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ORIGINAL ARTICLE
Distinct and complex bacterial profiles in
human periodontitis and health revealed
by 16S pyrosequencing
Ann L Griffen1,6, Clifford J Beall2,6, James H Campbell3,6, Noah D Firestone2,
Purnima S Kumar4, Zamin K Yang3, Mircea Podar3,5 and Eugene J Leys2
1
Division of Pediatric Dentistry and Community Oral Health, The Ohio State University College of Dentistry,
Columbus, OH, USA; 2Division of Oral Biology, The Ohio State University College of Dentistry, Columbus,
OH, USA; 3Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA; 4Division of
Periodontology, The Ohio State University College of Dentistry, Columbus, OH, USA and 5Genome Science
and Technology Program, University of Tennessee, Knoxville, TN, USA
Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem.
With advances in sequencing technologies, comprehensive studies to elucidate bacterial commu-
nity differences have recently become possible. We used 454 sequencing of 16S rRNA genes to
compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects
with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were
sequenced, yielding 1 393 579 sequences. They were identified by BLAST against a curated oral 16S
database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped
to cultivated species. Differences between health- and periodontitis-associated bacterial commu-
nities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis
showed distinct community profiles in health and disease. Community diversity was higher in
disease, and 123 species were identified that were significantly more abundant in disease, and 53
in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas
the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the
class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were
associated with disease. These results implicate a number of taxa that will be targets for future
research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction
of sequences as compared with previously identified targets. Elucidation of these differences
in community composition provides a basis for further understanding the pathogenesis of
periodontitis.
The ISME Journal advance online publication, 15 December 2011; doi:10.1038/ismej.2011.191
Subject Category: microbemicrobe and microbehost interactions
Keywords: oral microbiome; pyrosequencing; 16S rRNA; periodontitis; bacteria
a
Students t-test (assuming equal variance).
b
Fishers exact test.
c
Only thresholds recorded.
d
Linear mixed models.
and from shallow and deep pockets of 29 patients bacteria among samples, for the top 12 disease-
with chronic periodontitis. Clinical metrics for the associated and health-associated species, the PCoA
participants are detailed in the Table 1. No was replotted and data points were scaled by the
significant differences were observed between the abundance of species of interest in each sample
healthy and periodontitis group for age or smoking (Figure 3).
status, although the periodontitis group included a
larger fraction of males. As expected, periodontal
indices including a marker of inflammation (bleed- Cultivation status
ing on probing) and probing depths were greater for Figures 4a and b show the abundance of cultivated
deep sites in the group with periodontitis than for and uncultivated species in common and rare phyla.
healthy sites. The preponderance of species, especially in the
Amplicons from both the V1-2 and V4 hypervari- most common phyla, have been cultivated.
able regions of the 16S gene were sequenced. Figure 4c depicts the overall abundance of culti-
Breakdown by hypervariable region is detailed vated and uncultivated species by sample, showing
below. A total of 16 phyla, 106 genera and 596 that there is considerable sample-to-sample varia-
species-level taxa were detected. Cumulative counts tion, but overall a tendency to have more unculti-
were normalized and averaged for the two vated species in the disease condition. Counting
sequenced regions, and expressed as percent of species without factoring abundance, over half
total. Differences between health- and periodontitis- (25/47) of the disease-associated species shown in
associated bacterial communities were observed at Figure 2 are currently uncultivated, but only one-
all phylogenetic levels. The phyla Spirochaetes, third (8/23) of the health-associated species are
Synergistetes, and Bacteroidetes were highly asso- uncultivated.
ciated with disease, whereas the Proteobacteria
were found at higher levels in healthy controls. Gram-stain status
Within the phylum Firmicutes, the class Bacilli was We surmised the probable Gram stain reaction of
health-associated, whereas the Clostridia, Negativi- organisms in our database by literature searches,
cutes and Erysipelotrichia were associated with and used this information to apply to the clinical
disease. A phylogenetic tree at the level of genus sequences. Figure 5 depicts a graph of the percen-
is shown in Figure 1. Overall abundance and the tage of Gram-negative organisms in each sample.
magnitude of the difference between health and Again, there is substantial variation between sam-
disease are indicated by bars. Figure 2 shows levels ples, but also a trend toward higher levels of Gram-
of the most abundant genera and species that negative organisms in disease (P 0.002, control vs
differed in health and disease. deep pocket, t-test with unequal variance).
Figure 3 shows 2-dimensional graphs of a princi-
pal coordinates analysis (PCoA) of the weighted
UniFrac distance between each pair of samples. Diversity
Bacterial community profiles were highly signifi- To determine how completely the community
cantly different among healthy subjects and shallow diversity was represented by our sequencing efforts,
(healthy) sites and deep (disease) sites in subjects rarefaction curves are shown in Figure 6. Figure 6a
with periodontitis. shows a tendency towards increased species
The ANOSIM test on the UniFrac distances richness (number of species per sample) in subjects
showed Po0.0001 for all pair-wise comparisons with periodontitis relative to healthy controls
among the three groups (one-way comparisons (P 0.00003 for control vs shallow pocket,
between subjects and two-way nested comparisons P 0.00022 for control vs deep pocket, t-tests with
within subjects). To visualize the distribution of unequal variance). The Shannon diversity index
Novosphingo cter
ium
Sphingomonas
Des
Methylobacter
Caulob ium
as
Mo udom terium
Paracoccus
Rhodo vibrio
Acetoba us
Brev acaulis
ulfom obulb ter
Desu robium
Asticc acter
Rhizobium
Car undimon
Des yloba nas
Ca
e
Ac raxel onas
Azorhizob
Po
ea
un
mp mo la
iac
rph anne ete a
Pse diobac
ulf
c.
vari
H sc En as a
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ter
Ba Pre iba ales
Al inet la
A ishe obac
yro
on ell
T roid ell r
un
gr o hi bac
cte vo cte
c. ingo
Sp
Ag aemheri tero
Od tero teri
us
Ba ba
h
eg ph a
rel s
or id um
ib s
c
r
te
at ilu
c
ac
t
Ca W Be as
E c.
pn au rg
c
on o
e
Cy ocy ter ye m bri
an top sie lla t ho nivi
ob ha lla n io
ac g Xa rop ella
ter a P ing eria
SR ia K eiss ella lus
TM 1 N ken ophi lus ae
7 Ei thyl bacil terace
Me thylo lobac
e
M . Ox a
Stre
ptoc unc tonia
Lacto occus Rals holderia ales
c Burk Burholderi
Entero occus ceae
coc unc. omamonada
Granulic cus unc. C
atella a
Abiotrophia Lautropi r
Leuconostoc Achromobacte
Treponema
Lactobacillus Thermodesulfovibrio
Aerococcus Nitrospira
Gemella Deinococcus
cilli
unc. Ba us Chlorofle
c Synerg xi
h ylococ um
S ta p
cteri Pyra istes
x ig uoba acillus Jonq midobac
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Pae ulleidia
B rium Ato enella
cte thrix Cr pobiu
oba Sla yptob m
Sol sipelo ceae
Ery tricha asm a
a C cki acte
pl P oryn a rium
lo
ipe hole plas eae
m Bi ropio eba
Phylum/class Er y s
Ac yco llac ella a A fid n cte
M ne ok ed Ac ctin o./S ibac rium
Bacteroidetes tin om ca teri
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Ve ph iste s
ob yc rdo um
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ac ba ac us
Deltaproteobacteria ac es via
eg D mo
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Ra erm a
c C
hy ct te
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Alphaproteobacteria u um
ba eri r
ep tost ctor
R hro chia
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Art totri erium
Se
Le obac mona
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er
u
Fus tropho us
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tridia les C
iu
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Parv les B
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Pepto onas
Betaproteobacteria
unc. lfotomaculu
ramiba us
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cter
Clostrid thia
unc. Lachnospirac s A
Shuttlewor
eae A
Oribacterium
Johnsonella
Catonella
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to
niphil
Nitrospira
ia
ac
ridiale
im
er
t
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Synergistetes
str
Mo
u
Clos
pto
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unc. Clost
Pseudo
Fusobacteria
pirac
unc
Clostridia
m
eae
Negativicutes
Mollicutes
B
Erysipelotrichia
Bacilli
Figure 1 Circular maximum likelihood phylogenetic tree at level of genus. The inner band shows genera colored by phylum or class
(see key for taxa with multiple members), the next band shows significant mean differences between healthy controls and deep pockets
(colored green for genera higher in healthy pockets and red for genera higher in disease pockets), and the outer band shows overall
relative abundance. The tree was constructed in iTOL (Letunic and Bork, 2007).
(Shannon, 1948) (that reflects both richness and genus and species level for several abundant taxa. Good
evenness) also increased with disease (P 0.002 for agreement was seen between data from the two regions,
control vs shallow pocket, P 0.0000006 for control except in the case of the genus Prevotella, for which
vs deep pocket, t-tests with unequal variance). there was very little V4 data. Figure 7 also shows
abundance data from the V1-2 and V4 regions for the
most prevalent phyla and Venn diagrams of the total
Comparison of data derived from the V1-2 and the number of taxa found in each of the data sets at the
V4 regions levels of species, genus and phylum.
Figure 7 shows a comparison of community profiles
obtained from V1-2 and V4 amplicons. Yield was
516 478 sequence reads from the V1-2 region and Discussion
877 101 from the V4 region. The mean number of
sequences per sample was 55202030 (s.d.) for the V1- Recent advances in DNA sequencing and bioinfor-
2 region, and 97372303 for the V4 region. Overall matics technologies have made possible two orders
93% of the V1-2 reads and 97% of the V4 reads were of magnitude higher resolution of bacterial commu-
identified, to species OTU level as defined in the CORE nity composition. This study, carried out using 454
database (Griffen et al., 2011). Figure 7 shows scatter pyrosequencing of subgingival samples from a group
plots with fitted lines and R2 values for all samples at of individuals with chronic periodontitis and
Figure 2 Differences between health and disease at level of phylum, genus and species. Pie charts show the number of taxa that were
significantly different. The graphs show levels for genera that were X0.1% different and species that were X0.2% different in health and
periodontitis samples. Taxa were sorted according to magnitude of change. Pp0.05 after FDR correction for all taxa shown.
matched periodontally healthy controls, showed of samples were highly significantly different, and
fundamental and clear differences in community healthy periodontal sites in patients with disease
composition that were not evident using less-power- were intermediate between healthy gingiva in
ful approaches. This work confirms previous find- disease-free subjects and deep pockets in patients.
ings that certain species are more common in We were able to make additional general observa-
disease, but provides a much broader picture of tions about the bacterial population shifts accom-
overall community differences and a much deeper panying periodontitis. Measures of diversity,
look at community complexity, thereby expanding both the number of species and the combination of
knowledge of putative pathogenic species. richness and evenness reflected in the Shannon
Global differences between health- and disease- index, were higher in periodontitis than in health
associated communities were clearly visualized by (Figure 6). Health-associated species were
the separation of groups in PCoA using the UniFrac not generally lost, but accounted for a smaller
distance (Figure 3) comparing the phylogenetic fraction of the total community. Considered from
similarity of bacterial communities. All three groups the bacterial point of view, this more diverse
80
p=0.00054 p=0.00056 p=0.00016
Treponema o.t. 230 Prevotella intermedia Desulfobulbus R004
% cultivated
Health-associated species:
60
40
p=0.0006 p=0.00026 p=0.00037
S. mitis group Streptococcus sanguinis Moraxella osloensis
20
Control Shallow Deep
V4 Region Abundance
Streptococcus
80
60%
R2 = 0.566
Percent
R2 = 0.511
60 40%
40 20%
R2 = 0.180
0%
20 0% 20% 40% 60% 80% 100%
Health Shallow Deep V1 Region Abundance
Disease 50%
Species
Figure 5 Gram status by disease status. A small but significant
shift to more gram-negative status in periodontitis is seen in this
scatter plot with means and 95% CI. 40% R2 = 0.465
V4 Region Abundance
R2 = 0.685
# Species Shannon Diversity 30%
300 4.5
250
4.0 20% R2 = 0.797 F. nucleatum ssp. ff.
V. atyp/disp/parv.
S-OTUs
3.5
H
200 S. mitis/pneu/inf/ora
10%
3.0 T. denticola
150
2.5 R2 = 0.903
0%
100
0% 10% 20% 30% 40% 50%
Health shallow deep Health shallow deep
V1 Region Abundance
Disease Disease
V1 + V4 Region Data
180 Synergistetes
TM7 V4 region
Weighted Mean: S-OTUs/sample
160
V1 region
140 Spirochaetes
120 Actinobacteria
100 Bacteroidetes
80 V4 Healthy Controls
V4 Patient Healthy Sites
Fusobacteria
60 V4 Patient Disease Sites Proteobacteria
V1 Healthy Controls
40 V1 Patient Healthy Sites Firmicutes
V1 Patient Disease Sites
20
0% 10% 20% 30% 40% 50%
0
0 2000 4000 6000 8000 10000 12000
Species Genera Phyla
Number of Sequences