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Uses and Methods for Preventing And/Or Treating Caries Caused by Mutans Streptococci

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(19) TZZ Z¥Z_T (11) EP 2 094 830 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 1/20 (2006.01) A61K 35/74 (2006.01) 10.04.2013 Bulletin 2013/15 A23L 1/03 (2006.01) (21) Application number: 07866245.9 (86) International application number: PCT/EP2007/011127 (22) Date of filing: 18.12.2007 (87) International publication number: WO 2008/074473 (26.06.2008 Gazette 2008/26) (54) USES AND METHODS FOR PREVENTING AND/OR TREATING CARIES CAUSED BY MUTANS STREPTOCOCCI ANWENDUNGEN UND VERFAHREN ZUR VORBEUGUNG UND/ODER BEHANDLUNG VON DURCH MUTANS STREPTOCOCCI VERURSACHTER KARIES MÉTHODES ET MOYENS A PRÉVENIR ET/OU TRAITER DES CARIES CAUSÉES PAR MUTANS STREPTOCOCCI (84) Designated Contracting States: • SOOKKHEE S ET AL: "Lactic acid bacteria from AT BE BG CH CY CZ DE DK EE ES FI FR GB GR healthy oral cavity of Thai volunteers: Inhibition HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE of oral pathogens" JOURNAL OF APPLIED SI SK TR MICROBIOLOGY, vol. 90, no. 2, February 2001 (2001-02), pages 172-179, XP002321894 ISSN: (30) Priority: 19.12.2006 EP 06026301 1364-5072 • KRUGER C ET AL: "In situ delivery of passive (43) Date of publication of application: immunity by lactobacilli producing single-chain 02.09.2009 Bulletin 2009/36 antibodies" NATURE BIOTECHNOLOGY, vol. 20, no. 7, July 2002 (2002-07), pages 702-706, (83) Declaration under Rule 32(1) EPC (expert XP002321895 ISSN: 1087-0156 cited in the solution) application • CHUNG J ET AL: "Isolation and characterization (73) Proprietor: BASF SE of Lactobacillus species inhibiting the formation 67056 Ludwigshafen (DE) of Streptococcus mutans biofilm." ORAL MICROBIOLOGY AND IMMUNOLOGY. JUN 2004, (72) Inventors: vol. 19, no. 3, June 2004 (2004-06), pages 214-216, • REINDL, Andreas XP002321896 ISSN: 0902-0055 cited in the 68199 Mannheim (DE) application • LANG, Christine • WEI H ET AL: "Stability and activity of specific 10625 Berlin (DE) antibodies against Streptococcus mutans and • BÖTTNER, Mewes Streptococcussobrinus in bovine milk fermented 10407 Berlin (DE) with Lactobacillus rhamnosus strain GG or •VEEN,Markus treated at ultra-high temperature." ORAL 84453 Altmühldorf (DE) MICROBIOLOGY ANDIMMUNOLOGY, vol. 17, no. 1, February 2002 (2002-02), pages 9-15, (56) References cited: XP002321897 ISSN: 0902-0055 EP-A- 1 312 667 EP-A- 1 634 948 • WHATMORE ADRIAN M ET AL: "Re- evaluation of WO-A-2004/067729 WO-A-2006/027265 the taxonomic position of Streptococcus ferus" US-A- 4 746 512 US-A1- 2004 101 495 INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 52, no. 5, September 2002 (2002-09), pages 1783-1787, XP002492129 ISSN: 1466-5026 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 094 830 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 094 830 B1 • LOESCHE W J: "Role of Streptococcus mutans in human dental decay." MICROBIOLOGICAL REVIEWS, vol. 50, no. 4, December 1986 (1986-12), pages 353-380, XP002492130 ISSN: 0146-0749 cited in the application 2 EP 2 094 830 B1 Description [0001] The present invention relates to the use of a microorganism belonging to the genus Lactobacillus or a mutant or derivative (as defined below) thereof, characterized in that it is capable of specifically binding to a bacterium belonging 5 to the group of mutans Streptococci, wherein the specific binding is (i) resistant to heat treatment, wherein said heat treatment is carried out at a temperature of more than 95 °C for at least 20 minutes; and (ii) resistant to protease treatment, wherein said protease treatment is treatment with a protease selected from the group consisting of pronase E, proteinase K, trypsin and chymotrypsin; and (iii) calcium-dependent; and (iv) formed within a pH range between 4.5 and 8.5; and (v) formed in the presence of saliva, for the preparation of an anticariogenic composition for the treatment or prevention 10 of caries caused by Streptococcus sobrinus. [0002] Preferably, the specific binding can be assayed as follows: (a) growing said microorganism to stationary phase; (b) mixing said microorganism with a bacterium belonging to the group of mutans Streptococci which has been 15 grown to stationary phase; (c) incubating the mixture obtained in step (b) under conditions allowing the formation of aggregates of said micro- organism and a bacterium of the group of mutans Streptococci; and (d) detecting aggregates by the occurrence of a pellet. 20 [0003] Another aspect in connection with the present invention is a method of prophylaxis or treatment of caries caused by mutans Streptococci other than Streptococcus mutans, comprising administering a microorganism belonging to the group of lactic acid bacteria characterized in that said microorganism is capable of specifically binding to a bacterium belonging to the group of mutans Streptococci or a mutant, derivative or fragment of said microorganism. [0004] Mutans Streptococci colonize the host after the first teeth erupt (Carlson et al., Caries Res. 9 (1975), 333-339). 25 They are localized on the surfaces of the teeth, and their abundance in the plaque is highest over intitial leisions (Duchin and van Houte Arch. Biol. Biol. 23 (1978) 779-786). Their level of colonization within the plaque is increased by sucrose consumption (Staat et al., J. Dent. Res. 54 (1975) 872-880). They are able to synthesize certain macro- molecules from sucrose that foster their attachment to the teeth (Tanzer et al., Infect. Immun. 10 (1974) 197-203). Mutans Streptococci are rapid producers of acid from simple carbohydrates, including sucrose, and are tolerant to low pH (Edwardsson, Arch. 30 Biol. 13 (1968) 637-646). Furthermore, they are essentially alwas recovered on cultivation of intial and established carious lesion sites (Littleton et al., Arch. Oral. Biol. 15 (1979) 461-463). Interest in them grew after the demonstration of their potent induction and progression of carious lesions in a variety of experimental animals, including mono- infected gnotobiotes. Their virulence expression is strongly associated with consumption of carbohydrates, especially sucrose. [0005] The role of further bacterial species that are connected to caries development like lactic acid bacteria or actin- 35 omycetes is not conclusive. These bacteria are often found in cariotic lesions, but only in association mutans Streptococci. According to present knowledge the presence of mutans Streptococci is an indispensable condition of cariogenesis (Tanzer et al., J. Dent. Educ. 65 (2001) 1028-1037). The group of mutans Streptococci has been defined as comprising at least S. mutans, S. sobrinus, S. cricetus, S. ratti, S. ferus and S. macacae (Loesche et al., Microbio. Rev. 50 (4) (1986) 353-380). Due to the fact that Streptococcus mutans is the most abundant representative of mutans Streptococci in 40 humans, most microbiological caries research as well as anti- caries measures concentrate on this specific species. [0006] The initial binding of S. mutants to the surface of the teeth occurs via two mechanisms. The first mechanism is binding of S. mutans via the streptococcal antigen I/II (SA I/II) - a surface protein also known by the synonyms B, IF, P1, SR, MSL-1 or PAc - to the pellicle, a layer of saliva proteins on the teeth surface. Antibodies against this protein have been shown to prevent the adhesion of S. mutans in vitro. 45 [0007] Accordingly, the streptococcal antigen I/II (SA I/II) is a target for vaccination. In different recombinant combi- nations - the complete antigen, the saliva binding region, the protein coupled to cholera toxin or expressed on the surface of an avirulent Salmonella strain - a successful immunization of animals has been shown. This resulted in high IgA titers and a reduction of S. mutans colonization (Huang et al., Infect. Immun. 69 (2001), 2154-2161). Comparable results have been achieved using a DNA- vaccine coding for SA I/II (Fan et al., J. Dent. Res. 81 (2002), 784 - 787). Passive immunity 50 has been achieved by recombinant expression of anti-SA I/II antibodies on the surface of lactic acid bacteria. These lactobacilli aggregate S. mutans and administration of the bacteria to rats led to a reduction of caries development (Krueger et al., Nature Biotechnology 20 (2002), 702 - 706). [0008] WO 06/027265 provides lactic acid bacteria capable of binding to S. mutans with the aim to suppress adhesion to the teeth. 55 [0009] The most important binding partner of the streptococcal antigen is the salivary agglutinin, a protein similar to the lung glycoprotein gp-340 from the scavenger receptor cysteine-rich superfamily (Prakobphol et al., J. Biol. Chem. 275 (2000) 39860-39866). [0010] The role of agglutinin in cariogenesis is not entirely understood so far. It can lead to the adhesion of S. mutans 3 EP 2 094 830 B1 when present bound to surfaces, and it can lead to an aggregation of S. mutans when present in a soluble state. The latter might result in a removal of aggregated S. mutans from the mouth by saliva flow. A high agglutinin concentration in saliva leads in vitro to an increase in the adhesion of S. mutans, whereas in vivo there is no clear correlation between the agglutinin concentration in saliva and the risk for caries (Stenudd et al., J. Dent. Res. 80 (2001), 2005-2010). 5 [0011] Monoclonal antibodies against agglutinin completely block the binding of S.
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  • Patterns of Horizontal Gene Transfer Into the Geobacillus Clade

    Patterns of Horizontal Gene Transfer Into the Geobacillus Clade

    Imperial College London London Institute of Medical Sciences Patterns of Horizontal Gene Transfer into the Geobacillus Clade Alexander Dmitriyevich Esin September 2018 Submitted in part fulfilment of the requirements for the degree of Doctor of Philosophy of Imperial College London For my grandmother, Marina. Without you I would have never been on this path. Your unwavering strength, love, and fierce intellect inspired me from childhood and your memory will always be with me. 2 Declaration I declare that the work presented in this submission has been undertaken by me, including all analyses performed. To the best of my knowledge it contains no material previously published or presented by others, nor material which has been accepted for any other degree of any university or other institute of higher learning, except where due acknowledgement is made in the text. 3 The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 4 Abstract Horizontal gene transfer (HGT) is the major driver behind rapid bacterial adaptation to a host of diverse environments and conditions. Successful HGT is dependent on overcoming a number of barriers on transfer to a new host, one of which is adhering to the adaptive architecture of the recipient genome.