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Prevotella Intermedia The principles of identification of oral anaerobic pathogens Dr. Edit Urbán © by author Department of Clinical Microbiology, Faculty of Medicine ESCMID Online University of Lecture Szeged, Hungary Library Oral Microbiological Ecology Portrait of Antonie van Leeuwenhoek (1632–1723) by Jan Verkolje Leeuwenhook in 1683-realized, that the film accumulated on the surface of the teeth contained diverse structural elements: bacteria Several hundred of different© bacteria,by author fungi and protozoans can live in the oral cavity When these organisms adhere to some surface they form an organizedESCMID mass called Online dental plaque Lecture or biofilm Library © by author ESCMID Online Lecture Library Gram-negative anaerobes Non-motile rods: Motile rods: Bacteriodaceae Selenomonas Prevotella Wolinella/Campylobacter Porphyromonas Treponema Bacteroides Mitsuokella Cocci: Veillonella Fusobacterium Leptotrichia © byCapnophyles: author Haemophilus A. actinomycetemcomitans ESCMID Online C. hominis, Lecture Eikenella Library Capnocytophaga Gram-positive anaerobes Rods: Cocci: Actinomyces Stomatococcus Propionibacterium Gemella Lactobacillus Peptostreptococcus Bifidobacterium Eubacterium Clostridium © by author Facultative: Streptococcus Rothia dentocariosa Micrococcus ESCMIDCorynebacterium Online LectureStaphylococcus Library © by author ESCMID Online Lecture Library Microbiology of periodontal disease The periodontium consist of gingiva, periodontial ligament, root cementerum and alveolar bone Bacteria cause virtually all forms of inflammatory periodontal diseases Periodontial diseases can be grouped broadly: Gingivitis (inflammation of gingiva, it does not affect of the attachment apparatus of the teeth) Periodontitis (denotes destruction of connective tissue attachment and adjacent alveolar bone) Each can be divided according to: Disease activity and severity Age of onset Related systemic disorders © by author Other factors ESCMID Online Lecture Library Cultivable subgingival microflora % of microflora 100 80 60 40 20 © by author 0 ESCMIDHealth Online Gingivitis Lecture periodontitis Library anaerobes facultatives G-negatives G-positves Cultivable subgingival microflora % of microflora 80 70 60 50 40 30 20 © by author 10 0 ESCMIDHealth OnlineGingivitis Lecture LibraryPeriodontitis Gram+rods Gram+cocci Gram-rods Gram-cocci Subgingival morphotypes % of total cells 70 60 50 40 30 20 10 © by author 0 ESCMIDHealth Online Gingivitis Lecture PeriodontitisLibrary cocci non-motile rods motile rods spirochetes Chronic gingivitis Microflora in chronic gingivitis is predominated by Gram-positive and facultative organism (55%) Gram-neg. and anaerobic organisms are almost abundant (45%) Predominant microorganisms: Actinomyces viscosus Fusobacterium nucleatum Actinomyces naeslundii P. intermedia Streptococcus mitis Veillonella parvula P. micra © by Wolinella, author Haemophilus ESCMID Online Lecture Library Pregnancy gingivitis From the second to the eight month of pregnancy The gingiva is fiery red and enlarged Associated with Prevotella intermedia Which may „bloom” in the periodontial pocket as a result of inceased gingival crevicular fluid levels of the growth factors estradiol and progesterol © by author ESCMID Online Lecture Library Terh © by author ESCMID Online Lecture Library Acute necrotizing ulcerative gingivitis (ANUG) ANUG represents one of the few acute infections of the marginal periodontium. ANUG displays a characteristic hystopathological picture four zones: Bacterial zone: variety of bacteria, similar to the subgingival microflora of periodontal lesions Neutrophyl zone: leukocytes Necrotic zone: cell debris, spirochetes© by, Gram-negativeauthor rods Invasion zone: infiltration of medium- and large-sized spirochetes ANUG lesions harboured high numbers of spirochetes and P. intermedia.ESCMID Online Lecture Library Small propotions of Fusobacterum sp. © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Adult periodontitis (AP) Periodontal disease has now become a major concern in dentistry and 80% of adults in the USA have had periodontal disease at some point in their lives Lesions contain high proportions of anaerobes (90%), Gram-negative organisms (75%) and spirochetes (30%) Important: the composition of periodontopatic microflora can differ markedly from patients to patients and from pocket to pocket in a given patient Microflora of AP (Slots et al.) Highest occurance of active lesions % Organism ACTIVE INACTIVE T. forsythia © by author6.5 0.5 P. gingivalis 12.7 0.4 P. micra 8.5 1.2 ESCMIDA. actinomycetemcomitans Online Lecture2.2 Library0.03 P. intermedia 7.4 0.7 W. recta 1.6 0.5 Rekha Seshadri, et al:. 2004. “Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes. Proc Natl Acad Sci USA 101, No. 15. Periodontitis © by author ESCMID Online Lecture Library The grouping of bacteria into complexes to reflect their relationship with the host in health and periodontal disease Members of the ‘yellow’, ‘green’ and ‘purple’ complexes were generally associated with healthy sites © by author ESCMID Online Lecture Library The ‘red complex’ is found most frequently in deep periodontal pockets, and their presence was usually preceded by members of the ‘orange complex’ Localized juvenile periodontitis Afflicts 0.1% to 1% of subjects of US and EU Involves alveolar bone destruction around permanent incisors and first molars in otherwise healthy individuals The onset of disease is associated with relatively little dental plaque and gingival inflammation The disease progresses rapidly during the early stages Close association between© Agregatibacter by author (Actinobacillus ) actinomycetemcomitans and localized juvenile periodontitis. AmericanESCMID Academy of Periodontology Online (1999). "Consensus Lecture report: Aggressive PeriodontitisLibrary". Ann. Periodontol. 4 (1): 53 © by author ESCMID Online Lecture Library Clinical and microbiological findings in a localized juvenile periodontitis patient at the time of breakdown Slots J. and DiRienzo JM, 1991, Univ. Of Pennsylvania, School of Dental Medicine 80 70 60 50 40 30 % of% flora 20 10 © by author 0 A. actinomycetemcomitans A. actinomycetemcomitans 12 13 14 15 16 ESCMID Online Lectureyears Library patients with LJP LJP control healthy control Periodontitis in medically compromised patients HIV-periodontitis Often exhibits rapid onset and progression HIV-periodontitis lesions reveal: spirohetes Fusobacterium sp. A. actinomycetemcomitans each averanging 5-20% W. recta P. micra P. intermedia Some HIV-periodontitis lesions© by yield author organisms not usually recovered from common forms of adult peritonitis: B. fragilis, F. necrophorum, Eubacterium aerofaciens, Clostridium sp., enterococciESCMID, P. aeruginosa, Online various enteric Lecture rods, C. albicans Library HIV © by author ESCMID Online Lecture Library Periodontitis in medically compromised patients Diabetes mellitus: insulin-dependent DM is generally associated with higher frequency of periodontitis when compared to age-matched nondiabetic controls. Capnocytophaga sp. have been related to the P. intermedia initial breakdown in DM W. recta A. actinomycetemcomitans P. gingivalis Neutropenia: frequently show© byadvanced author generalized periodontitis, Np. may enhance subgingival microbial colonization A. actinomycetemcomitans Black-pigmentedESCMID Gram-negative Online anaerobic Lecture rods Library Fusobacterium sp. spirochetes Microbial species associated with periodontial disease Organism GI AP LJP HIV PI A. actinomycetemcomitans - ++ +++ + + Porphyromonas gingivalis - +++ - + - Prevotella intermedia ++ ++ + ++ ++ T. forsythia - +++ - ? ? Fusobacterium sp. +++ ++ - + + P. micra - +++ - ++ ++ Wolinella recta + © by ++ author - ++ + Streptococcus sp. +++ - - - - Actinomyces sp. +++ - - - - GI: gingivitis,ESCMID AP: adult periodontitis, Online LJP: localizedLecture juv.period . HIVLibrary Period. PI: periimplantitis Isolation and identification of oral pathogens Have been complicated by the large number of species Despite of this complexity only a few are clinically associated with the diseases Microbial diagnosis of oral infections is performed by using culture, direct microscopic examination, immunoserological identification, and nucleic acid- based methods Traditional cultivation based isolation-identification is very difficult Rapid ID methods, MALDI-TOF Molecular methods DNA-DNA hybridization© by author 16S rRNA gene sequencing, denaturing gradient gel electrophoresis ESCMIDterminal restriction Online fragment length Lecture polymorphism Library denaturing high–performance liquid chromatography and pyrosequencing cloning and Sanger sequencing „OMICS” Porphyromonas gingivalis P. gingivalis belongs to the phylum Bacteroidetes and is a nonmotile, asaccharolytic, Gram -, rod-shaped, obligatory anaerobic It forms black colonies on blood agar (production porphyrin pigment) Characteristic: large amount of cell-associated protoheme It is found: Normally: humans, dogs, cats, nonhuman primates Oral cavity, where it is implicated in certain forms of periodontal disease Upper gastrointestinal tract, respiratory tract, and in the colon P. gingivalis is divided into K-serotypes based upon capsular antigenicity of the various types © by author Virulence factors: Gingipains: cystein proteinases (active against the a broad spectrum of host proteins) CapsularESCMID polysaccharide Online Lecture Library
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