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Metagenomic analysis of the microbial changes following non-surgical periodontal therapy in aggressive periodontitis

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Metagenomic analysis of the microbial changes following non-surgical periodontal therapy in aggressive periodontitis

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Content METAGENOMIC ANALYSIS OF THE MICROBIAL CHANGES FOLLOWING
NON-SURGICAL PERIODONTAL THERAPY IN AGGRESSIVE PERIODONTITIS

by

Theresia Laksmana

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)

August 2011

Copyright 2011 Theresia Laksmana

ii

Table of Contents

List of Tables ........................................................................................................ iii

List of Figures ....................................................................................................... iv

Abstract ................................................................................................................... v

Chapter 1: Introduction ........................................................................................... 1
Metagenomics ........................................................................................... 10
Pyrosequencing ......................................................................................... 17
Real-time PCR .......................................................................................... 20
Objective & Hypothesis ............................................................................ 23

Chapter 2: Materials & Methods........................................................................... 25

Chapter 3: Results ................................................................................................. 31

Chapter 4: Discussion ........................................................................................... 40

Chapter 5: Conclusion........................................................................................... 47

References ............................................................................................................. 49

iii

List of Tables

Table 1: Discriminative power among variable regions of 16S rRNA genes .... 11

Table 2: Primer sequences ................................................................................. 28

Table 3: Primer sequences for real-time PCR .................................................... 29

Table 4: Proportion of the 27 most frequently present subgingival species/
phylotypes before and after treatment ................................................. 36

Table 5: Real-time PCR for P. gingivalis using 16S universal primers ............ 38

iv

List of Figures

Figure 1: Pyrosequencing .................................................................................... 18

Figure 2: Full mouth clinical changes ................................................................. 31

Figure 3: Comparison of the subgingival microbiota between patients before
(a) and after (b) treatment .................................................................... 32

Figure 4: Cumulative % of total reads with each addition of ranked species/
phylotypes ............................................................................................ 34

Figure 5: Comparison of quantitative changes in the subgingival microbiota
after treatment between patients .......................................................... 35

Figure 6: Real-time PCR standard curve for P. gingivalis using 16S rDNA
specific primers .................................................................................... 38

Figure 7: Comparison of metagenomics and real-time PCR proportions of
P. gingivalis ......................................................................................... 39

v

Abstract

Background: Metagenomics is the sequencing of DNA from whole communities of
organisms taken directly from the environment. As opposed to culturing methods which
focus on individual species, metagenomics allows for the characterization of intact
community genomes, including the identification of noncultivable bacterial species. The
objective of this pilot study was to test the feasibility of a metagenomic approach in
combination with a novel design of primers to analyze the pre- and post-treatment of
subgingival plaque in two aggressive periodontitis patients.
Methods: Periodontal examinations and microbial sampling were performed before and
after non-surgical initial phase therapy. DNA was extracted from the subgingival plaque
samples and subjected to PCR amplification using primers that amplified the c2-c4
regions of the 16S rDNA. Specific bar codes were included in the primers to identify
individual samples. The PCR products were pooled and sequenced for the v4 region of
the 16S rDNA using the pyrosequencing 454 FLX standard platform. The results were
analyzed for species identification in the Human Oral Microbiome Database (HOMD)
and Ribosomal Database Project (RDP) database. The observed proportional changes of
the subgingival species after therapy were verified by testing the pre- and post-therapy
levels of Porphyromonas gingivalis by real-time PCR.
Results: The sequencing of the amplicons resulted in 24,673 reads and identified 208
species/phylotypes. Of those, 129 species/phylotypes were identified in both patients but
their proportions varied. While >120 species/phylotypes were identified in all samples,
vi

85-90% of the species/phylotypes were represented by ≤100 sequencing reads (≤0.2% of
the total reads). Similar levels of subgingival P. gingivalis were found based on the
numbers of sequencing reads or quantitative Real-time PCR analyses. Pre- and post-
therapy samples showed significant quantitative and distributive species changes along
with clinical improvements. Quantitatively, many known pathogenic species went from
easily detectable to zero counts while the opposite occurred for many known commensal
bacteria. Additionally, several novel, pathogenic and health-associated species were
found.
Conclusion: High throughput metagenomic analysis is applicable to assess the changes
in subgingival microbiota after nonsurgical periodontal therapy in aggressive
periodontitis. The examined subgingival microbiota are characterized by high species
richness (>120 species/phylotypes) dominated by a few species/phylotypes. The
composition of the subgingival microbiota appears to be distinct between individuals
before and after the treatment. The considerable variety of known and novel
species/phylotypes detected supports further research to better understand the microbial
etiopathogenesis of periodontitis.

1

Chapter 1: Introduction

Periodontal disease is an inflammatory disease of the periodontium, the
supporting and surrounding structures of teeth, which can lead to bone loss and eventual
loss of teeth. Periodontitis is characterized by clinical attachment loss, alveolar bone loss
and bleeding upon probing (Flemmig, 1999). It is most often classified as chronic or
aggressive, localized or generalized, and is further distinguished by the severity or extent
of destruction (Armitage, 1999). The breakdown of the periodontium affects not only the
condition of the oral cavity, but also overall quality of life. Surveys in the United States,
as well as around the world, have revealed that periodontitis affects people of all
ethnicities, ages and socioeconomic standings. In conjunction with regular in-office
maintenance, proper home hygiene and sometimes surgical therapy, treatment is often
successful. However, in some cases the disease may relapse and in refractory cases, the
disease may persist despite all efforts.
The most recent report from the National Health and Nutrition Examination
Survey (NHANES) in 1999-2004 revealed that the prevalence of moderate or severe
periodontitis amongst Americans is up to 10.73% (Dye, 2007). However, a recent critique
of the survey found that the prevalence of the disease reported by the NHANES may in
actuality underestimate its true prevalence. Eke et al. (2010) evaluated 456 subjects by
both a full mouth periodontal exam (FMPE) and a partial mouth periodontal exam
(PMPE) as per the parameters of the NHANES 2001-2004 exam, using the same CDC
definition of periodontal disease and comparing the percent of subjects diagnosed with
2

periodontitis based on the different types of measurement. The researchers found that the
prevalence using the FMPE on their sample population was 22.4% versus the 9.7%
prevalence found using the PMPE of the NHANES 2001-2004 protocol such that the
estimated prevalence of periodontal disease as reported using the 2001-2004 NHANES
parameters underestimated the true prevalence of periodontitis by 12.7% for a relative
bias of 56.9%. According to the latest report from the surgeon general (May 25, 2000,
Oral Health in America: A Report of the Surgeon General), the proportion of adults with
moderate periodontitis ranged from 3.0-64.6% with severe periodontitis ranging from
0.2%-29.5% (U.S. Department of Health and Human Services, 2000). In both cases, the
lowest incidence was found in the youngest age group (18-24 yrs old) while the greatest
incidence occurred in the oldest age group (>75 yrs old).

Impact
Araujo et al. (2010) evaluated the effects of periodontal disease on quality of life
measures using the Oral Health Impact Profile, simplified version (OHIP-14) and found
that the disease could have a damaging affect on quality of life. Functional limitation,
represented as difficulty pronouncing words and changes in taste perception, was the
most impacted measure, affecting 91.5% of patients. This was followed by psychologic
discomfort (89.3%), gauged by embarrassment and difficulty relaxing due to problems
with teeth, mouth or dentures. Physical pain, the third most impacted measure (87.3%),
was found to most greatly affect the ability to eat. O‟Dowd et al. (2010) performed semi-
structured interviews on patients diagnosed with periodontitis to evaluate the
3

psychosocial impact of the disease. Recurrent themes in all subjects descriptions were
categorized into seven groups: impairment, functional limitation, discomfort, disability,
handicap, stigma, and retrospective regret.
More recently, associations with other systemic diseases such as diabetes,
pregnancy and cardiovascular disease have been reported. Grossi and Genco (1998)
discussed the possible two-way relationship between diabetes and periodontitis in which
uncontrolled diabetes may increase the severity of periodontitis, while periodontal
disease itself may also contribute to poor glycemic control in diabetics. Following an
extensive literature review, Beck et al. (1998) stated that, although causality could not be
concluded, periodontitis could be considered a risk factor for atherosclerosis/coronary
heart disease.
Dasanayake (1998) found that poor periodontal health was a potential risk factor
for low birth weight babies. In a transversal study on women with preterm labor, Gauthier
et al. (2011) sequenced the strains of the putative periodontal pathogen Fusobacterium
nucleatum found in the amniotic sludge of three of the women and compared them to
those found in the oral cavities of the same women or their partners. They discovered a
100% sequence match between the strains suggesting that the intra-amniotic F.
nucleatum could have originated from the oral cavity.

Etiology
Overwhelming evidence has shown that periodontal disease is an infection of
mixed bacterial species. Several putative pathogens have been studied extensively using
4

in vitro culturing methods. Socransky et al. (1998) suggested categorizing bacteria into
five complexes, each designated by a different color based on the community association
of the species in subgingival plaque. Based on order of colonization, the yellow and
purple complexes are believed to be early colonizers while the green, orange and red
complexes are believed to be secondary colonizers. With regards to periodontal disease,
members of the red, orange and green complexes are believed to be putative periodontal
pathogens.
The red complex, composed of Porphyromonas gingivalis, Tannerella forsythia,
and Treponema denticola, have been found to have the largest association with increased
probing depths and bleeding on probing. The larger, though slightly less strongly
associated orange complex, includes the species Prevotella intermedia, Prevotella
nigrescens, Fusobacterium nucleatum subspecies, Micromonas micros (previously
Peptostreptococcus micros), and associated species Campylobacter rectus,
Campylobacter showae, and Eubacterium nodatum. Although both complexes are
considered to be secondary colonizers, species of the red complex rarely exist in the
absence of orange complex species, and most often are only present after colonization by
orange complex species. Although also pathogenic, species of the green complex,
Eikenella corrodens, Capnocytophaga spp., and Aggregatibacter actinomycetemcomitans
serotype a, show little association with the red and orange complexes in disease.
Still some putative pathogens exist that are not otherwise categorized in any of the
five complexes. A. actinomycetemcomitans has one of the strongest associations with
destructive periodontal disease, particularly localized aggressive periodontitis, and when
5

found in conjunction with other members of the red complex, has been shown to have a
synergistic effect on periodontal breakdown. Listgarten (1994) included Selenomonas
species and other Eubacterium species as pathogens in “adult periodontitis”.
Haffajee and Socransky (1994) found that P. gingivalis and A.
actinomycetemcomitans, when found together, increased the risk of disease progression
to 6.7 fold compared to the 2.2 and 3.2 fold risk seen for each species individually
(respectively). P. gingivalis, A. actinomycetemcomitans, and Streptococcus intermedius
were detected more frequently at progressing sites while C. rectus, P. intermedia, and P.
gingivalis were also found elevated at visits at which attachment loss over the previous
two months had been observed.
The most common isolate in subgingival samples is the orange complex species
F. nucleatum (Bolstad et al. 1996; Socransky et al. 1998). Though perhaps less virulent
than other pathogens when cultured in isolation, of greater importance is the relationship
of F. nucleatum to other species. P. intermedia, for example, is rarely seen in the absence
of F. nucleatum (Socransky et al. 1998). Similarly, T. forsythia is most often found
concurrently with F. nucleatum. Its role in periodontal disease appears to be two-fold:
first, it seems important in the initial adhesion of the biofilm to the tooth structure, and
second, its key role in the coaggregation with other putative pathogens seems essential
for the growth of the biofilm complex (Bolstad et al. 1996).
Conversely, Listgarten (1994) noted species more predominant in periodontal
health including the Gram-positive species Streptococcus sanguinis (previously S.
sanguis), Streptococcus mitis, Actinomyces naeslundii, Actinomyces viscosus, Rothia
6

dentocariosa, and the Gram-negative species Veillonella parvula. Socransky & Haffajee
(1992) described four beneficial species: S. sanguinis I & II, Capnocytophaga ochracea,
and V. parvula. The production of H
2
O
2
by S. sanguinis has been shown to kill A.
actinomycetemcomitans. Similarly C. ochracea was shown to reduce the destructive
effects of P. gingivalis when present concurrently in high numbers.

Treatment
Treatment of periodontal disease can be categorized as non-surgical versus
surgical therapy. The basic approach of non-surgical periodontal therapy is a non-
specific, mechanical debridement of all supra- and subgingival plaque and calculus. To
this end, scaling and root planing has become the “gold standard” in non-surgical
treatment. Its effect can be seen in the subsequent shift in subgingival flora, from a
predominantly Gram-negative microbial community to a more Gram-positive community
as is more typically associated with gingival health. The concept of “critical mass”
suggests that another benefit is the overall reduction of the quantity of bacterial plaque
down to a level that can be managed by the host (Cobb, 2002).
Lindhe et al. (1984) showed that after scaling and root planing, a significant
decrease in probing depths and a significant increase in clinical attachment could be
noted, and that these changes could be maintained after 5 years. Haffajee et al. (1997)
and Cugini et al. (2000) evaluated its efficacy on the clinical and microbiological
parameters in 57 patients with adult periodontitis and found that at 3 months, 6 months,
and 12 months, a significant decrease in probing depths, bleeding on probing, plaque
7

levels and attachment levels was observed. Moreover, a reduction of the prevalence and
levels of T. forsythia, P. gingivalis and T. denticola was detected.
Additional non-surgical, combination therapies aimed at targeting specific
bacterial species have also been employed. The most common approach is to supplement
scaling and root planing with systemic antibiotic therapy. In order to evaluate for the
presence or absence of specific periodontal pathogens, clinicians most commonly use a
method termed microbial sampling. It is a method by which paper points are placed
subgingivally into the sulcus of a diseased periodontal pocket in order to collect bacteria
that can be subsequently analyzed in a laboratory by anaerobic culture (Hartroth et al.
1999). This culture-based test evaluates for the presence of specific periodontal
pathogens and can additionally test for antibiotic susceptibility of those particular strains
(Pacini et al. 1997). Laboratory reports sent to the clinician thus indicate the percent
presence or absence of certain known putative pathogens along with recommendations
for type of antibiotic use.
For supplemental therapy using systemic antibioitics in infections associated with
A. actinomycetemcomitans, van Winkelhoff et al. (1992) suggested the use of amoxicillin
plus metronidazole. Haffajee et al. (1994) suggested tetracycline when the predominant
flora was comprised of P. gingivalis, P. intermedia, P. nigrescens and T. forsythia while
Slots et al. (1990) recommended ciprofloxacin in situations where enteric rods have been
identified. Non-specific, non-systemic approaches have also been developed in the form
of locally administered antibiotics like Arestin and antimicrobials like PerioChip for
direct placement into periodontal pockets (Salvi et al, 2002).
8

A significant limitation of the current „gold standard‟ method for microbial
sampling by clinicians, however, is that it tests for cultivable species. Many bacteria can
be difficult to culture due to their specific living conditions. Bacteria require specific
temperatures, atmospheric pressures, gas compositions, and nutrient preparations for
growth, some of which have yet to be determined or are not currently feasible to provide.
Other bacteria may be obligate pathogens which cannot be grown as pure cultures while
still others may produce toxins as a byproduct and, therefore, be self-limiting (Tringe &
Rubin, 2005). Additionally, a substantial amount of starting sample is required in order to
replicate these bacteria in vitro. These challenges limit the data that can be provided.

Microbial testing methods
Currently one of the most widely used culture-based detection methods is DNA-
DNA hybridization. DNA-DNA hybridization is a method in which digoxigenin-labeled
whole genomic DNA probes are attached to a membrane to which sample DNA is added.
DNA probes are created from pure cultures of known species and are composed of
complementary nucleic acid sequences with which extracted DNA from the plaque
samples can hybridize. Hydrogen bonds are formed between complementary nucleotide
base pairs such that a DNA strand with a higher amount of complementary base pairs will
have a tighter degree of non-covalent bonding. Once the membrane is washed, loosely
bonded strands will wash off. The resultant double strands can be measured by
chemiluminescence. The intensity of the fluorescent signal is dependent on the amount of
9

DNA strands present at that site. Thus, using DNA probes of known species, the relative
amounts of those particular species present can be determined.
Haffajee et al. (1998) performed checkerboard DNA-DNA hybridization on
periodontally healthy, well maintained and adult periodontitis patient plaque samples.
Using DNA probes for 40 subgingival species, they were able to detect the relative
amounts of those species above a certain threshold present in the given samples. In this
study, they reported a sensitivity of 10
4
cells of a given species. The main limitations of
this method are that it requires the fabrication of probes from cultivable species only, and
it requires a threshold amount of DNA from any given species present in the sample.
Additionally, each assay is limited by the amount of probes that can be placed on a single
checkerboard.
Using conventional culture-based detection methods, a limited number of
subgingival bacterial species or phylotypes have been indicated as major etiologic agents
of periodontitis. Despite the wealth of information gained from these culture-based
studies, however, it is estimated that less than half of the species of the oral cavity have
been isolated by in vitro culture. Thus, there is a significant need to identify the
remaining uncharacterized species of oral bacteria. More recently, advancements in
microbial research, specifically in culture-independent studies, have allowed for closer
analyses of these complexes of bacteria.

10

Metagenomics
More recently, metagenomic analysis has become one of the most widely used
culture-independent methods for microbial analysis. Metagenomics has addressed some
of the remaining gaps of knowledge about the subgingival microbial community in
periodontal health and disease. Using this method, researchers have shown that there are
over 700 species of bacteria in the oral cavity, 400 of which can be found in the
periodontal pocket, with each individual harboring approximately 100-200 species
(Paster et al., 2006).
Metagenomics is the direct extraction of DNA from intact communities of
organisms in their natural environment rather than as individual species (Tringe & Rubin,
2005). Metagenomic analysis is a culture-independent approach in which genomic DNA
is extracted and amplified for the 16S rRNA gene, a highly conserved sequence amongst
all cellular organisms, and, therefore, a commonly used phylogenetic marker. The 16S
rRNA gene is part of the small (30s) subunit prokaryotic ribosome present in all
prokaryotes, which, between highly conserved sequences, contains small highly variable,
unique regions (Woese, 1977; Woese 1987).
Using PCR and specifically designed primers to amplify both conserved and
variable sequences, the intervening unique sequences can be used to readily distinguish
different species within a complex. Each hypervariable region has a distinctive specificity
that can be used to preferentially target different classes of bacteria (table 1). Thus, it can
provide not only information about each species present, but also conceivably give an
idea of the different species interaction (Tringe et al. 2005).
11

Table 1. Discriminative power among variable regions of 16S rRNA genes
Adapted from Chakravorty et al, 2007.

The process of metagenomic analysis begins with the acquisition of an intact
microbial sample taken directly from its natural ecosystem. In periodontal work,
subgingival plaque samples can be obtained using the same paper point method as with
traditional culture-based microbial sampling. DNA is then extracted from the sample by
lysis of the cell wall and precipitation of the DNA. This can be routinely performed using
a number of manufacturer prefabricated kits.
Next, the DNA undergoes PCR amplification using primers designed to target one
of the nine conserved regions, designated c(1-9), of the 16S rRNA gene. Interspersed
between each of the nine conserved regions are the nine hypervariable regions,
designated v(1-9), with each region typically ranging from 50-100 base pairs (Petrosino,
2009). Therefore, depending on the number of bases sequenced, multiple conserved and
variable regions can be analyzed. Multiple forward and reverse primers can be used to
target several regions at once, and likewise, multiple samples can be combined and
distinguished using bar-coded reverse primers.
12

Once the targeted 16S rRNA regions are annealed and amplified, the PCR
amplicons can be used for analysis using a variety of methods, including terminal
restriction fragment length polymorphism (T-RFLP) analysis, denaturing gradient gel
electrophoresis (DGGE), cloning and sequencing, microarray analysis, and most recently,
pyrosequencing analysis. Each method has been used extensively and each presents with
its own advantages and limitations. Combined, these techniques have enabled further
advancement in the identification of bacterial species contributions to periodontal health
and disease.
Denaturing gradient gel electrophoresis (DGGE) is a method by which same-
length DNA strands, such as those from 16S rDNA amplification, are placed into an
electrophoresis gel and subjected to a series of increasingly higher concentrations of
chemical denaturing agents. Denaturation of the strands at sites specific to the DNA
sequence dramatically decreases the migration rate of the DNA fragments at specific
points along the gel. The resultant banding patterns can then be used to identify distinct
species present.
Zijnge et al. (2006) evaluated the efficacy of using DGGE as a diagnostic tool in
periodontal microbiology, and showed that using DGGE, multiple samples could be
examined simultaneously to check for a limited number of species, in particular for this
study, A. actinomycetemcomitans, P. gingivalis, P. intermedia and T. forsythia. DGGE is
limited, however, in that only a few species can be tested for in one run. Additionally, it
requires that the banding profiles of different species be known prior to examination;
thus, unknown or only recently identified species are less likely to be examined.
13

Terminal restriction fragment length polymorphism (T-RFLP) is a similar, but
alternative molecular approach in which fluorescence-labeled PCR amplicons are
digested with restriction enzymes that cut DNA at specific recognition nucleotide
sequences at the labeled end (Liu et al. 1997). The labeled terminal fragments are then
separated by fragment length by use of polyarylamide gels. The varying fluorescence
released by the fragments is then measured so that an electrocephelogram outputs the
different lengths and their respective amounts. Using a clonal library, the different
lengths can indicate which species type are present and in what relative abundance. To
overcome the limitation of identical terminal fragment lengths amongst some species,
several restrictive enzymes can be used on one sample to provide a more unique
fingerprint of each species present.
Sakamoto et al. (2004) used T-RFLP analysis of 16S rRNA genes from samples
before and after scaling and root planing therapy in three subjects with periodontitis. In
one patient, the investigators identified 47 species, present either before or after
treatment. As with DGGE, T-RFLP also requires that the fragment length profiles of
different species be known prior to examination; thus, as before, unknown or only
recently identified species are less likely to be examined. Additionally, the number of
clones and species retrieved per subject are still well below the 100-200 species believed
to be present in each individual.
Cloning and sequencing is a method by which the resultant 16S rRNA gene
amplicons are cloned into E. coli, cultured on agar plates, purified and subsequently
sequenced. Paster et al. (2001) and Aas et al. (2005) cloned 16S rDNA amplicons into E.
14

coli and subsequently sequenced the 16s rDNA inserts using an ABI sequencer.
Approximately 50 to 100 clones of 500 base pairs each were available per subject in the
study by Paster et al. (2001) and 42 to 69 clones per subject in the study by Aas et al.
(2005).
While the use of the ABI sequencer bypasses some of the limitations of using
DGGE and T-RFLP, cloning and sequencing is still limited by the amount of clones
retrieved per subject. Even if each clone from one subject represents a different species,
the number of species detected per patient is still below the estimated 100-200 species
present per individual. Therefore, if more species are present in the patient, as has been
conjectured, they could go undetected.
The application of microarray multiplex technology to metagenomics has greatly
increased the amount of information obtainable per any given subject or sample. A
microarray chip can consist of potentially thousands of DNA probes on a solid surface
chip. Similar to DNA-DNA hybridization in which labeled DNA samples hybridize to
known DNA probes and are measured by chemiluminescence, labeled 16S rDNA PCR
amplicons hybridize to the 16S rRNA-based oligonucleotide reverse-capture
hybridization probes on the surface of the microarray chip (Colombo et al., 2009).
Colombo et al. (2009), using a microarray chip based on over 300 bacterial taxa
from the Human Oral Microbiome Database (HOMD), called the Human Oral Microbe
Identification Microarray (HOMIM), analyzed the subgingival plaque samples of 67
patients. A total of 400 probes were used to assess over 300 bacterial taxa, including
universal probes for positive controls as well as probes for negative controls. The absence
15

or proportion presence of a given species was determined by the different measured band
intensities on a 5 point scale from 0 to 5+.
Currently, however, the HOMIM technology is available only at the Forsyth
Institute in Cambridge, Massachusetts. Additionally, it is offered only for the purposes of
research and not for diagnostic purposes.

Etiology revisited
Utilizing 16S rRNA gene metagenomic analysis, researchers have identified a
growing number of previously unknown oral microbial species. The studies further
suggest the existence of lesser known but sometimes frequently found potentially
pathogenic and health-associated periodontal species. Some of the species from three of
the previously mentioned studies, Paster et al. (2001), Aas et al. (2005), and Colombo et
al. (2009), are mentioned below.
Using 16s rRNA gene cloning and sequencing, Paster et al. (2001) detected
significant trends in species present in disease. In addition to the more well known
putative pathogens, less commonly identified species associated with diseased were
identified. These included Eubacterium saphenum, Filifactor alocis, Catonella morbid,
Megasphaera sp., Dialister sp., Streptococcus constellatus, Fusobacterium animalis, and
Selenomonas sputigena. In total, the researches detected 347 species in their sample
population. Using estimation calculations, they predicted that approximately 68
additional unseen species were present, thus suggesting that the best estimate of total
species diversity in the oral cavity is between 500-600 species, with 415 subgingival
16

species. In this study 215 phylotypes were identified, 33 of which were cultivable but not
yet characterized species and the remaining 182 were represented only by clones.
Similarly, Aas et al. (2005), using 16S rRNA gene cloned inserts and ABI
sequencing on a patient population of five periodontally healthy subjects, noted
significant species trends. Of the most commonly found subgingival species
Streptococcus mitis, S. sanguinis, S. gordonii, S. intermedius, Gemella sanguinis,
Gemella haemolysans, Abiotropha defectiva, Granulicatella elegans, Granulicatella
adiacens, Rothia dentiocariosa, Camplyobacter gracilis and Prevotella melaninogenica
predominated.
Colombo et al. (2009), using the HOMIM microarray chip, looked at the
subgingival plaque samples of 47 periodontitis patients after non-surgical and surgical
therapy, those that responded well (n=30) and those who appeared to have recurrent
periodontitis (n=17), and 20 periodontally healthy controls. More unusual species
associated with recurrent periodontitis included Selenomonas infelix, Selenomonas spp.,
Fusobacterium naviform, F. alocis, Dialister invisus, Dialister pneumosintes, G.
adiacens, E. saphenum, Veillonella atypica, Desulfobulbus sp., G. sanguinis, and P.
stomatis. Of the species more commonly detected in periodontally healthy controls, less
common species included Capnocytophaga sputigena, Cardiobacterium hominis, R.
dentiocariosa, R. mucilaginosa, Neisseria elongata, Kingella oralis and G. elegans.

17

Pyrosequencing
In addition to the methods previously mentioned, another approach which takes
advantage of metagenomic 16S rRNA genes for species identification and quantification
is DNA sequencing by high throughput pyrosequencing analysis. Pyrosequencing is a
relatively new method developed in 1996 in which relatively large scale segments of
DNA are sequenced by machine. Previously termed sequencing by synthesis,
pyrosequencing is a DNA sequencing approach in which nucleotide incorporation during
DNA extension is detected by luminescence (Ronaghi, 1996).
Single strands of DNA are immobilized onto specifically designed DNA capture
beads. An excess amount of beads are added to over-saturate the reaction so that for any
given bead, a single unique strand of DNA is bound. Each bead is then processed in a
single well containing its own emulsification of enzymes. Thus, while hundreds of
thousands of beads are run in parallel per a 10-hour run, each strand is replicated in
exclusion from all other potentially contaminating or competing strands.
Additionally, as different DNA nucleotides are added and either rejected or
integrated into the growing strand, the enzyme apyrase degrades all excess nucleotides
after each addition. Apyrase thereby prevents the erroneous addition of non-
complementary nucleotides, and it facilitates the continuous addition of further
nucleotides without the necessity of constantly adding new enzymes to the system (Agah
et al, 2004). Combined, all of these measures allow for high quality, high throughput
processing.
18

In order to then determine the sequence of the nucleotides added, this technique
uses two enzymes, ATP sulfurylase and luciferase, to take advantage of the release of
inorganic pyrophosphate (PPi). For each round of DNA nucleotide integration into the
growing strand, PPi is released in an amount equal to the molarity of the specific
nucleotide added. The resulting PPi is converted to ATP and then to light by ATP
sulfurylase and luciferase, respectively. The light released is captured by chip and traced
onto a Pyrogram output (Petrosino et al, 2009). The height of each peak is proportional to
intensity of light which is determined by the amount of nucleotide incorporated into the
growing strand. Consequently, the nucleotide sequence can then be determined from the
nucleic acids added and their resulting signal peaks (Figure 1).

Figure 1. Pyrosequencing
As nucleotides are added, light is produced by the luciferase reaction and is then detected by the
program. The height of each light peak is proportional to the nucleotides incorporated.
Adapted from Petrosino et al, 2009.

This sequencing approach has been used both in the dental and medical fields in
recent years. Vickerman et al. (2007) observed that infected root canals had a more
diverse flora than previously believed. Aas (2008) stated that nearly half of all bacteria
19

from caries lesions were yet to be identified. In 2005, medical researchers studying the
intestinal flora found that approximately 62% of bacteria identified from the human
intestine were previously unknown species and that 80% of the bacteria identified were
non-cultivable species.
While there exist some degree of variation between the different pyrosequencing
systems, the fundamental principles remain the same. The method stated above follows
the 454 Life Sciences technology. Founded in 2000, 454 Life Sciences, a division of
Roche Applied Science, manufactures and commercializes Genome Sequencer machines
for pyrosequencing. The first generation pyrosequencing platform GS20, developed in
2005, yielded a mere 100 base pair reads for 30-60Mb/run. The second generation 454
FLX standard platform released in 2006 yields 250 base pair reads, approximately
150Mb/run, and the most recent, third generation 454 FLX titanium platform, updated in
2008, yields more than 400 base pair reads, approximately 400Mb/run. Both the 454 FLX
standard and titanium platforms yield sufficiently long reads to encompass multiple
hypervariable regions. The newest edition, to be expected soon, is said to potentially
generate read lengths approaching 1000 bases.

Sequence analysis
The resulting DNA sequences from the pyrosequencing analysis can then be
uploaded to a phylogeny-based database containing 16s rRNA gene sequences to help
identify species/phylotypes. The Ribosomal Database Project (RDP) is a web-based
database that allows for in-depth analysis of multiple, ultra-high-throughput, raw
20

sequence rRNA reads (Cole, et al. 2008). The RDP version 10, released in 2008, is
updated monthly from the International Nucleotide Sequence Database Collaboration and
currently contains nearly 1.5 million unique 16S rRNA gene reads. The RDP 10 is
currently on update 24, released on the RDP website on January 13, 2011
(http://rdp.cme.msu.edu).
The Human Oral Microbiome Database (HOMD) is another web accessible
database of 16s rRNA gene sequences (www.homd.org). The NIDCR supported database
launched in 2008 has currently identified 619 taxa under 13 phyla (Dewhirst, et al. 2010).
Pending validation, the database is expected to include another 434 novel named and
unnamed taxa. Output from both of these databases details the species present and the
corresponding quantity of sequence-matched clones.

Real-time PCR
Developed in 1983, polymerase chain reaction (PCR) is a novel technology which
has revolutionized molecular biology. It allows for the amplification and analysis of even
the most minute amounts of DNA. Through thermo-regulated cycling, a single copy of
DNA can be exponentially replicated to thousands or even millions of copies. Briefly,
double stranded DNA is denatured by heat and then rapidly cooled. Primers designed to
target specific sequences anneal to each strand, and using a heat tolerant DNA
polymerase enzyme such as Taq polymerase, replication occurs along the desired
sequence. Since the product of each cycle can be used as a template for the next, multiple
cycles of replication result in exponential amplification of the desired product.
21

More recently, real-time PCR was developed as a modification of the PCR
method. It is a technique in which the amount of DNA products generated is continuously
measured throughout each cycle of the PCR amplification process. While traditional PCR
is designed for measurement of the final product (endpoint detection), real-time PCR
allows for the quantification of the absolute or relative amounts of DNA initially present
in a sample.
The basis of real-time PCR is the quantification of DNA replication by means of
fluorescent signaling detection (Heid et al. 1996). Following the standard PCR protocol,
real-time PCR works with the addition of a fluorescent labeled probe. During the PCR
elongation process catalyzed by the Taq polymerase, the labeled probe is digested and the
fluorescent tag is cleaved thus releasing a fluorescent signal (Nadkarni et al 2002). For
each additional strand replicated, the degree of fluorescence increases. With each cycle,
the fluorescence is captured and plotted on a graph.
In theory, the amount of DNA doubles with every cycle of PCR, but as the
amount of reagents is limited, the reaction will eventually plateau. When the fluorescence
is plotted on a logarithmic scale, a straight line indicates the exponential phase of the
reaction. It is this linear region that can be used to measure the amount of DNA. By
setting a threshold line that passes through the linear phase of each line of the dilution
curve as well as the unknown sample, the threshold value (C
T
), or the point at which the
fluorescence crosses the threshold, can be measured. By then comparing the C
T
value of
the unknown sample to the threshold values of a standardized curve of known
22

concentrations, the rate of increase in fluorescent signaling can allow for the calculation
of the starting amounts of DNA.
Following the PCR synthesis and amplification cycles, the reaction is brought
down to a low temperature and slowly increased to determine the melting point of the
products present. Because DNA strands melt at varying temperatures dependant on the
length of the strand and base composition (GC content), melting temperature profiles can
be used to identify products and confirm uniform genotypes. All pcr products from a
particular primer pair should have the same melting temperature. Disparities would
indicate contamination, e.g. primer-dimers. At the appropriate melting temperature, the
double stranded DNA will separate and since the SYBR green fluoresces much stronger
with double stranded DNA than single stranded DNA, the drop in fluorescence will be
captured by the camera.
To further assure the reliability of the PCR amplification, samples and standards
are run in triplicate and mean values are calculated (Nadkarni et al. 2002). A standard
curve is obtained by using 10-fold serial dilutions of positive controls. To generate a
standard curve, the threshold cycle values of the standard dilution are plotted against the
known DNA concentrations.
Several studies have applied qPCR technology to the quantification of oral
microbial species. Among them, Lyons et al. (2000) used real-time PCR to determine the
amount of P. gingivalis and total bacteria in a series of human plaque samples. Using a
species-specific primer for P. gingivalis amplification and a universal primer for total
bacterial counts, they were able to calculate the total number of bacterial cells present and
23

the percentage of P. gingivalis in the entire sample. Nonnemacher et al. (2004) used real-
time PCR to quantify the levels of total bacteria and the pathogens A.
actinomycetemcomitans, P. gingivalis, P. intermedia, Dialister pneumosintes, and P.
micros in the subgingival plaque samples from periodontitis and periodontally healthy
patients. The method proved to have accurate sensitivity and specificity and was able to
provide a quantification range of 10
2
to 10
8
, thus providing a reliable and valuable
method for quantification.
In this study, real-time PCR was used as the standard by which to verify the
quantitative changes observed in P. gingivalis from both patients before and after
treatment using metagenomic analyses. Subgingival plaque samples were thus divided for
use in metagenomic analysis and high throughput sequencing as well as for real-time
PCR verification.

Objective & Hypothesis
Using conventional culture-based detection methods, approximately half of the
subgingival bacterial species have been identified, and of those, a limited number have
been indicated as major etiologic agents of periodontal disease. More recently, culture
independent metagenomic analysis has addressed some of the remaining gaps of
knowledge about the subgingival microbial community in periodontal health and disease.
Thus far, metagenomic studies have presented a static snapshot of the microbial flora
associated with different periodontal diseases and conditions. However, there is currently
insufficient data available to describe the microbial changes over time or following
24

treatment. The objective of this study is to examine the subgingival microbial community
in subjects diagnosed with aggressive periodontitis before and after nonsurgical initial
phase periodontal therapy. This is also intended as a pilot study to examine the feasibility
of metagenomic analysis and high throughput sequencing for the microbial analysis of
periodontal bacteria in clinical practice.

25

Chapter 2: Materials and Methods

Subject population
Two subjects with generalized aggressive periodontitis were recruited from the
graduate clinic of the Department of Periodontology of the Herman Ostrow School of
Dentistry of the University of Southern California. The ethics committee of the
University of Southern California approved the protocol. Both subjects signed an
informed consent prior to their enrollment in the study. A diagnosis of aggressive
periodontitis was determined based on the American Academy of Periodontology (AAP)
parameters. Generalized aggressive periodontitis patients had generalized interproximal
attachment loss of at least 3 permanent teeth not first molars and incisors due to
periodontitis (not orthodontic therapy or tooth brushing trauma). The subjects did not
have any systemic conditions that might contribute to their periodontal condition (ie
uncontrolled diabetes), nor had taken antibiotics within the past year for any reason, nor
had had any non-surgical or surgical periodontal therapy in the past year.

Clinical exam and treatment
A single clinician measured clinical periodontal parameters, delivered treatment
and obtained subgingival plaque samples. Clinical parameters measured were probing
depth (PD), bleeding on probing (BOP), gingival recession (GR) and attachment level
(CAL) on six sites per tooth for all remaining teeth. Both patients received Amoxicillin
26

(500 mg) and Metronidazole (500 mg) TID for 8 days in conjunction with full mouth
scaling and root planning completed over 2 visits.

Sampling method
Subgingival plaque samples were obtained at baseline and at the 8-week follow-
up so that a total of 2 plaque samples before treatment and 2 plaque samples after
treatment were analyzed. Samples were taken from both maxillary first molars, 2-3 sites
per tooth, each with initial probing depths ≥5 mm. Sampling sites were isolated using
cotton rolls after all supragingival plaque and calculus were removed using sterile Gracey
curettes. Teeth were air dried and a total of 5 paper points were inserted into the depth of
the pocket for 30 seconds. The paper points were then packed into one microcentrifuge
tube per patient containing 1.5 mL phosphate buffer solution (PBS). The samples were
vortexed to release the subgingival bacteria and stored at -70°C until ready for
processing.

16S rDNA based PCR amplification & 454 Pyrosequencing
DNA from plaque samples were isolated and purified using the Qiamp DNA
Mini-kit (Qiagen) according to the manufacturer‟s instructions. Briefly, bacteria were
pelleted by centrifugation and then suspended in Buffer ATL (all buffers provided by the
manufacturer). Twenty microliters of Proteinase K were added, and the mixture was
vortexed and incubated at 56°C on a rocking platform in a water bath. Buffer AL was
added, vortexed and incubated at 70°C for 10 minutes. Ethanol was then added, vortexed
27

and again centrifuged. The mixture was added to the QIAamp mini-spin column and
centrifuged for 1 minute and the eluate was discarded. The column was then transferred
into a clean 2 mL collection tube. After performing sequential rinses with Buffer AW1
and Buffer AW2, the column was placed into a new 2 mL collection tube and the eluent
Buffer AE was added. The final eluate was collected and stored at -70°C. The
concentration was determined by UV spectrometer (abs 260nm) and purity was
determined by the A260/A280 ratio.
PCR primers were designed to anneal to the conserved C2 and C4 regions so that
products would include the v4 regions (table 2). The final working concentration of the
combined forward primers was 2.0 uM and the reverse primer, 4.0 uM. Samples were
pooled for a ¼ plate on a single 454 FLX standard platform run. Barcoded primer tags
were designed to distinguish samples using barcode-linker bases attached to the reverse
primers. Using the Stratagene PFUTurbo hotstart kit, the primers were added to the
PFUTurbo hotstart DNA polymerase, dNTPs, and final 10x reaction buffer, all provided
by the manufacturer. After adding the DNA samples, the PCR cycler was set so that 30
cycles of melting at 95°C for 30 seconds, annealing at 55°C for 40 seconds and extension
at 72°C for 1 minute were performed. The final extension was at 72°C for 10 minutes.
The products of the 16S rRNA amplification were pooled and run on ¼ of a plate on the
454 sequencing FLX standard platform. The sequencing was performed from one end of
the amplicons for the hypervariable v4 region of the 16S rDNA.
28

Table 2. Primer sequences
The 3 forward primers are mixed in a 3:1:1 ratio and used with one of the reverse primers
(distinguishable by barcodes) for each sample.

Sequence analysis
Sequencing data were analyzed using the LibCompare and Pyrosequencing
pipelines from the Ribosomal Database Project (RDP) website (http://rdp.cme.msu.edu).
Sequences were sorted and designated according to the respective barcodes assigned to
each sample. A classifier checked for the proper orientation of the sequence and possible
chimeras. Assignments were made above a user-specified confidence threshold and
results were listed in hierarchy views which showed the summary of the assignments and
their respective significance values. Post processing was performed to assign sequences
to the species level when possible. These results were then crosschecked with the NIH-
supported Human Oral Microbiome Database (HOMD) project (http://www.homd.org).

29

Real-time PCR
The amounts of P. gingivalis and the total bacteria in each sample were
determined by real-time PCR using the protocols suggested by the manufacturer. Briefly,
2 µl of sample were added to 12.5 µl 2X Bio-Rad iQ SYBR Green supermix for real-time
PCR, which contained each primer at a concentration of 300 nM. Initial incubation was
set at 95°C for 10 minutes followed by 45 cycles of denaturation at 95°C, annealing for 5
seconds (at a primer-specific temperature, 52°/60°C) and amplification at 72°C for x sec
(where x equals the product size/25, 17s/30s; table 3). Fluorescent products were
captured by camera after annealing for each cycle. A melting curve analysis was
performed to determine the specificity of the PCR products. For the melting curve, the
final DNA products were denatured at 95°C for 1 minute and then incubated at 5° below
the annealing temperature for 1 minute before the temperature was increased to 95°C at a
ramp rate of 0.5°/10 seconds.

Primer Target Sequence Size
Ann
temp
Amp
time
Universal-
16S -733F/R
Forward: 5‟-GAT TAG ATA CCC TGG TAG TCC AC-3‟
733bp 52°C 30s
Reverse: 5‟-TAC CTT GTT ACG ACT T-3‟
P. gingivalis-
16S - 404F/R
Forward: 5‟-AGG CAG CTT GCC ATA CTG CG -3‟
404bp 60°C 17s
Reverse: 5‟-ACT GTT AGC AAC TAC CGA TGT-3‟

Table 3. Primer sequences for real-time PCR. Obtained from Kirakodu et al, 2008.

30

Ten-fold dilutions of the pure DNA (from 0.001 to 100 ng in 2 µl ) of P.
gingivalis-ATCC-33277 genomic DNA were used to generate a standard curve. Two
standard curves were generated: one for the quantification of P. gingivalis using species-
specific 16S rDNA primers, the other for the quantification of total bacteria using
universal primers. Linear regressions were obtained by plotting the Log10 (ng input
DNA) against the CT (cycle threshold) value. The slope of each standard curve was used
to determine the reaction efficiency by the following equation: efficiency=10-1/slope - 1.
Using the linear regression obtained by the standard curve, the amount of DNA in the
unknown samples was calculated. All pure DNA and unknown samples were run in
triplicate to confirm the reproducibility of the data. CT values were then averaged for
each triplicate.

31

Chapter 3: Results

Clinical Observations
Both patients showed significant clinical improvements (Figure 2). At teeth #3
and #14, where samples were obtained, an average reduction of 2 mm was observed at all
surfaces initially probing ≥5 mm. Following treatment, there was a decrease in bleeding
on probing (BOP) and number of deep, ≥6mm periodontal pockets in both patients.
Additionally, there was an increase in shallow, 1-3mm pockets in patient D119. Patient
D114 initially presented with 24 sites with pre-treatment periodontal probing depths
greater than or equal to 7mm. After treatment, the number of sites reduced to 15 sites. In
patient D119, of 30 teeth (one 3
rd
molar bony impaction), 17 sites were recorded with
PD≥7mm. After treatment, the number of sites reduced to 9 sites.

A

B

Figure 2. Full-mouth clinical changes
Bleeding on probing (BOP) and probing depth (PD) noted in patients D114 (a) and D119 (b) after
non-surgical periodontal therapy.

D114

D119
32

Pyrosequencing
The sequencing of the amplicons resulted in 24,673 reads with an average length
of 252 base pairs. Among these reads 22,852 reads were of sufficient quality for
species/phylotypes identification, and included 5,820 (122 species/phyotypes) and 5,946
(133 species/phylotypes) reads from patient D114 before and after treatment, and 5,994
(133 species/phylotypes) and 5,092 (100 species/phylotypes) reads from patient D119
before and after treatment. In total the analysis identified 208 species/phylotypes; 168
were found in D114 and 171 in D119. Of those, both patients presented with the same
129 species. While a majority of the species/phylotypes were detected in both patients
(before or after treatment) their proportions varied between patients (Figure 3).

A

B

Figure 3. Comparison of the subgingival microbiota between patients before (a) and after (b)
treatment
Each dot represents the number of a specific sequencing read (matched to species/phylotype)
identified in the subgingival samples. A total of 121 species/phylotypes were shown in the
analysis after data were filtered to include only reads that reached a count of >10 in at least one of
the 4 samples.
33

While more than120 species/phylotypes were identified in all samples, 85-90% of
the species/phylotypes were represented by less than 100 sequencing reads (≤0.2% of the
total reads). Such unevenness (i.e. a profile dominated by a few species/phylotypes) is
also illustrated in the cumulative percent of total reads in Figure 4. Fifty percent of the
total reads was accounted for by 6 species/phylotypes in patient D114 before treatment
and 42 species/phylotypes after treatment. The same amount was accounted for by 6
species/phylotypes before and after treatment in patient D119. Similarly, 90% of the total
reads, were accounted for by 28 species/phylotypes in D114 before treatment and 42
species/phylotypes after treatment. In patient D119, 36 species/phylotypes before and 32
species/phylotypes after treatment accounted for 90% of the total reads.

34

A

C

B

d

Figure 4. Cumulative % of total reads with each addition of ranked species/phylotypes
Each species added in order from highest to lowest read counts. (a) & (b), D114 before and after
treatment; (c) & (d), D119 before and after treatment.

Changes in species/phylotypes after treatment
Figure 5 shows the log
2
(fold change) for the 121 commonly found
species/phylotypes in the subjects after treatment. While significant microbial changes
occurred after periodontal therapy, there were no consistent patterns in the levels of
changes between subjects. A more detailed analysis of the changes for the 27 most
35

frequent species before and after periodontal treatment is presented in Table 4. In general,
half of the species/phylotypes increased while the other half decreased by more than 2-
fold after treatment. Red complex bacterial species (P. gingivalis, T. denticola and T.
forsythia) were among the putative pathogenic species that showed the greatest decrease
after treatment. In contrast, several Gram-positive Streptococcus, Rothia, Actinomyces
and the Gram-negative Veillonella were among the bacteria that showed the greatest
increases after treatment.

Figure 5. Comparison of quantitative changes in the subgingival microbiota after treatment
between patients
Each dot represents the log
2
(fold change) of a species/phylotype in D114 (x-axis) and D119 (y-
axis).

36

Table 4. Proportion of the 27 most frequently present subgingival species/phylotypes before and
after treatment
*For non-detectable bacterial species (0%) 1 sequence read was used to calculate fold-change

37

Real-time PCR
The standard curve for the real-time PCR analysis of P. gingivalis is shown in
Figure 6. The linear regressions based on the serial dilutions using both the P. gingivalis-
specific primer and the universal primer had a correlation coefficient (R
2
) of 0.99 with a
linear range of 6 orders, and the efficiency of the reactions was 99%. The melting curve
analyses showed a clear melting peak and no formation of unspecific products (data not
shown). Similarly, the linear regression generated from the standard curve using the
universal primers was used to calculate the total amounts of bacterial DNA in the
samples. Table 5 shows the Ct values of P. gingivalis and total bacteria and the derived %
P. gingivalis in each sample.
The change in the levels of P. gingivalis after treatment in patient D114 was
23.27% to 1.88% and in patient D119 was 11.2% to 0.01%. The magnitude of the change
seen by real-time PCR were comparable to those calculated from sequencing reads which
for patient D114 was 11.92% to 5.3% and for patient D119 was 13.56% to 0.12% for
D119 (figure 7).

38

Log
10
(ng input genomic DNA)

Figure 6. Real-time PCR for P. gingivalis using 16S rDNA specific primers
A standard curve was built by plotting the log input of the DNA (0.0014-140ng) against the CT
value. The regression line from the standard curve was used to determine the P. gingivalis
concentration of the unknown samples. All standard dilutions were assayed in triplicates, each
point representing the average value.

Primer
Patient D114 Patient D119
Mean
Ct
[Pg]
% P.
gingivalis
Mean
Ct
[Pg]
% P.
gingivalis
Before Tx
Pg-
specific
20.66 2.51
23.27%
17.91 16.54
11.19%
Universal 21.72 10.77 17.77 147.81
After Tx
Pg-
specific
23.59 0.34
1.88%
27.23 0.03
0.01%
Universal 20.92 17.87 17.16 204.35

Table 5. Real-time PCR for P. gingivalis using 16S universal primers

39

Figure 7: Comparison of metagenomics and real-time PCR proportions of P. gingivalis

40

Chapter 4: Discussion

This study provides an opportunity to examine patient- and sites-specific
subgingival microbiota and their changes after treatment. The results show that the
subgingival microbiota are high in species richness and dominated by a few species.
Significant shifts of microbiota occurred after treatment. Also, the compositions of the
subgingival microbiota appear to be highly individualized.
It is difficult to compare the results of this study to other metagenomic studies due
to differences in study design, experimental protocols and data interpretation. However,
the observed species richness in the subgingival microbiota is in general agreement with
other studies. Approximately 100 to 130 species/phylotypes were identified in each
sample; the magnitude of species richness is in general agreement with previous studies.
Kroes et al. (1999) examined two sites (teeth #3 and #30) of a single patient and
estimated that a total of 84 phylotypes could be identified in the sites. In this study 6 to
11 species accounted for 50% of the total subgingival bacteria. While there is a lack of
information in the evenness of subgingival microbiota, our results are in general
agreement with the previous estimates.
Pre- and post-therapy samples showed significant quantitative and distributive
species changes. Quantitatively, many known pathogenic species went from easily
detectable to zero counts while the opposite occurred for many known health-associated
bacteria. For example, the red complex species, T. forsythia, P. gingivalis, and T.
denticola, were present in both patients as were other well known virulent pathogens, A.
41

actinomycetemcomitans, P. nigrescens, P. intermedia, and P. micra. In almost all cases,
the counts of these known pathogens decreased significantly, with some species, such as
P. gingivalis, T. forsythia, T. denticola, and P. micra decreasing to almost zero counts.
Conversely, known commensals such as S. sanguinis and K. oralis increased after
treatment, ranging from 2-fold increases up to 100-fold increases.
Other suspected periodontopathogens, as those found in greater abundance in
refractory periodontitis patients by Colombo et al. (2009) and periodontally diseased
patients by Paster et al. (2001), including Fusobacterium naviforme, Dialister invisus,
Filifactor alocis, Eubacterium saphenum, and Desulfobulbus sp. were also found to have
significantly decreased in both patients after treatment. The potentially pathogenic
species Desulfovibrio fairfieldensis observed by Loubonix et al. (2002) decreased
appreciably in both patients while the suspected pathogen observed by Paster et al.
(2001), Fusobacterium nucleatum ss. animalis, surprisingly increased in both patients.
Likewise, other suspected pathogens, Selenomonas infelix and Bacteroides
zoogleoformans (Van Dyke et al., 1988), appreciably increased after treatment. Of the
more recently suspected commensals, Cardiobacterium hominis, Capnocytophaga
sputigena, Campylobacter showae, Corynebacterium matruchotii, and Rothia
muclaginosa (Aas et al., 2005; Colombo et al., 2009) all increased, on average, 30-fold in
patient D114.
The assignments of bacterial species to either pathogen or commensal were not
always consistent in the literature. Aas et al. (2005) found Gemella sanguinis to be part of
the normal subgingival flora of healthy subjects while Colombo et al. (2009) noted it as
42

being more prevalent in refractory periodontitis patients. Similarly, Campylobacter
gracilis and Granulicatella adiacens were deemed pathogenic by Colombo et al. (2009),
while Aas et al. (2005) described them as some of the most predominant subgingival
species found in health. Rothia dentocariosa was described by both Aas et al. (2005) and
Colombo et al. (2009) as being more predominant in health while Paster et al. (2001)
noted it more commonly in disease. In patient D114, counts of Gemella sanguinis,
Granulicatella adiacens, and Rothia dentiocariosa significantly increased after treatment,
as did Camplyobacter gracilis in patient D119, indicating that, as clinical parameters
improved after treatment, perhaps these species may be considered more beneficial than
pathogenic.
Of the more abundant but lesser known species detected in these samples,
associations with other environments or diseases have been noted. Pseudomonas
pseudoalcaligenes, the fourth most prevalent species both before and after treatment in
patient D114, can be found in environmental literature in reference to cyanide and
metalwork. Leptotrichia sp., present in significantly large numbers after treatment in both
patients, has been frequently studied with regard to the cervicovaginal microflora (Ling et
al., 2011). Chloroflexi can be found most commonly in the literature regarding
wastewater treatment sludge (Bjornsson et al., 2002). The Lachnospiraceae spp., which
generally increased after treatment, have previously been associated with caries-free
children (Kanasi et al., 2010). Cardiobacterium hominis is the third species of the
HACEK organisms which, along with Haemophilus spp., Aggregatibacter spp., E.
43

corrodens, and Kingella spp., is a group of oropharyngeal organisms frequently believed
to cause endocarditis in young children (Colombo et al., 2009).
Based on the published data regarding the 619 taxa represented in the HOMD
version 10 (Chen et al., 2010), several of the “species” identified by the present analysis
were, at the time of publication, still uncultured and unnamed bacteria. More specifically,
the database library contains 47% validly named species, 18% cultured but unnamed, and
35% unnamed and uncultured phylotpyes known primarily from 16S rRNA sequence
data. Of these uncultured and unnamed taxa, Treponema sp. was the third most abundant
species in patient D119 before treatment and, was completely eliminated after treatment.
The taxa Synergistetes [G-3] sp. was the fourth most abundant species in patient D114
before treatment and also significantly decreased after treatment. Present in both patients
D114 and D119 was the seventh and fifth most abundant species after treatment, the taxa
Leptotrichia sp.
Of the completely uncultured and unnamed phyla TM7 and Chloroflexi, the taxa
TM7 [G-1] sp. and Chloroflexi [G-1] sp. were both present, TM7 [G-1] sp.increased with
treatment in patient D119 but decreased in patient D114 and Chloroflexi [G-1] sp.
decreased to zero in patient D119 after treatment. Lastly, the taxa Desulfobulbus sp.,
observed to be potentially pathogenic by Colombo et al. (2009) was the 13
th
most
abundant species in patient D119 and decreased to zero after treatment.

44

Limitations
Metagenomics operates under certain assumptions which in reality may or may
not be completely met (Parahitayawa et al., 2009). First, it is assumed that the 16S rRNA
primer amplification is completely unbiased. The second assumption is that enough
genomic DNA can be recovered from a clinical sample, and third, it assumes that the
database sequences to which the unknown sequences are compared are accurate.
The question of primer and amplification bias has more than one implication. To
address the first assumption, in this study we compared the metagenomics data with that
from a real-time PCR for the percent change in P. gingivalis after treatment in both
patients. If the 16S rRNA amplification was biased towards certain genera (Kroes et al.,
1999), we would expect there to be differing numbers regarding the changes noted in P.
gingivalis, either it being present in greater numbers or in lesser numbers than what
would be found with the RT-PCR. However, in this study, the changes noted in both
patients were consistent using the metagenomics data as compared with that from the RT-
PCR using different sets of primers. While the possibility of a bias cannot be ruled out,
the comparison with the RT-PCR supports the belief that the 16S rRNA amplification
was not biased.
Several authors have published universal primer sequences to exploit distinct
conserved regions of the 16s rRNA gene. However, in a review of the 16s rRNA gene of
E. coli, Baker et al. (2003) noted that, in fact, few totally conserved sequences actually
exist. Within a sample of 500 bases of E. coli, roughly 10% of the bases can be
considered totally conserved amongst all species. Furthermore, continuous sequences of
45

those totally conserved bases are typically less than 4 sequential bases. While this means
that no primer of sufficient length could be designed as a “100% match universal
primer,” the majority of conserved sequences that are commonly exploited show some
variability between species, and evidence shows that a 70% identity is sufficient for
successful amplification and identification of distinct species. Thus, while a universal
primer has itself some innate variability, it is more likely that the sequence similarity in
the conserved regions and sequence difference in the hypervariable regions is sufficient
to identify and differentiate species.
Concerning the second assumption, while no specific guideline has been
determined for microbial sampling, the placement of medium-sized ISO 45 endodontic
paper points into the sulcus for a minimum of 10 seconds and up to 60 seconds, has been
shown to be the most efficient method of microbial sampling (Hartroth et al., 1999).
While the use of curettes has been indicated due to the greater amounts obtained per
sampling site, Kiel & Lang (1983) showed that the percentage of viable organisms per
sample was significantly higher when using paperpoints. In this study, over 160 species
were detected in both patients which is consistent with the estimate that each individual
harbors approximately 100 to 200 species of bacteria in the oral cavity (Paster et al.,
2006). Given the number of species detected, and that a single species was detectable
from a single pyrosequencing read, it is reasonable to believe that the amount of
recovered DNA from the plaque samples was sufficient for a thorough species analysis.
Lastly, the accuracy of the sequences on the HOMD and RDP databases must be
scrutinized. Phylotypes of the HOMD library are determined by a 98.5% similarity
46

(Dewhirst et al., 2010). Thus, sequences with a greater than 98.5% similarity are grouped
within the same phylotype and merged into single taxa. Conversely, sequences with less
than 98.5% similarity are placed into distinct phylotypes. Thus, the likelihood that a
sequence is incorrectly identified is minimized. Additionally, the HOMD version 10 has
753 reference sequences for 619 phylotypes, indicating that even for specific species,
some sequence variability has been accounted for (Chen et al., 2010). While this database
is constantly being updated, it is possible that some sequences may be re-categorized at a
later date. Therefore, there still remains the possibility that the sequences can be
mistakenly assigned to the wrong species at this time.

47

Chapter 5: Conclusion

There are many advantages to the techniques used in this study. The use of 16S
rDNA amplification facilitates the detection of species previously undetectable by
cultivation-based methods. The use of high-throughput sequencing using the 454 FLX
platform obviates the need for cloning before sequencing. This allows for larger-scale
analyses with a shorter wait time.
In terms of clinical applicability, the method employed in this study can be very
practitioner friendly. While most methods for microbial analysis require a laboratory set-
up with substantial equipment and solutions, the contribution of the clinician in this
situation is as simple as what has been routinely used in practice. The paperpoint
sampling technique used in this study is the same technique that has been used in routine
microbial sampling. From there, samples can be sent to a lab where the turnover rate for
amplification and pyrosequencing can be completed in roughly 2 to 3 days. While the
current costs are still high, approximately $1000/per sample, it is speculated that the costs
could be reduced to $100/sample in the next 5-10 years, thus making it practical for
routine microbial diagnosis in the management of periodontal disease.
In conclusion, this is the first study to our knowledge to use high-throughput
metagenomic analysis to evaluate subgingival microbial changes in conjunction with
clinical changes after non-surgical periodontal treatment. The examined subgingival
microbiota were characterized by high species richness dominated by a few
48

species/phylotypes. The composition of the subginigival microbiota appeared to be
distinct between individuals before and after the treatment. Due to the small sample size
of this study, no conclusions can be made regarding the potentially pathogenic or
commensal nature of these species; however, it is notable that the results from this study
support the current literature. The considerable variety of known and novel
species/phylotypes detected supports further research to better understand the microbial
etiopathogenesis of periodontitis.

49

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Abstract (if available)

Abstract Background: Metagenomics is the sequencing of DNA from whole communities of organisms taken directly from the environment. As opposed to culturing methods which focus on individual species, metagenomics allows for the characterization of intact community genomes, including the identification of noncultivable bacterial species. The objective of this pilot study was to test the feasibility of a metagenomic approach in combination with a novel design of primers to analyze the pre- and post-treatment of subgingival plaque in two aggressive periodontitis patients. ❧ Methods: Periodontal examinations and microbial sampling were performed before and after non-surgical initial phase therapy. DNA was extracted from the subgingival plaque samples and subjected to PCR amplification using primers that amplified the c2-c4 regions of the 16S rDNA. Specific bar codes were included in the primers to identify individual samples. The PCR products were pooled and sequenced for the v4 region of the 16S rDNA using the pyrosequencing 454 FLX standard platform. The results were analyzed for species identification in the Human Oral Microbiome Database (HOMD) and Ribosomal Database Project (RDP) database. The observed proportional changes of the subgingival species after therapy were verified by testing the pre- and post-therapy levels of Porphyromonas gingivalis by real-time PCR. ❧ Results: The sequencing of the amplicons resulted in 24,673 reads and identified 208 species/phylotypes. Of those, 129 species/phylotypes were identified in both patients but their proportions varied. While >120 species/phylotypes were identified in all samples, 85-90% of the species/phylotypes were represented by ≤100 sequencing reads (≤0.2% of the total reads). Similar levels of subgingival P. gingivalis were found based on the numbers of sequencing reads or quantitative Real-time PCR analyses. Pre- and post-therapy samples showed significant quantitative and distributive species changes along with clinical improvements. Quantitatively, many known pathogenic species went from easily detectable to zero counts while the opposite occurred for many known commensal bacteria. Additionally, several novel, pathogenic and health-associated species were found. ❧ Conclusion: High throughput metagenomic analysis is applicable to assess the changes in subgingival microbiota after nonsurgical periodontal therapy in aggressive periodontitis. The examined subgingival microbiota are characterized by high species richness (>120 species/phylotypes) dominated by a few species/phylotypes. The composition of the subgingival microbiota appears to be distinct between individuals before and after the treatment. The considerable variety of known and novel species/phylotypes detected supports further research to better understand the microbial etiopathogenesis of periodontitis.

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Creator Laksmana, Theresia (author)

Core Title Metagenomic analysis of the microbial changes following non-surgical periodontal therapy in aggressive periodontitis

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 06/02/2011

Defense Date 05/24/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aggressive periodontitis,bacterial changes,metagenomics,microbial profile,non-surgical periodontal therapy,OAI-PMH Harvest,periodontal microorganisms,pyrosequencing,real-time pcr

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chen, Casey (committee chair), Nowzari, Hessam (committee member), Rich, Sandra (committee member)

Creator Email TLaksman@usc.edu,TSMLaksmana@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c127-615348

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