Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2018; 63: 14–24

doi: 10.1111/adj.12565

Studying the human oral microbiome: challenges and the

evolution of solutions
AML Benn,* NCK Heng,† JM Broadbent,† WM Thomson†
*Southern District Health Board, Dunedin, New Zealand.
†The University of Otago – Sir John Walsh Research Institute, Dunedin, New Zealand.

ABSTRACT
Since the pioneering work of van Leeuwenhoek in 1684, subsequently built upon by other renowned microbiologists
Robert Koch, Willoughby Miller and GV Black, oral microbiology has developed innovative techniques to study the oral
microflora (now termed the ‘oral microbiome’). The advent of molecular techniques such as DNA–DNA hybridization,
polymerase chain reaction and DNA sequencing has created an array of opportunities to construct a comprehensive pic-
ture of the diversity and composition of the oral microbiome. Approximately 700 oral bacterial species have been identi-
fied, of which 50% have yet to be cultivated, and some of these are known only by their signature DNA sequences. The
synergism of ever-evolving culture-based and state-of-the-art culture-independent molecular techniques has facilitated in-
depth understanding of the dynamics, acquisition and transfer of oral bacteria, along with their role in oral and general
health and disease. Further research is needed to not only analyse but also to make sense of the ever-increasing volumes
of data which these molecular techniques (especially high-throughput DNA sequencing) are generating, as well as why
particular bacteria are present and what they are ‘actually doing’ there. This review presents a comprehensive literature
search of oral microbiology-related methods currently used to study the oral microbiome.
Keywords: DNA–DNA hybridization, next-generation DNA sequencing, oral microbiology, oral bacteria, polymerase chain reaction.
Abbreviations and acronyms: FISH = fluorescence in situ hybridization; NGS = next-generation DNA sequencing; PCR = polymerase
chain reaction.
(Accepted for publication 23 August 2017.)

In 1684, Antonie van Leeuwenhoek stated in a let-

INTRODUCTION
The oral cavity is home to one of the most complex,
dynamic and diverse microbial collections in the
human body. This collection, termed the ‘oral micro-
biome’, comprises mainly (eu)bacteria, viruses, fungi,
protozoa and archaebacteria (Archaea). It is also the
oldest recognized microbial ecosystem. Current esti-
mates suggest that up to 1000 bacterial species are
present in the mouth, inhabiting several distinct
microbial niches. These include saliva, the teeth, the
gingival sulcus, the attached gingiva, the tongue, the
cheek, the lip, and the hard and soft palate.1–3 Within
each oral habitat, the microbes can be found growing
in a distinct community, or biofilm, a functionally
and structurally organized, matrix-enclosed aggregate
of microorganisms which adheres to surfaces such as
tooth enamel. That the oral microbiome is readily
accessible and easily sampled has resulted in it being
the most-studied human microbiome, and it serves as
a model for biofilm biology in general.3–7
ter to the Royal Society of London that ‘The number
of these Animals [bacteria] in the scurf of a man’s
Teeth are so many that I believe they exceed the num-
ber of Men in a kingdom’.8–11 Van Leeuwenhoek
examined plaque samples and observed that the type
and numbers of different ‘Animals’ varied among indi-
viduals. His drawings were among the first to illus-
trate the main morphological types of bacteria – that
is, coccus, rod, vibrio and spirillum – in this complex
community.9–12
Almost two centuries later, Robert Koch’s develop-
ment, in 1881, of a solidified culture medium was a
momentous advance in microbiology. He developed a
readily reproducible technique for growing and isolat-
ing pure cultures of microorganisms using gelatin and
eventually agar. This technique enabled researchers to
cultivate, identify, name and classify different
microbes,9 including many of those causing serious
infections in humans.13 Koch’s postulates for identify-
ing the causative agent for a given infectious disease,

14 © 2017 Australian Dental Association


together with his culture medium innovation, not only
earned him the 1905 Nobel Prize, but also resulted in
a microbiological world divided into the ‘cultured’
and the ‘yet-to-be cultured’.14
Credited as being one of the first oral microbiolo-
gists was WD Miller, a dental scientist and Professor
of Operative Dentistry who worked with Koch at the
University of Berlin.11 In 1890, 9 years after Koch’s
innovative developments and formulations, Miller
published his seminal work ‘Micro-organisms of the
human mouth: the local and general diseases which
are caused by them’. He reported isolating more than
100 bacterial types of bacteria from the juices and
deposits in the mouth.15 He also noted and high-
lighted microbiological issues that are still pertinent
today, namely that numerous oral bacteria are resis-
tant to cultivation, and that the oral cavity is an open
system, with the number and variety of bacteria con-
tinually being augmented by new microbes from air,
food and drink.15 Concurrently, GV Black (then Pro-
fessor of Oral Pathology at the Missouri Dental Col-
lege) reported culturing and identifying bacteria from
samples of carious tooth material.16
CULTIVATION
Historically, guided by Koch’s postulates, the quest
has been to identify and characterize cultivable
bacteria that were associated with various oral dis-
eases. However, culture has limitations in revealing
the actual diversity of the oral microbiome. In 1890,
Miller himself commented on his inability to culture
all the bacteria he observed.6,16,17 This discrepancy
between population sizes and variety as estimated
from microscopy and culturing is known as the ‘great
plate count anomaly’.14,18,19 Hence, laboratory-based
culture per se is unable to fully characterize the diver-
sity and complexity of bacterial communities such as
those found in the oral cavity.20 Consequently, cultur-
ing and functional analysis of as-yet uncultivated
microorganisms remains a challenge in microbiological
research.19,21
It is estimated that more than 99% of bacterial
species on Earth have yet to be cultured in the lab-
oratory17,20 but the endeavours of numerous
researchers (building on the work of Miller and
Black) have now isolated, cultivated, identified,
characterized and classified approximately 50% of
the estimated 700 bacterial species which commonly
colonize the human mouth.13,19,22 However, the
remaining 50% of oral bacterial species identified
through more recently-developed culture-independent
DNA sequencing approaches remain resistant to
cultivation.6,13,16,17,19,22
Current concepts of oral disease are based largely
on knowledge obtained from fragments of bacterial
© 2017 Australian Dental Association
Studying the human oral microbiome
communities, using methods biased towards those
bacteria which survive transportation in a sample, can
grow easily or rapidly in the laboratory, and that are
amenable to genetic modification.20,23 Despite the
abundance of knowledge about opportunistic patho-
gens within the oral microbiota, it is limited and fun-
damentally incomplete. The ‘overlooked’ bacteria,
known by their molecular signatures, may be respon-
sible for several oral and general diseases.13 Indeed,
without studying the several hundred uncultivable spe-
cies found in healthy and diseased sites in the human
mouth, many aspects of microbial dynamics in the
oral cavity may never be understood.17,19,24 Hence,
one of the major challenges facing oral microbiology
is the ability to culture the as-yet-to-be cultivated
50% of oral species.
Advances in knowledge of oral microbial ecology
and dynamics have enabled development of novel cul-
ture techniques. The requirements for the cultivation of
numerous oral bacteria species include distinct condi-
tions such as an anaerobic (oxygen-free) environment,
incubation in a variety of temperatures, chemically-
defined media containing specific amounts of nutrients,
cytokine networks and microbial co-colonizers.13,24
Despite these advances, many organisms may remain
uncultivable in the conventional manner because they
exist in obligate metabolic associations with other
organisms. In reality, oral bacteria do not live in isola-
tion but in complex communities called biofilms, char-
acterized by multiple growth dependencies, synergies
and antagonisms, along with mutual reliance for
growth and survival.3,24 In vitro cultivation possibly
remains elusive for many species because of a lack of
essential nutrients, growth factors and/or signalling
molecules, overfeeding conditions, lack of cross-feeding
partners, culture media toxicity and disruption of bac-
terial quorum-sensing and other signalling systems.17
There may be other, unknown reasons for some species
not yet having been cultivated.
Conventional cultivation of bacteria samples requires
taking a sample, transferring the sample to an appropri-
ate medium for transportation, and storage following
collection. This is followed by dispersion and plating
the bacteria onto various culture media in the labora-
tory. The bacteria are then isolated and characterized
by their colony morphologies (appearance) and bio-
chemical testing. Species which do not grow are natu-
rally overlooked. Molecular techniques have enabled
an more in-depth investigation of mixed bacterial com-
munities. In turn, these techniques have resulted in dif-
ferent and more focused approaches to sample
cultivation. Initially, the species in the sample may be
identified by culture-independent molecular methods.
Cultivation can then be attempted using information
such as the site or sites (subgingival, supragingival) and
distinct conditions (substrate availability, co-colonizing
15
AML Benn et al.
species) under which the bacteria occur in the mouth.
However, many bacteria remain resistant to cultivation
despite this advance.22
Contemporary strategies for cultivation include cul-
ture media with few or no added nutrients, long-term
cultivation, serial dilution of slow-growing bacteria,
addition of specific growth factors to media, and
in vivo incubation. In addition, the availability of
sequenced genomes has created the possibility of using
computer modelling of metabolic networks to aid in
the development of bespoke culture media.17 For
example, Sizova et al.19 used a combination of some
of these novel strategies and traditional cultivation
techniques to isolate and culture 10 strains previously
known only by their molecular signature, as well as
20 new species. Similarly, using a variety of molecular
and traditional microbiological approaches, Soro
et al.24 and He et al.21 were able to culture and char-
acterize members of phylum TM7, a relatively new
phylogenetic group of bacteria previously identified by
next-generation DNA sequencing (NGS) and largely
believed to be unculturable.
Cultivation remains the cornerstone of oral microbi-
ology in characterizing phenotypically and formally
naming species, as well as describing the physiology
and pathogenicity of particular species. However, it is
now enhanced and informed by the development and
advances in molecular techniques, thereby enabling
and enriching the study of complex host-associated
bacterial communities such as those in the oral cavity.
MOLECULAR ORAL MICROBIOLOGY
The discovery of the double helical structure of the
DNA molecule by Nobel laureates James Watson and
Francis Crick in 1953 is arguably one of the most
important scientific discoveries of the 20th century.25 It
explained the mechanism of base pairing by which
genetic information is stored and copied in living
organisms. This ground-breaking work paved the way
for those seeking to detect, identify, type and under-
stand microorganisms in order to develop more sophis-
ticated culture-independent, nucleic acid-based
molecular technologies and techniques. These collec-
tively have revolutionized the field of oral microbiology
because they have revealed a degree of biodiversity
among oral bacteria which has exceeded expectations.
The dynamics of the oral microbiota are now better
understood because of the identification of patterns of
acquisition and transmission of bacteria.20,26
In addition, the identification of universally-con-
served DNA sequences such as the 16S rRNA gene
made information about evolutionary relatedness
among various groups of organisms (phylogenetics)
available. In turn, this has provided a universal system
for the identification and categorization of bacteria.
16
The process of categorizing bacteria phylogenetically
is closely related to taxonomy and is the means of
classifying organisms in an ordered hierarchical sys-
tem showing evolutionary relationships. It aims to
improve the understanding of bacterial function in
community structures, biodiversity and environments.
Species is the basic taxonomic group in bacterial sys-
tematics; in ascending order, each species belongs to a
genus, family, class, phylum/division and domain.20,27
The oral cavity has a complex microbial ecology
and a rich biological setting with a number of distinc-
tive niches, each of which provides a unique habitat
for microbial colonization. These niches include the
teeth, the gingival sulcus, the tongue, the floor of the
mouth, the cheek, the hard and soft palates, the ton-
sils, the throat and the saliva.2,5,7,28 The most com-
monly-used molecular research techniques used in
oral molecular microbiological research include DNA
hybridization, polymerase chain reaction (PCR) and
DNA sequencing. In the past, they were conducted as
stand-alone techniques but, at the present time, PCR
and DNA sequencing are invariably intertwined.
A consequence of research using culture-independent
approaches was the generation of thousands of sequences
of cloned human oral bacterial 16S rRNA genes, which
were deposited into DNA sequence databases such as
GenBank without any taxonomic reference points. In the
absence of any taxonomic (naming) scheme, researchers
were publishing findings using isolated 16S rRNA gene
sequences (clones) as provisional taxonomic names.
Moreover, in order to phylogenetically place an oral
clone, investigators had to manually align sequences and
generate their own phylogenetic trees.5
The recognition of this problem led to researchers
such as Chen and Dewhirst creating a standardized,
universal taxonomic scheme, namely the Human Oral
Microbiome Database (HOMD; www.homd.org), a
curated phylogeny-based database of 16S rRNA gene
sequences. Because 16S rRNA gene sequencing is
insufficient for formal species assignment of bacteria,
the taxonomic scheme that was developed is a provi-
sional one; the guidelines governing formal naming of
microbial species still require isolation of a pure cul-
ture and full phenotypic, and preferably genomic,
characterization. The HOMD was the first description
of a human-associated microbiome. It enables
researchers to relate sequence information to specific
organisms in a taxonomic framework which is key to
understanding the role of the microbiome in health
and disease.5 To date, approximately 46% of the
HOMD 16S rRNA gene reference sequence taxa are
validly-named species, 14% are unnamed (but culti-
vated), and 32% are unnamed and uncultured taxa
known primarily from 16S rRNA sequence informa-
tion. The number of species residing in the human
oral cavity ranges from 700 to over 1000.2,5
© 2017 Australian Dental Association

CONTEMPORARY MOLECULAR TECHNIQUES TO
PROFILE THE ORAL MICROBIOTA
Culture independent nucleic acid-based molecular
analysis techniques are used for detecting and identify-
ing individual or multiple bacteria, bacterial commu-
nity diversity and bacterial typing.20 Essentially,
molecular techniques need a macromolecule of inter-
est (DNA, RNA or protein), method-specific reagents,
automated sample handling such as those handled by
robots and, ideally, a bioinformatics platform which
allows computer-aided analyses of large molecular
datasets to be carried out.29 The molecular techniques
routinely used in oral microbiology are largely DNA-
based and can be divided fundamentally into three
broad categories, namely: (i) DNA hybridization; (ii)
PCR; and (iii) DNA sequencing. The techniques avail-
able today are essentially more advanced high-
throughput approaches which incorporate combina-
tions of the three categories.
DNA–DNA hybridization
The discovery of the hybridization reaction, which
is the spontaneous pairing of two complementary
(i.e. matching sequences) strands of a nucleic acid
double-helix, led to the development of DNA–
DNA hybridization techniques for the study of
microorganisms. These techniques utilize single-
stranded ‘DNA probes’ labelled with either a
radioactive isotope, fluorescent or chemiluminescent
tag that bind to complementary bases of a target
DNA strand to form a duplex and can then be
detected using an appropriate instrument or combi-
nation of chemical reagents. The probes used in
the annealing process are either whole-genome
DNA probes or short (15–30-base) oligonucleotide
probes with DNA sequences which complement
their target sequence.20,30
Whole-genome probes are more likely to cross-react
with non-target bacteria than the more specific
oligonucleotide probes such as those binding to
regions of the 16S rRNA gene. The 16S rRNA gene
enables precise taxonomic positioning of bacteria
because, in species with 70% or more genomic simi-
larity, the 16S rRNA gene is usually a more than
97% gene sequence match; this is the universally-
accepted cut-off for species assignment.20,30,31
Hybridization techniques used in oral microbiological
research include whole-genome checkerboard DNA–
DNA hybridization, reverse-capture oligonucleotide
hybridization (modification of the checkerboard
method), fluorescence in situ hybridization (FISH) and
DNA microarray technology. Table 1 presents a sum-
mary of the principles, applications, advantages and
limitations of these methods.
© 2017 Australian Dental Association
Studying the human oral microbiome
Checkerboard DNA–DNA hybridization was first
described in 1994 by Socransky et al.32 In this tech-
nique, DNA standards representing the target species
are immobilised on a nylon membrane in an array
format, then simultaneously cross-hybridized with
(usually) radioactively-labelled genomic DNA probes.
However, cultivable bacteria are needed in order to
provide the genomic DNA to construct the probes.
Checkerboard DNA–DNA hybridization provides use-
ful quantitative data and is useful in the study of the
complex biodiverse plaque microbiota in both small-
and large-scale studies. Initially used in the study of
periodontal disease, the technique has since been
employed to study the oral microbiota in diverse areas
such as microbial ecology, general health and disease,
smoking, cariology and endodontics.33–40
Reverse-capture oligonucleotide hybridization is a
modification of the checkerboard method involving
PCR amplification of the sample’s 16S rRNA and spe-
cies-specific 16S rRNA probes. These probes can be
constructed to identify both cultivable and uncul-
tivable bacteria.41 In 2006, Paster et al.42 introduced
a further modification using a microarray format
(glass slides) instead of a nylon membrane. This
method, together with visualization scanners and
advanced computer-based analysis, can detect a large
number of species simultaneously.20,30,44 This even-
tually led to the development of the sophisticated
human oral microbe identification microarray
which allows the detection of approximately 300
bacterial species.20,43,44 Reverse-capture oligonu-
cleotide hybridization and modifications of the tech-
nique have been used in researching topics such as
dental caries, endodontics, oral cancer, HIV and
periodontitis.20,42,43,45
The FISH method, first introduced in 1980, makes
use of the whole cell.46 Fluorescently-labelled oligonu-
cleotides probes designed to target the rRNA gene are
hybridized to partially-fixed, whole cells. The probes
can then be directly visualized using fluorescent
microscopy or combined with flow cytometry for
analysis of biodiverse microbial samples.30,43 FISH
can be used for detection of cultivable and as-yet
uncultivated bacteria, in the study of morphology,
taxonomic identification, quantity, spatial organiza-
tion and biofilm architecture.12,20,30,43,47 The applica-
tion of FISH in oral microbiological research has been
in fields such as biofilm structure, genome–genome
interactions, subgingival organisms and endodon-
tics.12,30,47 As with all of the molecular methods,
FISH has continued to evolve. For example, it can
now be used to investigate the system-level taxonomic
spatial structure of complex biofilms such as plaque
because of the development of combinational labelling
and spectral imaging FISH, which expanded the num-
ber of different taxa distinguishable in a single field.12
17
AML Benn et al.

Table 1. DNA hybridization-based techniques

Technique Principle Application Advantages Limitations

Whole-genomic Hybridization of a selection of
checkerboard DNA radioactively- or dye-labelled
–DNA whole genomic DNA probes to
hybridization sets of sample DNA fixed on a
membrane
Reverse-capture Hybridization of a selection of
oligonucleotide labelled polymerase chain
hybridization reaction (PCR)-amplified 16S
rRNA gene segments from sets
of samples to species-specific
oligonucleotide probes fixed on
a membrane. This is a
modification of the checkerboard
technique
Fluorescence in situ In situ hybridization of
hybridization fluorescent-labelled 16S rRNA
(FISH) oligonucleotide probes to
bacterial call rRNA in the
sample
DNA microarray Hybridization of labelled DNA
sequences in the sample to
target-specific oligonucleotides
fixed onto a nylon
(polyvinylidene difluoride)
membrane or a glass slide
12,20,30,36
Adapted from references .
Screening for selected Rapid, direct, sensitive,
bacteria relatively inexpensive
Enumeration of species in analysis of large number
complex systems of samples for high level
of different bacteria
Screening for selected Highly selective under
bacteria stringent conditions
Precise hybridization of
complementary target
sequences
Precise taxonomy
Detection, direct Identification and
visualization and quantification of several
identification of single species in same sample
microbial cells
Spatial organization and
detection when used with
confocal laser scanning
microscopy
Detection and Wide applicability
identification of
pathogens and bacteria in Vast amount of
mixed infections or information
contaminations Simultaneous analysis of
extensive number of
different bacteria
Lower specificity than
oligonucleotide probes
Requires cultivable
bacteria
Low sensitivity when
low levels of target
bacteria in complex
samples with bacterial
and human DNA
Loss of quantitative
assessment
Labour-intensive
Requires 16S rRNA
gene sequence data
Small number species
distinguishable in a
sample
Expensive
Hybridization
specificity
Quantification of
signals – noise ratios
Lack of probes for
unknown bacteria
Detection of target
bacteria present at
low levels

Methods using PCR thermocycling apparatus. Depending on the extension

period, DNA fragments of more than 20 kbp in
In 1985, Kary Mullis invented a process for the
length can be generated. Because amplification of the
amplification of specific DNA fragments from any
target DNA fragments is exponential, minute amounts
DNA molecule. This process, called the PCR, eventu-
of DNA can be multiplied billions of times in a matter
ally earned Mullis the Nobel Prize in 1993. Since its
of hours. PCR is extremely sensitive and highly spe-
inception, PCR has become a central technique in a
cific.30,48–51 However, there are limitations, particu-
myriad of molecular techniques. It can be used either
larly within heterogeneous bacterial samples such as
as a stand-alone method or as the first step in generat-
oral biofilms. These include: (i) errors from the need
ing starting material for other molecular techniques
for different methods for lysis of gram-negative and
such as DNA sequencing. The basic PCR technique,
gram-positive bacteria; (ii) oral samples containing
as practised today, relies on two specific oligonu-
amplification inhibitors such as blood; (iii) DNA
cleotide primers (usually 20–30 nucleotides long) that
sequence differences leading to unequal DNA denatu-
frame the target DNA sequence, namely the template,
ration and annealing causing amplification bias; and
which is then amplified by a thermostable DNA poly-
(iv) erroneous findings from the amplification of con-
merase enzyme. The dsDNA template is first heat-
taminating DNA.20 In addition, PCR is potentially
denatured (>94 °C) and then quickly cooled to 50–55
time-consuming for complex biodiverse systems
°C to allow the primers to bind (anneal) to the tem-
because primers and amplification conditions may
plate. After a period of annealing (usually 30–60 s),
have to be optimized for each target fragment.52
the temperature is raised to 68–72 °C to allow the
Pertinent information relating to conventional PCR
DNA polymerase to synthesize new DNA strands
and its derivatives is presented in Table 2. Conven-
(also known as extension). Each newly-synthesized
tional PCR and its many variants have been used in
DNA strand then acts as a new template in the next
oral microbiology research, examples of which include
cycle. The cycles of denaturation, annealing and
periodontal health and disease, epidemiology, cariol-
extension are then repeated up to 40 times in a
ogy, genotype diversity, micro-ecology, endodontics,
18 © 2017 Australian Dental Association
 Studying the human oral microbiome

Table 2. Polymerase chain reaction (PCR)-based methods

Method Principle Application Limitations Advantages

Conventional PCR Amplification of a single
(PCR) gene region using two
oligonucleotide primers
(multiplex PCR amplifies
several genetic regions in
a single reaction)
Real-time quantitative Quantitative monitoring of
PCR (q-PCR, qRT- the information of PCR
PCR) product in real time by
detection of incorporated
fluorescent dyes of
specific wavelengths
PCR-denaturing Electrophoretic separation
gradient of PCR-amplified 16S
gel electrophoresis rRNA gene fragments
(PCR-DGGE) through a polyacrylamide
gel containing an
increasing gradient of
denaturants
Random amplified PCR amplification of
polymorphic DNA/ genomic DNA using a
arbitrarily primed single random primer
PCR (RAPD/AP-PCR) under low-stringency
conditions and
subsequent size separation
by agarose gel
electrophoresis
Repetitive element- PCR amplification using
based PCR (Rep-PCR) consensus primers for
randomly dispersed
palindromic sequences in
genomic DNA and size
separation by agarose gel
electrophoresis
Multilocus sequence Comparison of sequences
typing (MLST) of PCR amplified
housekeeping gene
between test and
reference strains
PCR-restriction Restriction digestion of the
fragment PCR amplified target and
length polymorphism fragment size separation
(PCR-RFLP) by gel electrophoresis
Terminal restriction Restriction digestion of
fragment length PCR amplified 16S rDNA
polymorphism (T- using fluorescent-labelled
RFLP) forward primer and
detection by DNA
sequencer
20,26,39,64–67
Adapted from references .
Detection and
identification of bacteria
Single species detection in
epidemiological studies
Studying bacterial diversity
(detection and
identification of all
bacteria)
Monitor community
dynamics
Identify predominant
species in a community
Distinguishing condition/
disease-associated species
Genotype profile analysis
Distinguishing strains of a
known species
Genotyping
Community profiling
Monitoring changes over
time or after known
alterations
Formation of non-specific Extremely small sample can
products be used
Amount of amplified Identification of cultivable
product correlates poorly and non-cultivable bacteria
with original DNA
template amount
Laborious and expensive PCR product monitoring as
in early stages of the reaction happens
development Closed system reduces risk
May underestimate species of amplicons release into
diversity in a community laboratory
sample Provides quantitative data
No phylogenetic Fast
identification Semi-quantitative
Bias introduced by PCR
Poor reproducibility of No previous sequence DNA
band patterns information required
Easy to use
Primer annealing non-
stringent
Enables annealing to
unknown DNA sequences
of low complementarity
Reproducibility may be High discriminatory power
unreliable Inexpensive
Expensive High discriminatory power
Low throughput Portable
Standard nomenclature
No ambiguity of nucleotide
sequencing
Mass of gel bands Requires small amount of
resulting in difficulty in sample DNA
comparing strains Fast
No phylogenetic Semi-quantitative
identification Inexpensive
PCR bias Prior testing using sequence
data of restriction enzyme
discrimination capacity
and restriction fragment
sizes
Underestimate species Provides knowledge of
diversity in community taxonomic position of test
sample bacteria

bacterial communities (subgingival, supragingival and
saliva) and antimicrobial susceptibility.20,39,53–57
DNA SEQUENCING: THE NEXT GENERATION
In 1977, Sanger and Gilbert developed a DNA
sequencing method (commonly referred to as ‘Sanger
sequencing’) that has been the gold standard for four
© 2017 Australian Dental Association
decades.58–60 The prototype method was expensive,
labour-intensive and potentially dangerous. It
involved the use of a heat-labile DNA polymerase
enzyme, radioactively-labelled chain terminators and
X-ray films. ‘Reading’ a DNA sequence had to be
done visually. Over the decades, the Sanger technique
has been extensively improved, automated and minia-
turized by combining a PCR-based step for the DNA
19
AML Benn et al.
synthesis and chain termination (with dye-labelled
dideoxynucleotides) reactions, capillary electrophore-
sis, detection of the fluorescent dyes, and computer-
aided base-calling into a single bench top apparatus.
Even the all-important purification of the DNA tem-
plate to be sequenced can be automated by using
robotic instruments.60,61 The first bacterial genome
sequence, that of the pathogen Haemophilus influen-
zae, was completed in 1995 using Sanger sequenc-
ing.59 In the early decades of molecular oral
microbiology, Sanger sequencing was used in combi-
nation with PCR, sometimes accompanied by cloning,
to study the microbiota associated with various oral
sites, in health and in association with disease condi-
tions such as dental caries, periodontal diseases,
endodontic infections, peri-implantitis and halitosis.61
However, the main limitation of these techniques,
especially when investigating the microbial composi-
tion of complex biofilms, was the requirement for
cloning genes of interest in the laboratory workhorse
Escherichia coli. This created ‘cloning bias’, whereby
not all genes could be cloned due to incompatibilities
with the E. coli host.
In the last decade, advances in miniaturization and
ingenious molecular detection methodologies have
resulted in an unprecedented range of so-called NGS
technologies. These technologies are based on either
the DNA-by-synthesis approach – that is, still based
on the Sanger principle but incorporating PCR – or
more recently, the detection of changes in DNA prop-
erties during the sequencing reaction. One of the first
NGS technologies was pyrosequencing (also known as
‘454’), which featured an etched glass array contain-
ing over 1 000 000 individual nanolitre ‘PCR vessels’,
each capable of effecting a single PCR-based sequenc-
ing reaction.60,61 The basis of pyrosequencing was
detection of light upon incorporation of a fluores-
cently-labelled nucleotide. Computer software bundled
with the 454 instrument (the prototype GS20 fol-
lowed by the GS-FLX and GS-FLX+) would analyse
and provide the user with the DNA sequence reads,
each averaging up to 450 bp. Variants of this princi-
ple with different detection schemes emerged, includ-
ing the Illumina HiSeq/MiSeq, SOLiD and
semiconductor (Ion TorrentTM) sequencing. For exam-
ple, Ion Torrent sequencing detects base incorporation
by measuring the subtle change in pH due to the
release of a proton when a base is added. More
recently, single DNA molecule (Pacific Biosciences
Single Molecule Real-Time [SMRT])62 and MinION63
sequencing have emerged as potentially cost-effective
DNA sequencing alternatives due to their very long
and reliable sequence read lengths (>5000 to
30 000 bp).62,63 The chemistry, advantages and limi-
tations of Sanger sequencing and the most commonly-
used NGS sequencing technologies available today are
20
summarised in Table 3. At the time of writing, the
454 pyrosequencing and SOLiD platforms have since
been discontinued due to their non-competitive cost-
effectiveness.
Their truly culture-independent nature means that
NGS systems enable hypothesis-driven studies on
previously unknown and unclassified microorgan-
isms.23 These methods result in considerably greater
depth (number of sequences per sample) and breadth
(number of samples/individuals analysed) than San-
ger sequencing. NGS technology offers much lower
costs, avoids cloning biases and offers the possibility
of bacterial genome or profiling analyses in a matter
of hours or days rather than months or years.23,59,61
However, it suffers from sequencing errors, referred
to as ‘sequencing noise’, which can result in either
overestimation of sample diversity or, after removal
of the errors/sequencing noise, underestimation of
sample diversity. Researchers also have to consider
the compromise between generating huge amounts
of data (billions of sequence reads) but with short
read lengths (100–800 bp) and longer read lengths
(up to 30 000 bp) but with significantly fewer (sev-
eral million) sequence reads. The shorter read
lengths limit the identification of bacterial species
because reliable identification of new species and
some known species ideally requires sequencing of
the entire 16S rRNA gene, which is approximately
1550 bp long.59 This limitation, however, may even-
tually be circumvented by future higher-throughput
versions of ultra-long-length sequencing systems such
as SMRT and MinION.62,63 Moreover, technologies
such as SMRT and MinION facilitate the sequenc-
ing of whole microbial genomes (i.e. bona fide
metagenomics) within a given sample of interest,
thus potentially yielding much more meaningful data
on not only what microbes are present but also on
their genomic secrets (e.g. metabolic pathways, viru-
lence factors).
To date, investigations into the bacteria of the oral
cavity and its various unique niches using NGS have
been wide-ranging, but they are beyond the scope of
this review. Reported in the literature have been
research topics as diverse as the oral microbiome in
health and disease (paediatric, adult and geriatric),
dental plaque, supra- and subgingival bacteria, peri-
odontal health and disease, endodontic infections,
microbiota of root canals (pre- and post-treatment),
cariology, oral cancer, microbiota of saliva and sys-
temic diseases associated with oral bacteria.44,55,61
Without a doubt, as NGS technologies are further
improved and refined to (i) be more cost- and time-
efficient, (ii) generate fewer amplification biases and
sequencing noise, and (iii) capture data in real-time,
this will open up endless possibilities for further oral
microbiological research.23,59
© 2017 Australian Dental Association
 Studying the human oral microbiome

Table 3. The evolution of DNA sequencing

Technique Year of Chemistry Advantages Limitations
introduction

First-generation sequencing
Sanger (dideoxy) 1977 Dye-labelled Gold standard for the last three decades Expensive ($5–10 per sequence read)
sequencing dideoxynucleotide Validation of next-generation sequencing Laborious
chain terminators results Cloning bias
Long contiguous DNA sequence reads Fairly low throughput
(up to 900 bp of reliable sequence data) Scalability
Fairly quick run times (3 hours) Speed
Resolution
Next-generation Open-ended view on whole breadth of Short length read
sequencing (NGS) microbiome Low taxonomic resolution (most to
Increase depth and sensitivity of bacterial genus level)
profile DNA and amplification bias
Enables rapid sequencing of large Sequencing errors
stretches of DNA spanning entire
genomes
Pioneer or discontinued NGS platforms
Roche/454 GS 2005 Pyrosequencing Can generate 1.2 million DNA sequences Costlier than the Illumina systems
(600 million base pairs) Relatively high error rates (>5%)
Long read length (~450 base pairs) Repetitive sequences can be its
Relatively high speed Achilles heel
Excellent for genome sequencing
Assembling scaffold for metagenomics
Life Technologies 2006 Sequencing by Very high (>99%) accuracy Short read length (50 bp)
SOLiD ligation Very high yields (60 billion base pairs) Long run times (7–14 days)
Colourspace mapping of DNA
sequence
Currently-available NGS platforms
Illumina MiSeq/ 2006 Reversible Very established system with majority Relatively short length read (up to
HiSeq terminators market share 300 bp)
Very high yield (up to 15 billion base
pairs)
High accuracy (>95%)
Reduced hands-on time
Short run times (56 h)
Lowest reagent cost
Life Technologies 2010 Change in pH upon Intermediate yields (relative to Illumina Relatively short read lengths (200–
PGM base incorporation and SMRT) of up to 1 billion base pairs 400 bp)
(Ion Torrent) Up to 5 million sequences can be Error rates of 5%
generated Prone to base homopolymer runs,
Very high accuracy (>99%) which could lead to misassemblies
Fast run times (<8 hours)
Single-molecule 2009 Dye-modified Fast cycle time High raw error rates (>5%)
real-time nucleotides Very long reads (up to >30 000 base Low throughput (hundreds of
sequencing pairs) thousands of sequences)
(SMRT) Simple preparation Highly-specialised sensitive
Low reagent costs instrument
Ideal for whole-genome sequencing and Difficult set-up
metagenomics
MinION 2008 Change in current Read length (>5 kb pairs) Relatively low throughput
(Nanopore upon base Portability – essentially ‘sequencing on a High translocation velocity
sequencing) incorporation USB stick’ Lack of nucleotide specificity
Speed
No fluorescent modification of bases
results in reduced costs and biases
Ideal for sequencing in the field (e.g.
infectious disease outbreaks)
20,59,61–63,68
Adapted from references .

Coping with DNA sequence data overload: enter the
bioinformatic tools
Up until a decade ago, DNA sequence data genera-
tion, despite being automated with output as readable
text files, still required verification by the human
researcher or teams of bioinformaticians. With the
advent of NGS technologies that are now powerful
© 2017 Australian Dental Association
enough to generate up to millions or billions of indi-
vidual DNA sequences (Table 3), ‘denoising’ and
ensuring the integrity of these gigantic sequence data-
sets can be done only by using sophisticated and pow-
erful computing resources. What used to be
achievable with a desktop computer now requires
massively-parallel multi-processing computing arrays
21
AML Benn et al.
comprising tens or hundreds of processing units to do
it in the same time. These super computing arrays,
however, would be ineffective if there were not the
software packages to analyse the data and generate
results that would be meaningful to the researchers.
Many software packages have been developed over
the years that aim to denoise sequence data, classify
16 rRNA gene sequences into species, and assemble
whole genome sequences from single-species or
metagenomic data. One of the most common software
suites used to profile bacterial communities from a
variety of environments is QIIME (Quantitative
Insights Into Molecular Ecology),69 which is regularly
updated, and offers many types of analyses from basic
species assignment to the measurement of diversity
(such as the Chao1 and Shannon diversity indices).
Another software package is Galaxy,70 a cloud-based
platform which features workflow options from
denoising through to diversity analyses. Post-analysis
software may include packages such as Bioconductor
and those allowing principal component analysis. As
NGS technologies have evolved to become more
affordable and mainstream, the composition of groups
undertaking oral microbiological research has evolved
from small specialist teams to larger multidisciplinary
groups.
Impact of molecular microbiology on the oral
pathogen ‘rogues gallery’
For several decades now, students of dentistry and
other oral health-related professions have been taught
that the two most prevalent oral diseases in the world,
dental caries and periodontitis, are caused by the
gram-positive Streptococcus mutans and the gram-
negative Porphyromonas gingivalis, respectively. This
was largely due to the ability to culture these organ-
isms using blood agar media and to these species
being amenable to genetic modification. To this day,
much research is still being carried out on the viru-
lence of these organisms. With the use of checker-
board DNA hybridization, Socransky categorized
periodontal pathogens into complexes with P. gingi-
valis being in the Red Complex and organisms
exhibiting lower pathogenicity, such as Aggregatibac-
ter actinomycetemcomitans, in the Orange and Yellow
Complexes.32,36
More recently, the use of 16S rRNA gene sequenc-
ing, either as cloned amplicons or analysed by NGS,
has allowed the identification of new genera and new
species, such as Scardovia wiggsiae, in dental caries
lesions.71,72 NGS, due to its superlative sequence
yields, can also detect rare species (those representing
<0.001% of the microbial population in a given
sample). Subsequent analyses using packages such as
QIIME and Galaxy can provide quantitative
22
information about species of interest in cases of dis-
ease cases such as severe early childhood caries. Our
own research utilizing the 454 and Ion Torrent plat-
forms has revealed the presence of new genera such as
Granulicatella, Leptotrichia, Rothia, Veillonella and
phylum TM7, some of which remain recalcitrant to
conventional culture methods.73,74 Some genera
appear to exhibit site specificity; for example, Gran-
ulicatella can be detected in buccal swabs but not in
dental plaque samples.73 Interestingly, S. wiggsiae has
not been detected in oral samples of New Zealand
children with severe dental caries, and, in some cases,
Streptococcus sanguinis (and not S. mutans) has been
found to be the prevalent species in caries lesions.73,74
Furthermore, we observed relatively high levels of
Neisseria bacilliformis and Neisseria flavescens in
severe dental caries samples, an observation
previously unreported but which remains to be repli-
cated elsewhere.73,74 Similarly, our own 454-based
NGS-based bacterial profiling of severe periodontitis
cases has shown that Prevotella spp. (an Orange
Complex genus), rather than P. gingivalis, was
frequently detected in diseased samples.73 Our
findings, albeit from relatively small samples, are
nevertheless supported by reports from other
groups.7,23,30,42,45,53,56,75,76 Current molecular oral
microbiological strategies, therefore, have evolved to
the point where we have to discard completely the
notion that oral diseases are caused by a single patho-
gen (or groups of pathogens) in favour of a concept
of a ‘healthy’ versus ‘disease-associated’ oral micro-
biome. Using NGS systems such as SMRT, which is
ideal for metagenomic analyses, it is only a matter of
time before the roles of oral microbes in health and
disease will be elucidated.
CONCLUSION
Since van Leeuwenhoek’s initial discoveries, the fun-
damental microbiological question remains ‘what is
there?’. Most often, the answer has been simplistic
and rather opportunistic, resulting in as-yet-to-be cul-
tivated bacteria and those that are present in low
numbers being overlooked or ignored. Molecular
methods have yielded a more comprehensive picture
of composition and diversity of the oral microbiota.
However, these methods have also resulted in a deluge
of data, leading to the need to develop increasingly
sophisticated computational and statistical tools in
order to analyse and make sense of it.
Further research is needed to reveal why particular
bacteria are present and, more importantly, what in
fact they are ‘actually doing there’. Understanding
how bacteria interact with each other and their envi-
ronment in an open system such as the oral cavity is
key to the ultimate aim of unravelling the complexity
© 2017 Australian Dental Association

of the oral microbiome and to providing a better basis
for understanding the important role oral bacteria
play in both oral and general health and disease.
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Address for correspondence:
Professor William Murray Thomson
The University of Otago – Sir John Walsh Research
Institute
PO Box 647, Dunedin 9054
New Zealand
Email: murray.thomson@otago.ac.nz
© 2017 Australian Dental Association