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

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 Lecture Staphylococcus 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

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

 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.

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 ESCMID A. actinomycetemcomitans Online Lecture 2.2 Library 0.03 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

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

 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  Fimbriae  Evasion of host defenses and immune responses  Community activist

Porphyromonas gingivalis

 Proteins the main primary energy source  The major fermentation end products of growth on complex carbohydrates and proteins are n-butyronate, propionate and acetate  Isovaleriate, isobutyrate, succinate, phenylacetate  Malodour associated oral infections  Isolation:  Anaerobic condition, 37C, 48 h, FAA agar, VKLB agar, selective mediums: smooth, shiny, convex, raised© by colonies, author first white to cream  With time: colonies darken from their edge towards to the center to ESCMIDdeep red to black Online Lecture Library  Identification: API, ATB, VITEK, MALDI-TOF….

 Prevotella species are present in humans, often existing in the oral cavity, gastrointestinal tract, and vagina  In humans, they can act as an opportunistic pathogen, causing periodontal and tooth problems such as gingivitis and periodontitis  Virulence factors:  Enzymes: collagenase, neuraminidase, deoxyribonuclease, haparinase, and proteases  Cysteine protease, interpain A: to destroy the bacterial killing activity of human serum  Exopolysaccharides (EPS)  In the oral cavity, P. intermedia secretes salivary IgA proteases that break down IgA1 and IgA2, which facilitates proliferation© by authorof P. intermedia and subsequent diseases such as gingivitis and periodontitis  P. intermedia is known to synergize with P. micra ESCMID Online Lecture Library Finegold, SM. "General Concepts." Anaerobic Gram-Negative Bacilli. U.S. National Library of Medicine 1996. Marcotte, Harold, and Marc C. Lavoie. "Oral Microbial Ecology and the Role of Salivary Immunoglobulin A." Microbiology and Molecular Biology Reviews 62.1 (1998): 71-109. 

 Gram -, obligately anaerobic, nonsporeforming, nonmotile, pleomorphic rods  Colonies on blood agar:  Translucent, opaque, grey, light brown, or black, hemolysis is variable.  The glucose utilization varies between 30 and 90%  Use steroids as growth factors, so their numbers are higher in pregnant women Hemin, vitamin K and menadione are required for growth  Growth is inhibited by 20% bile  The major fermentation products: acetic and succinic acids; lower levels of isobutyric, isovaleric, or lactic© acid by author  Malate dehydrogenase and glutamate dehydrogenase are present; glucose 6- phosphateESCMID dehyrogenase Online and 6-phosphogluconate Lecture dehydrogenase Library are absent  Proteolytic activity is variable, not ferment amino acids  Indole negative  Nitrate is not reduced to nitrite Prevotella intermedia/nigrescens

 Fusobacterium nucleatum: Gram-, obligate anaerobic spindle-shapedbacilli, not motile anaerobes belonging to the family Bacteroidaceae  Heterogeneous , five subspecies:  F. nucleatum  F. polymorphum  F. vincentii  F. animalis  F. fusiforme  Requires a media containing trypticase, peptone, or yeast extract  Produce butyric acid as a major product of fermentation of glucose and peptone: differentiates Fusobacterium ©species by from author other Gram-, rod-shaped bacterium  One of the few non-sporulating anaerobic species that utilize amino acid catabolism to provide energy  UsesESCMID glutamate, histidine, Online and aspartate Lecture Library  Does not use glucose as its main energy source instead is used for the biosynthesis of intracellular molecules and not energy metabolism  Bennett, K. W., A. Eley. 1993. Fusobacteria: new taxonomy and related diseases. J. Med. Microbiol. 39:246-54. Fusobacterium nucleatum

 Predominant member of the human oral flora  Associated with a wide range of human and animal infections (Lemierre syndrome….)  Due to its versatile adhesion properties, F. nucleatum can bind several host proteins as well as coaggregate with many other potential oral pathogens  F. nucleatum possesses many virulence factors:  Porin  Outer membrane proteins  A potent lipopolysaccharide © by author  Further: toxic metabolites, e.g., butyrate, propionate, and ammonia, are considered to be important in the virulence of these organisms ESCMID Online Lecture Library 1

 T. forsythia: anaerobic Gram-, fusiform, fastidious bacilli, member of the Cytophaga- Bacteroides family, initially described as Bacteroides forsythus by Tanner et al. later reclassified as Tannerella forsythia by Sakamoto et al.  Associated more frequently and/or in higher levels with disease:  Gingivitis, chronic and aggressive periodontitis  Skin abscesses in rabbits, mice as well as alveolar bone loss in mice and rats  Colonies: pale speckled-pink, circular, entire and slightly convex  The growth inhibited on BBE (20% bile), but hydrolysed aesculin  Indole variable, trypsin activity is positive  Virulence factors: © by author  Trypsin-like and PrtH proteases  Sialidases SiaH and NanH  Leucine-rich repeat cell-surface-associated and secreted protein BspA  Apoptosis-inducingESCMID activity Online Lecture Library  Alpha-D-glucosidase and N-acetyl-beta-glucosaminidase  Hemagglutinin components of the bacterial S-layer and methylglyoxal production  Tanner ACR, Listgarten MA, Ebersole JL, Strzempko MN. Bacteroides forsythus sp. nov., a slow growing, fusiform Bacteroides sp. from the human oral cavity. Int J Syst Bacteriol. 1986;36:213–221. © by author ESCMID Online Lecture Library

Blood agar with N-acetyl muramic acid (NAM) disks (10 mg/l), prolonged incubation: 7-10 days. Treponema denticola

 T. denticola is a Gram- anaerobic bacterium from the Spirochetes family that is motile, slender, helically shaped and flexible  Consists of periplasmic flagella: allows for mobility by using a proton motive force to cause thrusting through rotation (Its habitat is and host-associated)  Found: human oral cavity, specifically in subgingival dental plaque, periodontal disease  Culturable-unculturable  It primarily utilizes:  Serine, alanine, cysteine, and glycine  Generates fermentation products:

 Acetate, lactate, succinate, formate, pyruvate, ethanol, carbon dioxide, hydrogen sulfide (H2S), ammonia  Virulence factors: © by author  Motility and chemotaxis, which enable the bacterium to rapidly colonize new sites, penetrate deep periodontal pockets, and penetrate epithelial layers  AbilityESCMID to interact synergistically Online with other periodontalLecture pathogens Library on several levels  Ability to produce cytotoxic metabolites  Ability to form biofilms and a range of cell-surface proteins to dysregulate the host defense to help protect the subgingival biofilm and cause host tissue destruction

 Aggregatibacter actinomycetemcomitans (Aa, previously Actinobacillus actinomycetemcomitans) is a Gram-negative facultative non-motile rod (described by Klinger 1912)  Oral commensal often found in association with localized aggressive periodontitis, a severe infection of the periodontium, also associated with non- oral infections  Virulence factors:  Leukotoxin; kills PMNs and monocytes  Cytolethal distending toxin  Immunosuppression factors that inhibit blastogenesis, antibody production, activate T-suppressor cells  Inhibition of PMNs functions © by author  Resistant to complement mediated killing  Lipopolysaccharides  ESCMIDSurface antigens Online Lecture Library  Heat shock proteins  Antimicrobial resistance

A. actinomycetemcomitans

 A. a.: small, fastidious, non-motile, non-encapsulated, slow-growing, capnophilic, Gram- coccobacillus  A. actinomycetemcomitans grows slowly at 37oC, aerobically or anaerobically provided there is an atmosphere of approximately 5% CO2  No requirement for either X or V factor. In liquid media the organism tends to grow in small granules adhering to the walls of the bottle: this property is behind the name of the new genus Aggregatibacter  Colonies become visible after 24 hours reaching approximately 3mm diameter after several days. Initially smooth, domed and translucent they become corrugated, star-shaped, adherent and may pit the agar.  Catalase positive and oxidase negative  Reduces nitrates to nitrites and is ONPG,© ureaseby and author indole negative.  Ferment carbohydrates including glucose, fructose, maltose and mannose, and variably xylose, and mannitol.  Unable to ferment galactose, lactose, raffinose, sorbitol, sucrose, trehalose and glycerol  SubsequentlyESCMID several biotypes andOnline serotypes have beenLecture described: Library  a : frequently in oral cavity, variable leukotoxin expression.  b : most frequently in localized aggressive periodontitis, leukotoxin expression.  c: low leukotoxin.  serotypes d, e

 Many of the bacteria present in the oral cavity are viable but not culturable, which severely limits laboratory analysis  However, with the advent of new molecular techniques, the microbial makeup of oral biofilms can be better identified  DNA-based methodology for the identification and detection of specific bacteria and viruses represents a remarkable advantage in time and cost as compared with cultivation techniques  This has led to a dramatic increase in the number of samples that can be examined and the number ©of microorganisms by author enumerated ESCMID Online Lecture Library Thank You for attention © by author ESCMID Online Lecture Library