Patent Application: US-201213644357-A

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:
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 . 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 . 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 . 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 . in the present description are defined as upper respiratory tracts the nasal and paranasal cavities , the pharynx , the larynx . 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 . said compositions preferably comprise the bacterial strain streptococcus salivarius filing number dsm 23307 . 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 . the amount of bacteria in said composition is preferably in the range between 10 3 and 10 10 cfu for each gram of composition . 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 . said compositions may include also carriers idoneous to improve the bioavailability , the stability and the endurance of the microrganism . 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 ). 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 . for administration through the respiratory tract in the present description it has to be intended nasal or by inhalation administration . examples of suitable pharmaceutical forms are cream , lotion , gel , ointment , solution , suspension , emulsion , capsule , tablet , powder , granules , sprays , drops . preferably compositions may be formulated to be administered through the respiratory tract in a nebulizer , with or without propellants . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 %) ( fig1 ). 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 . 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 . in the table 3 below are identified the species to which belong the strain isolated from the analyzed nasal and pharyngeal swabs : 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 . grown on columbia agar with 5 % horse blood at 37 ° c ., 5 % co 2 the strain is not haemolytic and has the following morphology 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 . 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 16s and soda gene sequence analysis have demonstrated that the identified strain belongs to the species s . salivarius ( 99 . 8 % identity ). 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 1 . altschul s f , madden t l , schäffer a a , zhang j , zhang z , miller w , lipman d j . 1997 . gapped blast and psi - blast : a new generation of protein database search programs . nucleic acids res . 1 ; 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