www.kcl.ac.uk

Implications of characterisation

of the oral microbiome for oral

care

William Wade

www.kcl.ac.uk

Outline

• Composition of the human oral

microbiome

• Role of oral bacteria in health

• Role in disease – changing concepts

of the pathogenesis of tooth decay

and gum disease

• Implications for oral care

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The oral microbiome

• Includes fungi, viruses and protozoa

• Bacteria predominate:

• Saliva - 108 / ml

• All oral surfaces colonised by

dense biofilm

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Impact of culture-independent

analyses

• Oral bacteria are typically slow-growing and

fastidious

• Comparison of microscopic and viable counts

show that 50% are uncultivable

• 16S rRNA-based sequencing methods revealed

the extent of the diversity of oral bacterial

populations

• Next generation sequencing led to analyses of

greater depth

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Oral Bacteria and Archaea

Bacterial phyla: Archaea:

Actinobacteria Minor component

Bacteroidetes All methanogens:

Firmicutes Methanobrevibacter

Fusobacteria Methanobacterium

Proteobacteria Methanosarcina

Spirochaetes

Synergistetes

Tenericutes

SR1

TM7

GN02

Acidobacteria

Chlamydiae

Chloroflexi

Deinococcus-Thermus

www.kcl.ac.uk

The Human Oral Microbiome (Dewhirst et al., J. Bacteriol., 2010)

• Release 12 update (Feb 2013):

- 663 bacterial species

- 6 phyla – Firmicutes, Bacteroidetes,

Proteobacteria, Actinobacteria,

Spirochaetes and Fusobacteria contain

96% of species

- 69 % of species cultured

- 116 cultured but un-named species

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The phylum Synergistetes

(Jumas-Bilak et al. 2009) Synergistes BH017 Synergistes JV006

Synergistes DO84

Synergistes JV023 Synergistes E2

Synergistes D006

Synergistes BH007 Synergistes JV001

Synergistes W028

Synergistes E3

Synergistes W090 Jonquetella anthropi

Synergistes RMA 14551 Pyramidobacter piscolens

Dethiosulfovibrio peptidovorans Dethiosulfovibrio russensis Dethiosulfovibrio acidaminovorans Dethiosulfovibrio marinus

Aminobacterium mobile

Aminobacterium colombiense

Anaerobaculum mobile

Anaerobaculum thermoterrenum Aminiphilus restrictus

Thermovirga lienii

Thermanaerovibrio velox

Aminomonas paucivorans

Synergistes jonesii

Synergistes RMA 14605 Synergistes RMA 16290

Thermanaerovibrio acidaminovorans

Cluster A - “uncultivable”

Cluster B - cultivable

www.kcl.ac.uk

www.homd.org

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Health benefits of the oral

microbiota

• Colonisation resistance

• Normal development of structures

and systemic and local immunity.

Evidence available for distal gut - ?

for mouth.

• Nitrogen metabolism

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Nitrate and health (Lundberg et al. Cardiovascular Res 2011;89: 525-

532)

• 25% of ingested nitrate returned to

mouth by entero-salivary circuit

• Oral bacteria reduce nitrate to nitrite

• Nitrite absorbed and converted to

nitric oxide (NO)

• NO essential for vascular health

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Nitrate and health

• Nitrate supplements lower blood pressure

(Kapil et al. Hypertension 2010; 56: 274-

281)

• Increase in plasma nitrite markedly

reduced by antimicrobial mouthrinse

(Govoni et al. Nitric Oxide 2008; 19: 333-

337)

• Blood pressure lowering effect of nitrate

abolished by mouthrinse in rats

(Petersson et al. Free Rad Biol Med

2009;46: 1068-1075)

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Mouthwash use raises blood

pressure (Kapil et al. Free Radical Biol Med 2013; 55:93-100)

• 19 volunteers, cross-over design

• 7-day use of 0.2 % chlorhexidine

mouthwash

• Oral nitrite reduced by 90 %, plasma

nitrite by 25 %

• Blood pressure significantly raised after

mouthwash use: systolic 3.5 mm Hg;

diastolic 2.2 mm Hg, correlated with

plasma nitrite

• ? Implications for oral care

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Aci netobacter C1 Aci netobacter junii

Act inomyces C1

Act inomyces C2

Act inomyces C3 Act inomyces georgiae

Act inomyces odontolyticus

Bi fidobacteriaceae C1

Bi fidobacterium dentium Bra chybacterium C1

Capnocytophaga C1

Centipeda periodontii

Corynebacterium matruchotii E. coli

Flavobacteriaceae C1

Flexist ipes C1

Fusobacterium C1 Fusobacterium C2

Ko curia krist inae

Lachnospiraceae C1

Lactobacillus buchneri Lactobacillus C1

Lactobacillus C2

Lactobacillus C3

Lactobacillus casei Lactobacillus colehominis

Lactobacillus crispatus

Lactobacillus fermentum

Lactobacillus gasse ri/johnsonii Lactobacillus oris

Lactobacillus pentosu s/plantarum

Lactobacillus reuteri/panis

Lactobacillus salivarius Lactobacillus vaginalis

Leptotrichia C1

Megasphaera C1

Micrococcus luteus Neisse ria mucosa

Olsenella C1

Pa rasca rdovia denticolens

Pe ptonophilus C1 Pre votella C1

Pre votella C2

Pre votella veroralis

Pro pionibacterium C1 Pse udomonas sp.

Rothia dentocariosa

Sca rdovia C1

Sca rdovia inopinata Se lenomonas C1

Se lenomonas C2

Se lenomonas noxia

Sh uttleworthia satelles Staphylococcu s haemolyticus

Staphylococcu s hominis

St reptococcus C1

St reptococcus C10 St reptococcus C2

St reptococcus C3

St reptococcus C4

St reptococcus C5 St reptococcus C6

St reptococcus C7

St reptococcus C8

St reptococcus C9 St reptococcus parasanguis

St reptococcus sobrinus

St reptococcus cristatus

Treponema denticola Ve illonella atypica

Atopobium parvulum

Atopobium rimae

Bu lleidia extructa Lactobacillus rhamnosus

Olsenella profusa

Pre votella denticola

Pre votella oris Pre votella oulorum

Pro pionibacterium acnes

Staphylococcu s epidermidis

St reptococcus mutans Act inomyces israelii

Act inomyces naeslundii

Neisse ria subflava

Pre votella intermedia Staphylococcu s warneri

St reptococcus constellatus

St reptococcus gordonii

St reptococcus intermedius St reptococcus mitis

St reptococcus salivarius

St reptococcus sanguinis

Ve illonella dispar Ve illonella parvula

Olsenella uli

Pe ptost reptococcus micros

Se lenomonas sputigena St reptococcus anginosus

Ba cteroidales E1

Ba cteroides forsythus

Clostridiales E1 Dialister E2

Eu bacteriaceae E1

Eu bacterium minutum

Firmicutes E2 Flexist ipes E1

Lachnospiraceae E2

Lachnospiraceae E3

Lachnospiraceae E4 Lachnospiraceae E5

Lactobacillus catenaformis

Neisse ria sicca

Pe ptoniphilus E1 Pe ptost reptococcus lacrimalis

Pre votella E1

Pre votella E2

Pre votella E3 Pre votella E4

Pre votella tannerae

Slackia exigua

So lobacterium moorei St reptococcus E1

St reptococcus oralis

Syn ergistes E1

Ba cteroidales E2a Ba cteroidales E2b

Ba cteroidales E3

Campylobacter graci lis

Dialister E1 Dialister pneumosintes

Eu bacteriaceae E2

Eu bacterium brachy

Eu bacterium nodatum Eu bacterium saphenum

Eu bacterium sulci

Filifactor alocis

Firmicutes E1 Fusobacterium nucleatum

Lachnospiraceae E1

Megasphaera P1

Mogibacterium sp . Pe ptococcus E1

Pe ptost reptococcus anaerobius

Pre votella bucca e

Pre votella nigrescens Ab iotrophia P1

Act inobaculum EL030

Act inomyces meyeri

Act inomyces P1 Act inomyces B27SC

An aeroglobus geminatus

Ba cteroidales P1

Ba cteroidales P2 Ba cteroides gracilis

Ba cteroidesAU 126

Bu tyrivibrio sp. oral clone DA074

Campylobacter concisus Campylobacter gingivalis

Campylobacter rectus / showae

Capnocytophaga BB1 67

Capnocytophaga gingivalis Capnocytophaga ochracea

Capnocytophaga P 1

Capnocytophaga DS0 22

Capnocytophaga AA0 32 Capnocytophaga BB1 67

Capnocytophaga BM058

Capnocytophaga X089

Capnocytophaga sputigena Capnocytophaga sputigena

Cardiobacterium hominis

Clostridiales P1

Clostridiales P3 Clostridiales P4

Corynebacterium P1

Desulfobulbus R004 / CH031

Desulfomicrobium orale Desulfovibrio P1

Eu bacteriaceae P2

Eu bacteriaceae P3

Eu bacterium BB1 42 Eu bacterium DA014

Eu bacterium DO016

Eu bacterium saburreum

Firmicutes AO068 Fusobacterium necro phorum

Gemella P1

Gemella 933 -88

Granulicatella adiacens Haemophilus P1

Kingella DE012

Lachnospiraceae P1

Lachnospiraceae P3 Leptotrichia P1

Leptotr ichia P2

Megasphaera BB166

Neisse ria P2 Neisse ria P3

Pa enibacillus sp.

Pe ptost reptococcus P3

Pe ptost reptococcus P4 Ph ylum P1

Po rphyromonas endodontalis

Po rphyromonas gingivalis

Po rphyromonas P1 Po rphyromonas P2

Pre votella dentalis

Pre votella oralis

Pre votella P2 Pre votella P4

Pre votella P5

Pre votella AA0 20

Pre votella BE0 73 Pre votella BU035

Pre votella DA058

Pre votella FM005

Pre votella B31FD Pro pionibacterium BN085

Sch warzia P2

Se lenomonas

Se lenonomonas periodontii Se lenomonas dianae

Se lenomonas flueggei

Se lenomonas infelix

Se lenomonas DS071 Se lenomonas AJ036

Se lenomonas DD020

Se lenomonas DY027 16S

Se lenomonas EQ054 Se lenomonas EW051

Se lenomonas EW079

Se lenomonasEW 084

Se lenomonas EY047 Se lenomonas P2

Se lenomonas P4

Se lenomonasC S015

Se lenomonas GAA14 St reptococcus P3

St reptococcus P4

St reptococcus sinensis

St reptococcus AY020 St reptococcus BE024

St reptococcus BW009

St reptococcus CH016

St reptococcus DN025 St reptococcus FN042

St reptococcus FN051

St reptococcus 7A

St reptococcus T1-E5 St reptococcus P2

Sy nergistes BA1 21

Sy nergistes BH017

Sy ntrophomonas P1 TM7 I025

TM7 P1

Treponema BZ013

Treponema socranski i Treponema 3:E:AT013

Treponema 9:A:D01

Treponema I:8:G57

Treponema I:G:T21 Treponema II:I:C53

Treponema P3

Treponema VI :G:G47

Eu bacterium PUS9 .170 Uncultured bacterium BH017

Uncultured bacterium D006

Uncultured bacterium W090

Specificity of oral disease-associated microbiota Species Caries Endodontic Periodontitis

• 3592 isolate/

clone

sequences

included

• 268 species

Wade WG. Interface

Oral Health Science

2007

www.kcl.ac.uk

Caries – “textbook view”

• Plaque bacteria produce acid from dietary

carbohydrates

• Acid demineralises teeth

• Streptococcus mutans and lactobacilli found in high

numbers in carious lesions

• Subjects with active caries have raised counts of S.

mutans

• S. mutans acidogenic and aciduric

www.kcl.ac.uk

Caries – combined cultural and culture-

independent studies (Munson et al. J Clin Microbiol 2004;42:3023-9

Tanner et al. J Clin Microbiol 2011;49:1464-74

Beighton et al. J Dent Res 2010;89:970-4)

• Complex bacterial community

• S. mutans frequently not present

• Other acidogenic species significantly associated

with carious lesions, including Scardovia

wiggsiae, Propionibacterium acidifaciens,

bifidobacteria and others

• Some lesions have high levels of lactobacilli,

some Prevotella species

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Extended ecological plaque

hypothesis (Takahashi and Nyvad 2008)

Dynamic stability stage

Acidogenic stage

Aciduric stage

Mild / infrequent

acidification

Moderate / frequent

acidification

Severe / prolonged

acidification

Net mineral gain

Net mineral loss

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Dynamic stability stage

• Acid produced by range of bacteria lowers pH of

plaque

• Followed by alkalinisation phase, caused by:

• diffusion of acid

• buffering by plaque constituents and saliva

• production of alkali by bacteria

• Production of alkali:

• ureolysis – ammonia production from urea by

urease

• arginine deiminase – dietary arginine catabolised to

ammonia

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Acidogenic stage

• Initiated by:

• Repeated, raised levels of sugar

intake

• Reduced salivary flow

• Poor oral hygiene

• Microbiota typically dominated by

non-mutans streptococci and

Actinomyces

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Aciduric stage

• After prolonged acidogenic stage, ecology

changes with:

- Selection of aciduric bacteria, particularly S.

mutans and lactobacilli and some

Bifidobacterium and Propionibacterium species

• Thus, the ecological change drives the change in

the composition of the microbiota and not vice

versa, but the presence of aciduric bacteria

compounds the disease state

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Caries - implications for

probiotics

• Most probiotics developed originally for

lower gut use – lactobacilli and

bifidobacteria

• Associated with aciduric phase

• Some probiotic strains shown to

antagonise S. mutans but S. mutans now

not thought to initiate carious lesions

• S. mutans a marker of, but not a cause of,

caries.

www.kcl.ac.uk

The plaque biofilm and

gingivitis

Microbiology non-specific - plaque load

and maturity of primary importance

www.kcl.ac.uk

Molecular basis of inflammation

in gingivitis

• Poorly understood

• LPS, toxins

• Recognition of bacterial components and

products by Toll-like receptors and

activation of NF-kappaB

• Role of microbial load under-investigated

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Periodontitis

Loss of attachment Chronic lesion

Diverse microbiota – highly variable between sites and

subjects.

www.kcl.ac.uk

Clusters associated with periodontitis

– the specific plaque hypothesis (Socransky et al. J Clin Periodontol 1998;25:134)

Purple Green Yellow Orange Red

A. odontolyticus Capnocytophaga S. mitis P. intermedia P. gingivalis

V. parvula C. concisus S. oralis P. nigrescens T. forsythia

E. corrodens S. sanguis P. micra T. denticola

A.a S. intermedius F. nucleatum

S. gordonii

Health Disease

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Periodontitis - specific

plaque hypothesis

• Relies on conventional view of host-

parasite interactions: pathogens, Koch’s

postulates etc.

• Complex diseases at mucosal surfaces,

involving normal microbiota, may not

conform to this model

• Disease may result from defect in normal

mechanisms that maintain homeostasis

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Proresolving lipid mediators (Van Dyke J Clin Perio 2011;38:119)

• Resolution of inflammation is active

process

• Periodontitis may be failure of resolution

as well as excess inflammation

• Mediators of inflammation resolution are

lipoxins produced from metabolism of

arachidonic acid and resolvins, which

have similar properties, and are receptor

agonists

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Effects of treatment with RvE1 (Hasturk et al J Immunol 2007;179:7021)

• Rabbit ligature periodontitis model

• Treatment with RvE1 resulted in complete

resolution of inflammation and restoration of soft

and hard tissues, compared to controls

• Controls had periodontal microbiota typical of

human disease; RvE1 treated animals had

normal healthy biota, without “pathogens”

• Successfully treating the inflammation changed

the microbial ecology

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Microbiota of progression of

periodontitis (Tanner et al. J Clin Perio 2007; 34:917)

• 117 healthy or with “slight periodontitis”

subjects monitored clinically and

microbiologically for 18 months

• Disease activity seen in 22 subjects and

“pathogens” detected

• Presence of specific bacteria did not

predict attachment loss

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Implications for oral care

• Emerging consensus that although dental caries

and periodontal diseases are bacterial diseases,

bacteria are not specific primary aetiological

factors

• Aim should be to maintain healthy balance

between commensal microbiota and host –

excessive use of antimicrobials may negatively

impact health

• Caries – diet and oral hygiene of primary

importance

• Gingivitis and periodontitis – primary role for

probiotics likely to be modulation of inflammation