Oral Flora I

Karen Ross

2007

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Anaerobic Bacteria of Clinical Importance

Genera Anatomic SiteBacilli (rod)Gram-negative Bacteroides

Colon

Fusobacterium* Mouth, Colon

Tannerella* Mouth

Prevotella* Mouth

Porphyromonas* Mouth

Gram-positive Actinomyces* Mouth

Lactobacillus Mouth, Vagina

Propionibacterium Skin

Eubacterium, Bifidobacterium and Arachnia Mouth, Colon

Clostridium Colon, also found in the soil

Cocci (spheres)

Gram-positive PeptostreptococcusColon

Gram-negative Veillonella* Mouth, Colon

Porphrymonas gingivalis

• Gram -ve coccobacilli• Anaerobic, dark pigmentation on media containing lysed blood.• Black pigmentation due to accumulated hemin used as an iron source

for growth• The diseases collectively termed periodontitis are bacterial infections

which begin with inflammation of the periodontium, and can progress to loss of teeth. Untreated infections lead to destruction of the periodontal ligament and alveolar bone. It is estimated that over 49,000,000 people in the United States have some form of periodontitis (Cutler et al., 1995). These diseases are infectious conditions which may progress over several years with episodes of exacerbation and remission. Periodontitis occurs with higher frequency in patients with systemic diseases such as diabetes mellitus, AIDS, leukemia, neutropenia, Crohn's disease, and Down's syndrome (Neville et al., 1995)

Virulence Factors of P.gMolecules and Organelles

Proteases -arginine and lysine specific cysteine proteases

Hemagglutinins -mediate the binding of Pg to epithelial cells and erythrocytes

LPS-very different composition from enteric bacteria lacks endotoxicity

Fimbriae -peritrichous (all directions), neccesary for infection

Outer membrane vesicles -outgrowth of outer membrane, platelet aggregation, precise delivery of virulence factors, can enter where Pg cannot

Polysaccharide capsule -six serotypes, reduced phagoscytosis

Cytotoxic metabolic end products -include butyrate, propionate, have low molecular weights which allows them to easily penetrate periodontal tissue and disrupt the host cell activity

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Tsute Chen, Department of Molecular Genetics,The Forsyth Institute.

MechanismsAdhesion, colonization and dental plaque

(biofilm) formationEpithelial and Endothelial cell invasionProteolysis - proteases degrade host proteins

including fibrinogen, plasma proteins, cytokines, can also activate neutrophils

Inflammatory response -can activate and suppress components of the innate immune response.

Bone resorption, bone destruction, and bone formation inhibition -LPS, fimbrial and outer membrane consituents play a major role. Eg. LPS causes release of bone resorption mediators (cytokines) from fibroblasts, macrophages and monocytes. These mediators also induce host cell proteases that destroy both bone and connective tissue and inhibit synthesis of collagen by osteoblasts.

(Lamont et al., 1995)

gingipainsgingipainsLPSLPS

CCR5CCR5

PAR 1-2PAR 1-2TLR 2-4TLR 2-4

EndosomeEndosome

Other HIV receptorsOther HIV receptors

CCR5CCR5

CCR5 tropic HIV-1CCR5 tropic HIV-1

CXCR4 tropic HIV-1CXCR4 tropic HIV-1

CCR5CCR5

Porphyromonas Porphyromonas gingivalisgingivalis

Oral Oral

keratinocyteskeratinocytes

CD4CD4

CCR5CCR5

HIV target HIV target CD4(+) T cellsCD4(+) T cells

Courtesy of Rodrigo Giacaman-Sarah; Herzberg Lab

Morphological changes were delayed in KB-MRP8/14.

Morphological changes of epithelial cells

after exposure to P. gingivalis

Morphological changes of epithelial cells

after exposure to P. gingivalis for 24 h

wild type

fimbriae-deficient mutant

P. gingivalis proteases

Cysteine proteases

• Arg-gingipain, RgpA, RgpB

• Lys-gingipain, Kgp

Cells expressing calprotectin are more resistant to cell detachment mediated by P. gingivalis proteases, Kgp and Rgp.

Strain Phenotype

ATCC33277 Wild type

KDP129 Kgp-deficient mutant

KDP133 RgpA, RgpB-deficient mutant

KDP136 Kgp, RgpA, and RgpB-deficient mutant

Morphological changes of epithelial cells after exposure to P. gingivalis wild type and protease-deficient mutants for 24 h

Gram -ve, filament shaped, non motile, non pigmentedAnaerobe

Virulence factorsHydrolases -produces a trypsin-like protease, and an arginine-specific

cysteine protease, and a sailase.Hemolytic activity -cyteine protease, iron acquisition from erythrocytesCo-aggregation -P. gingivalis, S. cristatis Adhesion -leucine rich surface protein (BspA), binding to RBCs,

fibroblasts, leukocytes, and epithelial cells (see above)

Seok-Woo Lee, School of Dental and Oral SurgeryColumbia University

Tannerella forsythia

Treponema denticola

Motile helical rodsIrregular (3-8) spiralsGram-ve cell wallAnaerobe, able to be grown in vitro

MoleculesMajor outer membrane protein(Msp) - 53-

kDa adhesin with pore forming activity, cytotoxic for epithelial cells and erythrocytes

Proteinases -Dentilisin/PrtP/CTLP involved in cell attachment, tissue destruction, tissue invasion

Hemin- and lactoferrin-binding proteins -iron acquisition and utilises lactoferrin from the saliva

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

'TREPONEMA DENTICOLA’by Joe Dixon, 2007

Treponema denticola

MechanismsMotility and chemotaxis - motility

mutants fail to infect their hosts. Maneuvering in viscous fluids, gingival crevice is highly viscous.

Hemagglutination and hemolytic activityAdhesion - Fibroblasts, extracellular

matrix, epithelial cells, endothelial cells coaggregation

Invasion-Tissue and cellular invasion, produce lesions in tissue models, penetrate epithelial and endothelial cell monolayers

Proteolytic activity Dentilisin/PrtP/CTLP disrupts cell junction and impairs epithelial monolayers

Immunosupppressive activity - lymphocyte proliferative responses to antigens and mitogens suppressed

T. d induces actin rearrangement and detachment of human gingival fibroblasts. Baehni et al., 1992

Fusobacterium nucleatum

Gram -ve, anaerobic, cigar-shaped bacilli with pointed endsRecovered mainly from periodontal pocketsIn combination with oral spirochaetes) causes fusospirochaetal

infections -acute necrotizing ulcerative gingivitis, vincents angina, cancrum oris or noma

Toxic metabolites -butyrate, proprionate, ammonium ionsCoaggregation -anchor, bridge formationAdhesion -binds to fibronectin

-binds PMNs, macrophages, lymphocytes, HeLa cells, fibroblasts, both periodontal ligament and gingival fibroblasts and buccal epithelial cells

Invasion -epithelial cells

Collaborative invasion

Edwards, Grossman, and Rudney 2006, Infect Immun 74: 654

Tissue culture experiment

F. nucleatum invades epithelial cells

S. cristatus does not invade cells

After coaggregation, S. cristatus is carried inside by F. nucleatum

Prevotella intermedia

Anaerobic, Non-motile, Short, round-ended, Gram-ve rods

VirulenceFimbriae -4 different typesHydrolasesHemolysin and hemagglutininCoaggregationAdhesionInvasion Induction of inflammatory lymphokines

Veillonella spp.

• V. parvula, V. dispar, V. atypica• Non-motile, nonsporulating, small, anaerobic Gram-ve

cocci• Commensals of the oropharynx, gastrointestinal tract

and female genital tract • Unable to use carbohydrates or amino acids, ferment

organic acids to propionic and acetic acids, CO2 and H2• Component of early plaque, may use lactic acid

produced by streptococci the early colonisers -metabolic cooperation

Capnocytophaga spp.

C. gingivalis C. ochraceaC. sputingenaC. granulosaC. haemolyticaHydrolasesTrypsin-like protease

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Actinomyces • Gram+ve anaerobic and microaerophilic• Filamentous, branching,gram-positive rods.

Older microbiologists sometimes referred to actinomyces as fungi because of the were long like fungal hyphae. However, they are only about a tenth as wide.

• They normally reside in the human mouth, throat, large intestine, vagina, and the crevices between teeth and gums, especially underneath dental plaque.

• Normal flora and is not transmitted from person to person. • Not usually virulent, reside as saprophytes in the body without

producing disease. • Problem only if they have the opportunity to grow on a surface

away from oxygen. They stick to one another, and eventually can break loose as a mass that is too big to be engulfed by the body's defense cells.

Actinomycosis• Actinomyces israelii• Tooth abscess or a tooth extraction and the endogenous organism becomes

established in the traumatized tissue and causes a suppurative infection(pus). • These abscesses are not confined to the jaw and may also be found in the

thoracic area and abdomen. • The patient usually presents with a pus-draining lesion, so the pus will be the

clinical material you send to the laboratory. • Yellow sulfur granules, characteristic of this organism, can be seen with the

naked eye. You can also see these granules by running sterile water over the gauze used to cover the lesion. The water washes away the purulent material leaving the golden granules on the gauze.

• Aggregation• Co-aggregation

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Fusobacterium nucleatum cells coaggregating with Porphyromonas gingivalis cells.

Fusobacterium nucleatum intergeneric coaggregations in the form of corn-cob

formations

Kolenbrander et al., 2000

Kolenbrander et al., 2006

Advantages of co-aggregationPhysical contact -attachmentMetabolic exchangeSmall-signal-molecule-mediated communicationExchange of genetic material

Streptococcus oralis in three-fold excess over partner Prevotella loescheii (arrows)

Fusobacterium nucleatum

P. loeschii does not coaggregate with F. nucleatum but S. oralis coaggregates with both and acts as a coaggregation bridge

Kolenbrander et al., 2006

Kolenbrander 2006

Reviews: light reading :)

Socransky and HaffageePeriodontal microbial ecologyPeriodontology 2000Vol 38:135-187, 2005

Kolenbrander et al.,Bacterial interactions and sucessions during plaque developmentPeriodontology 2000Vol 42: 47-79, 2006