Technological advancements in microbial analyses of periodontitis patients: focus on Illumina® sequencing using the Miseq system on the 16s rRNA gene: a clinical and microbial study

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Content TECHNOLOGICAL ADVANCEMENTS IN MICROBIAL ANALYSES OF
PERIODONTITIS PATIENTS:
FOCUS ON ILLUMINA
®
SEQUENCING USING THE MISEQ SYSTEM ON THE
16S RRNA GENE:
A CLINICAL AND MICROBIAL STUDY

By

Chloé Léa Cohen

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 2013

Copyright 2013 Chloé Léa Cohen
ii

DEDICATION
This manuscript is dedicated to my father Charles Cohen and to my mother
Caroline Cohen for being loving, supportive and awe-inspiring. Your support has helped
fulfill my dreams and achieve goals which at times seemed out of reach. I dedicate this
manuscript to my husband Dr. Brian Shachar Nadav for his love and commitment, and
for believing in me and supporting me when I couldn’t find the strength to do it alone,
you are my rock and my inspiration to become a better person with each day that passes.
I also dedicate this manuscript to my brothers Andrew Cohen for his words of
encouragement and support, and to Noah Cohen and Lyam Cohen for giving me a reason
to achieve greatness and serve as their role model. A special dedication to my extended
family, in particular my grandmother Marguerite Lasry and my mother-in-law Mazi
Moshé, and to my close friends who have believed in me and supported me from the
beginning of my academic endeavor to the end.

iii

ACKNOWLEDGEMENTS
Although this has been a long journey full of diligence and commitment, I did not
accomplish this great achievement alone. I am honored to have worked with the great
leaders of microbiology at the Ostrow School of Dentistry of Southern California as well
as San Diego State University and Argonne National Laboratory in Illinois.
First and foremost, I would like to thank my professor and principal investigator
Dr. Jorgen Slots who has pushed me to achieve well beyond what I had expected for
myself throughout the duration of this study. We are truly fortunate to have you as
faculty at the Ostrow School of Dentistry of Southern California, and my experiences as
your student are truly invaluable and undoubtedly will be applicable throughout the
extent of my career. You have made an everlasting scholastic impression on my life that
I will never forget.
Thank you to Dr. Sandra Rich for giving me the guidance and patience to see this
study through to its denouement. Without you I would not have been able to express and
see my ideas through which contributed advantageously to this research. I appreciate our
many discussions and your dedication and contributions devoted to this manuscript.
Thank you to Dr. Scott T. Kelley for spending countless hours educating me on
the fundamentals of microbiology and the principles of microbial sequencing. I
appreciate your patience and your simplified approach to teaching which makes
education exciting; a skill which not many professors possess. Thank you for being
accessible and accommodating, and also for being the liaison between Ostrow School of
Dentistry of Southern California and Argonne National Laboratory. Thank you to
iv

Bonnie Le and Rosalin Le for being organized, positive, and welcoming. We could all
benefit from committed and knowledgeable researchers such as the both of you.
Thank you to Argonne National Laboratory, namely Dr. Jack A. Gilbert, Dr. Sarah
Owens, and especially Dr. Sean Gibbons for your dedication and hard work with the
QIIME software and High-Speed, Multiplexed 16S Microbial Illumina
®
Sequencing on
the Miseq
®
system.
Thank you to my director Dr. Homa Zadeh for your indispensable advice, your
time, and your patience. For giving me the proper guidance to present my research to the
periodontal and dental communities, I thank you from the bottom of my heart.
Thank you to my co-residents, Dr. Maria Galvan and Dr. Stephanie Gonzalez. This has
been a two year journey and I could not have done it without your patience, dedication,
and diligence to the very end.
Thank you also to my other committee members Dr. Mahvash Navazesh and Dr.
Michael Paine for being a part of my achievement of this milestone as I make my way
into the next chapter of my life.

TABLE OF CONTENTS
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abstract viii
Chapter 1: Introduction and Background 1
Chapter 2: Materials and Methods 33

Chapter 3: Results 40

Chapter 4: Discussion 58

Chapter 5: Conclusion 67

Bibliography 69

vi

LIST OF TABLES

Table 1: Haffajee et al. 2008 4
Table 2: Paster et al. 2001 6
Table 3: Adapted from Socransky et al. 1998 7
Table 4: Socransky et al 2005 23
Table 5: Adapted from Liu et al. 2012 27
Table 6: Methodologies Previously Employed 29
Table 7: Patient Distribution by Pocket Depth Group 40
Table 8: QIIME Raw Data 44
Table 9: Statistically Significant Bacterial Distribution 49
Table 10: Non-Statistical, Noteworthy 53
Table 11: Yamada and Sekiguchi 2009 63

vii

LIST OF FIGURES

Figure 1: Boone et al., 2005 2
Figure 2: Preparation of Plaque and Saliva Samples 36
Figure 3: Epifluorescence Microscopy 42
Figure 4a: Legend of the Phyla Taxonomy Plot 46
Figure 4b: Phyla Taxonomy Plot 47
Figure 5: Bar Graph - Bacterial Abundance 50
Figure 6: Line Graph – Bacteria by Pocket Depth 51
Figure 7: Principal Coordinates Plot 52
Figure 8: Non-Statistical Bacterial Abundance 57
Figure 9: Paster et al. 2006 60

viii

ABSTRACT
Technological advances have been made over the past decade to ameliorate
bacterial taxonomy in phylogenetics. Such an instance would best be illustrated by the
unearthing of the 16S rRNA gene and its ability to provide what is known today as the
bacterial genome. Because of this discovery, we have evolved technologically from
being able to run 500,000 DNA sequences at one time with 454 pyrosequencing, to 30
million sequences at one time with Illumina
®
sequencing. This allows for differentiation
and association of species in the oral microbiome that exist supragingival and
subgingival to the periodontal pocket.
Previous studies have shown that different species exist above and below the
periodontal pocket. This study stands to accomplish two main goals: to assess the
microbial profile of sites with distinct periodontal status, and to apply high-speed,
multiplexed 16s Microbial Illumina
®
sequencing on the Miseq
®
system for analysis as a
discovery base for potential new taxa within and supragingival to the periodontal pocket
in a diseased state.
In this double-blind randomized clinical trial, a total of 21 otherwise healthy adult
patients with periodontitis were included. Whole unstimulated saliva samples were
collected to determine the relative abundance and ratios of bacteria in the mouth at
baseline through epifluorescence microscopy. Three separate subgingival samples from
the deepest pockets were obtained for culture-independent 16s rRNA analysis followed
by Illumina
®
sequencing.
ix

Fifteen statistically significant (P < 0.05) bacteria were identified. Select
pathogens, both anticipated and non-anticipated, were discovered at statistically
significant levels. Supragingival plaque group yielded the highest abundance in
Leptotrichia sp. (p < 0.028). The subgingival plaque samples were divided into three
groups: the 5-6 mm pocket group, the 5-8 mm pocket group, and the 5-9 + mm pocket
group. The 5-6 mm pocket group yielded the lowest in abundance for all bacterial
groups. The 5-8 mm pocket yielded Filifactor sp. (p < 9.90 x 10
-6
) , Bacteroidales (p <
0.021 and p< 0.038) of two unidentified families, and Clostridia (p < 0.024) in highest
abundance. The 5-9+ mm pocket range group proved most abundant in the highest
number of bacterial groups tested: Treponema sp. (p < 4.06 x 10
-6
) , Firmicutes clone
RF3:ML615J-28 (p < 2.04 x 10
-6
), Desulfovibrio sp. (p < 0.00024), Synergistetes clone
TG5 (p < 0.0045), Chloroflexi clone SHD-231(p< 0.0059), Porphyromonas sp. (p <
0.0068), Clostridiales (p < 0.0067), Desulfobulbus sp. (p < 0.0076), Leptotrichiaceae (p
< 0.0074), and Clostridiaceae (p < 0.022).
High-speed, multiplexed 16s microbial Illumina
®
sequencing on the Miseq
®

system throughput has proven to be a valuable assay to further understand the oral
microbiome. This method is both cost effective and efficient for providing data that
supports the discovery of microorganisms pertaining to the oral cavity that have not been
identified to date.
1

CHAPTER 1: INTRODUCTION AND BACKGROUND

Chronic periodontitis is caused by a multitude of microorganisms that progress
both supra- and subgingivally through a series of events initiated by bacterial adhesion
and maturation, and succeed by either co-adhesion or co-aggregation to the tooth’s
surface forming dental plaque and biofilm. An abundance of dental plaque will result in
chronic inflammation of the periodontal tissues and may elicit a host response
accountable for the irreversible loss of alveolar bone, characteristic of periodontitis.
Dental calculus is considered to be a secondary etiologic factor for periodontitis. This
can be explained because calculus; which is equivalent to plaque in a mineralized state,
is incessantly encompassed by biofilm composed of viable bacterial plaque(Lindhe et al.
2008). However, bacteria present in the oral cavity are not only responsible for initiation
and progression of disease, they also promote and sustain health by forming an
ecosystem that maintains health when in equilibrium (Zarco et al. 2012). A paper
published by Zarco (2012) summarized concepts developed by Socransky and Haffajee
et al. (1992) by describing key ways in which healthy microbiota protect the
periodontium from pathogenesis: prevention of adherence of pathogens onto specific
surfaces by occupying the niche preferred by a pathogen, prevention of a pathogen from
occupying a site, hindrance of pathogen’s abilities to multiply, and degradation of
pathogen’s virulence factors.
The oral microbiome has proven to be extremely complex, affected by many
factors including but not limited to location, temperature, pH, oxygen, mechanical
2

abrasive forces, and fluid flow (Lindhe et al. 2008). Although many oral microbiological
advances have come to fruition through various clinical and laboratory based techniques,
the oral microbiome is relentlessly evolving, leaving scientists continuously perplexed.
According to Bergey’s manual of Systematic Bacteriology (Boone et al. 2005),
pathogens responsible for periodontal disease include seven different phyla: Firmicutes,
Proteobacteria, Bacteroidetes, Actinobacteria, Spirochaetes, Fusobacteria, and
Deferribacteres which are illustrated in Figure 1. Dewhirst, et al. (2010) states that these
major phyla account for 96% of the taxa, and the remaining 4% is made up of
Euryarchaeota, Chlamydiae, Chloroflexi, SR1, Synergistetes, Tenericutes, and TM7.

Figure 1 Phylogenetic tree of the 7 major phyla associated with periodontopathogenic bacteria.

Although periodontopathogenic bacteria can exist in both health and disease, it is
the bacterial load of the organism that will determine the harmful or beneficial effects on

3

the periodontium. Extensive literature has been published to support the identification of
three major pathogens; present in high levels, to be responsible for periodontitis:
Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella
forsythus (Holt Ebersole 2005, Haffagee Socransky 1994). In addition to these
pathogens, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum,
Eubacterium, Campylobacter rectus, and Eikenella corrodens are amongst the few other
organisms also recognized to have pathogenic roles in the oral microbiome (Dzink et al.
1985, Socransky et al. 2004). Slots and Listgarten (1988) have also reported on the
presence of yeasts, enteric rods, and pseudomonads in approximately one third of
refractory periodontitis patients. Moreover, supra- and subgingival pockets seem to be
comprised of similar species with a few differences.

SUPRAGINGIVAL PLAQUE

Moore et al. (1983) found that the major taxa most highly associated with
supragingival plaque were: Steptococcus, Actinomyces, Capnocytophaga, Veillonella
parvula, Leptotrichia buccalis, Selenomas and Rothia dentocariosa. Haffajee et al.
(2008) published a study whereby 4475 biofilm samples of supragingival plaque were
analyzed using DNA-DNA hybridization methods. This study also showed that sites
with bleeding on probing yielded higher bacterial counts, particularly of the ‘red’ and
‘orange’ complexes. The ‘yellow’ complex bacteria appeared to be more associated with
supragingival plaque than subgingival plaque, explained by streptococcal species being
replaced over time by the actinomyces species as the biofilm matured. ‘Orange’ complex
4

bacteria yielded the highest bacterial counts rendering this group of organisms (Table 1)
the largest complex found in supragingival plaque. Supragingival plaque exhibited
higher proportions of ‘green’ and ‘purple’ complexes as well as actinomyces species
(Ximenez-Fyvie et al. 2000). Thus, supra- and subgingival plaque harbor similar
bacteria composition, however the main difference between these groups continues to be
higher bacterial counts in subgingival plaque as the supragingival plaque accumulates
and consequently migrates apically into the subgingival pocket.

Complex Bacteria
Red T. denticola, P. gingivalis, T. forsythia, E. nodatum
Orange
F. periodonticum, F. nuc. ploymorphum, F. nuc. nucleatum, F.
nuc. vincentii, P. intermedia, P. nigrescens, P.
melaninogenica, C. gracilis, S. noxia, C. ochracea, C. showae,
C. rectus
Yellow
S. anginosus, S. constellatus, S. intermedius, S. mitis, S. oralis,
S. sanguinis, S. gordonii, P. micros, E. saburreum
Green C. sputigena, E. corrodens, C. gingivalis
Blue
A. naeslundii, A. israelii, A. odontolyticus, A. gerencseriae, A.
naeslundii
Purple N. mucosa, V. parvula

Table 1 is adapted from Haffajee et al. (2008) listing bacteria present in supragingival plaque in patients
prior to periodontal therapy (baseline) and their corresponding complex classification.

SUBGINGIVAL PLAQUE
Moore et al. (1983) studied taxa associated with subgingival plaque and reported
its major constituents to be: Fusobacterium, Peptostreptococcus, Eubacterium,
5

Campylobacter rectus, Porphyromonas gingivalis, and Prevotella species. Socransky et
al. (1994) and Ximenez et al. (2000) later showed that subgingival samples exhibited a
significantly higher proportion of red and orange complex species.
Paster et al. (2001) published a study which aimed to identify bacteria present in
subgingival plaque using culture independent PCR amplification of 16S rRNA in
otherwise healthy periodontitis patients with pockets of >4mm. Unlike Bergey’s Manual
of Systematic Bacteriology (Boone et al. 2005) which identifies the seven
aforementioned phyla associated with periodontitis, Paster was able to identify one
additional phyla: TM7. This study offered a major breakthrough in oral microbiology
stating that approximately 79.3% of the total species of subgingival plaque from
periodontitis patients were novel phylotypes that were identified in diseased subjects
seen in Table 2 (Paster 2001).

6

Table 2 Adapted from Paster et al. (2001) shows the putative pathogens associated with subgingival
plaque and the novel phylotypes associated with the identified bacterial groups.

It has repeatedly been reported that supra- and subgingival plaque are comprised
of similar bacterial organisms differing in the proportion of bacterial load. Such an
example would be the ‘red’ complex bacteria (Socransky et al. 1998, Ximenez-Fiyvie et
al. 2000). Research suggests that supragingival plaque does influence the bacterial
composition of the subgingival pocket, especially as the disease progresses
(Kolenbrander et al. 2006). These microorganisms, particularly those inhabiting the
subgingival portion of a diseased periodontal pocket, receive their nutrients mostly from
the surrounding blood supply and components found in the gingival fluid necessary for
metabolic reactions to take place and for growth which enables these pathogens to thrive
(Lindhe et al. 2008). Of these organisms, it has been reported that 96% of bacteria are
comprised of Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes and
7

Fusobacteria (Dewhirst et al 2010). Table 3 summarizes the pathogens associated with
subgingival plaque.

Complex Bacteria
Red T. denticola, P. gingivalis, T. forsythia
Orange
P. intermedia, P. nigrescens, P. micros, F. nuc. vincentii, F. nuc.
nucleatum, F. nuc. ploymorphum, F. periodonticum, E. nodatum, C.
gracilis, C. showae, C. rectus, S. constellatus
Yellow
Streptococcus sp., S. intermedius, S. gordonii, S. mutans, S. oralis,
S. sanguis
Green C. sputigena, E. corrodens, C. gingivalis
Purple A. odontolyticus, V. parvula

Table 3 is adapted from Socransky et al. (1998) listing bacteria present in subgingival plaque and their
corresponding complex classification.

ACTINOBACTERIA

This phylum is the most prevalent taxa in both supra- and subgingival plaque
(Ximenez-Fiyvie et al. 2000). Actinobacteria are Gram-positive bacteria with high G+C
content; meaning they contain high amounts of guanine and cytosine within the structure
of their DNA. This mostly aerobic and filamentous phylum can be found in both
periodontal health and disease and include 3 orders: Actinomycetales, Bifidobacteriales,
and Coriobacteriales associated with 8 genera: Actinomyces, Atopobium,
Bifidobacterium, Corynebacterium, Olsenella, Propionibacterium, Rothia, and Slackia
(Dewhirst et al. 2010, Paster et al. 2001, Siqueira Jr. Rocas 2010). Some research has
8

deduced that Actinobacteria are mostly associated with health and other research has
argued the opposite (Abusleme et al. 2013 and Griffen et al. 2011 respectively).
Although the results are inconclusive, one thing is clear, this phylum is present in both
healthy and diseased periodontal states.

FIRMICUTES

First described by Gibbons and Murry in 1978, this phylum is mostly made up of
either coccoid or rod shaped, Gram-positive bacteria; belonging to the low G+C group,
that are categorized into one of three main classes: Bacilli (obligate or facultative
aerobes), Clostridia (anaerobic), and Erysipelotrichia. This spore-forming phylum is
either coccoid or bacilli in shape. The Bacilli class includes 86 taxa including the largest
group; the Streptococci. These organisms exist both in health and disease however, S.
constellatus and Gemella haemolysans are mostly associated with diseased sites (Paster
et al. 2001). The Clostridia include the largest family; the Veillonellaceae, as well as the
Peptostreptococcaceae, and the less studied Lachnospiraceae. The Erysipelotrichia
include Bulleidia extructa, Solobacterium moorei, and the more commonly recognized
Lactobacillus catenaformis, some of which have been linked to endocarditits. The major
periodontal bacteria associated with this phylum are the Streptococcus (Bacilli),
Peptostreptococcaceae (Clostridia), Lactobacillus (Erysipelotrichia), and Veillonellaceae
(Clostridia, Gram-negative) (Paster et al. 2001, Dewhirst et al. 2010, Boone et al. 2005).

9

BACTEROIDETES

This phylum of anaerobic Gram-negative bacteria can be divided into eight
genera: Prevotella, Bacteroides, Porphyromonas,Tannerella, Bergeyella,
Capnocytophaga, and eight that are unnamed. Bacteroidetes are anaerobic bacilli that
have mostly been cultivable through bacterial plaque analyses. Twenty-one known
species as well as 37 novel phylotypes have been detected amongst this phylum whose
genera include: Prevotella, Bacteroides, Porphyromonas, Tannerella, Bergeyella,
Capnocytophaga, and eight that are unnamed (Dewhirst et al. 2010). Species that are
highly associated with periodontitis in this phylum include Porphyromonas gingivalis
and Tannerella forsythia, although they are not as commonly detected as Prevotella
denticola or Capnocytophaga gingivalis. Prevotella, being the largest group containing
approximately 50 species, includes periodontally associated organisms such as
Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, and uncultivated
phylotypes (Paster et al. 2001, Siqueira Jr. Rocas 2010).

PROTEOBACTERIA

This group of Gram-negative organisms, which are classified into the high G+C
group, is named for its ability to alter the appearance of its form. Most of these bacteria
are considered to be aerobes that may self-attach onto other colonizing bacterial species
by coaggregation. This phylum is comprised of 5 classes: Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and
10

Epsilonproteobacteria containing taxa detected in the human oral cavity (Dewhirst et al.
2010). Proteobacteria account for the largest of all bacterial groups.
Aggregatibacter actinomycetemcomitans (gammaproteobacteria) is a member of this
phylum and one of the major pathogens associated with periodontitis first discovered by
Dr. Jorgen Slots. Also classified under the gammaproteobacteria is the family of enteric
gram-negative rods (Enterobacteriaceae). P. mirabilis, Proteus mirabilis, Klebsiella
pneumoniae, Klebsiella oxytoca, Serratia marcescens, Escherichia coli, Acinetobacter
calcoaceticus, E. cloacae, K. pneumoniae, P. aeruginosa, Enterobacter cloacae,
Enterobacter agglomerans and unidentified sp. bacteria were identified in a study by
Slots et al. (1988). A subsequent study by Slots et al. (1990) in periodontitis patients
revealed that E. cloacae, K. pneumoniae, P. aeruginosa, Enterobacter cloacae, and
Enterobacter agglomerans accounted for 50% of all of Gram-negative enteric rods
isolated from 427 of 3,050 individuals. These findings suggest that 5% of severe
periodontitis lesions may harbor non-oral, gram-negative, facultatively anaerobic rods
(Slots et al. 1990).
Other bacteria associated with periodontitis belong to the genera Campylobacter
(deltaproteobacteria), Eikenella (betaproteobacteria), and Neissera (betaproteobacteria).
These organisms have been easily cultured as reported by previous literature
(Hugenholtz Stackebrandt 1998).

11

SPIROCHAETES

Spirochaetes are easily identified due to their helically coiled structure and
presence of flagella however this organism is also hard to culture. This phylum is
comprised of anaerobic, Gram negative, and highly motile organisms. To date, one
genera from this phylum has been associated with periodontitis: Treponema. Currently,
there are 60 oral treponemal species or phyloypes (Paster 2001). This genus has been
associated with higher levels present in subgingival plaque versus supragingival plaque
and in periodontal disease versus gingivitis or healthy sites (Socransky et al.1998,
Moore et al. 1983). Over 70% of the 49 identified taxa thus far resist cultivation
(Dewhirst et al 2010). Studies have proven that an increase in pocket depth increases the
bacterial counts of this organism, while healthy sites exhibit virtually no counts of this
organism (Lindhe et al. 2008). Species included in this genus include the most common
Treponema denticola; present in health and disease, as well as Treponema medium,
Treponema socranskii, Treponema maltophilum, and four predominant novel phylotypes
proving to be periodontopathogenic (Paster et al. 2001).

FUSOBACTERIA

The Fusobacteria most frequently associated with the oral microbiome belong to
the genera Fusobacterium and Leptotrichia and are considered to act as coaggregation
bridges between early and late colonizers (Socransky et al. 1998). There have been 47
clones detected in association with this bacteria (Dewhirst et al. 2010). This phylum is
12

comprised of obligate anaerobic Gram-negative bacilli that are often quite difficult to
detect with a number of microbiological testing methods, however technological
advancements have been made that enable this phyla to become more detectable. Some
species belonging to this phylum which have been reported to show association with the
oral cavity via 16S rRNA testing methods include: Fusobactium nucleatum,
Fusobactium alocis, Fusobactium periodonticum, and Fusobactium simiae (Lawson et
al. 1991). Fusobacteria are found in large quantities in the oral cavity in health and
disease states.
In the human oral microbiome, 700 taxa have been identified to date, with over
57% of these species originating from the periodontal pocket. An individual is estimated
to have anywhere from 14-29% of these taxa present, making the combination of taxa
per individual virtually limitless (Paster et al. 2006). Nevertheless, substantial
breakthroughs have been made in the field of oral microbiology which enable us to both
differentiate and disease associate microorganisms of the oral microbiome, aiding us to
further our cognizance on such an expansive and evolving discipline. Deciding which
form of analysis to opt for when analyzing bacterial samples can often be difficult,
however understanding the intent for which this assessment is being carried out can
alleviate such confusion. Three main objectives for evaluating bacterial samples in the
oral microbiome are: to culture for specific target bacteria, to detect antibodies, and to
detect microbes present whether it be specified or non-specified. Such techniques
include, but are not limited to: culture sampling, microscopic assessment,
immunoassays, enzymatic assays, checkerboard DNA-DNA hybridization technique,
13

DNA probe testing, polymerase chain reaction assays, Sanger method, and next
generation sequencing. To date, there is no form of bacterial analysis that has been able
to identify all pathogens responsible for periodontal disease.

CULTURE SAMPLING TECHNIQUES

Culture methods continue to be referred to as the gold standard to which evolving
microbial assays should achieve or surpass. The most commonly used sampling
techniques when cultivating plaque samples for analysis are dental curettes and
endodontic paper points. Several studies have shown that curettes yield higher
pathogenic colony-forming units than paper point samples. The reason for this is simply
due to the design of a dental curette, allowing facile accessibility, thus obtaining a more
veritable representation of plaque in a particular pocket, as opposed to mere superficial
layers when employing endodontic paper points (Loomer 2004, Socransky et al. 1998,
Kiel Lang, 1983, Moore et al., 1983). It has been inferred by many microbiologists, that
sites with deeper periodontal pockets and concurrent bleeding upon probing, are more
likely to harbor pathogenic bacteria (Socransky Haffajee, 2005, Mombelli et al. 1991;
Savitt et al, 1991). Thus, when retrieving plaque from the periodontal pocket, harvesting
from the deepest pocket with bleeding should theoretically provide the most periodontal
pathogens in the highest amounts, ensuring the most thorough cultivation of plaque per
sample for analysis.
Favero, et al. (1968) stated that there are four basic methods for enumerating
microorganisms on surfaces: the swab rinse, the rinse, the agar contact and direct surface
14

agar plating (DSAP). Direct surface agar is the most commonly used technique when
analyzing bacteria in the oral microbiome. Pure microbiological cultures were first made
available in the early nineteenth century, when microbiologist Dr. Robert Koch
revolutionized bacteriology utilizing solid media to isolate target bacteria. Prior to this
breakthrough, liquid cultures using bacterial broth and nutrient medium were used,
making it virtually impossible to obtain an axenic culture. The rationalization behind
Koch’s approach was to use a Petri dish consisting of solid agar growth medium
inoculated with the target bacteria and allowing it to thrive at a favorable temperature
over a desired period of time. The colonies were then counted at the location of the agar
surface interface to attain the microbial count of the target bacteria (Favero et al. 1968).
Compared to all other microbiological methods, bacterial culturing offers detection of
antimicrobial or antibiotic sensitivity, providing a unique advantage over other
techniques. Organisms routinely examined for by culture include the following:
Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis,
Tannerella forsythia, Prevotella intermedia group, Fusobacterium nucleatum,
Campylobacter rectus, Parvimonas micra (Peptostreptococcus micros), Streptococcus
constellatus, Streptococcus intermedius, Enterococcus faecalis, enteric gram-negative
rods/pseudomonads, Staphylococcus aureus and other staphylococci, and Candida
species (www.usc.edu/hsc/dental/OMTL/OMTL_P0.html).
Nevertheless, much like all other methods of microbiological testing, there are
limitations to this technique. Apart from the high costs accrued, it is technique sensitive,
labor intensive, and time-consuming. Microbiological culturing also requires the need of
15

viability of perio-pathogenic bacteria making it extremely time sensitive. Another issue
with this technique is the inability to distinguish closely related bacteria from one
another. Transportation of the samples poses yet another problem in that high
reproducibility of some bacteria results in inaccurate bacterial counts and certain bacteria
unable to multiply at a rapid rate may remain undetected. (Loomer 2004, Jervøe-Storm
et al. 2005, Kiel Lang 1983, Socransky Haffajee 1992). Finally, culturing methods are
able to recover many pathogenic organisms that can be grown under specific
circumstances but are unable to detect many of the recently discovered microorganisms
discovered by next generation technology. The target pathogen(s) must be specified to
achieve an optimal growing environment depending on the type of bacteria being
cultured.

MICROSCOPIC ASSESSMENT

Microscopy is recognized as one of the major scientific advancements to have
revolutionized the fields of medicine and dentistry. Dark field and phase-contrast
microscopy are amongst the earliest different modalities most commonly employed for
the distinction of microbes in plaque samples. Microscopy is achieved by visually
inspecting these microbes and discerning between characteristics that may pertain to one
specific group. For example, we can observe a trend from healthy to diseased bacterial
states in a patient if rods, cocci and vibrios of acceptable counts become more abundant.
Rods in substantial proportions can be representative of periodontopathogens such as red
complex bacteria namely P. gingivalis. Spirochetes are also easily identified by their
16

corkscrew appearance and can represent periodontal destruction responsible for bleeding
and inflammation.
Scanning electron microscopy (SEM), introduced in the late 1930’s, has become
more valuable in the field of periodontology. Higher resolution and magnification can be
accomplished with the use of SEM as opposed to dark field or phase contrast
microscopy. This technique provides an effortless approach to more precise bacterial
counts, shape, and composition extracted from patient plaque samples. Expansive
documentation has been published regarding bacterial trends observed on both tooth and
implant surfaces, allowing the field of microbiology a means to further understanding
the oral microbiome (Wilkinson Maybury 1973, Goldstein et al. 2003).
Light microscopy provides ease of in office use and has proven to be useful for
observing trends and distributions of bacteria as well as proven practical for patient
education. That said, microscopy is incapable of identifying specific bacterial species.
Furthermore, antibiotics and antimicrobials should not be administered based solely on
the findings of microscopic identification (Loomer 2004, Listgarten Loomer 2003).

IMMUNOASSAYS

Immunoassays include immunofluorescence microscopy, latex agglutination,
enzyme-linked immunosorbent assays (ELISA), flow cytometry, and
immunoprecipitation. In general, immunoassays are tests that involve the use of
antibodies or immunoglobulins to aid in the detection of an analyte; or the target
antibody or antigen. Labels are employed to allow for detection of these antibodies or
17

antigens, and are available in many different forms depending upon which immunoassay
is being conducted.
Fluorescence microscopy is used in the field of dental microbiology and is
different from other microscopic techniques because it uses fluorescence as opposed to
reflection or absorption to produce an image. Each sample analyzed must either be
stained or tagged with a fluorescent protein. Because this technique is stain specific, one
can only see the structures that have been tagged or stained. Slots et al. (1985) described
the importance of immunofluorescence and how it can be a useful and rapid diagnostic
method in the detection of periodontopathogens such as Porphyromonas gingivalis and
Aggregatibacter actinomycetemcomitans whose species possess one set of surface
antigens and three different serotype determining surface antigens, respectively. These
antigens also confer little or no cross-reactivity with surface antigens of other oral
bacterial species (Slots 1984, Zambon et al. 1983). Drawbacks of this technique are
being addressed as technology is evolving, such an example would be that of microtitre
fluorescence assays which accelerate the data acquisition from the plate reader after the
processing and loading has been completed (Filoche et al. 2007).
Another technology that has been applied to periodontal samples and
commercialized is the latex agglutination test introduced by Zambon et al. 1986. This
technique involves a series of steps whereby antibody-bound latex is mixed with a supra-
or subgingival plaque sample, suspended, and agitated. A positive test is visualized by
the clumping of the mixture, which occurs when the target microorganisms form
18

complexes with the species-specific antibodies. The major problem is that these tests
have a low sensitivity and provide only qualitative results (Zambon et al. 1986).
Enzyme linked immunosorbent assay (ELISA) is another method employed in the
detection of bacteria through antibodies. This method differs from the other
immunoassays in that in utilizes color change for the identification of the target bacteria.
The principles of ELISA are based on an antigen which is use to bind to an antibody
which is linked to an enzyme which then reacts with the substances containing the
enzyme, and a color change ensues. This technique is often employed for the study of
immunologic responses of the periodontium to putative periodontal pathogens (Zambon
et al. 1986).
Flow cytometry can be used to further understand the progression of periodontal
disease. This method can offer rapid, high throughput processing of multiple samples
through polyclonal or monoclonal antibodies against species-specific surface antigens to
identify target bacteria (Baehni Guggenheim 1996). The methodology behind this
technique involves light scatter and fluorescence emission whereby particles are counted
and visually inspected through a process of fluid suspension using monochromatic lasers
and fluorescent detectors for illumination of these particles. This method however is not
used frequently for the analysis of plaque samples due to the difficulty in transforming a
bacterial plaque composed largely of cell clumps into a single cell suspension suitable
for analysis. (Zambon et al. 1986, Quirke Dyson 1986).
Immunoprecipitation applies the same concepts as the other immunoassays with
one minor difference, the antigen is precipitated out by using an antibody that is able to
19

bind to the target bacteria. Chromatin immunoprecipitation is a type of
immunoprecipitation method used in next generation sequencing to study the interaction
between protein and DNA in a cell (Liu Faller et al. 2012).

N-BENZOYL-DL-ARGININE-2-NAPHTHYLAMIDE (BANA) ASSAY

Enzymatic Assays are among the fastest, inexpensive, and user-friendly
techniques for the evaluation of dental plaque samples. The BANA test was developed
by Loesche in the late eighties, and published in 1990 (Loesche Bretz Kerschensteiner et
al. 1990, Loesche Giordano et al. 1990). This assay was commercialized for the chair-
side detection of microbial groups by placing a plaque sample possibly containing
trypsin-like enzymes such as Treponema denticola, Tannerella forsythia, and
Porphyromonas gingivalis onto a paper strip impregnated with N-benzoyl-DL-arginine-
2-naphthylamide and diazo dye. If the enzyme is present within the plaque sample, the
BANA enzyme is hydrolyzed and produces B -naphthylamide, which in combination
with the diazo dye will turn the paper strip blue in color over a period of approximately
24 hours. This short incubation period renders this exam highly efficient. The more
intense the color appears on the paper strip, the higher the concentration of one or all of
the bacteria present. However, this test does not give quantitative results, it is only meant
for the detection of the soundly documented aforementioned putative
periodontopathogenic bacteria, causing this test to lack specificity due to all three
bacterial species’ ability to cleave the same substrate, making it indistinguishable among
the three species.
20

False-positive reactions may occur due to a derivation from the host causing
unwanted enzyme activity. (Loesche Bretz Kerschensteiner et al. 1990, Loesche Bretz
Lopatin et al. 1990, Loesche Giordano et al. 1990, Loomer 2004).

NUCLEIC ACID HYBRIDIZATION

Prior to multiplexed 16s rRNA sequencing, hybridization techniques were mostly
used to achieve microbial advancements in periodontology. This technique is based on a
spontaneous reaction whereby two or more complementary strands of nucleic acids
become unified. Prior to the pairing of the these nucleic acids, denaturing and annealing
processes take place, allowing for the double helices to separate and subsequently bond
to a complementary strand of nucleic acids. Three major methods involving
hybridization have been utilized extensively to broaden our understanding of
periodontopathogenic bacteria found in plaque: checkerboard DNA-DNA hybridization,
and oligonucleotide microarray technology.
The checkerboard DNA-DNA hybridization technique was first applied in the
field of periodontology by Socransky in 1994 and enabled the hybridization of 45 DNA
samples against 30 DNA probes on a single nylon membrane using chemofluorescence
procedures (Paster Dewhirst 2009, Socransky et al. 1994). This very high throughput
technique allows for thousands of samples to be analyzed at a rapid rate resulting in
exhaustive amounts of quantitative data being generated. The DNA-DNA hybridization
method permits the simultaneous determination of the presence of multiple bacterial
species in single or multiple dental plaque samples, thus suggesting its usefulness for a
21

range of clinical or environmental samples (Socransky et al. 1994). This technique
incorporates the annealing of complementary bases of two single-stranded DNA
molecules. The DNA of a plaque sample is extracted and labeled with digoxigenin so it
can be utilized as a hybridization probe. The labeled probes from a variety of species are
hybridized to a large number of denatured DNA samples on a nylon membrane. Then,
the hybridization is detected and quantified using anti-digoxigenin antibody with a
chemifluorescent substrate (Socransky 1994, Siqueira Jr. Rocas 2010). Apart from being
efficient and quantitative, this technique is also inexpensive and sensitive as proven by
Socransky et al. in 2004 when the methodology was applied to study complex microbial
ecosystems.
A potential drawback however, is the unknown cross reactivity with unknown
taxa present in the sample. Additionally, the technique can only provide information on
known culturable taxa and while very valuable does not address the unculturable
proportion of any sample (Spratt 2004).
Oligonucleotide microarray technology was originally developed to detect
predominant bacterial species including species that have not yet been discovered or
cultured (Paster Dewhirst 2009). Its major advantage is that it has the ability to generate
massive quantities of data in one single run. The way this method is carried out entails
the use of many oligotide probes which are fixed onto a glass slide; referred to as chips,
which allows for the accumulation of tens to hundreds of thousands of probes on a single
high-density microarray which are then imaged and analyzed (Huyghe et al. 2008). The
data is then analyzed by comparing signals of individual intensities to the average
22

signals from universal probes. To date, there has been limited research published with
regard to the identification of bacterial in the oral microbiome. A minor drawback may
be that probes on these high-density microarrays target mainly family level and genus
level, rather than species level distinctions (Paster Dewhirst 2009). This methodology
has been used in the comparison of bacterial composition found in subgingival plaque in
patients with refractory periodontitis, severe periodontitis, and periodontal health, and is
one of the more promising advances made in bacterial identification of plaque (Columbo
et al. 2009).

POLYMERIZED CHAIN REACTION (PCR) ASSAY

PCR is the most widely documented assay in the detection of bacteria within the
oral microbiome. This form of testing involves denaturing DNA molecules to near
boiling temperatures of 95 °C in order to cause the strands of the double helix to
separate. Once this is accomplished, the temperature is then lowered to either 72 °C or
60 °C depending on the desired rate of the reaction. This allows for the pair of
oligonucleotide primers to anneal. The enzyme known as Taq polymerase is added; due
to its thermostable properties which can sustain high temperatures of near boiling, and is
then directed by primers to synthesize a complementary strand of DNA from free
nucleotides. This entire process is then repeated multiple times over and only the DNA
containing the target sequences are copied over. This is possible due to the Taq
polymerase only being able to copy molecules that have a primer attached. Two partially
double strands of DNA molecules are formed and the temperature is again raised to 95
23

°C initiating the reoccurrence of the reaction. At the end of this chain reaction, over a
million copies of the short fragments of target DNA that are produced are referred to as
amplicons. These amplicons may have built in adapters depending on the oligos
incorporated into the polymerized chain reaction in order to perform higher generation
sequencing such as 454-pyrosequencing or high-speed, multiplexed 16s microbial
sequencing with the Illumina
®
system (Rogers 2008).

Table 4 is adapted from Socransky et al. (2005) and summarizes the applications, strengths, and
weaknesses of important methodologies used in oral microbiology for the detection of putative periodontal
pathogens.
24

THE SANGER METHOD

The chain termination method of DNA sequencing, more commonly referred to as
Sanger sequencing has been in existence for over 30 years. Sanger sequencing was
introduced by Fredrick Sanger and originally employed the use of radioactively labeled
synthesis-terminating dideoxynucleotides in combination with DNA polymerase and X-
ray films. This method has been modified by the use of fluorescence detection
instrumentation which has allowed for >900 bp of reliable nucleotide sequence data
(Heng Stanton 2010). However, this method is seldom being employed today for the
purposes of bacterial detection of the oral microbiome mainly do to the high costs
incurred, low throughput produced, and laborious approach (Table 5).

454-PYROSEQUENCING

The sequencing by synthesis method of DNA sequencing, more commonly
referred to as 454-pyrosequencing utilizes the detection of pyrophosphate release as a
complementary nucleotide gets incorporated onto a single strand of the DNA that has
been denatured. Then a luminescence signal is emitted in order to identify the DNA
sequence by the identification of DNA polymerase as the nucleotides are incorporated
into the sequence. This whole process is then repeated until the DNA sequence is
revealed, reading 300-500 base pairs per run within a 24 hours period (Pozhitkov et
al.2011, Liu et al. 2012).
25

Some potential drawbacks of this method include but are not limited to individual
reads being shorter than the 800-1000 nucleotides obtainable with the Sanger method
described above , the high error rate associated with more than 6 bp, the high cost, and
low throughput.

METAGENOMICS

Metagenomic sequencing has also been given more attention as a diagnostic tool
for detection of bacteria in the oral cavity. This technique samples all genes present in a
habitat rather than just 16S rRNA, thereby providing clues to the functional capacity of a
community rather than just its phylogenetic composition (Tringe). However, small
studies may find this methodology to be excessive due to unnecessary strain
identification and overwhelming quantitative data achieved.

ILLUMINA
®
SEQUENCING: HIGH-SPEED, MULTIPLEXED 16S rRNA GENE
MICROBIAL SEQUENCING

The 16S rRNA gene is the ‘gold-standard’ of markers for culture-independent
microbial studies. The reason this technology has proven to be effective is that it allows
for association and differentiation of species in the oral microbiome; in particular supra-
and subgingival periodontally involved pockets, which allows for the detection of
cultured and uncultured bacteria as well as massive amounts of quantitative data.
26

Developed by Manteia Predictive Medicine and acquired by Solexa (Hayward, CA) then
later by Illumina
®
(San Diego, CA), high-speed, multiplexed 16S rRNA microbial
sequencing was invented in 1996 by Dr. Pascal Mayer and Dr. Laurent Farinelli, and
was introduced to the public in 1998 (Mayer et al. 1998).
This technology is known as sequencing by synthesis which works through dye
terminators that enable the identification of single bases as they are introduced into DNA
strands through fluorescent light emission in a process involving three principal steps
that constitute the genome analyzer workflow: 1. library preparation, 2. cluster
generation, and 3. sequencing. Prior to these steps taking place, the samples must first be
prepared. This process is initiated by extracting the DNA molecules by way of
denaturation. Library preparation is initiated by obtaining extracted DNA from a dental
plaque sample and attaching an adaptor to it, preparing the sample for integration onto a
flow cell. This flow cell is comprised of a lawn of oligonucleotides, which allows for
acceptance of the DNA sample by way of the adaptor. Copies are then made through
bridge amplification resulting in hundreds of unique generated clusters. The template
DNA is then washed away, and the ends of the copied DNA are then blocked and ready
for the third and final step; sequencing. Here, a detectable fluorescent color specific to
one of the four bases is then visible. These four bases naturally compete to bind to the
DNA. This ensures the highest accuracy. The process is then repeated until the full DNA
molecule is sequenced. The final product is a series of colors allowing for sequence
coding which is then translated into bacterial identification via the Quantitative Insights
Into Microbial Ecology (QIIME) software, and matched to a corresponding code in the
27

genome bank. Should a code fail to match a corresponding bacterial sequence in the
genome bank, one of two possibilities can be responsible for this occurrence. Either a
new taxa has been identified in the sample analyzed, or the analyzer cannot identify the
bacteria on a specific taxonomic level (http://www.illumina.com/literature.ilmn).
One disadvantage to using Illumina
®
sequencing is that it lacks the ability for
precise quantification because only an end-point determination can be analyzed
(http://www.illumina.com/literature.ilmn). Although Illumina
®
sequencing works with
35-50 bp, it allows for millions of reads and is proven to have a low error rate, high
throughput, and is inexpensive (Caporaso et al. 2011, Caporaso et al. 2012).
Table 5 compares the aforementioned next generation sequencing systems to one another
based on multiple variables.

Sequencer 454-
Pyrosequencin
g
(GS FLX
®
)
Illumina
®

Sequencing
(Miseq
®
)
Sanger Sequencing
3730xl
®
Reads (bp) 700 bp 30-50 bp 400-900 bp
Time/run 24 hours 4-48 hours
(depending on bp
read)
20 minutes - 3 hours
Output/data 0.7 Gb 600 Gb 1.9-84 Kb
Advantage Read length, fast High throughput,
low cost
High quality, long
read length
28

Sequencer 454-
Pyrosequencin
g
(GS FLX
®
)
Illumina
®

Sequencing
(Miseq
®
)
Sanger Sequencing
3730xl
®
Disadvantage Error rate with
more than 6 bp,
high cost, low
throughput
Short read assembly High cost, low
throughput

Table 5 Adapted from Liu et al. 2012 Summary of next generation sequencing systems including
advantages and disadvantages for bacterial sequencing.

In a comparison study by Luo et al.(2012) Illumina
®
sequencing appears to have
less of an error rate than 454-pyrosequencing, yielding longer and more accurate contigs
despite the shorter read length. Moreover, Illumina
®
sequencing costs 75% less than
454-pyrosequencing making this method more appropriate for studies like the present
study as well as metagenomic studies. However, both next generation technologies were
proven to be effective methods of bacterial identification (Luo et al. 2012).
Technological advances have been made over the past decade to ameliorate
bacterial taxonomy in phylogenetics. Such an instance would best be illustrated by the
unearthing of the 16S rRNA gene and its ability to provide what is known today as the
bacterial genome. Because of this discovery, we have evolved technologically from
being able to run 500,000 DNA sequences at one time with 454 pyrosequencing, to 30
million sequences at one time with high speed, multiplexed 16S rRNA microbial
sequencing using the MiSeq
®
system. This technology has allowed us to differentiate
and associate species in the oral microbiome that exist supragingival and subgingival to
the periodontal pocket in health and disease.
29

Author(s) Methodology Major Findings
Socransky et al
2004
DNA-DNA checkerboard
hybridization using Gracey
curette sampling
40 common subgingival species were
tested in a checkerboard hybridization
format in 8887 subgingival plaque
samples from 79 periodontally healthy
and 272 chronic periodontitis subjects
and 8126 samples from 166 subjects
taken prior to and after periodontal
therapy proving to be a useful
diagnostic tool for target bacteria.
Ximenez-Fyvie et
al.
2000
Whole genomic DNA probes
and DNA-DNA checkerboard
hybridization using Gracey
curette sampling
22 periodontally healthy and 23 adult
periodontitis subjects were sampled for
supra and subgingival plaque for a
total of 2358 samples from the mesial
aspect of all teeth excluding 3rd
molars in each subject. Examined for
the presence and levels of 40 bacterial
taxa. Porphyromonas gingivalis,
Bacteroides forsythus and Treponema
denticola could be detected in
supragingival plaque samples of both
healthy and periodontitis subjects.
Actinomyces species were the
dominant taxa in both supra- and
subgingival
plaque from healthy and periodontitis
subjects. Increased proportions
of P. gingivalis, B. forsythus, and
species of Prevotella, Fusobacterium,
Campylobacter and Treponema were
detected subgingivally in the
periodontitis subjects. P. gingivalis, B.
forsythus and T. denticola were
significantly more prevalent in both
supra- and subgingival plaque samples
from periodontitis subjects.
30

Author(s) Methodology Major Findings
Kumar et al.
2003

PCR amplification using
endodontic paper point
sampling
66 subjects with chronic
periodontitis and 66 age-matched
healthy patients were sampled.
Associations with chronic periodontitis
were observed for uncultivated clones
D084 and BH017 from the
Deferribacteres phylum, AU126 from
the Bacteroidetes phylum,
Megasphaera clone BB166, clone
X112 from the OP11 phylum, and
clone I025 from the TM7 phylum, and
the named species Eubacterium
saphenum, Porphyromonas
endodontalis,
Prevotella denticola, and
Cryptobacterium curtum. Species or
phylotypes more prevalent in
periodontal health included two
uncultivated phylotypes, clone W090
from the Deferribacteres phylum and
clone BU063 from the Bacteroidetes,
and named species Atopobium rimae
and Atopobium parvulum.
Griffen, et al.
2011
454 pyrosequencing using
endodontic paper point
sampling
Identified 123 taxa more abundant in
disease and 53 taxa more abundant in
health from 29 diseased and 29 healthy
patients.
Keijser, et al.
2008
454-pyrosequencing using
sterile DNA-free wooden
toothpick sampling
Saliva and supragingival plaque were
sampled from 71 and 98 exclusively
healthy adults, respectively.197,600
sequences generated yielded about
29,000 unique
sequences, representing 22 taxonomic
phyla. OTU (6%) yielded 3621 and
6888 species-level phylotypes in saliva
and plaque, respectively.19,000
phylotypes found.
31

Author(s) Methodology Major Findings
Lazarevic et al.
2009
Metagenomics through
Illumina
®
sequencing using
expectoration and cotton swab
sampling
Sampled 3 healthy patients sampled
for saliva and oropharyngeal culture.
Identified 135 genera or higher
taxonomic ranks from the resulting
1,373,824 sequences. Abundances of
the most common phyla (Firmicutes,
Proteobacteria, Actinobacteria,
Fusobacteria and TM7). Bacteroidetes
were less present. Achieved a much
greater depth of coverage and thirty-
four taxa have not been identified in
previous studies of oral
microbiota such as low-abundance
genera as well as putative members of
the candidate divisions BRC1, OP10,
OP3. BRC1 and OP10 sequences were
also identified.
Liu et al.
2012
Metagenomics through
Illumina
®
sequencing using
Gracey curette sampling
5 patients were sampled: 15
exclusively subgingival plaque
samples, four from each of two
periodontitis patients, and the
remaining samples from three healthy
individuals were used to partially
reconstruct several oral microbes and
to preliminarily characterize some
systems-level differences between the
healthy and diseased oral microbiomes
which were achieved.

Table 6 summarizes the major findings of previous authors utilizing advanced sequencing methodologies
and the type of instrument employed for cultivation

In the past, research has analyzed supragingival plaque separately from
subgingival plaque (Socransky Haffajee 1998 and , Griffen et al. 2011, Tringe
Hugenholtz 2008, DeSantis et al. 2006, Slots1984, Slots et al 1988, Slots et al. 1985).
32

Studies have also detected bacteria in sites of healthy versus diseased periodontium
using a vast array of microbial diagnostic technologies over time (Table 6). However, to
our knowledge, no studies have compared the differences between supra- and
subgingival plaque samples of exclusively diseased sites and at different depths of the
periodontal pocket prior to any therapy delivered.
This study stands to accomplish two main goals: to assess the microbial profile of
sites with distinct periodontal status, and to apply multiplexed 16S Illumina
®
sequencing
through the MiSeq
®
system for throughput analysis as a discovery base for potential
new taxa within and supragingival to the periodontal pocket in a diseased state.

33

CHAPTER 2: MATERIALS AND METHODS

This study was approved by the Herman Ostrow School of Dentistry of the
University of Southern California Institutional Review Board (IRB# HS-10-00509).
Clinical assessment and microbiological sampling were collected with informed consent
at baseline from a total of twenty-two otherwise healthy adult patients with
periodontitis. The patients were enrolled in the study in a staggered fashion on the basis
of the following inclusion criteria:
• 18 years of age or older regardless of sex and/or race
• diagnosed with chronic or aggressive forms of periodontitis
• current full mouth radiographs on record
• at least 4 teeth with periodontal pockets of 6mm or more

Exclusion criteria were as follows:

• treatment planned to undergo, or were undergoing, standard periodontal therapy
including scaling and root-planing within the last 6 months
• antibiotic treatment within the last 6 months
• medically-compromised and unable to comply with protocol
• smoking habit of >10 cigarettes/day
• emergency dental care indicated for dental caries or periodontitis
• periodontal abscesses and/or acute periodontitis

The experimental design of the study was a double blind randomized clinical trial.
34

CLINICAL ASSESSMENT

Upon admittance into the study, each patient was given a medical questionnaire
and full mouth oral examination. Each examination form was dated and numbered for
identification purposes. Number of teeth, plaque index, gingival index, bleeding upon
probing, probing depths in mm, recession in mm, furcation involvement, mobility, and
suppuration were all assessed upon completion of the oral examination. Patients were
then made aware of their periodontal condition and were given the opportunity to ask
questions. Instruction in oral hygiene was given to each patient using visual aids and
verbal explication.

MICROBIOLOGICAL SAMPLING

Whole un-stimulated saliva samples were collected to determine the relative
abundance and ratios of bacteria in the mouth at baseline via two sterile polypropylene
centrifuge tubes measuring 3ml and 1ml independently. A third sample of expectorate
was collected in a sterile polypropylene centrifuge tube containing 0.5 ml Brain Heart
Infusion (BHI) broth media for cultivation of fastidious and non-fastidious organisms
with.
A pooled sample consisting of four supragingival sites (2 lingual and 2 facial)
with high plaque accumulation was chosen for cultivation using a Hu-Friedy® 3A
diagnostic explorer and placed into a sterile polypropylene sterilized graduated micro-
35

centrifuge tube containing 100 µL phosphate buffered saline (PBS) solution.
Three subgingival samples from the deepest periodontal pockets of the dentition were
individually obtained using separate ends of two sterile stainless steel Gracey curettes
and placed in their respectieve microcentrifuge tubes containing 100 µL PBS. An
additional sample of pooled subgingival plaque and calculus (2 lingual and 2 facial) was
collected using a sterile Gracey curette tip and placed into an agar containing vile for
susceptibility testing of fourteen different microbiota associated with periodontal
disease: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis,
Prevotella intermedia, Tannerella Forsythia, Campylobacter species, Eubacterium
species, Fusobacterium species, Peptostreptococcus micros, enteric gram negative rods,
Beta hemolytic streptococci, yeast, Eikenella corrodens, Staphylococcus species, and
Dialister pneumosintes.
All saliva and plaque/calculus samples were labeled with the date, patient
identification number, and letter corresponding to the recorded site of the extrapolated
sample. The samples were then placed in a Styrofoam box filled with ice for immediate
transfer to a -20°C freezer until prepared for microbiological analysis.

EPIFLUORESCENCE MICROSCOPY OF WHOLE UN-STIMULATED SALIVA

Following the collection of saliva samples, each specimen was individually
fixated, filtered, and hybridized using a synergy brands (SYBR) gold stain; enabling the
detection of single or double stranded DNA or RNA in electrophoretic gels using
standard ultraviolet transilluminators (Figure 2). The presence of the dye in stained gels
36

at standard staining concentrations does not interfere with Taq polymerase or many other
enzymes. The SYBR stain is readily removed from the DNA or RNA samples by ethanol
precipitation followed by the application of ammonium acetate, which allows for
additional investigations to be carried out using the same purified sample. Once this
process is completed, and the gel is illuminated with the ultraviolet transilluminators, the
gel is photographed for visual inspection of ample presence of bacteria prior to screening
for inserts using PCR (http://probes.invitrogen.com/media/pis/mp11494.pdf).

Figure 2 Preparation of plaque and saliva samples for epifluorescence microscopy in order to screen for
bacterial abundance prior to PCR amplification.

37

CULTURE-INDEPENDENT 16S rRNA GENE ANALYSIS OF PLAQUE

DNA was extracted from all microbes in the plaque and calculus samples and
purified using a MoBio PowerSoil™ DNA Isolation Kit. A partial amount (100 µL) of
the plaque and calculus samples were added to the PowerBead tubes for homogenization
and lysis accompanied by gentle mixing via vortex. A Sodium Dodecyl Sulfate
containing solution (C1) was then added in the amount of 60 µL and brief vortex was
applied for mixing. Once the PowerBead Tubes were horizontally secured, the sample
was then placed into the vortex for 10 minutes at maximum speed in order to break open
the cells and complete the homogenization and lytic process. Once the supernatant was
obtained, it was transferred to a 2ml collection tube and 250 µL of Solution C2 (Inhibitor
Removal Technology®) was added and vortexed for 5 seconds. The sample was then
incubated for 5 minutes at 4°C and centrifuged at room temperature for 1 minute. The
sample without the pellet was transferred to another clean 2 ml collection tube and 200
µL of Solution C3 (Inhibitor Removal Technology®) was added. The mixture was
briefly vortexed followed by another 5 minute incubation period at 4°C. The sample was
centrifuged for 1 minute at room temperature and then transferred to a clean 2 ml
collection tube. A high concentration salt solution labeled C4 (1200 µL) was added to
the supernatant and vortexed to 5 seconds to allow for binding of DNA to silica. This
step was followed by a series of loading supernatant onto the spin filter followed by
centrifugation in 1 minute intervals eliminating all remaining impurities and thus
generating only DNA bound to the membrane. Solution C5 is an ethanol based wash
38

solution that was used to further exclude residual foreign matter, salt, and humic acid.
Removal of the wash contaminated with impurities was then discarded and the filter was
centrifuged for 1 minute at room temperature. The filter was placed into a clean 2 ml
collection tube and 100 µL of Solution C6 (sterile elution buffer) was added to the filter
membrane. Once the sample was centrifuged one last time for 1 minute, the filter was
discarded and the DNA was stored in a -20°C freezer until ready for PCR.
Once the samples were thawed, the master mix was prepared using water,
Lucigen® (10X buffer, MgCl
2
, dNTPs, and Taq98™ DNA polymerase), 27F primer,
and 805R primer. UV light was applied onto all tubes for 10 minutes. The DNA
extrapolated from the plaque and calculus samples was added to the PCR tubes followed
by the master mix. The PCR tubes were gently vortexed to avoid any bubbles prior to
programming the PCR machine on the TaqMM2 setting. Meanwhile, the 1% agarose gel
was made using a gel mode and comb application. The gel was fabricated utilizing 0.6 g
of agarose that was added to a flask followed by 60 ml 1X TAE buffer and microwaved
for 30 seconds. The microwave was stopped to circulate the contents of the flask
followed by an additional 15 seconds in the microwave and recirculation was initiated to
cool down the constituents inside of the flask. To the melted agarose, 4 µL of ethidium
bromide were incorporated for fluorescence under UV light, and the agarose was then
deposited into the mold. Once gel was solidified, the comb and tape were removed, and
the mold was placed into a rig filled with 1X TAE buffer allowing the mold to be fully
immersed. The samples and loading dye were then loaded onto the gel. A lid along with
red and black wires were connected to the rig and machine was allowed to run the gel to
39

about half of the mold. The gel was placed into the UV machine and the images were
focused prior to capturing. All images were saved onto a secure digital card, and gels
were disposed of properly.

ILLUMINA
®
SEQUENCING ON THE MISEQ
®
SYSTEM VIA ARGONNE
NATIONAL LABORATORY (ILLINOIS)

Once proper extraction of the 16S rRNA was complete with annealed adaptors,
the samples were run on the Miseq
®
system at Argonne National Laboratories in Illinois
according to the aforementioned protocol described. QIIME raw data was recovered and
prepared for analysis.

DATA ANALYSIS

Non-parametric statistics were applied by using the one way ANOVA test to determine
statistical differences between the groups for bacterial measurements. False Discovery
Rate (FDR) correction was used to analyze microbial significance and achieve multiple
comparisons. This value allows for more flexibility of the range in p-value than does the
Bonferonni correction by accounting for the distribution of false positives attained,
making this value more desirable in this particular type of analysis. A p-value of equal
to or less than 0.05 was used to determine significance.

40

CHAPTER 3: RESULTS

In total, 21 otherwise healthy adult patients with chronic or aggressive forms of
periodontitis each provided one whole unstimulated saliva sample, one supragingival
plaque sample, and 3 subgingival plaque samples at baseline. A total of 21 whole saliva
samples were sent for epifluorescence microscopy and 84 plaque samples were sent for
high-speed, multiplexed 16S rRNA gene Illumina
®
sequencing through the MiSeq
®

system. 12 patients included for analysis presented with subgingival periodontal pockets
ranging from 5-9+ mm, 3 patients presented with a range of 5-8 mm pockets, and the
remaining 6 patients presented with a range of 5-6 mm of periodontal pocket depth.
These samples were included for epifluorescence microscopy of plaque and saliva,
followed by high-speed, multiplexed 16S Illumina
®
sequencing through the MiSeq
®

system of supra and subgingival plaque, in this double-randomized clinical control trial.
The patient distribution is summarized in Table 7. The male to female ratio of
participants in this study was 13:8 with a mean age of 45.5 years.

Patient (n) Gender Age in Years
Subgingival Pocket
Group
1 male 25 5-9 + mm
2 female 54 5-9 + mm
3 male 38 5-9 + mm
4 male 51 5-6 mm
5 female 51 5-9 + mm
6 male 70 5-9 + mm
41

Patient (n) Gender Age in Years
Subgingival Pocket
Group
7 female 32 5-9 + mm
8 male 44 5-9 + mm
9 male 43 5-6 mm
10 male 64 5-6 mm
11 female 38 5-9 + mm
12 male 30 5-6 mm
13 female 36 5-9 + mm
14 female 55 5-9 + mm
15 male 52 5-8 mm
16 female 51 5-6 mm
17 female 48 5-8 mm
18 male 43 5-8 mm
19 male 48 5-9 + mm
20 male 30 5-9 + mm
21 male 52 5-6 mm

Table 7 Patients were enrolled in no particular order and assigned to the study once all inclusion and
exclusion criteria were evaluated.

EPIFLUORESCENCE MICROSCOPY ANALYSIS

Each of the whole unstimulated saliva, supra- and subgingival plaque samples
were analyzed for bacterial abundance prior to sequencing. Figure 3 shows one plaque
sample stained and photographed after microscopy preparation with an abundance of
42

bacteria represented by fluorescent light emission. Each sample at baseline yielded
adequate bacterial abundance, therefore qualifying for analysis through high-speed,
multiplexed 16S Illumina
®
sequencing through the MiSeq
®
system. The purpose of
epifluorescence microscopy in this study was purely observational, no further data was
withdrawn or used for analysis with this assay.
Figure 3 Epifluorescence microscopy of a subgingival plaque sample at baseline. This allowed for the
observation of abundant bacteria illustrated in the areas arraying fluorescent light emission. This sample
seen here has ample bacteria present to yield data after sequencing.

QUANTITATIVE INSIGHTS INTO MICROBIAL ECOLOGY (QIIME)
SOFTWARE ANALYSIS

Results withdrawn from high-speed, multiplexed 16S Illumina
®
sequencing
through the MiSeq
®
system were uploaded into the QIIME software program for
operational taxonomic unit (OTU) identification as well as abundance ratios per sample.
This software allows for comparison and analysis conversion of microbial communities
43

from the raw data received through sequencing. Table 8 represents the 15 statistically
significant bacterial taxa from the samples of supra and subgingival plaque samples
included in this study as well as the non-significant data extracted via the QIIME
software. The results of this study indicate that the 15 statistically significant taxa in
supra and subgingival plaque highlighted in Table 8: Leptotrichia sp., Filifactor sp.,
Bacteroidales of two unidentified families, Clostridia, Treponema sp., Firmicutes clone
RF3:ML615J-28, Desulfovibrio sp., Synergistetes clone TG5, Chloroflexi clone SHD-
231, Porphyromonas sp., Clostridiales, Desulfobulbus sp., Leptotrichiaceae,
Clostridiaceae.

44

Table 8 QIIME raw data after Illumina
®
sequencing has terminated analyses.

45

PHYLA TAXONOMY PLOT

Once the QIIME data has been uploaded onto the excel sheet, a taxonomy plot is
then fabricated using layering software and color-coding. Figure 4a lists the taxa
uncovered through analysis with the five predominant bacterial groups highlighted by
the red circles: Actinobacteria, Fusobacteria, Spirochaetes, Bacteroidetes, and
Firmicutes. Proteobacteria, Synergistetes, Tenericutes and TM7 were also present as
well as other bacteria uncommonly reported such as Acidobacteria, Chloroflexi,
Gemmatimonadetes, Elusimicrobia, Cyanobacteria and Verrucomicrobia represented in
the taxonomy plot at the phyla level (Figure 4b). It is at the phyla level that abundance
ratios can clearly be observed. As the taxa are plotted on a more specific level, taxonomy
plots become more puzzling and hard to differentiate between taxa.
This taxonomy plot shows highest abundance of Firmicutes in the supragingival
and 5-6 mm subgingival plaque samples, Bacteroidetes in the 5-8 mm subgingival
plaque group, and Spirochaetes in the 5-9+ mm subgingival samples.
Firmicutes and Proteobacteria were present amongst all plaque groups, however
the abundance ratio tended to decrease as the periodontal pocket increased. The
Spirochaetes, Actinobacteria, Synergistetes, and Bacteroidetes tended to have the
opposite trend; increasing as the periodontal pocket deepened. Fusobacteria were fairly
consistent in both supra and subgingival plaque groups at different pocket depths.

46

Figure 4a Legend for the phyla taxonomy plot with encircles abundant bacteria for all groups analyzed.

47

Figure 4b Taxonomy plot on a phyla level illustrating abundance distributions of bacteria identified per
group analyzed.

SIGNIFICANT FINDINGS

Select pathogens, both anticipated and non-anticipated, were discovered at
statistically significant levels. Non-parametric statistics were applied by using the one
way ANOVA test to determine statistical differences between the groups for bacterial
measurements. FDR correction was used to analyze microbial significance. A p-value of
equal to or less than 0.05 was used to determine significance.
Supragingival plaque group yielded the highest abundance in Leptotrichia sp. (p <
0.028). The subgingival plaque samples were divided into three groups: the 5-6 mm
Supragingival 5-9+mm 5-8mm 5-6mm
Fusobacteria
Firmicutes
Proteobacteria
Bacteroidetes
Spirochaetes
48

pocket group, the 5-8 mm pocket group, and the 5-9 + mm pocket group. The 5-6 mm
pocket group yielded the lowest in abundance for all bacterial groups. The 5-8 mm
pocket yielded Filifactor sp. (p < 9.90 x 10
-6
) , Bacteroidales (p < 0.021 and p< 0.038)
of two unidentified families, and Clostridia (p < 0.024) in highest abundance. The 5-9+
mm pocket range group proved most abundant in the highest number of bacterial groups
tested: Treponema sp. (p < 4.06 x 10
-6
) , Firmicutes clone RF3:ML615J-28 (p < 2.04 x
10
-6
), Desulfovibrio sp. (p < 0.00024), Synergistetes clone TG5 (p < 0.0045), Chloroflexi
clone SHD-231(p< 0.0059), Porphyromonas sp. (p < 0.0068), Clostridiales (p < 0.0067),
Desulfobulbus sp. (p < 0.0076), Leptotrichiaceae (p < 0.0074), and Clostridiaceae (p <
0.022). Summarized statistically significant data are summarized below (Table 9).

Bacteria identified
FDR
Correction
(p value <
0.05)
Supra-
gingival
Group
distributio
n
5-6 mm
Group
distributio
n
5-8 mm
Group
distributio
n
5-9+ mm
Group
distribution
Leptotrichia sp. p <0.028 0.05201 0.03548 0.02200 0.00851
Filifactor sp. p < 9.90 x 10
-6
0.00187 0.00368 0.03476 0.01255
Bacteroidales p < 0.021,
p < 0.038
0.00015,

0.00448
0.00015,

0.00870
0.00101,

0.02931
0.00053,

0.01651
Clostridia p < 0.024 0.00093 0.00422 0.01330 0.00860
Treponema sp. p < 4.06 x 10
-6
0.02261 0.08505 0.14838 0.23867
49

Bacteria identified
FDR
Correction
(p value <
0.05)
Supra-
gingival
Group
distributio
n
5-6 mm
Group
distributio
n
5-8 mm
Group
distributio
n
5-9+ mm
Group
distribution
Firmicutes clone
RF3:ML615J-28
p < 2.04 x 10
-6
0.00018 0.08504 0.14838 0.23866
Desulfovibrio sp. p < 0.00024 0.00078 0.00079 0.004560 0.03207
Synergistetes clone
TG5
p < 0.0045 0.00269 0.02036 0.01918 0.05398
Chloroflexi clone
SHD-231
p< 0.0059 7.95 x 10
-
05

0.00064 0.00185 0.00600
Porphyromonas sp. p < 0.0068 0.01195 0.025969 0.05697 0.08591
Clostridiales p < 0.0067 0 0.00032 0.00117 0.00217
Desulfobulbus sp. p < 0.0076 0.00013 0.00154 0.00334 0.00697
Leptotrichiaceae p < 0.0074 0.00314 0.00023 0.01902 0.03428
Clostridiaceae p < 0.022 0.00011 0.000718 0.00180 0.01100

Table 9 Distribution of the statistically significant bacteria identified along with the abundance ratios
between the groups. p < 0.05 for all taxa shown. Periodontal pocket groups with highest abundance per
plaque sample collected is highlighted in green.

50

The distribution of statistically significant bacteria represented per supra- and
subgingival plaque sample is summarized in Figures 5 and 6.

Figure 5 Bar graph of the abundance ratios between the groups per bacteria identified. p < 0.05 for all taxa
shown.
51

Figure 6 shows the distribution of the statistically significant bacteria identified along with the abundance
ratios between the groups. p < 0.05 for all taxa shown.

PRINCIPAL COORIDATES ANALYSIS

The results outlined in this manuscript display distinct dissimilarities between
supra- and subgingival plaque at different depths of a diseased periodontal pocket, in
particular the 5-9+ mm group. This is observed in Figure 7, whereby irrefutable
dissociation between the 5-9+ mm pocket group and the other groups is presented. This
52

dissociation is very clearly illustrated in this diagram and confirms differences in
bacterial genetic makeup between supra- and subgingival plaque, and between
subgingival plaque existing at different levels of the diseased periodontal pockets. This
is represented in the scatter plot (Figure 7) by aggregation of plaque samples belonging
to the same group. The supragingival group along with the 5-6mm pocket group shows
the most overlap between groups. There is also some overlap between the 5-8mm pocket
group and the 5-9+ mm pocket group. The 5-6 mm pocket group also shows the most
variation aggregating the least between these plaque samples. These visual
considerations are purely observational, and are to be analyzed in conjunction with the
aforementioned quantitative data.

Figure 7 PCoA plot of the Unifrac distance between all plaque samples by pocket depth. Clustering of
individual periodontal pocket groups is illustrated by a circle of corresponding color.

53

NON-STATISTICAL, NOTEWORTHY FINDINGS

Although there were only 15 statistically significant bacteria revealed through this
diagnostic methodology, a detailed inventory of noteworthy bacteria were also observed
(Table 10). This small sample of patients included major periodontopathogenic bacteria
as well as bacteria associated in health, bacterial clones that have not been cultured, and
unidentified or unclassified bacteria.

Bacteria Identified FDR Correction
unidentified bacteria 0.10
Clostridiaceae 0.12
Selenomonas sp. 0.23
Streptococcus sp. 0.31
Oribacterium sp. 0.32
Cardiobacterium sp. 0.31
Actinomycetales 0.30
Bacilli 0.35
Bacteroides sp. 0.35
Johnsonella sp. 0.36
Streptococcaceae 0.37
Veillonella sp. 0.36
Actinomyces sp. 0.36
Corynebacterium sp. 0.45
54

Bacteria Identified FDR Correction
Capnocytophaga sp. 0.45
Atopobium sp. 0.44
Lachnospiraceae 0.44
Comamonadaceae 0.47
Fusobacteriales 0.48
Mycoplasma sp. 0.47
Acinetobacter sp. 0.49
Pasteurellaceae 0.48
Rothia sp. 0.55
Bulleidia sp. 0.54
Catonella sp. 0.55
Clostridia 0.55
Aggregatibacter sp. 0.57
Fusobacterium sp. 0.56
Flavobacteriaceae 0.56
Prevotella sp. 0.55
Actinomycetaceae 0.55
Tannerella sp. 0.58
Lautropia sp. 0.57
Veillonellaceae 0.59
unclassified bacteria 0.61
Neisseriaceae 0.60
55

Bacteria Identified FDR Correction
Clostridiales 0.59
Actinomycetaceae 0.60
TM7 clone: TM7-3 0.60
Megasphaera sp. 0.59
Abiotrophia sp. 0.60
Bacteria clone: GN02
BD1-5
0.61
Parascardovia sp. 0.64
Neisseriaceae 0.64
Bacillaceae 0.65
Coriobacteriaceae 0.65
Campylobacter sp. 0.66
Haemophilus sp. 0.66
Prevotella sp. 0.66
Firmicutes 0.74
Lactobacillae 0.80
Bacteria clone: SR-1 0.80
Enterococcus sp. 0.79
Neisseria sp. 0.78
Peptostreptococcaceae 0.77
Gemellaceae 0.79
Hylemonella sp. 0.79
Veillonellaceae 0.84
56

Bacteria Identified FDR Correction
Ruminococcaceae 0.83
Moryella sp. 0.84
Peptostreptococcus
sp.
0.85
Streptococcaceae 0.88
Mogibacterium sp. 0.88
Kingella sp. 0.88
Pseudoramibacter sp. 0.88
Dialister sp. 0.87
Peptococcus sp. 0.91
Mollicutes
clone:RF39
0.90
TM7 clone: TM7-
3,I025, Rs-045
0.94

Table 10 Non-statistically significant bacteria identified through Illumina
®

.

57

Fig 8 shows the distribution of the non-statistically significant bacteria identified along with the abundance
ratios between the groups. p > 0.05 for all taxa shown.

58

CHAPTER 4: DISCUSSION

Patients accepted into this study excluded patients that were not diagnosed with
either chronic or aggressive forms of periodontitis. Consequently, patients with healthy
periodontium were excluded from this study, resulting in the absence of a traditional
control group. Previous literature has extensively documented differences between
supra- and subgingival plaque between patients with periodontal health versus disease,
however studies have yet to explain the complex discrepancies that exist between
microorganisms existing at different depths of a wide range of the periodontal pocket
exclusively in a diseased state.
Sterile stainless steel curettes were employed for harvesting plaque samples at
different depths of the periodontal pocket. However, due to the advanced progression of
periodontal disease present, particularly in the 5-8 mm and 5-9+ mm groups, it is likely
that adequate retrieval of plaque was not achieved. Perhaps mini-curettes may have
provided more accessibility to the base of the pocket for a more accurate sample
retrieval. Using paper points, endodontic barb broaches, DNA-free wooden toothpicks
and swabs are other methods of collecting microbial samples that have previously been
documented. Paper points and sterile curettes are among the various methods that are
most widely used. Nevertheless, there is evidence to support the usage of both paper
points and curettes, and that one technique is not superior to the other (Jervøe Storm et
al. 2007). Ultimately, the precision of any diagnostic device used to analyze
59

microbiological samples will only be as accurate as the quality of the sample that is
collected from the patient.
Apart from sampling techniques utilized in this study, the sample size consisted of
a total of 21 patients at baseline. This could explain why the results presented above
show many microorganisms present in the absence of statistical significance. Sampling
one subgingival pocket, or collecting supra- and subgingival plaque samples of the same
tooth to receive an accurate representation of the bacteria present in the diseased mouth
as a whole is somewhat of a gamble that may be attenuated statistically by a larger
sample size. Further studies comprised of a larger population and number of samples
collected may yield more statistically significant results of these bacteria known to be
present supra- and subgingival to a diseased periodontal pocket.

60

Figure 9 Adapted from Paster et al. 2006) this figure represents a phylogenetic tree in which both cultured
and uncultured bacteria have previously been identified through next generation sequencing.

Another possible drawback of Illumina
®
sequencing on the Miseq
®
system as well
as other next generation sequencing methodologies as stated by Wade (2013) is,
insufficient resolution for confident identification of species comprising some of the
61

important and predominant oral genera due to recombination within the 16S rRNA gene,
making the interpretation of phylogenetic relationships derived from this gene difficult.
As observed in this study, all bacteria identified were at a genus level or higher, making
species identification merely a presumption from previous literature.
High-speed, multiplexed 16S rRNA Illumina
®
sequencing on the Miseq
®
system
has proven to be a valuable test to further understand the oral microbiome. This method
is both cost effective and efficient for providing data that supports the discovery of new
microorganisms pertaining to the oral cavity that have not been identified to date. Based
on the evidence outlined throughout this manuscript, high-speed, multiplexed 16S
Illumina
®
sequencing on the Miseq
®
system offers a multitude of advantages in the
detection of bacterial complexes of the oral microbiome in patients diagnosed with
periodontitis on a more profound sequencing level.
To our knowledge, there have been no prior publications that exclusively include
patients with advanced stages of disease (both aggressive and chronic forms of
periodontitis), exhibiting deeper periodontal pocket depths from which subgingival
plaque samples were harvested from at different levels of the pocket within the same
patient. Samples collected from subgingival plaque at various depths of the periodontal
pockets harvested were also evaluated for association and differentiation. Principal
coordinates analysis plots were accomplished through a process called multidimensional
scaling (MDS) which allows for visualization of similarities and differences of data
being analyzed. The aforementioned data was first presented in an exceedingly high
dimensional space that is not visible to the human eye, then reduced to a lower
62

dimensional space, safeguarding the data through mathematical equations, and
conveying this data in the form of a two-dimensional scatter plot via the Unifrac method.
The results were clearly able to prove that there are distinct dissimilarities between
supra- and subgingival plaque at different depths of a diseased periodontal pocket;
particularly in the 5-9+ mm group, and even in small sample groups. Similar groups
generally tend to cluster together and the further apart the level of the pocket is, the
further apart the dissociation becomes. This observation is remarkable due to its ability
deduce that there are distinct microbial profiles from different ecological oral sites. The
principal coordinates scatter plot only represented the plaque samples of the 21 patients
enrolled in this study at baseline. However it is important to keep in mind that this was a
small study with a limited number of patients due to stringent inclusion and exclusion
criteria. With a much larger yet similar study, we may be able to see many more bacteria
present with similar dissociation.
Illumina
®
sequencing has also allowed for identification of relatively new taxa
associated with the oral microbiome; namely Chloroflexi Anaerolineae, enabling future
microbiological advancements in periodontology to emerge. Little is known of
Chloroflexi Anaerolineae, however there have been a few studies that have detected this
filamentous monoderm that mostly stains Gram negative . The phylum is named for it’s
‘green bending’ characteristics and formerly known as ‘Green non sulfur bacteria’
(Yamada Sekiguchi 2009, Hugenholtz et al. 2008). According to Yamada and Sekiguchi
(2009), Chloroflexi can be characterized into seven species in six genera: Anaerolinea,
Levilinea, Leptolinea, Bellilinea, Longilinea, and Caldilinea. These six genera represent
63

two classes: Anaerolineae and Caldilineae. Chloroflexi Anaerolineae is part of the
subphylum I, which is comprised of the most diverse environmental clones, responsible
for >70% of sequences identified with Chloroflexi. Previous reports have identified
Chloroflexi in natural and artificial habitats (Table 11).
Table 11 Adapted from Yamada and Sekeguchi (2009) Chlorflexi identified in natural and artificial
habitats namely sediment, soil, hot springs, freshwater, hypersaline lakes, wastewater treatments, lagoons,
mine drainages, and microbial fuel cell systems. Oral microbiota is absent from this summary in a
subphyla categories

Few authors have been able to identify Chloroflexi as present in the oral
microbiome (Griffen et al. 2011, Kiejsov et al. 2008, Dewhirst et al. 2010).
Furthermore, there has not been extensive literature focusing on quantitative bacterial
analysis of the oral microbiome using next generation sequencing. This relatively new
finding should be taken seriously given that previous groups as well as the present study
recognizes Chloroflexi as a significant finding amongst samples of plaque from
periodontitis patients. This bacterium may need to be considered as pathogenic to the
periodontium. Further studies are needed and preferentially culture isolates are to be
64

obtained in order to study the pathogenic potential of the bacteria in functional assays
such as tissue cultures and animal models.
Next generation sequencing also offers a non-biased technique that is highly
specific and accurate without requiring specifications for target bacteria. In addition,
Illumina
®
sequencing is highly efficient in its ability to read over 30 million DNA
sequences at one time as opposed to 500,000 DNA sequences at one time using 454-
pyrosequencing; significantly reducing the cost and time per specimen sampled (Tringe
Hugenholtz 2008, Caporaso et al. 2012, Luo et al. 2012). The present study was able to
confirm that organisms found utilizing diagnostic methodologies are in concurrence with
higher generation sequencing with additional organisms found. Illumina
®
sequencing in
the present study was able to identify major putative periodontal pathogens such as
Treponema sp., Porphyromonas sp, Synergistetes, Spirochaetes, and unidentified
Bacteroidetes in statistically significant amounts amongst these plaque samples (Griffen
et al. 2011, Socransky Haffajee 1998, Paster et al. 2001, Paster et al. 2006, Ximenez-
Fyvie et al. 2000, Moore et al. 1983). Other periodontopathogenic bacteria were also
identified; albeit in non-statistical amounts, namely Tannerella sp., Fusobacterium,
Peptostreptococcus, Clostridiales, Campylobacter sp., and Prevotella sp. Bacterial
organisms found in lower amounts in health and supragingival plaque were also in
accordance with previous literature namely the Proteobacteria and Leptotrichia sp. in
statistically significant amounts and the Steptococcus sp., Actinomyces sp.,
Capnocytophaga sp., Veillonella sp., Selenomas sp. and Rothia sp. (Moore et al. 1983,
65

Haffajee Socransky 1994, Ximenez-Fyvie et al. 2000). These results clearly show an
association between bacterial species and clinical periodontal status.
If we compare next generation sequencing methods to one another, we can see
advantages and disadvantages for each method (Table 5), however there is evidence to
support that Illumina
®
sequencing can provide a lower error rate, lower cost, and higher
throughput than 454-pyrosequencing (Caporaso 2011). Metagenomics was another
methodology considered for bacterial identification, however due to small sample size
and high cost per run, this method was less preferable for the purposes of this study.
Also, we did not need to look at these bacteria on a strain specific level beyond species.
This method would have given us overwhelming amounts of quantitative data not
pertinent to the realm of this study. For all of the aforementioned reasons, Illumina
®

sequencing was an appropriate method to use for the aims of this study.
In order for the findings outlined in this manuscript to have meaning they must
establish a causal relationship between the suspected causative microbe found and
periodontal disease. Koch’s postulate is based on four criteria that confirms such a
relationship. These criteria as described by Carter in 1987 are as follows: 1. the agent
must be isolated from every case of the disease, 2. it must not be recovered from cases of
other forms of disease or non-pathogenically, and 3. after isolation and repeated growth
in pure culture, the pathogen must induce disease in experimental animals. Socransky
later revised these criteria in 1977 and again with Haffajee and Socransky in 1994 to
include the presence of pathogenic bacteria at the site of active disease to have higher
numbers than in healthy sites in the same individual, examination of microbial changes
66

during progression periodontal disease from to normal to the periodontitis state,
elimination of suspected pathogens followed by monitoring for progression of disease,
host response possibly providing clues to a pathogenic role of an organism, virulence
factors, animal studies suggesting similar trends in pathogenicity of the human may be
observed, and transmission (risk of periodontal disease progression conferred by the
presence of an organism at given levels may be assessed) such as husband to wife, as
opposed to causing harm in an individual for the purposes of advancing microbiological
research (Socransky 1977, Haffajee Socransky 1994).

67

CHAPTER 5: CONCLUSION

The present manuscript details an efficacious, highly safe, minimally invasive,
practical and inexpensive method to further explore the complex nature of the oral
microbiome. Distinct microbial profiles from different ecological oral sites were found
and summarized in this manuscript. The results of the present study were able to reveal
15 statistically significant bacteria: Leptotrichia sp., Filifactor sp., Bacteroidales of two
unidentified families, Clostridia, Treponema sp., Firmicutes clone RF3:ML615J-28,
Desulfovibrio sp., Synergistetes clone TG5, Chloroflexi clone SHD-231,
Porphyromonas sp., Clostridiales, Desulfobulbus sp., Leptotrichiaceae, and
Clostridiaceae. Suggestive and remarkable findings were also observed by the
recognition of over 60 other bacteria present in these samples, although not statistically
significant namely Tannerella sp., Fusobacterium, Peptostreptococcus, Clostridiales,
Campylobacter sp., Prevotella sp., Steptococcus sp., Actinomyces sp., Capnocytophaga
sp., Veillonella sp., Selenomas sp. and Rothia sp.
This study has effectively illustrated the capabilities of Illumina
®
sequencing on
the Miseq
®
system of providing data that supports the discovery of relatively new taxa
pertaining to the oral cavity. Illumina
®
sequencing has proven to be a valuable test to
further understand the oral microbiome in an efficient and cost effective manor. It is well
known that pathogenesis of periodontal disease becomes more complex as the disease
progresses. As more bacteria become progressively identified as putative pathogens
through technological advancements in bacterial diagnostics, the oral microbiome
68

becomes less of a quandary and more of a recognized entity. Increasing knowledge and
further similar studies of such a perplexing ecosystem can allow the fields of
microbiology and periodontology to advance towards eliminating these pathogenic
microorganisms by way of an effective and accessible treatment.

69

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Abstract (if available)

Abstract Technological advances have been made over the past decade to ameliorate bacterial taxonomy in phylogenetics. Such an instance would best be illustrated by the unearthing of the 16S rRNA gene and its ability to provide what is known today as the bacterial genome. Because of this discovery, we have evolved technologically from being able to run 500,000 DNA sequences at one time with 454 pyrosequencing, to 30 million sequences at one time with Illumina® sequencing. This allows for differentiation and association of species in the oral microbiome that exist supragingival and subgingival to the periodontal pocket. ❧ Previous studies have shown that different species exist above and below the periodontal pocket. This study stands to accomplish two main goals: to assess the microbial profile of sites with distinct periodontal status, and to apply high-speed, multiplexed 16s Microbial Illumina® sequencing on the Miseq® system for analysis as a discovery base for potential new taxa within and supragingival to the periodontal pocket in a diseased state. ❧ In this double-blind randomized clinical trial, a total of 21 otherwise healthy adult patients with periodontitis were included. Whole unstimulated saliva samples were collected to determine the relative abundance and ratios of bacteria in the mouth at baseline through epifluorescence microscopy. Three separate subgingival samples from the deepest pockets were obtained for culture-independent 16s rRNA analysis followed by Illumina® sequencing. ❧ Fifteen statistically significant (P < 0.05) bacteria were identified. Select pathogens, both anticipated and non-anticipated, were discovered at statistically significant levels. Supragingival plaque group yielded the highest abundance in Leptotrichia sp. (p < 0.028). The subgingival plaque samples were divided into three groups: the 5-6 mm pocket group, the 5-8 mm pocket group, and the 5-9 + mm pocket group. The 5-6 mm pocket group yielded the lowest in abundance for all bacterial groups. The 5-8 mm pocket yielded Filifactor sp. (p < 9.90 x 10⁻⁶), Bacteroidales (p < 0.021 and p < 0.038) of two unidentified families, and Clostridia (p < 0.024) in highest abundance. The 5-9+ mm pocket range group proved most abundant in the highest number of bacterial groups tested: Treponema sp. (p < 4.06 x 10⁻⁶), Firmicutes clone RF3:ML615J-28 (p < 2.04 x 10⁻⁶), Desulfovibrio sp. (p < 0.00024), Synergistetes clone TG5 (p < 0.0045), Chloroflexi clone SHD-231(p< 0.0059), Porphyromonas sp. (p < 0.0068), Clostridiales (p < 0.0067), Desulfobulbus sp. (p < 0.0076), Leptotrichiaceae (p < 0.0074), and Clostridiaceae (p < 0.022). ❧ High-speed, multiplexed 16s microbial Illumina® sequencing on the Miseq® system throughput has proven to be a valuable assay to further understand the oral microbiome. This method is both cost effective and efficient for providing data that supports the discovery of microorganisms pertaining to the oral cavity that have not been identified to date.

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The utility of bleeding on probing and 0.25% sodium hypochlorite rinse in the treatment of periodontal disease

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Creator Cohen, Chloé Léa (author)

Core Title Technological advancements in microbial analyses of periodontitis patients: focus on Illumina® sequencing using the Miseq system on the 16s rRNA gene: a clinical and microbial study

School School of Dentistry

Degree Master of Science

Degree Program Periodontology / Craniofacial Biology

Publication Date 08/05/2013

Defense Date 06/04/2013

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

Tag 16s rRNA gene,advanced periodontal pocket depth,Illumina sequencing,OAI-PMH Harvest,periodontitis,subgingival plaque,supragingival plaque

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Language English

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Advisor Slots, Jorgen (committee chair), Navazesh, Mahvash (committee member), Paine, Michael L. (committee member), Rich, Sandra (committee member)

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