Defining the Normal Bacterial Flora of the Oral Cavity

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J Clin Microbiol. 2005 Nov; 43(11): 5721–5732.doi: 10.1128/JCM.43.11.5721-5732.2005

PMC!: PMC1287824

Defining the Normal Bacterial Flora of the Oral Cavity

J"rn #. #$%&1&2&' rc* J. P$%+*r &1&3 ,$r*n N. +o*%&1 n$r l%*n&2 $nd lod . !*ir%+1&3

#+or in6or$+ion #r+icl* no+*% Co9ri+ $nd ,ic*n%* in6or$+ion

i% $r+icl* $% b**n ci+*d b o+*r $r+icl*% in PMC.

ABSTRACT

The oral cavity is comprised of many surfaces, each coated with a

plethora of bacteria, the proverbial bacterial biofilm. Some of these

bacteria have been implicated in oral diseases such as caries and

periodontitis, which are among the most common bacterial infections in

humans. For example, it has been estimated that at least 35% of dentate.S. adults aged 3! to "! years have periodontitis #$. &n addition,

specific oral bacterial species have been implicated in several systemic

diseases, such as bacterial endocarditis #', aspiration pneumonia #(),

osteomyelitis in children #*, preterm low birth weight #), (!, and

cardiovascular disease #(, 3'. Surprisingly, little is +nown about the

microflora of the healthy oral cavity.

y using culture-independent molecular methods, we previously detected

over 5!! species or phylotypes in subgingival plaue of healthy sub/ects

and sub/ects with periodontal diseases #($, necroti0ing ulcerative periodontitis in human immunodeficiency virus-positive sub/ects #(3,

dental plaue in children with rampant caries #3, noma #((, and on the

tongue dorsum of sub/ects with and without halitosis #$5. 1ther

investigators have used similar techniues to determine the bacterial

diversity of saliva #(5, subgingival plaue of a sub/ect with gingivitis

#$), and dentoalveolar abscesses #$!, (". 1ver half of the species

detected have not yet been cultivated. 2ata from these studies have

implicated specific species or phylotypes in a variety of diseases and oral

infections, but still only limited information is available on speciesassociated with health. ecently, 4ager et al. #$" demonstrated

significant differences in the bacterial profiles of '! oral cultivable

species on soft and hard tissues in healthy sub/ects. They also found that

the profiles of the soft tissues were more similar to each other than those

of supragingival and subgingival plaues.

1ur purposes were as follows #i to utili0e culture-independent molecular

techniues to extend our +nowledge on the breadth of bacterial diversity

in the healthy human oral cavity, including not-yet-cultivated phylotypes,

and #ii to determine the site and sub/ect specificity of bacterialcoloni0ation.

1

http://dx.doi.org/10.1128%2FJCM.43.11.5721-5732.2005

http://www.ncbi.nlm.nih.gov/pubmed/?term=Aas%20JA%5Bauth%5D

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1287824/





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http://dx.doi.org/10.1128%2FJCM.43.11.5721-5732.2005



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MATERIALS AND METHODS

S!"ect#$

Five sub/ects, representing both genders, ranging in age from (3 to 55

and with no clinical signs of oral mucosal disease were included in the

study. Sub/ects did not suffer from severe halitosis. The periodontia were

healthy in that all periodontal poc+ets were less than 3 mm deep with no

redness or inflammation of the gums. Sub/ects did not have active whitespot lesions or caries on the teeth. 1ur results were consistent with these

clinical observations in that species typically found in caries sub/ects

were not detected #3. The sub/ects had not used antibiotics for the last )

months.

Sam%le collection$

Samples from the following nine sites were analy0ed for each sub/ect

dorsum of the tongue, lateral sides of the tongue, buccal fold, hard palate,

soft palate, labial gingiva and tonsils of soft tissue surfaces, andsupragingival and subgingival plaues from tooth surfaces.

4icrobiological samples of supragingival and subgingival plaue

samples were ta+en with a sterile 6racey curette. 1ther samples were

collected with sterile swab brushes.

Sam%le ly#i#$

7laue samples were directly suspended in 5! 8l of 5! m4 Tris buffer

#p9 :.), $ m4 ;2T<, p9 *, and !.5% Tween (!. 7roteinase = #(!!

8g>ml? oche <pplied Science, &ndianapolis, &@ was added to themixture. The samples were then heated at 55AB for ( h. 7roteinase = was

inactivated by heating at "5AB for 5 min. 2etection of species is

dependent upon obtaining 2@< that can be amplified. Thus, a difficult-

to-lyse bacterium may not be detected. 9owever, by using our lysis

techniue, we were able to detect many hard-to-lyse species, such as

species of Actinomyces and Streptococcus.

Am%lification of &'S rRNA gene# !y (CR an) %rification of(CR %ro)ct#$

The $)S r@< genes were amplified under standardi0ed conditions using

a universal primer set #forward primer, 5C-6<6 <6T TT6 <TD 4T6

6BT B<6-3C? reverse primer, 5C-6<< 66< 66T 6ET BB< BB

6B<-3C #($. 7rimers were synthesi0ed commercially #1peron

Technologies, <lameda, B<. The 7B primers do not necessarily

include all bacterial species. @evertheless, a wide range of phylogenetic

types were obtained in this study and our previous studies by using this

universal primer set #3, $5, ($-(3. 7B was performed in thin-walled

tubes with a 6ene<mp 7B system ":!! #<&, Foster Bity, B<. 1ne

microliter of the lysed sample was added to a reaction mixture #final

volume, 5! 8l containing (! pmol of each primer, '! nmol of

2

















deoxynucleoside triphosphates, and $ of 7latinum Taq polymerase

#&nvitrogen, San 2iego, B<. &n a hot-start protocol, the samples were

preheated at "5AB for ' min, followed by amplification under the

following conditions denaturation at "5AB for '5 s, annealing at )!AB for

'5 s, and elongation at :(A for $.5 min, with an additional $5 s for each

cycle. < total of 3! cycles were performed? this was followed by a final

elongation step at :(AB for $5 min. The results of the 7B amplification

were examined by electrophoresis in a $% agarose gel. 2@< was stained

with ethidium bromide and visuali0ed under short-wavelength light.Cloning %roce)re#$

Bloning of 7B-amplified 2@< was performed with the T171 T<

cloning +it #&nvitrogen according to the instructions of the manufacturer.

Transformation was done with competent Escherichia coli T17$! cells

provided by the manufacturer. The transformed cells were then plated

onto Guria-ertani agar plates supplemented with +anamycin #5! 8g>ml,

and the plates were incubated overnight at 3:AB. ;ach colony was placed

into '! 8l of $! m4 Tris. Borrect si0es of the inserts were determined ina 7B with an 4$3 #H(! forward primer and an 4$3 reverse primer

#&nvitrogen. 7rior to seuencing of the fragments, the 7B-amplified

$)S r@< fragments were purified and concentrated with 4icrocon $!!

#<micon, edford, 4<, followed by use of the I&<uic+ 7B

purification +it #I&<6;@, alencia, B<.

&'S rRNA gene #e*encing$

7urified 7B-amplified $)S r@< inserts were seuenced using an <&

7rism cycle seuencing +it #ig2ye terminator cycle seuencing +it with

<mpliTaq 2@< polymerase FS, 6ene<mp 7B system ":!!? <&. The

primers used for seuencing have been described previously #($. Iuarter

dye chemistry was used with *! 84 primers and $.5 8l of 7B product in

a final volume of (! 8l. Bycle seuencing was performed with a

6ene<mp 7B system ":!! #<& with (5 cycles of denaturation at

")AB for $! s, annealing at 55A for 5 s, and extension at )!AB for ' min.

The seuencing reactions were run on an <& 3$!! 2@< seuencer

#<&.

&'S rRNA gene #e*encing an) )ata analy#i# of nrecogni+e)in#ert#$

< total of (,5*" clones with an insert of the correct si0e of approximately

$,5!! bases were analy0ed. The number of clones per sub/ect that were

seuenced ranged from '( to )", with an average of 5:.5 and a standard

deviation of :.$. < seuence of approximately 5!! bases was obtained

first to determine identity or approximate phylogenetic position. Full

seuences of about $,5!! bases were obtained by using five to six

additional seuencing primers #$5 for those species deemed novel. For

identification of closest relatives, the seuences of the unrecogni0edinserts were compared to the $)S r@< seuences of over $!,!!!

3







microorganisms in our database and over $!!,!!! seuences in the

ibosomal 2atabase 7ro/ect #: and the 6enan+ databases. 1ur cutoff

for species differentiation was (%, or approximately 3! bases for a full

seuence. The similarity matrices were corrected for multiple base

changes at single positions by the method of Ju+es and Bantor #$3.

Similarity matrices were constructed from the aligned seuences by using

only those seuence positions for which data were available for "!% of

the strains tested. 7hylogenetic trees were constructed by the neighbor-

/oining method of Saitou and @ei #('. T;;B1@, a software pac+agefor the 4icrosoft Eindows environment, was used for the construction

and drawing of evolutionary trees #(*. Ee are aware of the potential

creation of $)S r@< chimera molecules assembled during the 7B #$*.

The percentage of chimeric inserts in $)S r@< gene libraries ranged

from $ to $5%. Bhimeric seuences were identified by using the Bhimera

Bhec+ program in the ibosomal 2atabase 7ro/ect, by treeing analysis, or

by base signature analysis. Species identification of chimeras was

obtained, but the seuences were not examined for phylogenetic analysis.

Ncleoti)e #e*ence acce##ion nm!er#$

The complete $)S r@< gene seuences of clones representing novel

phylotypes defined in this study, seuences of +nown species not

previously reported, and published seuences are available for electronic

retrieval from the ;4G, 6enan+, and 22J nucleotide seuence

databases under the accession numbers shown in Fig. Fig.$ $ to to" ".

F&6. $.

acterial profiles of the buccal epithelium of healthy sub/ects.

2istribution and levels of bacterial species>phylotypes among five

sub/ects are shown by the columns of boxes to the right of the tree as

either not detected #clear box, K$5% of ...

F&6. ".

acterial profiles of the subgingival plaue of healthy sub/ects.


sub/ects are as described for Fig. Fig.$. $. @ovel phylotypes identified in

this study are indicated in bold. 6enan+ ...

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RES,LTS AND DISC,SSION

&t has long been +nown that oral bacteria preferentially coloni0e different

surfaces in the oral cavity as a result of specific adhesins on the bacterial

surface binding to complementary specific receptors on a given oral

surface #$$, $(. &ndeed, the profiles of '! cultivable bacterial species

differed mar+edly on different oral soft tissue surfaces, saliva, and

supragingival and subgingival plaues from healthy sub/ects #$". The

purpose of this study was to define the predominant bacterial flora of the

healthy oral cavity by identifying and comparing the cultivable and thenot-yet-cultivated bacterial species on different soft tissues and supra-

and subgingival plaues.

ased on the analysis of (,5*" $)S r@< clones, the bacterial diversity

of the microflora from nine different sites of five clinically healthy

sub/ects was stri+ingLa total of $'$ different bacterial taxa representing

six different bacterial phyla were detected. The six phyla included

the Firmicutes #previously referred to as the low-6MB gram positives,

such as speciesof Streptococcus, Gemella, Eubacterium, Selenomonas,Veillonella, and

related ones, the Actinobacteria #previously referred to as the high-6MB

gram positives, such as species of Actinomyces, Atopobium, Rothia, and

related ones, the Proteobacteria #e.g., species

of Neisseria, Eikenella, Campylobacter , and related ones,

the Bacteroietes #e.g., species

of Porphyromonas, Pre!otella, Capnocytopha"a, and related ones,

the Fusobacteria #e.g., species of Fusobacterium and #eptotrichia, and

the T4: phylum, for which there are no cultivable representatives. <s

determined in our previous studies using culture-independent molecular

techniues #$5, ($, ((, over )!% of the bacterial flora was represented

by not-yet-cultivated phylotypes. Thirteen new phylotypes #see Fig.

Fig.( ( through through" " were identified for the first time in this study.

&n a comparable ongoing study of caries in permanent teeth, using the

same techniues, we detected ((' species in the analysis of $,(:5 $)S

r@< clones with about )!% as not-yet-cultivable phylotypes #J. <. <as,

S. . 2ardis, <. G. 6riffen, G. @. Sto+es, <. 4. Gee, &. 1lsen, F. ;.

2ewhirst, ;. J. Geys, and . J. 7aster. J. 2ent. es. 84:abstract (*!5,

(!!5. Bonseuently, it is important to identify both the cultivable andnot-yet-cultivated bacterial flora in a given environment before we can

ascribe association to health or disease status. There is no evidence to

suggest that the not-yet-cultivated segment is any less important than the

cultivable segment.

F&6. (.

acterial profiles of the maxillary anterior vestibule of healthy sub/ects.


5























this study are indicated ...

acterial profiles for each site tested for each sub/ect are shown in nine

phylogenetic trees #see Fig. Fig.$ $ throughthrough". ". &n these

dendrograms, the distribution of bacterial species or phylotypes in each

sub/ect can be observed at a glance. For example, it is clear

that Streptococcus mitis, S$ mitis bv. (, and Gemella hemolysans are the

predominant species of the buccal epithelium #Fig. #Fig.$. $. ;ach of

these species was detected in most or all of the sub/ects and represented

N$5% of the total number of assayed clones #Fig. #Fig.$. $. Similarly, the

predominant bacterial flora for the other eight oral sites was examined. &n

the maxillary anterior vestibule, S$ mitis,Granulicatella spp.,

and Gemella spp. predominated #Fig. #Fig.(. (. 1n the tongue dorsum,

several species of Streptococcus, such as S$ mitis, Streptococcus

australis, Streptococcus parasan"uinis, Streptococcus

sali!arius, Streptococcus sp. clone F7!$5, and Streptococcus sp. clone

F@!5$, Granulicatella aiacens, andVeillonella spp. were the

predominant species #Fig. #Fig.3. 3. 1n the lateral tongue surface #Fig.#Fig.', ', S$ mitis, S$ mitis bv. (, Streptococcus sp. clone

27!!", Streptococcus sp. clone F@!5$, S$ australis, G$ aiacens, G$

hemolysans, and Veillonella spp. predominated. &t is interesting that there

were considerable differences in the bacterial profiles of the tongue

dorsum and the lateral tongue surface. For example, S$ mitis bv. ( was

well represented at the lateral side of the tongue but not detected on the

tongue dorsum. Bonversely,S$parasan"uinis strain *5-*$ was present on

the tongue dorsum, but was absent on the lateral side. This was not

surprising, because these surfaces are +nown to be different inultrastructure and function. For instance, the lateral side of the tongue has

a smooth non+eratini0ed surface, in contrast to the dorsum of the tongue,

which is a +eratini0ed, highly papillated surface with a large surface area

and underlying serous glands. These anatomic differences li+ely influence

the ecology of these habitats and create microbial environmental

differences.

F&6. 3.

acterial profiles of the tongue dorsum of healthy sub/ects. 2istribution

and levels of bacterial species>phylotypes among five sub/ects are as

described for Fig. Fig.$. $. @ovel phylotypes identified in this study are

indicated in bold. 6enan+ ...

6
































F&6. '.

acterial profiles of the lateral tongue surface of healthy sub/ects.



this study are indicated in bold. ...

1n the hard palate, the predominant bacterial species included S$ mitis, S$

mitis biovar (, Streptococcus sp. clone F@!5$, Streptococcus

in%antis, Granulicatella ele"ans, G$ hemolysans, and Neisseria

sub%la!a #Fig. #Fig.5. 5. 1n the soft palate, S$ mitis, other cultivable and

not-yet-cultivable species of Streptococcus, G$ aiacens andG$

hemolysans were predominant #Fig. #Fig.). ). The tonsil bacterial flora

#Fig. #Fig.: : was rather diverseLsome sub/ects

had Pre!otella and Porphyromonas spp. #sub/ects $ and 5, and others

had Firmicutes species #sub/ects (, 3, and '. 1n the tooth surface,

several species of Streptococcus, including Streptococcus sp. clone

;=!'*, S$ san"uinis, and S$ "oronii, and Rothia entocariosa, G$

hemolysans, G$ aiacens, Actinomyces sp. clone G!!*, and Abiotrophia

e%ecti!a were often detected #Fig. #Fig.*. *. Finally, in subgingival plaue, several species of Streptococcus and Gemella were often found

#Fig. #Fig." ".

F&6. 5.

acterial profiles of the hard palate of healthy sub/ects. 2istribution andlevels of bacterial species>phylotypes among five sub/ects are as



F&6. ).

acterial profiles of the soft palate of healthy sub/ects. 2istribution andlevels of bacterial species>phylotypes among five sub/ects are as



7































F&6. :.

acterial profiles of the tonsils of healthy sub/ects. 2istribution and

levels of bacterial species>phylotypes among five sub/ects are as


indicated in bold. 6enan+ accession ...

F&6. *.

acterial profiles of the tooth surfaces of healthy sub/ects. 2istribution

and levels of bacterial species>phylotypes among five sub/ects are as



The number of cultivable and not-yet-cultivable species detected in eachsub/ect for each site is also shown in Fig. Fig.$ $ through through". ". For

example, in Fig. Fig.$, $, only four species #S$ mitis, S$mitis bv. (, A$

e%ecti!a, and G$ hemolysans were detected on the buccal epithelium in

sub/ect 5. For convenience, the number of bacterial species detected in

each oral site for each sub/ect is summari0ed in Table Table$. $.

Surprisingly, the highest number of different species was found on the

tonsils in sub/ect $, with (* predominant species? collectively, 5: species

were detected on the tonsils #Table #Table$? $? Fig. Fig.:. :. &n contrast,

the maxillary anterior vestibule had the lowest diversity of the bacterialflora compared with the other sites, with as few as three to nine species

detected among the sub/ects #Table #Table$? $? Fig. Fig.(. (. < common

uestion as+ed is how many bacterial species are present in the oral

cavity of a single individual. The last column of Table Table$ $ indicates

the number of predominant species in each healthy individual from all

nine different sites? thus, the numbers ranged from a low of 3' in sub/ect

' to a high of :( in sub/ect 3.

T<G; $.

@umber of predominant bacterial species per site and sub/ect

1verall, cultivable and not-yet-cultivable species

of Gemella, Granulicatella, Streptococcus, and Veillonellawere

commonly detected in most sites. S$ mitis was the most commonly foundspecies in essentially all sites and sub/ects #Fig. #Fig.$ $ through

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through". ". &n one sub/ect, :"% of the clones identified were S$

mitis #data not shown in figures. @ote that in this study, S$

mitis and Streptococcus pneumoniae have been grouped together. Some

members of the mitis group share more than ""% $)S r@< seuence

similarity, although 2@<-2@< similarity values for the entire

chromosomes are estimated to be less than )!% #$'. oth S$

mitis andStreptococcus oralis have been associated with bacterial

endocarditis, especially in patients with prosthetic valves #". &n addition,

they are now recogni0ed as freuent causes of infection inimmunocompromised patients, particularly immediately after tissue

transplants and in neutropenic cancer patients #3!. G$ aiacens, often

considered an opportunistic pathogen, was also detected at all sites in the

healthy oral cavity. G$ aiacens isolates have been reported from

bacteremia>septicemia in patients with infective endocarditis>atheroma

#33 and in primary bacteremia in patients with neutropenic fever #33. <

high mortality rate for endocarditis by G$ aiacens has been reported #5.

Figure Figure$! $! represents the overall summary of this studyLnamely,

that there are emerging bacterial profiles that help define the healthy oralcavity. <s already discussed, several species, such as S$ mitis and G$

aiacens, were detected in most or all oral sites, whereas several species

were uite site specific. For example, R$

entocariosa, Actinomyces spp., S$ san"uinis, S$"oronii, and A$

e%ecti!a appeared to preferentially coloni0e the teeth, while S$

sali!arius was found mostly on the tongue dorsum. Some species

appeared to have a predilection for soft tissue, e.g., S$ san"uinis and S$

australis did not coloni0e the teeth or subgingival crevice.S$

intermeius preferentially coloni0ed the subgingival plaue in most of thesub/ects but was not detected in most other sites. 1n the other

hand, Neisseria spp. were not found in subgingival plaue but were

present in most other sites. Simonsiella muelleri coloni0ed only the hard

palate. &ndeed, S$ muelleri was initially isolated from the human hard

palate #3(, although it has been isolated from a neonate with dental cyst

and early eruption of teeth #3$. Several Pre!otella species were detected

in most sites, but only in one or two sub/ects. For example, P$

melanino"enica and Pre!otella sp. clone ;!:3 were abundant in seven

out of nine sites of one sub/ect and were detected sporadically in othersub/ects #Fig. #Fig.$!. $!. Pre!otella sp. clone 9F!5! was found in the

maxillary anterior vestibule of one sub/ect, dominating the bacterial flora

as ''% of the clones #Fig. #Fig.( (and and$!. $!. This clone was also

found in lower proportions on the soft palate and tonsils of another

sub/ect #Fig.#Fig.), ), ,:, :, and and$! $!.

F&6. $!.

9










































Site specificity of predominant bacterial species in the oral cavity. &n

general, bacterial species or phylotypes were selected on the basis of their

detection in multiple sub/ects for a given site. 2istributions of bacterial

species in oral sites among ...

&n conclusion, there is a distinctive bacterial flora in the healthy oral

cavity which is different from that of oral disease. For example, many

species specifically associated with periodontal disease, such

as Porphyromonas "in"i!alis, Tannerella %orsythia, and Treponema

enticola, were not detected in any sites tested. &n addition, the bacterial

flora commonly thought to be involved in dental caries and deep dentin

cavities, represented byStreptococcus

mutans, #actobacillus spp., Bi%iobacterium spp., and Atopobium spp.,

were not detected in supra- and subgingival plaues from clinically

healthy teeth. < more detailed description of bacterial species associated

with oral disease is discussed elsewhere #$:. <s noted in this study on

healthy sub/ects, some species are site specific at one or multiple sites,

while other species are sub/ect specific. <s much as )!% of the species

detected are not presently cultivable. 1verall, there are still more speciesto be discovered, although the number of new species is beginning to

reach saturation.

<s we have previously asserted #($, to rigorously assess the association

of specific species or phylotypes with oral health or disease, it is

necessary to analy0e larger numbers of clinical samples for the levels of

essentially all oral bacteria in well-controlled clinical studies. The

bacterial complexes involved in periodontal disease as defined by

Socrans+y et al. #(: were based on the microbial analyses of $*5

sub/ects representing about $3,!!! plaue samples using 2@< probes to

'! bacterial cultivable species in chec+erboard hybridi0ation assays. Ee

are currently developing 2@< probes for approximately 5!! +nown

bacterial species and novel phylotypes for use in similar studies. These

2@< probes are being developed for use in the chec+erboard

hybridi0ation assay #3, ((, (3 and 2@< microarray formats #S. =.

oches, <. 4. Gee, . J. 7aster, and F. ;. 2ewhirst, J. 2ent.

es. 83:abstract (()3, (!!'. 1ur intent was to first establish the full

bacterial diversity of the oral cavity and then to determine variation and

reproducibility using the microarrays. Bonseuently, it will be relativelyeasy to compare the bacterial composition of a statistically significant

number of samples to more precisely identify those species that are

associated with health and with oral disease from different anatomic sites.

&t is necessary to first define the bacterial flora of the healthy oral cavity

before we can determine the role of oral bacteria in disease.

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AC-NO.LED/MENTS

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This study was supported by @&9 grant 2;-$$''3 from the @ational

&nstitute of 2ental and Braniofacial esearch and grants from the 2ental

Faculty, niversity of 1slo, @orway.

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