Oral Bacteria A Flashcards
1
Q
Isolation, classification, and identification of oral bacteria
A
> 700 different species present in oral cavity
less than half of these bacteria have been cultured
more species continue to be discovered
2
Q
Sampling oral bacteria
A
distinctive communities in different niches
tongue, teeth, buccal mucosa, gingival crevice, etc.
3
Q
Sampling oral bacteria
sampling methods:
A
collect saliva, tongue blade,
scrape from tooth surface, wick fluid from deep
pockets (endodontic paper)
4
Q
Identifying oral bacteria
Molecular techniques -
A
often target 16S rRNA genes
5
Q
Identifying oral bacteria
Molecular techniques
Often target 16S rRNA genes:
A
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.
6
Q
Why 16S rRNA gene?
A
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.
7
Q
Dental plaque
2
A
- Biofilm on tooth surface
* One of the highest concentrations of bacteria in the body
8
Q
Colonizing bacteria interact with
A
acquired pellicle
9
Q
Formation of dental plaque
A
A. Bacteria never come in contact with a clean tooth surface.
Tooth surface is coated with an acquired pellicle
10
Q
Formation of dental plaque
A. Bacteria never come in contact with a clean tooth surface.
Tooth surface is coated with an acquired pellicle (2)
A
•film deposited on tooth surface •film composition = molecules in saliva (predominant) material shed from bacterial cell surfaces polymers from gingival crevicular fluid
11
Q
B. Passive transport of bacteria to pellicle surface
Initial colonization is by — species
(4)
A
Streptococcus
Streptococcus gordonii
Streptococcus oralis
Streptococcus mitis
Streptococcus sanguis (now sanguinis)
12
Q
— on bacterial surface bind to receptors in the pellicle.
A
Adhesins
13
Q
Pellicle receptors =
A
polymers from saliva and bacteria
14
Q
Adhesion is usually —
A
irreversible
15
Q
Streptococcus Antigen (2) are important adhesins Bind human salivary (3)
A
1 & 2
glycoproteins, other bacteria and calcium
16
Q
C. Subsequent attachment of these species and other
bacterial species occurs by —
A
coaggregation
17
Q
C. Subsequent attachment of these species and other
bacterial species occurs by coaggregation (3)
A
•bacteria binding to other bacteria
•additional bacteria bind to early-binding bacteria
and to each other
•multiple species coaggregate
18
Q
C. Subsequent attachment of these species and other
bacterial species occurs by coaggregation
Important species at this stage: (3)
A
Actinomyces naeslundii
Actinomyces viscosus
Streptococcus gordonii
19
Q
D. Microenvironment created that supports additional species
3
A
Streptococcus mutans
Streptococcus sobrinus
Bacteria multiply in the developing biofilm.
20
Q
E. Glucan production
Streptococci produce —
A
glucosyltransferases
21
Q
E. Glucan production
Streptococci produce glucosyltransferases (2)
A
•extracellular enzymes
polymerize the glucose moiety of sucrose into
glucan polymers and other polysaccharides
22
Q
Glucans =
A
branched-chain polysaccharides
Alpha(1à6) linkage
Alpha(1à3) linkage
23
Q
Glucans are like
A
cement
Bacteria bind to glucans
Bacteria are bound to each other and to matrix of glucans
24
Q
F. Oxygen levels drop
Late colonizers include obligate anaerobes. (3)
A
Prevotella melaninogenicus
Prevotella oralis
Veillonella spp.
25
```
F. Oxygen levels drop
Especially between (2)
```
teeth and dental gingival crevice
26
G. Get some detachment of bacteria and colonization
| of new sites
Some bacteria will shed or degrade their adhesins
| to facilitate release
27
Altered properties of bacteria in a biofilm (3)
1. Up-regulation of genes for extracellular
polysaccharide synthesis
2. Increased resistance to antimicrobial agents
3. Metabolic interaction between closely spaced bacteria
28
2. 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
29
3. Metabolic interaction between closely spaced bacteria
Synergistic -
Antagonistic -
degradation of complex nutrients
bacteriocins (exclude susceptible
strains)
30
Plaque eventually reaches a microbial
homeostasis
| = stability in bacterial composition
31
Breakdown of homeostasis alters bacterial composition (2)
* reduction in saliva flow
| * increased consumption of sucrose
32
--- can result
Caries
33
Bacterial role in caries development
Mutans streptococci
34
Fermentation in biofilm produces acids:
lactic acid (as well as acetic acid and formic acid)
35
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
36
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
37
Dentin is protein rich/many different bacteria can grow (3)
Then disease rapidly progresses
Root canal becomes invaded
Abscess formation
38
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
```
39
Age and root surface caries (2)
Gingival recession occurs with age
| This fact changes the microbial homeostasis
40
Cementum surface of the root is exposed and made
vulnerable to bacterial colonization
60% of individual >-- years old have root caries
60
41
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)
42
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)
43
Group translocation -
molecule transported into the cell
| while being chemically altered
44
EI=
enzyme I
45
HPr=
heat-stable protein
46
IIA, IIB, IIC=
components
| of enzyme III
47
Production of extracellular and intracellular polysaccharides (2)
Glucans and Fructans (extracellular)
Intracellular storage - allows acid production even
when sucrose in not available
48
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.
49
•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
50
•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
51
Inside cell to Outside cell
```
ATP usage is coupled
to protons being
pumped out
(using ATP
synthesized by
glycolysis)
MS and lactobacilli
```
52
Outside of the cell to Inside cell
```
ATP synthesis is
coupled to protons
flowing back into
cell.
(protons excreted
by respiratory
catalysts)
```
53
Notable property of noncariogenic bacteria =
Alkali production
54
(2) are the major substrates
for alkali production via the generation of
ammonia (NH3)
Urea and arginine
55
Virulence factors of S. mutans
| SpaP (AgB,Agl/Il, PI)
Adherence, binding to saliva-coated tooth surfaces and
| salivary agglutinin
56
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
57
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
58
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
59
Virulence factors of S. mutans
| Fructanase
Hydrolysis of fructan polymers produced by Ftf; extends depth and
duration of acidification
60
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
61
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
62
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
63
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
64
Virulence factors of S. mutans
| Acid tolerance and adaptation
Allows organisms to acquire enhanced resistance and to grow more
effectively at low pH
65
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
66
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
```
67
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
68
Specific plaque hypothesis (continued) (2)
Consider both early-onset and adult forms of disease
No single bacterial species uniquely involved
polymicrobial infection
69
No single bacterial species uniquely involved
| polymicrobial infection
Porphyromonas gingivalis
Tannerella forsythia
Treponema denticola (& other spirochetes)
70
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.
71
Aggregatibacter actinomycetemcomitans Virulence Factors (3)
Leukotoxin
Invasins
Bacteriocin
72
Leukotoxin
• Cytotoxic to human PMNs, monocytes, and T-lymphoctyes
73
Invasins
– Aids in bacteria penetrating eukaryotic cells
74
Bacteriocin
• Inhibition of growth or killing of other bacterial species, streptococcus
sanguis and actinomyces viscosus
75
Aggregatibacter actinomycetemcomitans
| Immunoinhibitory virulence-associated characteristics (1)
Capsular polysaccharide
76
Capsular polysaccharide
• Resistance to phagocytosis by PMNs, reduction in complement dependent
response by PMNs, increase In bone resorption
77
Aggregatibacter actinomycetemcomitans Virulence Factors (1)
Phospholipase C
78
Phospholipase C
• Hydrolyzation of host cell membrane
79
Fusobacterium nucleatum Virulence factors (4)
* Capsule
* Hemolysin
* Leukocidin/leukotoxin
* Superoxide dismutase
80
Prevotella intermedia Virulence factors (3)
* The brown or black pigment
* Collagenase, hyaluronidase, and protease
* Hemolysin
