Kariessjukdomens mikrobiologi

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1 Kariessjukdomens mikrobiologi Margaret Sällberg Chen Frisk tand - S. mitis - S. salivarius - S. oralis - Veillonellae - Actinomyces Karies angripen tand - S. mitis - S. salivarius - S. oralis - Mutans streptococker* - Veilonellae - Actinomyces - Lactobaciller * - Scardovia wiggsiae * Lästips: Dental Caries, the Disease and its Clinical Management. Kap. 10 Oral Microbiology and Immunology. Kap 11 * Acidogena (syraproducerande) * Aciduric (syra-älskande) Scardovia wiggsiae (Bifidobacterium, Gr+) Ett nytt karies patogen isolerat från S mutans fria högkariesaktiva barn JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2011, p Vol. 49, No /11/$12.00 doi: /jcm Copyright 2011, American Society for Microbiology. All Rights Reserved. Cultivable Anaerobic Microbiota of Severe Early Childhood Caries A. C. R. Tanner, 1,3 * J. M. J. Mathney, 1 R. L. Kent, Jr., 2,3 N. I. Chalmers, 1,3 C. V. Hughes, 6 C. Y. Loo, 7 N. Pradhan, 7 E. Kanasi, 1,3,4 J. Hwang, 5 M. A. Dahlan, 6 E. Papadopolou, 1,6 and F. E. Dewhirst 1,2,3 Department of Molecular Genetics 1 and Department of Biostatistics, 2 The Forsyth Institute, 245 First Street, Cambridge, Massachusetts 02142; Department of Oral Medicine, Infection and Immunity, 3 Oral Health Policy and Epidemiology, 4 and Harvard School of Dental Medicine, 5 Harvard University, Boston, Massachusetts 02115; Pediatric Dentistry Department, Goldman School of Dental Medicine, Boston University, Boston, Massachusetts ; and Pediatric Dentistry Department, Tufts School of Dental Medicine, Tufts University, Boston, Massachusetts Kariesattacken Angreppsfaktorer Bakterier Vilka? Mängd? Bakteriernas substrat Kost, diet? Hur ofta? Resistensfaktorer Saliv Mängd? Kvalitet? Tandsubstansen kvalitet och fluor? Received 29 November 2010/Returned for modification 10 January 2011/Accepted 19 January 2011 Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (ph 5) agars. Isolates were purified, and partial sequences for the 16S rrna gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rrna isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rrna identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen. Sekvensering av S mutans arvsmassa blev klar år 2002 Early childhood caries (ECC), dental caries of the primary dentition, also known as nursing (bottle) caries, is the most common chronic infectious disease of childhood in the United States, affecting 28% of the population (6). Advanced forms of this disease, severe ECC, can destroy the primary dentition and is the major reason for hospital visits for young children (42). Severe ECC disproportionately affects disadvantaged ethnic and socioeconomic groups and can affect over 50% of the children in these groups (2, 19, 23, 38, 55). Dental caries ca is caused 2000 by an interaction gener between acidogenic bacteria, a carbohydrate substrate which is frequently sucrose, and host susceptibility (51). The acidogenic and acid-tolerant bacterial species Streptococcus mutans is recognized to be the ca 2 miljoner DNA baspar * Corresponding author. Mailing address: The Forsyth Institute, 245 First Street, Cambridge, MA Phone: (617) Fax: (617) annetanner@forsyth.org. Present address: Department of Pediatric Dentistry, University of Maryland Dental School, Baltimore MD. Present address: Department of Periodontology, Boston University Goldman School of Dental Medicine, Boston, MA. Present address: King Faisal Hospital, Makkah 21955, Kingdom of Saudi Arabia. Published ahead3 ofmiljarder print on 2 February DNA baspar " The authors have paid a fee to allow immediate free access to this article primary pathogen in early childhood caries (4, 8, 31, 50). S. mutans is detected in caries-free populations but is not detected in all cases of childhood caries (1, 27), suggesting that other species may be cariogenic pathogens. Studies of severe ECC using culture have historically focused on selected bacterial groups, particularly S. mutans and other Streptococcus species and Lactobacillus, Actinomyces, and Veillonella species (27, 31, 33, 52). Isolates were generally identified phenotypically, sometimes only to the genus level, and thus, their relationships to currently defined human oral taxa on the basis of a 16S rrna-defined taxonomy (11) are unclear. Culture studies demonstrated a strong association of S. mutans with ECC and severe ECC and also reported significant associations with selected Actinomyces and Lactobacillus species. Primary isolation on acid media has been used to select for acid-tolerant species that would be present in active carious lesions. Total counts were higher on acid agar from children with initial caries (45) and severe ECC (21) than from caries-free children. Acid broth enrichment was found to select for Streptococcus oralis, S. mutans, Actinomyces israelii, and Actinomyces naeslundii in severe ECC (31). PCR of the 16S rrna gene with cloning and subsequent sequencing has been used to 3 - evaluate 200 tusen the diversity baser of the microbiota of early childhood tal caries, and combined gener with use of species/taxon-specific probes to the 16S rrna gene to evalu- < 65% har känd funktion 15% associerade med transport mekanism gener" (Human genome project)" Viktiga kännetecknen S. Mutans Omvandlar en lång rad olika sockerarter till olösliga glukan polymerer som stödjer kolonisering av hårda tandytor Bildar organiska syror under kort tid Tolererar extremt låga ph, jonstyrkor och höga sockerkoncentrationer Producera extra-och intracellulära polysackarider Gram stain of S. mutans in thioglycollate broth culture" 1

2 S mutans Virulensfaktorer (PTS) SOCKER TRANSPORT SYSTEM glukos, fruktos, sukros, laktos, galaktos, mannos, maltos, raffinos, stärkelse, isomaltossackarid, mannitol, sorbitol Specific metabolic pathways and transport mechanisms in S. mutans EXOENZYMER Glukosyltransferaser (GTF) Fruktosyltransferaser (FTF), Glucan-binding proteiner (GBP), Fruktanaser, Dextranaser PROTEASER Bryter ned värdproteiner till näring IMMUNOSTIMULERANDE MOLEKYLER ADHESINER binder till saliv agglutiner, andra bakterier, epiteliala cell receptorer JONPUMPAR Höjer intracellular ph = 7.5 Antibakteriella molekyler bakteriociner Immunitet Resistens mot bakteriociner PTS: (sugar) phosphotransferase Copyright 2002 by the National Academy of Sciences Dragana et al. (2002) S mutans Virulensfaktorer EXOENZYMER Glukosyltransferaser (GTF) Fruktosyltransferaser (FTF), Glucan-binding proteiner (GBP), Fruktanaser, Dextranaser PROTEASER Bryter ned värdproteiner till näring IMMUNOSTIMULERANDE MOLEKYLER (PTS) SOCKER TRANSPORT SYSTEM glukos, fruktos, sukros, laktos, galaktos, mannos, maltos, raffinos, stärkelse, isomaltossackarid, mannitol, sorbitol ADHESINER binder till saliv agglutiner, andra bakterier, epiteliala cell receptorer JONPUMPAR Höjer intracellular ph = 7.5 Antibakteriella molekyler bakteriociner Immunitet Resistens mot bakteriociner Molekylär patogenes av tandkaries En 3-fas process! Processen leder till utsöndring av organiska syror från mikrober i dentala biofilmen Och konsekvensen blir dissociation av mineraler från emalj och dentin 1. Bindningsfasen S mutans Antigen I/II binder till saliv komponenterna I tandpellikeln GBP Antigen I/II GTF GTF: glukosyltransferas glukos till glukan(klister) GBP: glukan-bindande proteiner söker och binder till glukanklistret 2. Tillväxtfasen Mer extracellulära glukaner bildas!! Bakterie aggregat växer och blir större!! 3. Syraproduktionsfasen - Fortsatt ansamling bakterieaggregat - Produktion och sekretion av organiska syror ffa mjölksyra - Demineralisering tandsubstans när syramängd tillräckligt stor Taubman Nature Reviews Immunology

3 Slutprodukternas metabolisering Veillonella, Propionbacterium, Eubacterium bryter ned mjölksyra Eubacterium omvandlar ättiksyra till smörsyra och kaprinsyra Distribution av laktobaciller 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Kariesfria Lågkaries Högkaries >10000 Tre typer av laktobaciller (>125 species) Homofermenterare Producerar bara mjölksyra från socker - L acidophilus Heterofermenterare Bryter ned socker till mjölksyra + etanol och CO 2 L fermentum Fakultativa heterofermenterare har bägge möjligheterna L casei L plantarum Baele M. J Microbiological Methods.2002 Laktobaciller associerade med karies L casei kan fermentera xylitol ökar i antal om hög xylitoltillförsel L fermentum L plantarum L acidophilus Ökade mängder av cariogena bakterier innan kariesdiagnosen kan ställas Time (months) Before caries diagnosis Mean % of MS and L in fissure plaque Caries sites Filled sites Caries-free sites MS L MS L MS L < Loesche et al

4 Dentinkaries Nekrotiskt område (mot ytan): Mutansstreptokocker minskar! Lactobaciller, Veillonella, Eubacterium, Propionebacterium; bryter ned kollagen i dentinet (ca 20% består av protein) Delvis demineraliserat område (djupare): Färre mutansstreptokocker! Rotytekaries Skiljer sig antagligen mycket litet från emaljkaries Ingen specifik bakterie Låg syraproduktion kan ha betydelse högre kritiskt ph Actinomycesarter finns i hög frekvens dock oavsett kariesförekomst Vuxna och äldre Höga mängder Mutans streptokocker (>10 5 /ml saliv) och Lactobaciller har stark korrelation till aktiv rotkaries på parodontalt skadade tänder Höga mängder Lactobaciller ( /ml saliv) ökar risken för ny rotkaries Ravald & Birkhed Caries Res, SBU rapport 2007 Munhålan: Hostile Environment Saliv i vågor Sofistikerat och komplementärt immunförsvar Stora variationer i temperatur, ph, jonbalans Konkurrens med andra bakterier om plats och näring Bakteriociner & andra toxiska metaboliter Antimicrobial proteins and peptides in saliva Proteins Major target/function Non-Ig proteins Lysozyme, Lactoferrin Peroxidases, myeloperoxidases Statherin Defensins Agglutinins Parotid salivary glycoproteins Mucins, β-microglobin Ig proteins siga IgG, IgM Bacteria, candida, virus Antimicrobial, H 2 O 2 Adherence Broad-spectrum killing bacteria Agglutination/aggregation of microorganisms Inhibit adhesion Phagocytosis Utnyttjar Saliv Clearance till Adherence! Salivkomponenter utnyttjas av bakterier under kolonisation och aggregation på tandytan Orala streptokocker utnyttjar salivkomponenterna: agglutininer, α-amylas, statherin, mucin, immunoglobuliner Actinomyces utnyttjar statherin för adhesion till tand 4

5 Utnyttjar salivkomponenter för att ta upp näring Vissa orala streptokocker binder till saliv-amylas med hög affinitet Amylaset är enzymatiskt aktivt. Hjälper till att hydrolysera stärkelsen i vår mat till glukos för bakterien. 5