Oral Bacteria A Flashcards
Isolation, classification, and identification of oral bacteria
> 700 different species present in oral cavity
less than half of these bacteria have been cultured
more species continue to be discovered
Sampling oral bacteria
distinctive communities in different niches
tongue, teeth, buccal mucosa, gingival crevice, etc.
Sampling oral bacteria
sampling methods:
collect saliva, tongue blade,
scrape from tooth surface, wick fluid from deep
pockets (endodontic paper)
Identifying oral bacteria
Molecular techniques -
often target 16S rRNA genes
Identifying oral bacteria
Molecular techniques
Often target 16S rRNA genes:
Introduction In exploratory work, it is important to reveal the total diversity of bacteria present in the microbial ecosystem. Moreover, in specific diseases, medical interventions or dietary regimes it is essential to identify the characteristic changes to the species level. Investigation of bacterial composition by culturing techniques is laborious and prone to strong bias since the growth requirements of many bacteria are still unknown. Therefore, microbial identification and taxonomic classification based on 16S ribosomal RNA (rRNA) gene sequencing has become the gold standard in microbiology.
Why 16S rRNA gene?
Ribosomal RNA genes are outstanding molecules for evaluating phylogenetic relationships among microorganisms, since within the genes the degree of conservation
differs considerably. Conserved regions of the gene are identical for all bacteria while the variable regions contain specific sites unique to individual bacteria. The
uniqueness enables taxonomic positioning and identification of bacteria.
Dental plaque
2
- Biofilm on tooth surface
* One of the highest concentrations of bacteria in the body
Colonizing bacteria interact with
acquired pellicle
Formation of dental plaque
A. Bacteria never come in contact with a clean tooth surface.
Tooth surface is coated with an acquired pellicle
Formation of dental plaque
A. Bacteria never come in contact with a clean tooth surface.
Tooth surface is coated with an acquired pellicle (2)
•film deposited on tooth surface •film composition = molecules in saliva (predominant) material shed from bacterial cell surfaces polymers from gingival crevicular fluid
B. Passive transport of bacteria to pellicle surface
Initial colonization is by — species
(4)
Streptococcus
Streptococcus gordonii
Streptococcus oralis
Streptococcus mitis
Streptococcus sanguis (now sanguinis)
— on bacterial surface bind to receptors in the pellicle.
Adhesins
Pellicle receptors =
polymers from saliva and bacteria
Adhesion is usually —
irreversible
Streptococcus Antigen (2) are important adhesins Bind human salivary (3)
1 & 2
glycoproteins, other bacteria and calcium
C. Subsequent attachment of these species and other
bacterial species occurs by —
coaggregation
C. Subsequent attachment of these species and other
bacterial species occurs by coaggregation (3)
•bacteria binding to other bacteria
•additional bacteria bind to early-binding bacteria
and to each other
•multiple species coaggregate
C. Subsequent attachment of these species and other
bacterial species occurs by coaggregation
Important species at this stage: (3)
Actinomyces naeslundii
Actinomyces viscosus
Streptococcus gordonii
D. Microenvironment created that supports additional species
3
Streptococcus mutans
Streptococcus sobrinus
Bacteria multiply in the developing biofilm.
E. Glucan production
Streptococci produce —
glucosyltransferases
E. Glucan production
Streptococci produce glucosyltransferases (2)
•extracellular enzymes
polymerize the glucose moiety of sucrose into
glucan polymers and other polysaccharides
Glucans =
branched-chain polysaccharides
Alpha(1à6) linkage
Alpha(1à3) linkage
Glucans are like
cement
Bacteria bind to glucans
Bacteria are bound to each other and to matrix of glucans
F. Oxygen levels drop
Late colonizers include obligate anaerobes. (3)
Prevotella melaninogenicus
Prevotella oralis
Veillonella spp.
F. Oxygen levels drop Especially between (2)
teeth and dental gingival crevice
G. Get some detachment of bacteria and colonization
of new sites
Some bacteria will shed or degrade their adhesins
to facilitate release
Altered properties of bacteria in a biofilm (3)
- Up-regulation of genes for extracellular
polysaccharide synthesis - Increased resistance to antimicrobial agents
- Metabolic interaction between closely spaced bacteria
- Increased resistance to antimicrobial agents (4)
a. restricted penetration of agent into biofilm
b. inactivation of agents by enzymes concentrated in
biofilm
c. slow growth rate of bacteria in biofilm
d. expression of novel surface-associated phenotypes
- Metabolic interaction between closely spaced bacteria
Synergistic -
Antagonistic -
degradation of complex nutrients
bacteriocins (exclude susceptible
strains)
Plaque eventually reaches a microbial
homeostasis
= stability in bacterial composition
Breakdown of homeostasis alters bacterial composition (2)
- reduction in saliva flow
* increased consumption of sucrose
— can result
Caries
Bacterial role in caries development
Mutans streptococci
Fermentation in biofilm produces acids:
lactic acid (as well as acetic acid and formic acid)
Acid demineralizes teeth (2)
Solubilizes calcium and phosphate (produced from hydroxyapatite)
Get reprecipitation when pH increases (becomes less acidic)
Prolonged acidic environment created by regular snacking
on high sucrose foods
demineralization > remineralization
Acid demineralizes teeth (dentin vs enamel) (3)
Enamel dissolves slowly
Dentin more easily attacked and colonized by bacteria
Dentin is protein rich/many different bacteria can grow
Dentin is protein rich/many different bacteria can grow (3)
Then disease rapidly progresses
Root canal becomes invaded
Abscess formation
Mutans streptococci participate in the formation of biofilms
on tooth surfaces. These biofilms are known as dental
plaque(s). Sucrose is required for the accumulation of
mutans streptococci. Also required for this accumulation are
the enzymes —, which are
constitutively synthesized by all mutans streptococci. a |
Initial attachment of mutans streptococci to tooth surfaces.
This attachment is thought to be the first event in the
formation of dental plaque. The mutans streptococcal
— (known as —) interacts with -galactosides
in the saliva-derived glycoprotein constituents of the tooth
pellicle. Other moieties at the surface of mutans streptococci
include —, serotype carbohydrate
and GTFs. b | Accumulation of mutans streptococci on tooth
surfaces in the presence of sucrose. In the presence of
sucrose, GTFs synthesize extracellular glucans from glucose
(after the breakdown of sucrose into glucose and fructose),
and this is thought to be the second event in the formation of
dental plaque. The mutans streptococcal protein GBP is a
receptor-like protein that is distinct from GTFs, and it
specifically binds glucans. GTFs themselves also have a
glucan-binding domain and can therefore also function as
receptors for glucans. So, mutans streptococci bind preformed glucans through GBP and GTFs, and this gives rise to
aggregates of mutans streptococci. c | Acid production by
mutans streptococci. The metabolism of various saccharides
(including glucose and fructose) by the accumulated bacterial
biofilm results in the production and secretion of considerable
amounts of the metabolic end-product —, which can
cause demineralization of the tooth structure when present in
sufficient amounts in close proximity to the tooth surface.
This is thought to be the third event in the formation of
dental plaque, and it eventually results in a carious lesion
(that is, in dental caries).
glucosyltransferases (GTFs) adhesin antigen I/II glucan-binding protein (GBP) lactic acid
Age and root surface caries (2)
Gingival recession occurs with age
This fact changes the microbial homeostasis
Cementum surface of the root is exposed and made
vulnerable to bacterial colonization
60% of individual >– years old have root caries
60
Cementum surface of the root is exposed and made
vulnerable to bacterial colonization
60% of individual >60 years old have root caries
MS and lactobacilli likely pathogens
Actinomyces viscosus and Actinomyces naeslundii.
(Both of these Actinomyces species had been shown to
produce root surface caries in experimental animals)
Pathogenic properties of cariogenic bacteria
•Rapidly transport fermentable sugars/convert to acid
Rapid compared to other plaque bacteria
Cariogenic bacteria have multiple sugar transporters
Including PEP-PTS systems (group translocation system)
Group translocation -
molecule transported into the cell
while being chemically altered
EI=
enzyme I
HPr=
heat-stable protein
IIA, IIB, IIC=
components
of enzyme III
Production of extracellular and intracellular polysaccharides (2)
Glucans and Fructans (extracellular)
Intracellular storage - allows acid production even
when sucrose in not available
Production of extracellular and intracellular polysaccharides
Glucans and Fructans (extracellular)
Intracellular storage - allows acid production even
when sucrose in not available
FIGURE I Metabolism of sucrose by S. mutans. The disaccharide sucrose can be con-
verted to the polymers glucan or fructan by the enzymes glucosyltransferase (B, C, or
D) or fructosyltransferase, respectively. Energy is provided by splitting of the glucose-
vide monosaccharides for glycolysis inside the bacterial cell. Similarly, dextranase can
convert glucan into glucose for glycolysis. Intracellularly, glucose can also be poly-
merized into intracellular polysaccharide (IPS), which can be mobilized for glycolysis
as necessary.
•Ability to maintain sugar metabolism under extreme conditions
Acidic conditions more tolerated by MS and lactobacilli
so they are both acid-producing and acid-tolerant
•Ability to maintain sugar metabolism under extreme conditions
Acidic conditions more tolerated by MS and lactobacilli
(so they are both acid-producing and acid-tolerant) (3)
a. maintain a favorable intracellular environment
(pump out protons even into acidic surroundings)
b. bacterial enzymes have more acidic pH optima
c. produce acid-stress response proteins to protect cell contents
Inside cell to Outside cell
ATP usage is coupled to protons being pumped out (using ATP synthesized by glycolysis) MS and lactobacilli
Outside of the cell to Inside cell
ATP synthesis is coupled to protons flowing back into cell. (protons excreted by respiratory catalysts)
Notable property of noncariogenic bacteria =
Alkali production
(2) are the major substrates
for alkali production via the generation of
ammonia (NH3)
Urea and arginine
Virulence factors of S. mutans
SpaP (AgB,Agl/Il, PI)
Adherence, binding to saliva-coated tooth surfaces and
salivary agglutinin
Virulence factors of S. mutans
Glucosyltransferases GtfB,
-C,and -D
Production of a 1,3/k1,6-linked polymers of glucose from sucrose:
important for adherence and biofilm accumulation
Virulence factors of S. mutans
Glucan-binding proteins (GbpA,
B, and -C glucosyltransferases)
Binding of glucans produced by the glucosyltransferases; adherence
to teeth, biofilm accumulation
Virulence factors of S. mutans
Fructosyltransferase Ftf
Production of B2, 1/B2,6-linked polymers of fructose from sucrose
that can serve primarily as an extracellular reserve of carbohydrate;
possibly implicated in adherence
Virulence factors of S. mutans
Fructanase
Hydrolysis of fructan polymers produced by Ftf; extends depth and
duration of acidification
Virulence factors of S. mutans
Dextranase
Endo-hydrolytic cleavage of a 1,6-linked glucans; remodeling of glucar
polymers to make them more water-insoluble and releases glucose
from polymers that can be used to produce acids
Virulence factors of S. mutans
Intracellular polysaccharides
Glycogen-like polymer of glucose used as a storage polysaccharide
when exogenous sources are depleted; extends depth and duration
of acidification
Virulence factors of S. mutansPhosphoenolpyruvate
sugar:phosphotransferase (PTS)
Catalyzes high-affinity and high-capacity uptake of multiple different
sugars; critical for growth and acid production
Virulence factors of S. mutans
ATPase (F,F. ATPase
or H*-ATPase)
Large enzyme complex that uses ATP to pump protons from the
cytoplasm; critical in acid tolerance
Virulence factors of S. mutans
Acid tolerance and adaptation
Allows organisms to acquire enhanced resistance and to grow more
effectively at low pH
Basis for periodontal disease
Non-specific plaque hypothesis -
disease is due to the host
response to non-specific growth of bacteria on tooth surfaces
(inflammatory disease)
Non-specific plaque hypothesis
Traditional view
Bacterial complexity of dental plaque
Non-specific mechanisms of generating inflammatory response
LPS
Also: volatile fatty acids (butyrate, propionate, isobutyrate)
sulfides (hydrogen sulfide, methyl mercaptan)
Treatment dictates that flora be suppressed
continuously or periodically
Basis for periodontal disease
Specific plaque hypothesis -
disease is due to a limited number of
species which produce biologically active molecules that are
proinflammatory or antigenic (infection)
Key illustrative examples: Localized juvenile periodontitis (LJP)(now classified as aggressive periodontitis) 1-5 out of 1000 teenagers Aggregatibacter actinomycetemcomitans can invade gingival tissues produces a leukotoxin (LT) that inhibits neutrophils LJP is a treatable bacterial infection locally delivered antimicrobial agents systemic tetracycline treatment
Acute necrotizing ulcerative gingivitis (ANUG) (2)
Trench mouth of World War I
Spirochetes and Fusobacterium nucleatum
controlled by antibiotic mouth rinses with oxidizing agents
also systemic metronidazole (antibiotic) treatment
Specific plaque hypothesis (continued) (2)
Consider both early-onset and adult forms of disease
No single bacterial species uniquely involved
polymicrobial infection
No single bacterial species uniquely involved
polymicrobial infection
Porphyromonas gingivalis
Tannerella forsythia
Treponema denticola (& other spirochetes)
Low-abundance bacteria with communitywide effects that are critical for the
development of dysbiosis are now known
as —, the bestdocumented example of which is —
keystone pathogens
Porphyromonas gingivalis.
Aggregatibacter actinomycetemcomitans Virulence Factors (3)
Leukotoxin
Invasins
Bacteriocin
Leukotoxin
• Cytotoxic to human PMNs, monocytes, and T-lymphoctyes
Invasins
– Aids in bacteria penetrating eukaryotic cells
Bacteriocin
• Inhibition of growth or killing of other bacterial species, streptococcus
sanguis and actinomyces viscosus
Aggregatibacter actinomycetemcomitans
Immunoinhibitory virulence-associated characteristics (1)
Capsular polysaccharide
Capsular polysaccharide
• Resistance to phagocytosis by PMNs, reduction in complement dependent
response by PMNs, increase In bone resorption
Aggregatibacter actinomycetemcomitans Virulence Factors (1)
Phospholipase C
Phospholipase C
• Hydrolyzation of host cell membrane
Fusobacterium nucleatum Virulence factors (4)
- Capsule
- Hemolysin
- Leukocidin/leukotoxin
- Superoxide dismutase
Prevotella intermedia Virulence factors (3)
- The brown or black pigment
- Collagenase, hyaluronidase, and protease
- Hemolysin
