NZ536689A - Streptococcus salivarius strain and extract having anti-mutans Streptococci activity - Google Patents

Abstract

Disclosed are a biologically pure culture of Streptococcus salivarius strain Mia and an extract obtained from that strain having anti-mutans Streptococci (MS) activity. Compositions containing the disclosed extract are useful in inhibiting the growth of bacteria and in particular the therapeutic treatment of dental caries.

Description

New Zealand Paient Spedficaiion for Paient Number 536689 53 6 6 8 9 intellfcoium. phuperty office of N.Z. 19 MOV 2204 ^RECEIVED NEW ZEALAND PATENTS ACT, 1953 No: Divided out of 517398 Date: Dated 14 May 2003 COMPLETE SPECIFICATION ANTIMICROBIAL COMPOSITION We, BLIS TECHNOLOGIES LIMITED, a New Zealand company of c/o Jackson Valentine Ltd, 258 Stuart Street, Dunedin, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: NOW AMENDED ANTIMICROBIAL COMPOSIT1 FIELD OF THE INVENTION This invention relates to novel Streptococcus salivarixls, impositions containing same, and use of Streptococcus salivarius strains as antimicrobial /gents, particularly in the prevention or treatment of dental caries.

BACKGROUND Dental caries is a disease characterised by dissolution of the mineral portion of the tooth. As caries progresses, destruction of tooth enjim^i and dentine occurs followed by inflammation of pulp and periapical tissues.

The mutans streptococci (MS) afe A cluster of acidogenic, dental plaque-inhabiting streptococcal species that are conside/ed the principal causative agents of caries. Presently, seven different MS species (known as S. mutans, S. rattus, S. cricetus, S. sobrinus, S. ferus, S. macacae, and S. downei) are recognised. Of these seven species it is mainly S. mutans and S. sobrinus that are of significance m terms of human caries.

Over the years various inetnods have been developed and tried with varying results, to prevent or at least alley ate/the problem of dental caries. Treatments with antibiotics such as penicillin have been suggested and are effective but indiscriminately destroy both useful and harmful bacteria in pie mouth leading to microbial imbalances.

In order to minimise disruption to the mouth microflora, antibiotic producing organisms have been investigaied/ror their ability to inhibit caries. A group of organisms identified as having potential in Ahist regard are microorganisms producing bacteriocin-like inhibitory substances (BLIS). BUST producers of the genera Streptococcus, Staphylococcus and Enterococcus have been screened for potential application to prevention of dental caries (Balakrishnan, M. et al., Caries/He/. 2001; 35:75-80). 2 OFN.Z.

NOW AMENDED 1 7 MAR 2006 CEIVED What is sought is a non-virulent analog of the disease-causing Si mutans, or a so called effector strain. To serve as an effector strain in replacement therapy in bacterial infection, the microorganism must be non-virulent itself and able to /compete successfully with the pathogenic microorganism either via competitive action and/pr antibiotic action. S. mutans effector strains have been identified (Hillman et al., J Dealt lies. 1987; 66:1092-4; James and Tagg, N Z Dent J. 1991; 87:80-3) and show strong ami -Sr. mutans activity. A disadvantage with the use of S. mutans effector strains is the cariogenic/potential of these strains.

S. salivarius is an alternative streptococcus species /which avoids this disadvantage. In WO 01/27143 S. salivarius strains are identified which liave utility in the treatment of dental caries caused at least in part by <5. sobrinus. No activity was recorded against MS generally or S. mutans in particular. Similarly, in BalaMslman (supra), S. salivarius K3 is identified as active against S. sobrinus when grown on/ryr/ticase soy broth yeast extract calcium carbonate agar medium, but had no effect on S. miftan/.

S. salivarius TOVE-R (Tanzer, J.M/f ey'al.; Infect Immun., 1985, 48:44-50) is an antagonist strain and which brought about a /eduction in dental caries. There have been no reports of BLIS production by this strain.

The applicants have now idemified BLIS-producing S. salivarius strains with a broad spectrum of activity against MS dental caries causing organisms including S. mutans.

The present invention is broadly directed to these novel S. salivarius strains, and the use of anti-MS S. salivarius/str/ins in the treatment of dental caries, or at least provides the public 25 with a useful choice.

SUMMARY DF/THE INVENTION Accordingly,/in one aspect, the present invention may broadly be said to consist in a biologically pure culture of Streptococcus salivarius strain Mia on deposit at Deutsche Samimung von Mikroorganismen Und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124, BraUnschweig, Germany, Accession No. DSM 14685, or a culture having the identifying cnara • II Wl I IWU NOW AMENDED 17 MAR 2006 RECEIVED Strain Mia is a Salivaricin A2 producer, exhibits anti-MS activity, and for carbohydrate metabolism is positive for at least one of L-arabinose, imdliiy glycogen, xylitol, and p-gentiobiose use, or p-galactosidase production; and/or is negati/e for at least one of glycerol, a-methyl-D-mannoside use, or alkaline phosphoaase prodyctic Strain Mia is positive for each of L-arabinose, inulin, glycogen, xylitol, and p-gentiobiose use, or P-galactosidase production; and/or is negative for/ea^n of glycerol, a-methyl-D-mannoside use, or alkaline phosphatase production.

Also described is an extract obtainable from Salivfricin A2-producing strains of S. salivarius, which extract has anti-MS activity. In particular, the extract has anti-S. mutans activity. Conveniently, the extract is obtainable fron/S. Salivarius strains Mia or K12.

Accordingly, the invention also provic 15 has anti-mutans Streptococci (MS) activit extract obtained from strain Mia which extract In a further aspect, the present invention provides an antibacterial composition which includes S. salivarius strain Mia, or an extract as defined above.

In a still further aspect, the salivarius strain Mia, or excipient. iresgnt invention provides a therapeutic formulation comprising S. tract as defined above, together with a diluent, carrier and/or In one embodiment, yUie^composition or formulation further comprises one or more secondary 25 antibacterial agents In one embodiment, the therapeutic formulations are in the form of foods or drinks, preferably in the form pf 4. dairy product-based food or drink. Alternative forms are medicaments, lozenges arfd The invention further provides a method for at least inhibiting the growth of bacteria sensitive to S. /alirarius of the invention, the method comprising contacting the sensitive bacteria with an inhibitory effective amount of an S, salivarius, extract or composition or formulation of the invention, and wherein the method is not carried out in a human body.

NOW AMENDED / Preferably the sensitive bacteria are MS, and more preferably S. mutins.

The invention provides in another aspect a method for at leasft inhibiting the growth of MS or S. mutans, the method comprising contacting the MS or S. muthns with an inhibitory effective 5 amount of an S. salivarius strain Mia, extract, composiwoiyor formulation of the invention, and wherein the method is not carried out in a human be>dy/ In a further aspect, the invention provides a method of prophylactic or therapeutic treatment of dental caries caused at least in part by S. mutans in/a non-human individual in need thereof, the method comprising administering to said/individual S. salivarius strain Mia, extract, composition or formulation of the invention m an amount effective to at least inhibit growth of S. mutans in the oral cavity of the individual/ In a further aspect, the invention provides ^method of controlling the incidence or severity of 15 dental caries comprising introducing into the oral cavity of a non-human individual susceptible to dental caries, a dental/cames controlling amount of an S. salivarius strain Mia, extract, composition or formulatioii oythe invention.

In one embodiment the dental varies is caused by MS. In that instance, S. salivarius K12 or 20 an anti-MS active extract, or ^composition or formulation containing same may also be used.

Preferably, S. salivarius is administered as part of a food, drink or nutraceutical.

The methods of the invention may include the preliminary step of pre-treating the individual to at least reduce the normal microflora already present.

The invention also /elates to the use of S. salivarius strain Mia, or an extract as defined in the present inventior/ in the preparation of a composition or formulation for use in at least inhibiting the growth of bacteria sensitive to S. salivarius strain Mia.

NOW AMENDED In another aspect, the invention also relates to the use of S. salivarius or an extract of the present invention in the preparation of a composition or forcmjalation for use in at least inhibiting the growth of S. mutans bacteria. / / The invention also relates to the use of S. salivarius straan Mia, or an extract of the present invention, in the preparation of a composition or formulation for use in at least inhibiting the growth of mutans streptococci (MS) bacteria. / / The invention also relates to the use of S. salivariusstrain Mia, or an extract of the present 10 invention as in the preparation of a composition or/formulation for use in prophylactically or therapeutically treating dental caries caused at/ea/t in part by S. mutans.

The invention also relates to the use of S. salivarius, or an extract of the present invention in the preparation of a composition or fohnvllation for use in controlling the incidence and 15 severity of dental caries. / / Although the invention is broadly is described above, it will be appreciated by those persons skilled in the art that the invention Js not limited thereto but also includes embodiments of which the following description gives examples.

/ / DETAILED DESCRIPTION OF THE INVENTION Described herein are Streptococcus salivarius strains which produce Salivaricin A2 and which 25 exhibit anti-MS activity. When grown on TSBCaYE agar, the S. salivarius strains desirably exhibit activity against a broader spectrum of MS including S. mutans. Salivaricin A2 and an A2-producing SJsalivarius strain (strain K12) are described for example in WO 01/27143 incorporated herein by reference.

In one aspect/the present invention is directed to S. salivarius strain Mia and S. salivarius strains having the identifying characteristics thereof.

NOW AMENDED Glycerol L-arabinose a-methyl-D-mannoside Inulin Glycogen Xylitol P-gentiobiose + + + + + anaerobic , + aerobk Preferably, strains for use in the invention exhibit at/least one, preferably at least three, more 10 preferably at least six, and even more prefpafyiy all of the distinguishing biochemical characteristics of strain Mia.

Mia also exhibits stronger anti-MS, and/in ^particular stronger anti-51. mutans activity than K12.

S. salivarius strain Mia is a BLlS-producing strain with activity against other bacteria, particularly streptococci, and more particularly MS, including S. mutans. S. salivarius strain Mia was deposited with Deutsche Ssanmlung von Mikroorganismen Und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124/Braunschweig, Germany on 12 December 2001 and has been assigned Accession NovDSJm 14685.

As noted above MS ar^ considered the primary causative agents in dental caries with S. mutans being of particular Significance. While BLIS-producing strains of S. salivarius active against streptococci have/been reported previously, this is the first time that BLIS producing S. salivarius active against MS and S. mutans in particular, have been identified.

The S. salivarius Strains of the invention exhibit broad spectrum antibacterial activity, particularly when/grown on TSBCaYE agar media. The S. salivarius are therefore useful as antibacterial agents per se as well as therapeutically. In this context, "therapeutic" includes 30 prophylactic treatment. Therapeutic uses include the treatment or prevention of microbial infectioias, /^specially streptococcal infections. The salivarius' of the invention are particularly suitable for use against MS and S. mutans. Conditions amenable to treatment witlythi/strains or extracts of the invention include dental caries, sore throats, and bad breath.

PP NOW AMENDED Also described are extracts obtainable from salivaricin a2-prod/ucmg strains of S. salivarius and especially from strain Mia of the invention. The strain MiA extract forms a further aspect of the invention. These active extracts may similarly be used in/therapeutic formulations and methods. Extracts can be obtained using known art protpcop, conveniently by cell culture 5 and centrifugation.

A "therapeutic formulation" is a formulation appropriate/for administration of an S. salivarius strain or extract of the invention, to an individual ii/nepd of same, particularly a dental caries-susceptible individual. In general, therapeutic forniulations of the invention are composed of 10 S. salivarius strain Mia or extract of the invegtiop and an acceptable carrier, diluent and/or excipient.

An "acceptable carrier, diluent and/or /xc^ient" means a vehicle for delivery of a S. salivarius strain or extract of the inyenuon, to the individual, in which the vehicle is 15 compatible with bacterial cell viability, oi activity of the extract. Acceptable carriers suitable for use in the administration of viable 8. salivarius strains of the invention and extracts are well known to those skilled in the/art/ Suitable carriers are generally inert and can be either solid or liquid.

In one embodiment, the canier/is a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for use with the S. salivarius strains herein include, but are not limited to, water, buffered saline solutions (e.g., phosphate-buffered saline), pharmaceutically acceptable culture media (i.g. BACa, TSBCaYE agar), or other solutions which maintain the viability of the bacterium. Additionally, such pharmaceutically acceptable carriers may be 25 aqueous or non-aqueous solutions, suspensions, and emulsions. A variety of pharmaceutically acceptable carriers suitable for oral administration of viable or lyophilized bacteria are well known in the act (s®e, for example, Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed., 1990, lA/kcy Publishing Co., Easton, Pa., incorporated herein by reference; and the pharmaceutifcal/composition LACTINEX™, a commercially available formulation for oral 30 admimstrsftioii of viable lactobacilli). Suitable solid carriers known in the art include, for exampl/, m&gnesium carbonate; magnesium stearate; celluloses; talc; sugars such as fructose, sucrose, ipannitol, lactose; starches; flours; and skim milk, and similar edible powders, but are not Hmifed thereto. Carriers for administration of extracts are similarly well known. 8 intellectual property OFFICI of n.z. 17 NAf! 200S RECEiven NOW AMENDED Typical diluents, by way of example, are: starches; lactos6; /tiannitol; kaolin; calcium phosphate or sulphate; inorganic salts such as sodium chpri^fe; and powdered sugars or celluloses.

The compositions may also include excipients such as iab/eting aids; resins; fillers; binders; lubricants; solvents; glidants; disintegrants; preservatives; buffers; flavourings; colourings; sweeteners; and fragrances as appropriate. A preferred excipient for tablet flowability and compactability is ProSolv™ (Penwest, NY, USAyAypreferred sweetener is isomalt.

Typical binders include starch; gelatin; sugars/sucfn as lactose, fructose, and glucose; and the like. Natural and synthetic gums are /also convenient, including acacia; alginates; methylcellulose; polyvinylpyrrolidine trasacanth; and the like. Polyethylene glycol; ethyl cellulose; and waxes can also serve as/uinaers. A currently preferred binder is Emdex™ (Penwest, NY, USA).

Lubricants to prevent sticking to th^ di6 during formation include slippery solids such as talc, silica, magnesium and calcium ^ceayate, polyethylene glycol, stearic acid and hydrogenated vegetable oils.

Disintegrators are substances which swell when wetted to break up the lozenge and release the S. salivarius or extraqt. Jhe disintegrators include starches; clays; celluloses; algins and gums; more particularly cdm and potato starches; methylcellulose; agar; bentonite; wood ' cellulose; cation exchangeresins; alginic acid; guar gum; citrus pulp; carboxymethylcellulose; powdered sponge; aj/d spdium lauryl sulfate.

The S. salivarius strains or extracts of the invention can be formulated in any of a variety of compositions suitable for oral administration. For example, the S. salivarius strains can be formulated f0r administration as a lyophil or cell paste prepared from a S. salivarius culture, or can be/directly administered to the oral cavity. The strain or extract can also be 30 administered/ in the form of a mouthwash, mouth rinse, toothpaste, mouthspray, gargle, capsulf^ lo/enge, syrup, floss, chewing gum, or chewable tablet but the forms are not limited there# 9 NOW AMENDED Therapeutic formulations may include food, confectionary or cfrinJiL In one embodiment, the foodstuff or drink is a dairy product-based food or drink/inoluding by way of example, yoghurt, cheese, milk, milk power, milk biscuits, and flavoured milks. In the case of confectionary, the formulation can be a chewing gum gucl/as described in WO 00/05972. 5 One preferred formulation employs freeze dried S. salifariiis of the invention in milk powder formulations in a manner similar to that previously /reported for the preparation of Bifidus Milk Powder (Nagawa et al. (1988); J. Dairy Sci. 7l/l 7/7-1782).

One orally administrate formulation of S. saliyariLs is a blend of freeze dried S. salivarius 10 strains with skim milk powder or the like which has been flavoured to enhance palatability.

Presently preferred orally administrable /formulation of S. salivarius, or extracts of the invention are lozenges, chewable tablets/or/capsules. Lozenges are particularly preferred. A suckable lozenge according to the invention comprises an S. salivarius strain or extract of the invention, isomalt and emdex. The lo/enge may be prepared by direct compression, wet granulation, or dry granulation. /Thf lozenges may be coated according to well known pharmaceutical practice.

The therapeutic formulation o&n Additionally contain nutrients to maintain the viability of the 20 bacterium in the formulation. As noted above, the formulation can also contain flavouring agents, colouring agents,/fragrances, or other compounds which increase the palatability of the composition and/or/enhance patient compliance without compromising the effectiveness of the formulation. Methods for preparation of formulations for oral administration are well known in the art (^e,/for example, Remington's Pharmaceutical Sciences, 18th ed., supra, 25 incorporated herein by reference).

For general mtimicrobial use, formulations may also be produced for other methods of administration including topically administrable formulations but not limited thereto.

K12 ana active extracts thereof discussed above may similarly be prepared as above, including in the compositions and formulations discussed.

TW formulations and compositions of the invention may further comprise one or more s/ecopdary antibacterial agents. These secondary agents may, for example, be antibiotics, or NOW AMENDED other antibacterial agent or antibacterial producing microorgariisfris. Useful antibacterials include nisin, and other BLIS for example. Preferably, the secondary antibacterial agent is a BLIS or BLIS producing microorganism. The BLIS may bp ot/e or more of salivaricin A, Ai, A2 and B. Other antibacterial microorganisms include k^owh S. salivarius such as K12 and 5 K30.

S. salivarius strains of the invention are primarily fatingon the tongue surface. Combinations with S. salivarius that grow in dental plaque such p TJOVE-R (supra) would be useful.

The formulation and compositions of the inVenrfon may additionally comprise other anti-cariogenic agents, for example, xylitol, fluoriae, Manuka honey, and tannins.

In the treatment of dental caries, S. salivarius strains or extracts of the invention can be administered to any dental caries-susoeptiole individual, usually an individual in which S. mutans or MS colonises the oral cavity such that the dental caries is caused at least in part by S. mutans and more commonly by MS./ The term "individual" as used Herein includes humans, horses, dogs, cats, pigs, sheep, cattle, goats but is not limited thereto./ Preferably, the individual is a human. The S. salivarius 20 strains can be administered tof the individual at any age, e.g. childhood, adolescence, or adulthood.

The S. salivarius of thje indention or K12 can be orally administered in a variety of ways. For example, in the forth of compositions or formulations discussed above, or as suspensions, 25 sustained release fjorriflulas (e.g. an oral implant containing the S. salivarius strain) or lyophil powders. The S. fsalikarius strains can also be administered by direct application of a lyophil, culture, or cell/pasrce to the teeth or tongue of the individual. Any mode of administration is suitable as Jong as the therapeutic formulation is applied to the oral cavity. In one embodiment, tfte S. salivarius or extracts are administered by applying directly to the teeth of 30 the individual, e.g. by brushing and/or flossing.

In general, the amount of S. salivarius administered to the individual will be an amount effective for replacement of dental caries-causing MS strains, or at least S. mutans in the oral c/vit/ of the host. "An amount effective for replacement of dental caries-causing MS strains 11 NOW AMENDED or at least S. mutans in the oral cavity of the host" means an amoimt effective for oral cavity colonisation by the S. salivarius strain, and significant reduction/of the resident dental caries-causing S. mutans or MS strains (e.g. by competition between Jne bacteria for nutrients and/or by the production of BLIS by the S. salivarius strain).

The term "unit dose" when used in reference to a therapeutic formulation of the present invention refers to physically discrete units suitable/as unitary dosage for the individual, each unit containing a predetermined quantity of active material (viable S. salivarius or active extract thereof) calculated to produce the desiyed yOierapeutic effect in association with the required diluent, carrier, or excipient.

Specific dosages can vary widely according Ao various individual variables including size, weight, age, disease severity (e.g. the tenacity and/or number of dental caries-causing resident MS) and responsiveness to therapy (e/g. fne susceptibility of the individual's oral cavity to colonisation). Methods for determimWthe appropriate route of administration and dosage may be determined by the consumer as they deem appropriate, or on a case-by-case basis by an attending dentist or other climcism. Such determinations are routine to one of ordinary skill in the art (see for example^Remington's Pharmaceutical Sciences, 8th ed., Gennaro, ed., Mack Publishing Company, Eastern, Pa., 1990).

In general, the number ofS. salivarius administered to the individual will range from about 102 to 1015 bacteria, preferably from about 103 to 1014 bacteria, more preferably from about 105 to 1012 bacteria, normally about 109 to 1010 colony forming units (CFU) per dose. One lozenge formulation/employs 3.8 x 109 CFU/ml.

Multiple dosesy^fAhe S. salivarius strain can be administered to achieve oral cavity colonisation aria replacement of the resident, dental caries-causing MS strains, particularly S. mutans, of the individual. The S. salivarius strain or extract may need to be administered to the patient/on/e only or repeatedly. Repeat treatments may be once a month, once a week, once a d^y,/twice a day, or as may otherwise be required. Conveniently, the administration may b^effected as part of the patient's routine dental care, e.g. as a component of a lozenge, gumjioompaste, floss, or mouthwash. 12 NOW AMENDED To facilitate colonisation, in one embodiment the treatment method of the invention includes a preliminary step of pre-treating the individual to at leasjt reduce the normal microflora present in the oral cavity, including dental caries causing o/ganisms. This pre-treatment comprises the step of administering an antimicrobial /agent such as chlorhexidine, 5 lactoperoxidase, green tea, or pineapple juice (freeze dned), but not limited thereto, or may follow a prescribed course of antibiotics such as penioillin, erythromycin, or amoxycillin administered to said individual. S. salivarius of me invention or S. salivarius K12 is then administered to the depopulated environment to r^pojjmate same.

A currently preferred treatment protocol for dehtatf caries comprises pre-treatment by brushing teeth with chlorhexidine gel for 2 to 5 days, preferably 3 days. A lozenge is administered 1-4 ^ hours, preferably 2 hours after the gel. This/s followed by administration of a further 2-5, preferably 3 lozenges through the day at intervals of 1-4 hours, preferably every 2 hours. This protocol is followed for 2-4 days to faoilitafte colonisation. For maintenance purposes 1, 2, or 15 3 lozenges, usually 1 to 2 lozenges ace taken each day following ordinary tooth brushing. The regime is continued for as long as required.

Successful colonisation of the/individual's oral cavity by the S. salivarius strain can be established by culturing the /bacteria of the individual's oral cavity, and identifying the S. 20 salivarius strain by, for exappje, BLIS production or other methods well known in the art for bacterial strain identification./ The methods and uses pi the invention may further comprise the use of one or more secondary antibacterial /gents, and/or anticariogenic agents as discussed above.

Where the term /6or^prise, comprises, comprised or comprising are used in this specification, they are to be/interpreted as specifying the presence of the stated features, integers, steps or components /frefj/rred to, but not to preclude the presence or addition of one or more other features, i^teg^rs, steps, components or groups thereof.

Varioi^ aspects of the invention will now be illustrated in a non-limiting way by reference to the Mloxfang experimental section. 13 NOW AMENDED EXPERIMENTAL Identification Strain Mia was isolated from the oral cavity of a healtny aault human subject. It grows on Mitis salivarius agar at 37°C, 5% CO2 with morphology typical of S. salivarius as follows: Colony shape and size: round, 1-2 mm in diame/er t Margin (edge): entire (smooth) Elevation: convex Colour: blue Texture: mucoid On Blood agar [Columbia Agar Base (GIEJCGf) with 5% human blood] at 37°C, 5% CO2 it is not haemolytic, and exhibits the following morphology: Colony shape and size: round, Biochemical (Characterization Biochemical' characterization of S. salivarius MLA was conducted using the API 20 Strep kit (bioM^riejax) and API 50 CH (bioMerieux) which allows the study of the carbohydrate metaDoijsm. 14 NOW AMENDED The API 20 Strep results are as follows: Acetone production Hydrolysis 5 fi-glucosidase Pyrrolidonyl arylamidase a-galactosidase fi-gluuronidase fi-galactosidase 10 alkaline phosphatase Leucine arylamidase Arginine dihyrolase Ribose L-arabinose 15 Mannitol Sorbitol ' Lactose Trehalose Inulin 20 RafSnose Starch Glycogen fi-haemolytic positive negatfr fosrave Native isitive iegative ' negative negative negative negative positive positive negative positive weak positive negative negative The API 50 CH results are as follow^ Glycerol Erythritol D-Arabinose 30 L-Arabinose Ribose D-Xylose | Adonitol ? Methyl-xyloside 35 Galactose D-Glucose D-Fructose D-Mannose L-Sorbose 40 Rhamnose Dulcitol Inositol Mannitol Sorbitol 45 a Methyl-D^mannoside a Methyl-D-glucoside N-Acetyl/glucosamine Amjgd/line Art 50 E/cunn negative negative negative positive (anaerobic only) negative negative negative negative positive positive positive positive negative negative negative negative negative negative negative positive (anaerobic only) positive positive positive positive NOW AMENDED Salicin Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Inulin Melezitose D-Raffinose Amidon Glycogen Xylitol 6 Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2 ceto-gluconate ceto-gluconate positive positive positive positive positive (^ferobic only) positv positive po/itiye (anaerobic only) live Positive 'positive >sitive (anaerobic only) Positive (aerobic only) ' positive negative negative positive (anaerobic only) negative negative negative negative negative negative negative S. salivarius MIA is urease positive/when grown on Christenens urea agar.

Inhibitory Activity Deferred Antagonism Wst for BLIS Activity | When tested for bactewocm-like inhibitory substance (BLIS) production on the Blood agar-based medium, ColumWa agar Base (GIBCO) + 0.1% CaCOs +5% human blood (BACa) according to the d/fei/ed antagonism test of Tagg and Bannister the P-type designation of strain Mia is 677y P-type of S. saHvaiius MIA Producer typing/(P-type) describes the antimicrobial activity of bacteria against a set of standard indicators. The procedure was first described by Tagg and Bannister (J. Med. Microbiol/1979; 12: 397-411). 40 For P^yping S. salivarius MIA was grown as a diametric streak culture on a Blood agar + 0.1°// c/lcium carbonate plate or Trypticase soy-yeast extract-calcium carbonate agar (Tpypfcfcase soy broth, 30; yeast extract, 20 g; calcium carbonate, 2.5 g; agar, 15 g; distilled 16 NOW AMENDED water, 1000 ml), incubated at 37°C, 5% CO2 for 18 h. The growth/was then removed and the surface of the plate sterilised with chloroform. Nine indicator strains were then cross-inoculated. After incubation at 37°C, 5% CO2 for 18 h imiibition of growth was recorded. The inhibition patterns were recorded in a code form by ccmsidering the nine indicators as three triplets (eg, II, 12, 13, 14, 15, 16; 17, 18, 19). Po/itiye reactions against each indicator were given a score of 4, 2 or 1 depending on whether/the/indicator was, respectively, the first, second or third member of the triplet. No inhibition wsis recorded as zero. The total score of each triplet thus specified uniquely the reactions against the three indicators. The complete P-type code is written as a sequence of three npml/ers, consecutively defining the reactions within the three triplets.

S. salivarius MIA has a 677 P-type on Blood Agar + calcium carbonate, and a 111 P-type on Trypticase soy-yeast extract-calcium carbonate agar.

This corresponds to inhibition of all/9 hacteria in the panel of 9 indicator strains except for indicator 3. This pattern is typical or that given by salivaricin A- producing S. salivarius such as strain 20P3 (Ross et al Appl.Enyr. Microbiol. 1993; 59;2014). However, when tested on trypticase soy broth (BBL) +D/vis/Agar (1.5%) + 0.25% calcium carbonate + yeast extract (2%) [TSBCaYE] the P-type /s 7/7 (i.e. all 9 indicators are inhibited). Associated with this 20 increased activity against indicator 3 there is also additional activity against a variety of other bacteria when tested as indicators (Table 1).

Table 1: Antimicrobial a/tivity of strain Mia on BACa and TSCaYE media Bacteria Susceptibility when tested on BACa TSBCaYE Clostridium suorqgenes + + Clostridium perjringens + + Actinomyces viicosus T14 + + Ml 00 + + Actinomyces naeslundii 10301 + + Streptoaocqus sobrinus OMZ176 - + Streptococcus mutans ATCC10449 - + OMZ175 - + 633K - + H7 - + 13M - + 17 NOW AMENDED 40 E49 K58 K60 M46 MUTI MUTII Corynebacterium diphtheriae gravis Enterococcus faecalis 98 Enterococcus hirae 9790 Streptococcus agalactiae 21 IB P3 Streptococcus uberis 14 D618 Lactobacillus brevis Lactobacillus casei Lactobacillus acidophilus Streptococcus pneumoniae PK2 PK34 Moraxella catarrhalis Listeria monocytogenes Listeria monocytogenes Stomatococcus mucilagenosus Neisseria gonnorhoea Neisseria meningitidis Neisseria lactamica Haemophilus influenzae 30 Staphylococcus saprophytic Staphylococcus cohnii 20260 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Deferred Antagonism Test ofAnti-S. mutans Activity The anti-S. mutans stjfect/um of inhibitory activity of salivaricin A2 producer strains was established by use 6f i. deferred antagonism test, essentially as described by Tagg and Bannister [J. Med/ Microbiol. 1979; 12:397] . In brief, a 1-cm wide diametric streak culture of each produce]/strain was inoculated onto TSBCa and BACa media either with or without yeast extract (YE)/Supplementation. Following incubation in an anaerobic atmosphere for 24 hours at 37°u the macroscopic cell growth was removed with a glass slide and residual cells on the agaysurface were killed by exposure to chloroform vapours for 30 minutes. The agar surface W/as tnen aired for 30 minutes and the indicator strained inoculated from 18 hour Todd Hewitt Brqfti (THB) cultures across the line of the original streak culture with use of cotton swab$! After incubation for 18 hours in 5% (v/v) CO2 at 37°C the extent of inhibition of each indi£at«fr strain was recorded.

NOW AMENDED TSBCaYE medium (per 500 ml) Trypticase soy broth (BBL) Davis Agar Yeast Extract (Difco) 5 CaC03 BACaYE medium (per 500 ml) Columbia Blood Agar base (Difco) CaC03 Yeast Extract (Difco) The results are shown in Table 2.

Table 2: Comparison of anti-S. mutans activity of S. salivarius strains Mia and K-12 in deferred antagonism tests on BACa or TSBCa when supplemented with yeast extract Salivaricin status Test medium Inhibition of S. mutans strain ATCC 10449 OMZ 175 H7 13M K-56 K-60 M-46 MutI Mutll Mia A2\. ^^aCa +++ +++ ++ - - - ++ - - Mia ^^BataYE +++ +++ ++ ++ ++ ++ ++ ++ ++ Mia T5BCa^\ ++ +++ + - - - + + - Mia TSBCat^ +++ ++ + + + ++ + + K-12 A2 + B BaCa ++ - - - + + - - K-12 BaCaYE +++ ++ ++ ++ +++ ++ ++ K-12 TSBCa ++ - - - - ++ - - K-12 TSBCaYE ++ - + + ++ + - > m D m D NOW AMENDED Production of inhibitory activity in saliva fluid by strain Miat Saliva fluid was prepared as follows: Stimulated saliva was collected, heated at 60°C for 0.5 hr rfier{centrifuged at 12000 x g for 5 10 min. The supernatant was supplemented with 0.5% m^Jto^e, 0.5% CaCC>3 and 0.25 jag/ml cysteine (final concentrations).

The supplemented supernatant was used as a basal fluief medium for growth of strain Mia at 37°C. The inoculum was from an 18 h TSBYBCy culture at a ratio of 100|il per 2 ml 10 supernatant. Different aliquots were supplemented as indicated.

After 24 h incubation in an anaerobic atmosphere samples of the saliva cultures were tested for inhibitory activity against the indicators OMZ 175, Mutll, and II either neat or following lOx concentration by rotary evaporation/ The1Ox concentrated supernatant from the culture grown in saliva supplemented with Maltose, Cysteine and CaCC>3 had inhibitory activity against S. mutans strain OMZ175 as/sWwn in the table below. S. salivarius Mia therefore produces anti-S. mutans activity when erown in saliva.

Assay of inhibitory activity 20 Inhibitory activity was deterfninfed by end-point titration using a surface spot method in which 20 \xL drops of two-fold^serial dilutions of the test preparation in saline were spotted onto the surface of BloodAWr medium. When the drops had dried into the agar, the surface of the medium was sterilized by exposure to chloroform vapour for 30 minutes, aired and then inoculated by swattoing evenly from an 18 hour THB culture of indicator strain. 25 Following incubation, me titre of inhibitory activity in Arbitrary Units (AU) per mL was taken to be the reciprocal of the highest dilution to show definite inhibitory activity. The results are show# in/Table 3. 21 NOW AMENDED Table 3: Production of inhibitory activity in saliva fluid by strain Mia Supplement Activity (AU/ml) against indicator supernatant of saliva fluid culture: Unconcentrated )6ncentrated x 10 speed vac 11 OMZ175 Mutll / / 11 OMZ175 Mut II Saliva only 0 0 0 / / 1 0 0 M 0 0 0 / / 1 0 0 M, Cy 0 0 0/ / 1 0 0 M, Ca 2 0 7 / 2 0 0 Cy, Ca 0 0 0 0 0 M, Cy, Ca 4 0 /o / 4 2 0 M= 0.5% maltose; Cy = 0.5% cysteine/Ca/= 0.1% CaC03 In vivo activity of S. salivarius MIA against MS.

One subject brushed their teeth with/2%r chlorhexidine gel for 2 minutes on the first day and then sucked a tablet containing 3M yJ 109 colony forming units of S. salivarius MIA, two hours after the chlorhexidine treatmont and then a further three tablets at two hourly intervals. On the second day the subject/fen£ated the same procedure as for day one. The subject for the remaining 25 days of the /trial sucked one tablet after brushing their teeth with a commercial toothpaste, in tjae ^doming and at night.

Control subjects cleaned th6ir teeth once a day for three days with 2% chlorhexidine gel and for the remaining timer brushed their teeth with a commercial toothpaste.

Saliva samples were/collected from all subjects prior to starting the trial and at intervals 15 throughout the tnalio determine number of MS (colony forming units (cfu) per ml of saliva). The saliva sample; was diluted in sterile saline and spiral plated onto Mutans selective agar, and the plates mcubated under anaerobic conditions at 37°C for 2 days. The number of S. salivarius /MIA in the saliva sample was also determined for the subject taking the tablets. The diluted/ saliva sample is spiral plated onto Mitis-salivarius agar and the plates are 20 incubatedki 37°C, 5% CO2 for 18-24 hours. The number of S. salivarius colonies are then courted/ To determine the percentage of colonization with S. salivarius MIA the following 22 NOW AMENDED protocol is used. Fresh THB cultures of Micrococcus luteus kxsstf S. mutans OMZ175 are spread separately on the top of a Blood agar/calcium plates. Js. ialivarius colonies are then picked into both plates. The plates are incubated at 37°9, 5//o CO2 for 18-24 hours. S. salivarius MIA colonies produce zones of inhibition around the stab cultures on both plates.

Percentage colonization is determined as the number of joosjxive colonies divided by the total number of colonies picked.

Brushing teeth with a 2% chlorhexidine gel resulted'in A 1.7-2.4 log reduction in MS counts in both the control (Table 4) and the colonizing subject (Table 5). The MS cell counts in the 10 control subjects increased to pre-treatment levels, in the control subjects, between one to six days after brushing with the gel. The test subject/was 100% colonized after the pre-treatment with the gel and remained highly colonized for me remaining trial period (Table 5). Numbers of MS were still 1.7 log lower than p/e-t/eatment levels at 27 days. This shows that colonization with S. salivarius MIA is/enable of preventing the re-establishment of high 15 levels of MS.

Table 4. The effect of 2% chlorhexidine gel on the levels of MS in the control subjects Time Number ofM t (cfu/ml of saliva) (days) Subject 1 / Subject 2 Subject 3 Subject 4 Pre-treatment 1.4 xl/ I 1.3 x 105 .8 x 103 9.9 x 103 1 2.2J10/ 1.2 x 103 1.5 x 102 4.3 x 101 2 3.0 xJLO2 ns <102 4.0 x 103 I.5L. 102 3.3 x 104 2.3 x 103 3.7 x 104 8 / JA x 104 ns .1 x 104 ns 33 / / 2.7 x 103 ns 3.5 x 104 2.0 x 103 23 NOW AMENDED Table 5. Effect of colonization with S. salivarius MIA on MS Time % colonization with Number of MS / / (days) S. salivarius MIA (cfu/ml of saliva) / / Pre- 0 .8 x l/4 / treatment 2.5/10/ 1 \JxAO2 2 Ao/x 102 3 / / 100 / lOx 102 6 / / 98 / / 2.0 x 102 13 J I 100 / / 6.0 xlO2 16 / / 100 / / 8.0xlO2 95 / / 2.6 xlO3 23 100 / / 1.0 x 103 27 Preparation of anti-MS aotiv^ extract One hundred ml of molten/Trypticase Soy agar containing 2% yeast extract and 0.25% calcium carbonate was poured into a 1 L schott bottle. One ml of an overnight culture of S. salivarius MIA, grovyh yd Todd Hewitt broth at 37°C, in 5% CO2 in air, was added to the bottle. The culture was/incubated anaerobically at 37°C for 18-24 hours. One hundred ml of 10 Trypticase Soy broth/containing 2% yeast extract and 0.25% calcium carbonate was added to the bottle, which had been preincubated under anaerobic conditions. The culture was then incubated for & further 24 hours anaerobically at 37°C. The broth was centrifuged to remove the bacterial/celjfe and then ammonium sulphate was added to 50% (w/v) and incubated at 4°C for 18 hours. /The sample was then centrifuged and the pellet resuspended in 1 ml of milli-Q 15 water. Anti/MS activity of the sample was then tested using a well diffusion assay in Blood agar piktesf. Fifty |xl of the sample is added to each well and air-dried. The plates were then 24 NOW AMENDED chloroform treated. An overnight culture of the indicator strain ^asyspread over the top of the plate and incubated at 37°C, 5% CO2 in air, for 18-24 hours.

Zones of inhibition (distance from edge of the well to edg/of/inhibition of cell growth) were 5 recorded against all the indicator strains (Table 6).

Table 6. Well diffusion assay of S. salivarius MIA extract Indicator strain Zone of inhibition/ (mm) / / Micrococcus luteus T18 \J / Streptococcus anginosus T29 Streptococcus mutans H7 jl / Streptococcus mutans 10449 / y Streptococcus mutans Mutll Streptococcus mutans OMZ 175 / f1 DOSAGE FORM EXAMPLE Lozenge Ingredients / / Per 945 mg lozenge S. salivarius / / 3.8 x 109 CFU (freeze dried) Isomalt J J 600mg Emdex™ / / 150mg ProSolv HD ty1 50mg Magnesium/stear ate 15mg Flavouy / lOmg The ingredients are bieiyled and tablets produced using dry compression.

INDUSTRIAL .ICATION The results jroo/e demonstrate the antibacterial effect of S. salivarius strains, particularly 20 strain Mia/agafinst a broad spectrum of microorganisms, particularly streptococci. These strains ar;e the first BLIS producing S. salivarius to be identified which have activity against MS, ai0 rt)6re particularly S. mutans. The strains and related active extracts herein therefore have Application in methods of therapeutically treating individuals against the harmful effects of sra-etftococcus infection, especially in the oral cavity. These methods include treatment of NOW AMENDED dental caries in which MS or S. mutans are the primary causative/agent. The S. salivarius extracts and compositions of the invention also have applicatio/ ii/the treatment of bad breath and sore throats.

It will be appreciated that the above description is provided by way of example only and that variations in both the materials and techniques used wl^ich/are known to those persons skilled in the art are contemplated. 26 NOW AMENDED

Claims (1)

1. WHAT WE CLAIM IS: 1. A biologically pure culture of Streptococcus salivarius strain Mia on deposit at Deutsche Sanmilung von Mikroorganismen Und Zellbulturen GmbH, Mascheroder Weg 1 b, D-38124, Braunschweig, Germany, Acc^ssi^m No. DSM 14685, or a culture having the identifying characteristics thereof. 10 An extract obtained from S. salivarius strain/Mia as defined in claim 1 which extract has anti-mutans Streptococci (MS) activity/ An extract as claimed in claim 2 which nas^nti-S1. mutans activity. 4. An antibacterial composition which/includes an S. salivarius strain Mia as claimed in claim 1 or an extract as claimed iq/clqim 2 or claim 3. 15 5. A therapeutic formulation cornfpri/ing an S. salivarius strain Mia as claimed in claim 1 or an extract as claimed in daim 2 or claim 3, together with a diluent, carrier and/or excipient. 20 6. A composition or foimutation as claimed in claim 4 or claim 5 which further comprises one or mo/e s/condary antibacterial agents. 25 7. A composition fox /formulation as claimed in claim 6 wherein the secondary antibacterial agent/s selected from nisin, bacteriocin-like inhibiting substances (BLIS) and BLIS prgauoing microorganisms. 8. A composition or formulation as claimed in claim 7 wherein the BLIS is one or more BLIS selected from salivaricin A, Ai, A2 and B. 30 9. A o6mposition or formulation as claimed in claim 7 wherein the BLIS producing Acroforganism is K12 and/or K30. 27 intellectual property office of n.2. 2 7 FES 2006 RECEIVED NOW AMENDED 10. A composition or formulation as claimed in any one o/ claims 4 to 9 which further comprises one or more secondary anti-cariogenic agent 5 11. A composition or formulation as claimed in claim /0 wherein the secondary anti-cariogenic agent is selected from xylitol, fluoride/Manuka honey and tannin. 10 12. A composition or formulation as claimed ii/ai^ one of claims 4 to 11 which is an orally administerable composition or formumti/n. 13. A composition or formulation as claimed in any one of claims 4 to 12 wherein the compositions or formulations are included in a food or drink. 14. A composition or formulation as /lai/ied in claim 13 wherein said food or drink is a 15 dairy based food or drink. 15. A composition or formulati/n /s claimed in claim 14 wherein said food or drink is milk powder, milk biscuits/milk, flavoured milk, yoghurt or cheese. 20 16. A composition or formulation as claimed in any one of claims 4 to 12 wherein the compositions or formulations are in the form of medicaments, lozenges or confectionaries. 17. A compositior/or/formulation as claimed in claim 16 which is in the form of a 25 lozenge. 18. A composition or formulation as claimed in claim 16 which is in a mouthwash, mouth rinse, toothpaste, gargle, syrup, mouth spray, capsule, floss, chewing gum, or tablet. 28 intellectual property office of nl. 2 7 FEB 2006 NOW AMENDED 19. A composition or formulation as claimed in any one of claims 12 to 18 which is in unit dosage form. 5 20. A composition or formulation as claimed in claim 1/9 wnich contains from about 105 to 1012, preferably 109 to 1010 CFU of S. salivarius scmn Mia per dose. 21. A method for at least inhibiting the growth of bacteria sensitive to S. salivarius strain Mia as claimed in claim 1 the method comprasing contacting the sensitive bacteria 10 with an inhibitory effective amount of S. kalparius strain Mia as claimed in claim 1, an extract as claimed in claim 2 or claim/3,px a composition or formulation as claimed in any one of claims 4 to 20, and wfyerejn the method is not carried out in a human body. 15 22. A method as claimed in claim 2l/wh&rein the sensitive bacteria are MS. 23. A method as claimed in claiqaf 23/wherein the sensitive bacteria are S. mutans. 24. A method for at least inMbitihg the growth of mutans Streptococci (MS) or S. mutans 20 bacteria, the method comprising contacting the MS or S. mutans with an inhibitory effective amount of an SJ salivarius strain Mia, extract, composition or formulation as claimed in any one/of /laims 1 to 20, and wherein the method is not carried out in a human body. 25 25. A method of therapeutic treatment of dental caries caused at least in part by S. mutans in a non-hujhai/ individual in need thereof, the method comprising administering to said individual S. salivarius strain Mia, extract, composition or formulation as claimed in any one pi claims 1 to 20, in an amount effective to at least inhibit growth of S. mutans in/the oral cavity of the individual. 30 29 INTELLECTUAL property OFFicIl of n.z. 17 MAR 200S v tk VET NOW AMENDED 26. A method of controlling the incidence or severity/of/dental caries comprising introducing into the oral cavity of a non-human indivmua/susceptible to dental caries, a dental caries controlling amount of S. salivarius strai/1 Mia, extract, composition or 5 formulation as claimed in any one of claims 1 to 20. 27. A method as claimed in claim 26 wherein j/ne Rental caries is caused by mutans Streptococci (MS). 10 28. A method as claimed in any one of claims >61 to 27 wherein the S. salivarius strain Mia or extract is administered as part ofa food, drink or nutraceutical. 15 29. A method as claimed in any one of claims 21 to 27 wherein the S. salivarius strain Mia or extract is administered in/i composition or formulation as claimed in any one of claims 4 to 20. 20 30. A method as claimed in ar^ ofie of claims 21 to 29 which includes the preliminary step of pre-treating the i/diyldual to at least reduce the normal microflora already present. 31. A method as claimed in claim 30 wherein the pre-treatment is effected using an antimicrobial or antibiotic. 25 30 32. A method as claimed in claim 31 wherein the antimicrobial is chlorhexidene. 33. A method or formulation as claimed in any one of claims 4 to 20. 41. A biologically pure culture of a Streptococcus salivarius strain Mia as claimed in 30 claim /substantially as herein described with reference to any example thereof. Extract as defined in claim 2 substantially as herein described with reference to any Sample thereof. 31 ' 'MELUicTUAL PROPERTY of n.z. off;; I 7 MAO 1 1 ' C E f v £7 fr> NOW AMENDED 43. An antibacterial composition as defined in claim 4 substantially as herein described with reference to any example thereof. 44. A therapeutic formulation as defined in claim 5 subji reference to any example thereof. jdally as herein described with 10 45. A method as defined in claim 21 for at least inhibiting growth of bacteria sensitive to S. salivarius strain Mia as claimed in clainyl substantially as herein described with reference to any example thereof. 46. A method as defined in claim 24 for at/ea/t inhibiting the growth of MS or S. mutans bacteria substantially as herein described Xvith reference to any example thereof. 15 20 47. A method as defined in claim 25 for -treating dental caries caused at least in part by S. mutans in a non-human individual in need thereof substantially as herein described with reference to any examplefthereof. 48. A method as defined in caaim 26 of controlling the incidence or severity of dental caries substantially as herein described with reference to any example thereof. 49. A method as claimed claim 33 of controlling the incidence of severity of dental caries substantial!Vas herein described with reference to any example thereof. 25 50. The use of S. salwarius as claimed in any one of claims 34 to 40, substantially as herein described/with reference to any example thereof. 32 ' INTELLECTUAL property off"' of n.z. 1 1 MAS 2005