Oral microbiology
Vladana Woznicová
Dept. of Medical Microbiology, Faculty of Medicine, MU Brno
Lectures - Dentistry / spring 2013
IntroductionIntroduction
 The resident oral microfloraThe resident oral microflora
 Dental plaDental plaqueque
 Dental cariesDental caries
 Periodontal diseasesPeriodontal diseases
 Infectious diseases in the oral cavityInfectious diseases in the oral cavity
ConsequencesConsequences
 atherosclerosisatherosclerosis
 strokestroke
 diabetes mellitusdiabetes mellitus
 preterm birthpreterm birth
 oesophageal canceroesophageal cancer
I.I. The resident oral microfloraThe resident oral microflora
Leeuwenhoek 1632 –1723Leeuwenhoek 1632 –1723
(a) Original drawing A.L., (b) Campylobacter rectus, (c) Selenomonas sputigena , (d) oral cocci (e) Treponema denticola,
(f) Leptotrichia buccalis
The resident oral microfloraThe resident oral microflora
 One of the broadest microbial communities, over 700700
generagenera, some were not still described
 Resident – commensal, or transient
 Ecological system
 Biofilm formationBiofilm formation
 Influential factor of human health (both local and in
general)
 Etiology of dental caries and parodontitisdental caries and parodontitis
Sulcus gingivalisSulcus gingivalis
Colonizing bacteria – the key factor in development of parodontal
diseases, anaerobic environmentanaerobic environment
ANAEROBESANAEROBES
Aggregatibacter (Actinobacillus) actinomycetemcomitans
Actinomyces – A. gerencseriae, A. georgiae
Fusobacterium – F. nucleatum, F. alocis, F. sulci
Prevotella nigrescens
Porphyromonas gingivalis,Porphyromonas gingivalis, P. endodontalis
Treponema denticolaTreponema denticola, T. vincentii, pectinovarum, socranskii
Tannerella forsythiaTannerella forsythia
Wolinella succinogenes
Selenomonas sputigena
AEROBESAEROBES
Streptococcus anginosus, Streptococcus constellatus subsp.
constellatus, Streptococcus constellatus subsp. pharyngis,
Streptococcus intermedius
Sulcus gingivalis - microfloraSulcus gingivalis - microflora
StreptococcusStreptococcus
 α-hemolytic streptococci, divided into the following groups:
 S. mutans group:
S. mutans - the MOST FREQUENT, less often S. sobrinus, S.
cricetus, and S. rattus (rare), make acids from saccharides
 S. salivarius group:
S. salivarius, S. vestibularis - in saliva and on the tongue
surface, growth in mucous colonies, can cause endocarditis.
 S. mitis group:
Subacute bacterial endocarditis (SBE)
S. mitis, S. oralis a S. peroris – on mucous membranes and
dental plaque - the causative agent of SBE (S. mitis
exemption)
S. sanguinis and S. gordonii – the tongue, buccal mucous
membranes, dental plaque. S. sanguinis cleaves
secretorial IgA.
 S. anginosus group - growing in tiny colonies Dentoalveolar
and endodontal infections
S. anginosus (S. milleri in British texts), S. constellatus and
S. Intermedius, in nasopharynx, sulci gingivales
Zdroj: Socransky et al. 1998
P.gingivalis
T.forsythia
T.denticola
P.intermedia
P.nigrescens
P.micros
F.nuc.nucleatum
F.nuc.vincentii
F.nuc.polymorphum
F.periodontium
S.mutans
S.oralis
S.sanguis
E.corrodens
C.gingivalis
C.sputigena
C.ochracea
A.actinomyc.
V.parvula
A.odontolyticus
Bacterial communities in periodontitisBacterial communities in periodontitis
II. Dental plaqueII. Dental plaque
Dental plaque - biofilmDental plaque - biofilm
 AdherentAdherent microbial layer on the tooth surface =microbial layer on the tooth surface = livelive
andand dead bacteria + their products + host compoundsdead bacteria + their products + host compounds
(from saliva)(from saliva)
 It can NOT be washed, can be removed only mechanically
(tooth brushing)
 Composition dependent on its location and age
Location:
 Supragingival plaqueSupragingival plaque
 Subgingival plaqueSubgingival plaque
SSubgingival plaqueubgingival plaque
 Plaque of two typesPlaque of two types - adherent and
non-adherent one
 Adherent plaqueAdherent plaque – adherent to the
dental root, similar to supragingival
plaque = i.e. G+ rods, vlákna
(actinomycetes), and G+ cocci
 Non-adherent plaqueNon-adherent plaque – between
adherent plaque and gingival surface =
G- motile anerobes
Distribution of microorganismsDistribution of microorganisms
 Actinomyces sp.Actinomyces sp. isis the most frequent genus in both supra- and
subgingival plaque
 Supragingival plaqueSupragingival plaque – significantly higher amount of some
actinomyces sp., neisseriae, streptococci, and bacteria of "green" and"green" and
"purple""purple" complex
 Periodontal pathogens can be occasionally found in supragingival plaque
 Supragingival plaque – reservoir of infection in the subgingival area
 Subgingival plaque - significantly higher amount of Prevotella sp.,
Tannerella forsythia and P. gingivalis, i.e. "red" a "orange" complex
bacteria
(Ximénez-Fivye et al., 2000)
Zdroj: Socransky et al. 1998
P.gingivalis
T.forsythia
T.denticola
P.intermedia
P.nigrescens
P.micros
F.nuc.nucleatum
F.nuc.vincentii
F.nuc.polymorphum
F.periodontium
S.mutans
S.oralis
S.sanguis
E.corrodens
C.gingivalis
C.sputigena
C.ochracea
A.actinomyc.
V.parvula
A.odontolyticus
Bacterial communities in periodontitisBacterial communities in periodontitis
Development of dental plaqueDevelopment of dental plaque
LessLess
than 24than 24
hourshours
Streptococci of mutans, sanguis, and
mitis groups are prevalent in suprag. plaque
DaysDays
G+ rods and filamentous microorganisms
(lactobacilli, actinomycetes) accumulate
WeekWeek
Columns/microcolonies of coccoid microbes –
rods and filamentous microbes get attached on their surface
ThreeThree
weeksweeks
filamentous microbes are prevalent,
„corn-cob“ formation:
a central filament (Eubacterium yurii)
is encompassed by G+ cocci
Source: www.bact.wisc.edu
www.ncl.ac.uk
Biofilm on a catheter (stafylococci and candida):
a) - canaliculus, b) - porous structure
Photo: Dr. Veronika Holá, MÚ
Dental plaque developmentDental plaque development
 Glykoproteins – pelliculapellicula – receptores for G+ cocci and rods
 ExopolysacharidExopolysacharid production – the main part of the intercellular
matrix
 Bacterial metabolism in plaque – other species involvment,
development quicker in a presence of sacharsacharoseose
 In bottom layers calculus (tartar)calculus (tartar) is being formed– calcified dentalcalcified dental
plaqueplaque - 80 % minerals
 pHpH decreases as a result of bacterial metabolism to < 5.5 – enamel
demineralisation
 Subgingival calculusSubgingival calculus - G - mikroorganisms
 Calculus is porous – filamentous bacteria on the surface – deposits of
microbial compounds - toxictoxic for parodontal tissuesfor parodontal tissues
Dental plaque on dental platesDental plaque on dental plates
 In the area close to the mucous membranes
streptococci mutans and sanguinis are
prevalent + Candida is found very often !
 Anaerobes - G+ rods Actinomyces israelii and
veillonelae inclusive
 often staphylococci, especially Staphylococcus
aureus
III. Dental cariesIII. Dental caries
HistoryHistory
 Archeological findings conclude that
dental caries is very old disease
 Increase in number of caries lesions
during neolite was a result of
increaseing volume of saccharides in
a diet
 In the beginning of rice-growing in
South Asia as a results dental caries
increase was reported
 Sumerian texts about 5000 years B.C.
describe „dental worms“ causing
dental caries
(wikipedia.org)(wikipedia.org)
Microbiology of cariesMicrobiology of caries
 Dental caries – the most frequent current disease
 Definition - local destruction of the tooth tissuelocal destruction of the tooth tissue
 Microbiological point of view – chronic infection caused bychronic infection caused by
normal oral floranormal oral flora
 Destruction is a result of demineralisationdemineralisation of the tooth
caused by acids producted by microorganisms in the dental
plaque during metabolism of saccharides from foodsaccharides from food
Course of cariesCourse of caries
 Primary lesion of enamel (whitish spot) is reversible, it can
remineralise
 After destruction of enamel, the process spreads to dentin
and causes inflammation and necrosis
 Also development of periapical acute or chronic inflammation
Dental caries = multifactorial diseaseDental caries = multifactorial disease
1.1. endogenous factorsendogenous factors
2.2. foodfood
3.3. microbes in the dental plaguemicrobes in the dental plague
Endogenous factors:Endogenous factors:
 Tooth shape
 Enamel structure
 Saliva – volume, flow and composition (buffer)
Nutritious factors:Nutritious factors:
 saccharides intake
 Saccharose is the most cariogenic sugar
 Excellent solubility, diffund to the plaque easily cariogenic
streptococci change it to insoluble glucan
 glucan enables initial adhesion of microbes on the tooth
surface, is a source of nutrients and takes place in
intercellular matrix development
Role of microbesRole of microbes
 almost allalmost all microbes in the dental plague have cariogenicmicrobes in the dental plague have cariogenic
effecteffect thanks to their biochemical features
 the most important in caries development - streptococci
of the mutans group, lactobacilli, and actinomycetesmutans group, lactobacilli, and actinomycetes
 also combination of other microbes can start the
cariogenic process
StreptococcusStreptococcus
 α-hemolytic streptococci, divided into the following groups:
 S. mutans group:
S. mutans - the MOST FREQUENT, less often S. sobrinus,
S. cricetus, and S. rattus rarely, produce acids from
saccharides
 S. salivarius group:
S. salivarius, S. vestibularis - in saliva and on the tongue,
growth in mucous colonies, can cause endocarditis
 S. mitis group: S. mitis, S. oralis a S. peroris – on mucous
membranes and in the dental plaque - the causative agent
of sepsis lenta (S. mitis is an exemption)
S. sanguinis and S. gordonii – on the tongue, buccal
musous membranes, dental plaque. S. sanguinis cleaves
secretorial IgA
Both species are important cause of subacute bacterialsubacute bacterial
endocarditis (endocarditis (sepsis lentasepsis lenta))
 S. anginosus group - tiny colonies - S. anginosus
(S. milleri in British texts), S. constellatus with two
subspecies, constellatus and pharyngis, and S. intermedius.
 In nasopharynx, sulci gingivales, dentoalveolar and
endodontal infections
Caries andCaries and mutansmutans group streptococci Igroup streptococci I
In man usually: S. mutans (serotypes c, e, and f)
S. sobrinus (serotypes d and g)
Some strains seems to be more cariogenic.
Ethiological role - facts:
 Numberes in the plaque and saliva correlates with caries
prevalence and incidence
 Isolated from the tooth surface immediately before caries
 Immunisation of animals with S. mutans specif. serotypes
decreases caries incidence
 lesion progression and S.mutans numbers correlates
 are attached to the tooth surface and together by glucanes
formed from saccharose
 are the most efficient microbes in making caries in lab
animals
 able to form acids and multiply in low pH
 reach pH needed to enamel demineralization quicker than
other bacteria
 form reserves e.g. glycogen (in case of low levels of
saccharides in food)
Caries andCaries and mutansmutans group streptococci IIgroup streptococci II
Dental cDental caries and other microbesaries and other microbes II
LactobacilliLactobacilli
 in high numbers in dental caries
 their numbers in saliva (and dental plaque) and caries activity
correlate
 growth in pH lower than 5 + develop lactate
 biochemically active - extracellular and intracellular polysaccharides
from saccharose
 some strains cause caries in microbe-free animals
 in healthy teeth – low numbers of lactobacilli
LactobacilliLactobacilli
Lactobacilli
Dental cDental caries and other microbesaries and other microbes IIII
ActinomycetesActinomycetes
 Related to root caries – especially
Actinomyces viscosus
 The role of actinomycetes in caries
development is not elucidated
completely
Copyright © The Upjohn Company, Kalamazoo,
Michigan, U.S.A.
Source: www.bact.wisc.edu
Caries and other microbesCaries and other microbes
   Veillonela sp.Veillonela sp.
 in high numbers in supragingival plague of most people
 need lactatelactate, are NOT able to use saccharides and use
lactate developed by other microbes – transform it to
less cariogenic organic acids
……………… positive outcomes………?positive outcomes………?
Ecological plaque hypothesisEcological plaque hypothesis
Veillonelae in people with (A) and without (B)dental cariesVeillonelae in people with (A) and without (B)dental caries
 similar numbers (unsignificant differences), BUT:
 veillonellae in A less diverse,A less diverse, in B more diverse
 V. parvula, V. dispar, V. atypica in both groups
 V. denticariosa only in caries lesions
 V. rogosaeV. rogosae only in people without dental caries
 in A highly probable finding of one predominant V. species
 average number of genotypes in lesions lower than in fissurs or buccal
location
(Source: Arif, J Dent Res, 2008)
Dental plaque developmentDental plaque development
LessLess
than 24than 24
hourshours
Streptococci of mutans, sanguis, and
mitis groups are prevalent in suprag. plaque
DaysDays
G+ rods and filamentous microorganisms
(lactobacilli, actinomycetes) accumulate
WeekWeek
Columns/microcolonies of coccoid microbes –
rods and filamentous microbes get attached on their surface
ThreeThree
weeksweeks
filamentous microbes are prevalent,
„corn-cob“ formation:
a central filament (Eubacterium yurii)
is encompassed by G+ cocci
Microbiological testing of people at riskMicrobiological testing of people at risk
 Saliva sample is taken
 S.mutans a Lactobacillus sp. numbers
assessed by cultivation
 High riskHigh risk patients > 106
S.m. or/and L. 105
 Low risk patients < 105
S.m. or/and L. 104
Preventive factorsPreventive factors
 Milk, diary products, milk proteinsMilk, diary products, milk proteins - buffer, increase of
pH thanks to decarboxylation of aminoacids from casein
 Milk caseinMilk casein – adsorbtion on the tooth surface, casein layer
prevents S.mutans adhesion
 Calcium phosphateCalcium phosphate from casein boost enamel
remineralization
 FluoridesFluorides – boost tooth mineralization, diminish glykolyse,
impair CM, and inactivate enzymes
 XylitolXylitol – inhibition of bacterial growth
Treatment and preventionTreatment and prevention
 Standard treatment =
ablation ofablation of impairedimpaired tooth tissue, preparatooth tissue, preparation oftion of
ccavity aavity and fillingnd filling
 Preventive measures = 
change of diet (low-carbohydrate diet),,
aappplipliccation of fluorides andation of fluorides and properproper dental caredental care
 Ozone – low efficiency, Műller, Eur J Oral Sci,
2007
Review:
Azarpazhooh A, Limeback H. The application of ozone
in dentistry: A systematic review of literature.
J Dent. 2008 Feb;36(2):104-16.
IV. PeriodontitisIV. Periodontitis
PeriodontitisPeriodontitis
 Almost 80 % adults
 Inflammation of gums, scarcement ofscarcement of
dentogingival junctiondentogingival junction
 Resorption of alveolar bone tissueResorption of alveolar bone tissue
 A periodontal pocket develops in the
place of gingival sulci, there is bleeding, there is bleeding
on probing,on probing, purulent content
 Dental plaqe and calculus sediment on
the cervical surfacesurface
 A teeth starts to move
Source: Center for biofilm engineering at MSU-BozemanSource: www.zahnarzt-hilpoltstein.de
Gum reactionGum reaction
 Dental plaque in the gum margine - chronic
inflammation of the tissue around sulcus gingivalis =
marginal gingivitismarginal gingivitis
 Exsudation – chemotaxis of anaerobic and proteolytic
bacteria
 Increasing migration of leucocytes
 Inflammation breaks function of the junctional epithel,
plaque spreads apically to subgingival areaplaque spreads apically to subgingival area
 Symptoms much more intensive with older and thicker
plaque
Microbiology of chronic marginalMicrobiology of chronic marginal
gingivitisgingivitis
 Clinical symptoms - ocasional gum bleeding - inflamed,
hurtfullness is minimal
 Early stageEarly stage – after a one week course - number of capnofilenumber of capnofile
and strictly anaerobic microbesand strictly anaerobic microbes is growing (especially
Actinomyces sp. and anaerobic G- rods)
 Late stageLate stage – more microbes, anaerobes areanaerobes are prevalent (in
black colonies growing e.g. Porphyromonas gingivalis and
Prevotella intermedia, oral spirochetes)
 Bleeding from gumsBleeding from gums lead to multiplication of blackpigmented
anaerobic rods, blood is a source of haeminhaemin
Prevotella melaninogenicaPrevotella melaninogenica (black pigment)(black pigment)
http://pharmacie.univ-lille2.fr
Changes in the periodontal pocketChanges in the periodontal pocket
Redox potentialRedox potential DECREASE
Pockets / liquidPockets / liquid INCREASE = nutrient medium for the growth of anaerobes
releasing proteolytic enzymes, proteins are cleaved by proteolytic
bacteria
pHpH INCREASE from normal neutral values to 7,4 – 7,8 - it enhances
bacterial growth (e.g. Porphyromonas gingivalis)
MicrofloraMicroflora INCREASE
G- anaerobic rods = P. gingivalis, P. intermedia, F. nucleatum, T.
denticola, A. actinomycetemcomitans , and C. sputigena
Infuence of subgingival plaque - studiesInfuence of subgingival plaque - studies
 Strong correlation between plaque volume and prevalence and
severity of periodontal diseses
 Volunteers studiesVolunteers studies – poor dental hygiene = plaq growth and
gingivitis – after plaq removal gingivitis heals
 Local application e.g. chlorhexidine diminish plaq and prevent gingivitis
 Microbe-free animal modelsMicrobe-free animal models - bacteria of „red complex“ from human
plaq lead to parodontal infection and immunoinflammatory bone
resorption (Kesavalu 2007)
Etiology of parodontitisEtiology of parodontitis
 Specific plaque hypothesisSpecific plaque hypothesis
 Non-specific plaque hypothesisNon-specific plaque hypothesis
 Ecological plaque hypothesisEcological plaque hypothesis
Specific plaque hypothesisSpecific plaque hypothesis
 Etiology of parodontitis = specific microorganismsEtiology of parodontitis = specific microorganisms
 Necrotizing ulcerative gingivitis – key agens fusobacteria and
spirochetes
 Terapeutic success with antimicrobials inhibiting anaerobes – e.g.
metronidazole
 Rapidly progreding juvenile parodontitis - Aggregatibacter
actinomycetemcomitans – sensitive to tetracycline – treatment
Ecological plaque hypothesisEcological plaque hypothesis
 Endogenous infection is caused by opportunist speciesEndogenous infection is caused by opportunist species =
parodontitis caused by change in sulcar microflora based on changes
of environment
 In the beginning, there is plaque development and spreading toplaque development and spreading to
sulcus gingivalissulcus gingivalis = macroorganism reacts by inflammation
 Increasing production of sulcar fluid increases supply ofproduction of sulcar fluid increases supply of
proteinsproteins - catabolised by proteolytic G- anaeroby easily
 Změna in zastoupení bacterial species:Změna in zastoupení bacterial species:
number of G- anaerobes is growing, whereas facultative G+
anaerobes not – the first ones produce sufficient amount of virulence
factors and break host immunity – destruction is a result
Therapeutic strategiesTherapeutic strategies
 Specific plaque hypothesisSpecific plaque hypothesis – therapy focused on specific
pathogen removal, e.g. antibiotics administrationantibiotics administration
 Non- specific and ecological hypothesesNon- specific and ecological hypotheses - parodontal disease
can be treated by measurments aimed at reduction of plaquereduction of plaque
volumevolume
PreventionPrevention
 Regular removal of dental plaqueRegular removal of dental plaque byby proper cleaning of the
teeth
 Perfect removal of calculusPerfect removal of calculus
 Improvement of exogenous factorsImprovement of exogenous factors (… impaired prothetic
devices etc.)
Key pathogensKey pathogens
Treponema denticolaTreponema denticola
Zdroj: fr. wikipedia.org/wiki/Treponema
 Spirochete (a close cousin ofT.pallidum)
 Proteolytic
 Colonizing older children (6 – 12 let 50 %,
but 0,5 % microb. population) and adults
 Close relationship to P. gingivalis – growth
factors
Porphyromonas gingivalisPorphyromonas gingivalis
 Highly proteolyticHighly proteolytic
 FimbriaeFimbriae – adhesioin and colonisation
 Releases vesiclesvesicles containing parts of outer
membranes - proteins, LPS, capsule etc.
 Vesicles - transport of toxins and enzymes,
bacterial adherention and aggregation,
adherention of thrombcytes
 Black pigment = acummulated hemin – a source
of iron (a growth factor) Zdroj: www.pgingivalis.org
P.gingivalisP.gingivalis
Zdroj: www.pgingivalis.org
„Budding“ vesicles
Fimbriae
Tannerella forsythiaTannerella forsythia
 Interaction betweenInteraction between T. forythiaT. forythia aa P.P.
gingivalisgingivalis
 P. gingivalis supports adhesion to host cells andadhesion to host cells and
invasioninvasion
 Epitelia withEpitelia with invading bacteria are th source of
recurrent infectionrecurrent infection
Zdroj: www.acsu.buffalo.edu
Invasion of T. forsythia into cells (arrows), Inagaki
2006, confocal laser microscopy
Mutual relationships in„the red complex“Mutual relationships in„the red complex“
Tannerella forsythiaTannerella forsythia
Porphyromonas gingivalisPorphyromonas gingivalis
Treponema denticolaTreponema denticola
nutrition,
coagregation
Support of adhesion and
invasion
RED
COMPLEX
adherention
+
FUSOBACTERIA
Oral microflora in systemic diseasesOral microflora in systemic diseases
 Cardiovascular diseases - bacterial endocarditis, aterosclerosis - esp.
coronary arterias (Gotsman et al. 2007)
 Strokes (Pussinen et al. 2004)
 Pneumonias
 Diabetes mellitus (Mealey, Rethman 2003)
 Preterm births and low birth weight (Lin et al. 2007)
 Oesophagal carcinoma (Narikiyo et al. 2004)
MMechanismsechanisms
 Microbes from the mouthMicrobes from the mouth = metastatic infections (bacteremia
after tooth extraction - bacterial endocarditis)
 Bacterial enzymes and toxines fromBacterial enzymes and toxines from parodontal focuses =
metastatic damage (e.g. endotoxin G- bacteria from subgingival
biofilm)
 Antigens of oral bacteria and pro-inflammatory cytokinesAntigens of oral bacteria and pro-inflammatory cytokines
from inflamed parodont = metastatic inflammation (reaction Ag-Ab
where immunocomplexes)
Periodontitis - summaryPeriodontitis - summary
 Model polymicrobial disease
 Oral biofilm and bacterial interactions
 Porphyromonas gingivalis, Tannerella
forsythia, Treponema denticola
 Influencing human health in a broader
sense
MICROBIAL DISEASES IN THE MOUTHMICROBIAL DISEASES IN THE MOUTH
Koplik's spots / measles
Hand-foot-mouth disease
http://pathmicro.med.sc.edu/virol/picorna.htm
Mykotické infekce - formyMykotické infekce - formy
 Pseudomembranózní kandidóza - moučnivka (soor) nejčastější skvrnitě
zarudlá ústní sliznice a smetanově bílé pablány - u novorozenců,
u starých osob, u imunokompromitovaných nemocných probíhá chronicky
a zvl. u AIDS její ložiska mohou přecházet až do jícnu
 Erytematózni (atrofická) kandidóza -  akutní formě jako následek
dysmikrobie dutiny ústní při léčbě širokospektrými antibiotiky - sliznice
d.ú. Zarudlá, pálení v ústech.
 Velmi častá chronická forma se objevuje jako tzv. protetická stomatitida snímatelné
zubní náhrady, zvl. protézní lože – tvrdé patro a jazyk: na
sliznici je patrný erytém a edém, stačí snímat protézu na noc a pečlivě ji
mechanicky očišťovat a dezinfikovat
 Hyperplastická kandidóza - kandidová leukoplakie probíhá chronicky
ve formě ohraničených vyvýšených tuhých plaků, obvykle na vnitřní straně
tváří, prekanceróza
 Angulární kandidóza postihuje ústní koutky nebo provází jiné formy,
zvláště protetickou stomatitidu - únik sliny při výškově nevyhovujících
protézách
kmil.trios.cz/ObrLues/hutchin1.JPG
Hutchinson's teeth Moon's molar
Zdroj: Wikipedia
Thank you