weak adhesion: 100&lt;ADI&lt;500; no adhesion: ADI&lt;100. 
         [0051]    In the assay the ability to adhere to HEp-2 cell line of the  S. salivarius  strain DSM 23307 has been compared to that of strains  S. salivarius  K12 and  S. salivarius  4SMB. The results were expressed as percentage adherence comparing the initial inoculum, the initial cell count (10 6  CFU/ml) and the cells that adhered to HEp-2 cells after extensive washing with PBS. We found that between 50% and 57% of  S. salivarius  DSM 23307 remained attached to the HEp-2 monolayer, a similar percentage (50% to 60%) was found for  S. salivarius  K12, while  S. salivarius  4SMB showed the lowest percentage of adhesion (25%-30%) ( FIG. 1 ). The results on HEp-2 cell line adhesion was confirmed by microscopic examination. Therefore, the adhesion index of  S. salivarius  DSM 23307 and  S. salivarius  K12 (used as positive control) showed similar value of adhesion indicating good adhesion which can interfere with the adhesion of opportunistic bacteria and fungi on host cells. 
         [0052]    Table 2 summarizes adhesion indexes found in the assay. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Adhesion indexes 
               
             
          
           
               
                   
                   
                 AdI (Adhesion Index) 
               
               
                 Species 
                 Strain 
                 Number of bacteria/100 FaDu cells) 
               
               
                   
               
             
          
           
               
                 
                   S. salivarius 
                 
                 K12 
                 1059 
               
               
                 
                   S. salivarius 
                 
                 DSM 23307 
                 1362 
               
               
                 
                   S. salivarius 
                 
                 4SMB 
                 500 
               
               
                   
               
             
          
         
       
     
       Stability Test 
       [0053]    Stability tests have been performed by incubating the strain  Streptococcus salivarius  DSM 23307 for 18 hours at pH 8.0 in “Tryptic Soy” (TSB), Todd Hewitt and Brain Heart Infusion (BHI) media. 
       Results 
     Identification of Isolated Strain 
       [0054]    In the table 3 below are identified the species to which belong the strain isolated from the analyzed nasal and pharyngeal swabs: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Bacteriocins producer 
                 Molecular identification 
               
               
                   
                   
               
             
             
               
                   
                 3A-TF(1) 
                 
                   S. mitis 
                 
               
               
                   
                 3A-TF(3) 
                 
                   S. salivarius 
                 
               
               
                   
                 8A-TF 
                 
                   S. mitis 
                 
               
               
                   
                 11A-TF(2) 
                 
                   S. mitis 
                 
               
               
                   
                 14A-TF(2) 
                 
                   S. salivarius 
                 
               
               
                   
                 14A-TF(4) 
                 
                   S. salivarius 
                 
               
               
                   
                 15A-TF 
                 
                   L. cremoris 
                 
               
               
                   
                 19A-TF(1) 
                 
                   S. sanguis 
                 
               
               
                   
                 21A-TF(3) 
                 
                   S. mitis 
                 
               
               
                   
                 24A-TF(4) 
                 
                   S. salivarius 
                 
               
               
                   
                 25A-TF 
                 
                   L. cremoris 
                 
               
               
                   
                 25A-TN(2) 
                 
                   S. salivarius 
                 
               
               
                   
                 26A-TF(1) 
                 
                   S. mitis 
                 
               
               
                   
                 25A-TF(2) 
                 
                   S. mitis 
                 
               
               
                   
                   
               
             
          
         
       
     
         [0055]    Identification and Characterization of Strain  Streptococcus salivarius  DSM 23307 
         [0056]      Streptococcus salivarius  DSM 23307 has been isolated from the nosepharynx of a human subject. The strain growths on a “ Mitis salivarius ” medium at 35° C., 5% CO 2 , having the typical morphology of  S. salivarius  species. 
         [0057]    Colony shape and size: round, 1-2 mm diameter. 
         [0058]    Edge: continuous, smooth. 
         [0059]    Colour: Blue. 
         [0060]    Grown on Columbia agar with 5% horse blood at 37° C., 5% CO 2  the strain is not haemolytic and has the following morphology 
         [0061]    Colony shape and size: round, 1-2 mm diameter. 
         [0062]    Edge: continuous, smooth. 
         [0063]    Colour: White. 
         [0064]      Streptococcus salivarius  DSM 23307 strain has been analyzed by the commercial kit for the identification of streptococci API 20 Strep. After 24 hours incubation, according to the manufacturer&#39;s instruction, has resulted code 5070451, corresponding to the species  Streptococcus salivarius.    
         [0065]    Results obtained by API 20 Strep
       Acetoin production: positive   Hydrolisis: negative   β-glucosidase: positive   Pirrolinodil arilamidase: negative   α-galactosidase: negative   β-glucuronidase: positive   Alcaline phosphatase: positive   Leucin arilamidase: positive   Arginine dihydrolase: negative   Ribose: negative   L-arabinose: negative   Mannitol: negative   Sorbitol: negative   Lactose: positive   Trealose: positive   Inuline: negative   Raffinose: negative   Glycogen: negative   B-hemolysis: negative       
 
         [0085]    16S and sodA gene sequence analysis have demonstrated that the identified strain belongs to the species  S. salivarius  (99.8% identity). 
         [0086]    Activity of  S. salivarius  DSM 23307 
         [0000]    
       
         
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 
                   S. 
                 
                 
                   S. 
                 
                   
                   
                   
                   
               
               
                   
                 
                   pyogenes 
                 
                 
                   pyogenes 
                 
                 
                   S. 
                   pneu- 
                 
                 
                   H. 
                   influ- 
                 
                   
                   
               
               
                 Incu- 
                 2812A M- 
                 SF370 M- 
                 
                   moniae 
                 
                 
                   enzae 
                 
                 
                   S. 
                 
                 
                   B. 
                 
               
               
                 bation 
                 type 1 
                 type 1 
                 11A-TN 
                 3A-TF 
                 
                   aureus 
                 
                 
                   catarrhalis 
                 
               
               
                   
               
             
             
               
                 BACa 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
               
               
                 TSYE 
                 + 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                   
               
             
          
         
       
     
         [0087]    Adhesion Experiment 
         [0088]    Adhesion assays have demonstrated that the  Streptococcus salivarius  DSM 23307 strain has an excellent ability to adhere to HEp-2 cells, up to 57%, interfering with the adhesion of opportunistic bacteria and fungi. 
         [0089]    Formulations
       1 . Streptococcus salivarius  DSM 23307, saline.   2 . Streptococcus salivarius  DSM 23307, EG56 polymer, xanthan, carboxymethyl-cellulose, saline.   3 . Streptococcus salivarius  DSM2 3307, silicone, Vaseline, white soft, magnesium stearate.   4 . Streptococcus salivarius  DSM 23307, maltodextrin, mannitol, 18 beta-glycyrrhetinic acid, starch.   5 . Streptococcus salivarius  DSM 23307, glucose, deionized water.   6 . Streptococcus salivarius  DSM 23307, propylene glycol and/or glycerine.       
 
         [0096]    In conclusion, the present invention provides a new bacterial strain belonging to the species  Streptococcus salivarius  having biological features making it the one and different from other patented strains indicated for the treatment of the above referred infections. 
         [0097]    In particular, the strain  Streptococcus salivarius  DSM 23307 of the present invention inhibit even  S. pneumoniae  (the main pathogenic agent of AOM) in different culturing conditions (BACa and TSYE) and  S. pyogenes  (TSYE). 
         [0098]    This feature differentiates it from other described strains belonging to  S. salivarius  such as  S. salivarius  30 which has inhibitory ability only towards  S. pyogenes  in the two BACa and TSYE media, expanding its range of action only in assays carried out in TSYE. 
         [0099]    Furthermore, surprisingly as demonstrated by BLIS tests results, the strain  Streptococcus salivarius  DSM 23307 of the present invention inhibits even the important pathogens such as  S. pneumoniae  19A and  S. pyogenes  M-type 1, which are frequently isolated from the upper respiratory tracts. 
         [0100]    Finally,  S. salivarius  DSM 23307, shows some biological features, such as sensitivity to antibiotics, absence of virulence genes, and adhesive ability up to 57%, which make it the one, well characterized and distinguishable from other  S. salivarius  strains, in particular  S. salivarius  30. 
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