DOI: 10.1111/prd.
12350
REVIEW ARTICLE
Metatranscriptomic analyses of the oral microbiome
Ana E. Duran-Pinedo
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL, USA
Correspondence
Ana E. Duran-Pinedo, Department of Oral Biology, College of Dentistry, University of Florida, 1395 Center Dr., P.O. Box 100424, Gainesville, FL 32610, USA.
Email: aduran-pinedo@dental.ufl.edu
Funding information
National Institute of Dental and Craniofacial Research; National Institutes of Health, Grant/Award Number: 2R01DE021553
1 | I NTRO D U C TI O N
It is well known that the human body harbors trillions of microbes,
including bacteria, fungi, and viruses. A recent revision of the num-
ber of bacteria residing in the human body puts the total number
of bacteria in a 70 kg "reference man" as 3.8 × 1013.1 The Human
Microbiome Project, an initiative of the National Institutes of Health
Road map for Biomedical Research (http://nihro​admap.nih.gov), in-
creased our understanding of microbial diversity in humans and its
role in human health and disease, as well as the dynamic interactions
of microbes with host cells. Joshua Lederberg, in 2001, coined the
term microbiome "to signify the ecological community of commen-
sal, symbiotic, and pathogenic microorganisms that share our body
space and have been all but ignored as determinants of health and
2
disease." The term microbiome describes the whole community as a
"superorganism" in the context of their habitats.
The “oral microbiome” comprises the bacteria, fungi,3 viruses,4-6
archaea,7 and protozoa8 that inhabit the human oral cavity. This re-
view will focus on the bacterial part of the oral microbiome, and to
simplify matters for the reader, we will subsequently refer to the
bacterial part as the oral microbiome. The oral microbiome is com-
posed of more than 700 species of bacteria9 with no significant dif-
ference based on gender, race, age, or geographic location of host.
However, the oral microbiome is distinct from that of the gut, colon,
10
or skin. The communities of the oral microbiome can be classified
as indigenous or exogenous (which is also known as the transient
microbiome). The indigenous communities contain the oral bacte-
rial species found in virtually all human adults. On the other hand,
the transient exogenous communities include bacteria that are
present in the oral cavity, but whose natural niche is other than the
11,12
mouth. Differentiation between transient species and endoge-
nous species should be based on the frequency with which clones of
© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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28     
wileyonlinelibrary.com/journal/prd
a particular genus are detected. Thus, it is clear that we cannot rely
only on oral human studies because we need to compare the fre-
quency and association of bacteria in oral sites with those in extra-
oral sites to establish a specific oral-related species.9,13-18 As a result
of the pioneering work of scientists such as F. Dewhirst, B. Paster, S.
Socransky, and A. Hafajjee, among others, we are fortunate to have
solid knowledge on the specific composition of the indigenous flora
in the oral cavity.
The golden era of the oral microbiome started 4 decades ago, in
1968, with the discovery of a link between bacteria as a causative
agent of periodontal disease and caries. Worldwide, periodontitis
and caries are still the leading causes of tooth decay in adults, and
those with such diseases experience a marked decrease their quality
of life. Regarding periodontal diseases, the most recent data, from
the National Health and Nutrition Examination Survey 2009-2014,19
indicates that the prevalence of chronic periodontitis among adult
Americans is 42%, representing $14B expenditure on treatment in
the US, and that periodontitis affects 743 million people worldwide.
Moreover, an increasing number of studies suggest that periodontal
diseases can influence the risk for certain systemic conditions, such
as cardiovascular diseases, diabetes, and respiratory diseases, and
can affect pregnancy outcome. In a systematic analysis, Kassebaum
et al reported a prevalence of untreated caries in permanent teeth of
34.1%, affecting 2.5 billion people worldwide. 20
Current concepts of periodontal disease and caries etiology
highlight the importance of oral biofilms as multispecies communi-
ties. The focus has shifted from the role of individual species within
the biofilm to a more holistic view of how biofilms, as a whole, cause
and maintain chronic infections. The metabolism of a bacterium is
modulated by its environment and is modified by the bacteria to
which it is in close proximity. The functions of distinct species within
the oral microbiota are intimately intertwined with the rest of the
Periodontology 2000. 2021;85:28–45.
DURAN-PINEDO
microbial community, highlighting the relevance of examining the
expression profile of specific species of the oral microbiota in the
context of the other members of the biofilm in vivo, as well as the
expression pattern of the whole community.
However, we should not dismiss the importance of studying indi-
vidual species. Knowledge of the function of bacterial species within
a community provides more insight into pathogenesis than the sim-
ple enumeration of that community's gene content.
Nowadays, as a result of the development of next-generation se-
quencing techniques for DNA and RNA sequencing and other omics
approaches, we can address those questions and obtain in situ in-
formation on the shift in microbial community structure and met-
abolic activities that occur in the transition from health to disease.
Among the powerful omics approaches is metatranscriptomics. The
metatranscriptome refers to the expression of genes in the whole
microbial community. 21 Metatranscriptomics is more direct than
phylogenetic analysis. In contrast to 16S ribosomal RNA profiling,
metatranscriptomic analysis provides information about the species
that are metabolically active in a community during health or disease
states.
Metatranscriptomic approaches have been successfully ap-
22,23
plied to study diverse environmental microbial communities,
and the use of this approach to study human-associated microbi-
24-34
omes, such as periodontal diseases and caries, is increasing.
Metatranscriptomic analysis can answer crucial questions, such
as: “What is the gene-expression profile of specific bacteria and
the whole community?”; “What are the functions of the expressed
genes?”; “Which bacterial groups are expressing genes associated
with the types of activity observed?”; and “Are the genes expressed
related to known virulence factors?”
Moreover, metatranscriptomic gives us good insight into puta-
tive factors assumed to drive the community to a disease state. That
knowledge could also be applied to the development of new ther-
apies to maintain a healthy oral microbiome. Metatranscriptomic
analysis can also be applied to large sets of samples in longitudinal
studies and thus will provide a wealth of knowledge on populations.
Moreover, the usefulness of metatranscriptomic approaches is not
limited to the study of bacterial gene-expression profiles; it could
also be applied to the host, to determine the pathways of bacte-
ria-host communication.
2 |  CO M P OS ITI O N O F TH E O R A L
M I C RO B I O M E
The healthy oral human microbiome is predominantly composed of
members of the phyla Actinobacteria, Proteobacteria, Firmicutes,
Bacteroidetes, and Fusobacter and Spirochaeteae are present at
lower number.13,35-38 The composition of the oral community is
shaped by physical and chemical interactions, environmental pres-
sures (such as oxygen tension, pH, and availability of nutrients), an-
timicrobial peptides, and host factors in saliva and gingival crevice
fluid, including lysozyme, secreted antibodies, and other immune
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mediators.39 The variety of ecological niches provided by the soft
and hard tissues within the mouth also determine the diversity and
abundance of the oral microbiome.40 The tooth surface close to the
soft gingival tissue furnishes 2 major ecological niches: the suprag-
ingival area and the subgingival area. The supragingival area is ex-
posed to saliva, liquid, and solid food. The subgingival area is bathed
by the serum transudate (known as gingival crevicular fluid), a fluid
rich in nutrients and proteins involved in the immune response.40
The main difference between these 2 niches is the lower redox po-
tential, and the lower level of oxygen, in the subgingival area.41 Even
though the environmental conditions are different, it is not easy to
make a statistical distinction between supragingival and subgingival
samples because they harbor almost the same group of microbes but
at different proportions.13,42-46
Moreover, the intermixing within the oral cavity is influenced by
saliva that coats all the oral surfaces. Bacteria transported by sa-
liva could fit into cracks or crevices, building biofilms in areas far
from their site of origin. Besides, during sampling procedures (using
a swab, buccal brush, paper point, or scaler), microbes can become
mixed in the biomass being sampled.47
As a result of limitations in the ability to discriminate species
using 16S ribosomal RNA analysis, the vast majority of studies have
identified oral bacteria only to the level of genus.36 Nonetheless,
given the significant variations in the structure and composition of
genomes belonging to the same species, in the future, we should aim
to characterize the oral microbiome not even to species level but to
strain level, which will be accomplished with improvements both in
sequencing capabilities as well as in bioinformatics analysis.
Supragingival and subgingival plaque harbor 14 common gen-
era: Streptococcus, Corynebacterium, Capnocytophaga, Haemophilus,
Aggregatibacter, Fusobacterium, Prevotella, Leptotrichia, Veillonella,
Neisseria, Rothia, Actinomyces, Lautropia, and Porphyromonas.
Our current knowledge of the diversity of the oral microbiome
has been beautifully complemented with the direct observation of
their structures, which was made possible by pioneer scientists in
this field (J. M. Welch, A. Valm, and G. Borisy). They combined light
and electron microscopy to visualize the intact dental biofilm and
unveiled the structure and organization of the oral community, with
no random distributions of polymicrobial biofilms.48-51
Welch et al elegantly revealed the in vivo architecture of sam-
ples of supragingival dental plaque from healthy volunteers. By com-
bined use of fluorescence microscopy, antibodies, or oligonucleotide
probes52-55 and the multiplex fluorescence in-situ hybridization
technique, they visualized the 3-dimensional structure of undis-
turbed regions of supragingival biofilms.56 Large circular "hedgehog”
and "corncob" structures were repeatedly observed. Bacteria of the
genus Fusobacterium were at the core of the hedgehog arrangement
and were surrounded by bacteria from the genera Streptococcus,
Porphyromonas, Leptotrichia, Capnocytophaga, and Actinomyces, and
from the families Pasturellaceae and Neisseriaceae. The corncob
structures were formed by bacteria of the genus Streptococcus and
sometimes by those of the genus Porphyromonas. Bacteria of the
Pasturellaceae family were observed to decorate the apical tips of
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30       DURAN-PINEDO
A F I G U R E 1   Corncob structures form
B
the filaments from some Corynebacterium species in the "corncob"
structure (Figure 1). Visualization of subgingival plaque represents
an additional challenge. Maintaining the integrity of the structures
in subgingival plaque has been achieved by using carriers in the
dental pocket, thus permitting imaging of the biofilm at different
developmental stages; the spatial arrangement of the microbiota in
periodontal pockets was visualized using electron microscopy, im-
munohistochemistry, and fluorescence in situ hybridization.57 These
findings showed that spirochetes colonize deep within the periodon-
tal pocket and streptococci colonize at sites closer to the surface.
The initial colonizers were found at the surface of the probe and
later colonizers showed a patchy distribution.58,59 Noiri et al, on their
series of microbial localization within periodontal pockets, found
that bacteria were distributed in different regions: Campylobacter
rectus was present deep in periodontal pockets; Treponemes
and Fusobacterium spp. were present in unattached plaque; and
Actinomyces viscosus and Eikenella corrodens were found on plaque
attached to the tooth.60,61 Porphyromonas gingivalis was directly at-
tached to the biofilm.62 In supra- and subgingival biofilm, at the genus
level, Corynebacterium and Kingella represent a substantial fraction
of the microbiome, while members of the genus Granulicatella are
absent in those sites and present in other oral structures.36
In subgingival sites, the proportions of obligate anaerobic bac-
teria of the genera Fusobacterium, Prevotella, Porphyromonas, and
around Corynebacterium but do not form
around nearby Fusobacterium, Leptotrichia,
or Capnocytophaga. (A–C) Methacrylate-
embedded sections from 3 different
donors. Rectangles indicate the location
of the insets; ovals and circles are drawn
around representative corncob structures,
each of which has a Corynebacterium core.
Originally published by Mark Welch et al.
Treponema are increased, and the relative abundance of bacteria of
the genera Dialister, Eubacterium, Selenomonas, and Parvimonas is
decreased.35,56 In supragingival plaque, the relative abundance of
facultative anaerobic bacteria, of genera including Streptococcus,
Capnocytophaga, Neisseria, Haemophilus, Leptotrichia, Actinomyces,
Rothia, Corynebacterium, and Kingella, is significantly increased.
A significant fraction of bacteria present in the oral cavity has
not yet been cultured in the laboratory.63 Despite intensive efforts
by the community of microbiologists studying the oral microbiome,
one-third of oral bacterial taxa are as yet uncultivated in vitro.64 The
unculturable condition of a specific bacterium indicates simply that,
using our current knowledge, we are unable to grow it under labora-
tory conditions. Thus, “unculturable” does not indicate the inability
of particular organisms to grow in laboratory conditions but rather
reflects our current lack of knowledge of their biology during natural
growth.65 This “unculturability” could be responsible for underesti-
mation of the number of known species. As an example, a fraction
of unculturable cells has been reported in 2 easily culturable spe-
cies, Fusobacterium nucleatum and Porphyromonas endodontalis. This
finding suggests that some of those species included unculturable
biotypes, implying that the abundance of species already described
could be higher. That a significat fraction of unculturable taxa are
present in gingivitis,66 as well as in periodontitis67,68 has now been
confirmed by molecular analysis.
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The Tannerella genus comprises 3 species, 1 of which–Tannerella
forsythia–has long been known to be highly associated with chronic
periodontitis. The other 2 species of this genus–Tannerella spp.
BU045 (human oral taxon 808) and Tannerella spp. BU063 (human
oral taxon 286)–are associated with oral health.9,69 All three spe-
cies need external factors (provided either by helper species such
as Propionibacterium acnes and Prevotella intermedia or by purified
forms of N-acetylmuramic acid on enriched growth medium) for
growth.64,70,71 As a result of intensive work and use of single-cell
genome sequencing techniques, Tannerella spp. BU045 and BU063,
classically unculturable species, have also been culturable and
sequenced.71
According to gene content and average nucleotide identity,
Tannerella phylotypes BU045 and BU063 are more closely related.
However, 2 independent data sets of association with periodon-
titis, one based on 16S ribosomal RNA gene abundance and the
other based on metatranscriptomic data, showed that Tannerella
BU045 is more highly associated with disease than Tannerella
64,71
BU063. In contrast to what was believed, these findings sug-
gest that Tannerella BU045 is more prevalent in periodontitis than
in oral health. 63,64,71 Interestingly, Tannerella BU063 lacks the vir-
ulence genes present in T forsythia, including karilysin, prtH, and
64,70,71
bspA, which could explain its lack of pathogenicity.
Among the 30% of unculturable oral microbial species (http://
www.homd.Org) are members of the Saccharibacteria phylum
(formerly known as TM7). Recent studies using 16S ribosomal
RNA have identified Saccharibacteria in environmental samples
and from eukaryotic cells, including those from the gastrointes-
tinal tract, skin, oral mucosa during active infection, and female
72
genital tract. Within the oral cavity, at least 6 distinct groups of
Saccharibacteria have been described across anatomic sites.73,74
The use of new technologies is unveiling the potential role of
Saccharibacteria in periodontal disease. Saccharibacteria spp.
have been found both in healthy sites and in sites with severe peri-
odontitis.75 One study reports the presence of a high number of
oral Saccharibacteria76 , contradicting previous studies that show
very low abundance.77,78
Recently, a first approach to the unculturable of Saccharibacteria
was reported.79 Despite multiple efforts, the routine unculturable
of Saccharibacteria remains arduous and challenging. Nanosynbacter
lyticus type strain TM7x, a member of the Saccharibacteria phylum,
has been described as an epibiont of Actinomyces odontolyticus strain
(XH001) with a parasitic phase. Depending on environmental con-
ditions, TM7x can kill the host bacterium, a feature that may have
an impact on the ecology of the oral microbiome. It is an extremely
small coccus (200-300 nm). Its genome (705 kb), the first of a hu-
man-associated Saccharibacteria phylotype to be sequenced, com-
pletely lacks the genes required to synthesize amino acids, vitamins,
nucleotides, and cell-wall precursors, and therefore it relies on ex-
79
ternal sources to supply these factors.
Despite the fact that the genomic sequence of candidate division
Saccharibacteria was still incomplete, in 2014, Duran-Pinedo et al75
were able to identify genes of TM7 that were differentially expressed
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during periodontal disease. These genes included ones that encode
cell-division proteins (chromosomal replication initiator protein
DnaA, cell division protein FtsA, and ATP-dependent protease FtsH)
and chaperones (GroEL, GroES, DnaJ, and Hsp20). In addition, a
number of putative virulence-related genes were also identified that
encode the following: oligopeptide ABC transporter; periplasmic oli-
gopeptide-binding protein, which has been identified as a virulence
factor in group A streptococci;80 a hemagglutinin-related protein;
exoproteins involved in heme utilization or adhesion; a homolog of
LemA; a widely conserved 2-component regulatory system that has
been described as regulating virulence factors and toxin production
in Pseudomonas syringae;73 as well as proteins involved in iron acqui-
sition (hemagglutinin-related proteins and an exoprotein involved in
heme utilization of adhesion) and biosynthesis of pili (PilB and PilC).
Also, in periodontitis samples, Saccharibacteria upregulated genes
encoding a chitinase and a putative ROK-family transcriptional reg-
ulator, which regulates chitinase expression in the gram-positive
pathogen Listeria monocytogenes at the post-transcriptional level.81
These findings suggest a contribution of Saccharibacteria to peri-
odontal disease; however, the question of whether Saccharibacteria
have a role in the progression of periodontitis remains unanswered.
Finally, Duran-Pinedo et al75 also observed the over-representation
of genes involved in pili biosynthesis. Although type IV pili may fa-
cilitate the adherence of bacteria to epithelial cells, it has been sug-
gested that in the case of Saccharibacteria, they may be involved
in gliding motility.74 These results highlight the usefulness of in situ
metatranscriptomic studies in allowing us to acquire knowledge on
physiology and on structures, such as pili, in the absence of culti-
vation. By combining the power of single-cell genome sequencing
and metatranscriptomic analysis, we will be able to unveil the role of
uncultured bacteria and open the way to a new hypothesis.
2.1 | Pipeline of metatranscriptome analysis
The metatranscriptome identifies the gene-expression profiles of
complex microbial communities based on the set of transcripts being
synthesized under diverse growing conditions or environments, in
contrast to the metagenome, which only reveals the microbial phy-
logenetic composition of the community (Figure 2). One of the main
requirements for analysis of the metatranscriptome is availability
of complete genomes of the community to be studied. Fortunately,
the oral microbial community is one of the best characterized in the
human microbiome, which makes this type of analysis possible.
The first big challenge faced when measuring microbial gene
expression in vivo in oral samples is the low biomass. This problem
can be solved by 2 approaches: the first suggests pooling sam-
ples with similar clinical profiles to obtain the concentration of
RNA required for the analysis 82; and the second involves linear
RNA-amplification methods, which have been successfully used in
deep sequencing, providing high-quality coverage almost identi-
cal to that of nonamplified libraries. 83 In many cases, an ampli-
fication step is required in order to generate sufficient RNA for
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32       DURAN-PINEDO
next-generation sequencing processes. The amplified RNA is a di-
rect representation of the relative amount of mRNA found in the
original clinical sample. 84
Although the application of metatranscriptomics to characterize
the functions of microbial communities is increasing rapidly, there is
still no consensus on the best approach to perform this type of study.
The approach will depend on the amount and quality of mRNA, the
success in preparation of the libraries, and the bioinformatic pipeline
utilized in the analyses.
F I G U R E 2   Laboratory workflow for
metatranscriptome analyses of microbial
communities. The figure summarizes the
different processes that can be performed
to study the metatranscriptome of the
oral microbiome. mRNA, messenger
RNA; NGS, next-generation sequencing
techniques; rRNA, ribosomal RNA.
Originally published by Solbiati and Frias-
Lopez. 28
F I G U R E 3   Summary of bioinformatic
workflow for metratranscriptomic
analysis. Common steps in the
bioinformatic analysis of microbial
metatranscriptomes start with quality
control of the sequences, alignment of
these sequences against the genomes of
the microbial community, phylogenetic
assignment of the transcripts,
differential expression analysis, and
pathway enrichment analysis. The
software packages widely used for
metatranscriptomic analysis are shown
in blue. HOMD, Human Oral Microbiome
Database; HMO, Human Microbiome
Project; LefSe, linear discriminant analysis
effect size; rRNA, ribosomal RNA.
Originally published by Solbiati and Frias-
Lopez. 28
Bioinformatic analyses of the metatranscriptome allow large
data sets to be processed through specific steps, called workflow
or pipeline (Figure 3). As is essential to work only with sequences
of optimal quality, the following procedures are undertaken: first,
low-quality sequences are removed; second, sequences are aligned
against the genomes of the microbiome being studied; third, a differ-
ential expression analysis is performed to identify changes in physio-
logic activities; and, fourth, a phylogenetic analysis of the transcripts
is carried out to identify active members of the community.
DURAN-PINEDO
Along with the bioinformatic analyses of the mRNA sequences,
sample size estimation with statistical power is crucial for optimi-
zation of a metatranscriptomic experiment. In this case, we should
consider power wanted, sequencing depth, the coefficient of vari-
ation, single- or paired-end reads, effect size, and budget con-
85,86
siderations. The statistical power can be enhanced by using
paired-end reads, and by increasing the sample size and the se-
quencing depth.86,87 To increase the power, the increment in sample
size is more potent than sequencing depth, especially when the se-
86
quencing depth reaches 20 million reads. Unfortunately, in many
clinical trials it is not possible to obtain a large number of replicates.
Nonetheless, the statistical power of deep sequencing provides a
robust output that can provide highly informative, statistically signif-
icant data. Interestingly, in model organisms, statistical power can be
reached with a fairly small number of replicates. For instance, Ching
et al compared 5 differential expression analysis packages, evaluat-
ing their efficiency using power and other metrics; they found that
after achieving a sequencing depth of 20 million paired-end reads, all
packages reached a power of > 0.8 with only 10 replicates per con-
dition.86 Using a similar analysis, Heart et al found that the biological
coefficient of variation of the data sets was a significant driver in
calculating sample size, but with low coefficients of variation, the
number of samples per group was also small. With a coefficient of
variation of 0.4, only 10 samples per condition and 10 million se-
quences were needed to reach a power of 0.8. However, with a co-
efficient of variation of 1.2, 40 samples per condition were needed
to achieve the same statistical power.85
3 |  M I C RO B I A L M E TATR A N S C R I P TO M E I N
H E A LTH
The oral cavity is the first contact of the digestive tract with the en-
vironment and is exposed to countless numbers of microbes through
breathing, eating, and drinking. The commensal oral microbiota may
play a role in directing the foreign microbes ingested to flushing sa-
liva, and once in the stomach, the extremely low pH will do its part.88
The commensal microbiome may also modulate colonization of
the oral epithelium by extraoral microbes and pathogens through
"colonization resistance" or "bacterial interference" processes.89
Diverse mechanisms, including competition for nutrients, could also
be responsible for colonization resistance through modulating the
microenvironment to inhibit potential competitors and by secretion
of antimicrobials against these species.89 A significant function of
the commensal microbiome is to stimulate development of the im-
mune system to maintain a controlled balance between pro- and
anti-inflammatory responses to bacteria in the absence of active in-
fection.90 As a response to colonization resistance, pathogenic bac-
teria release their arsenal of virulence factors.
Currently, because of next-generation sequencing approaches
and the availability of genomes for oral microbes, we are able to as-
sess the functional role of the oral microbiome in different environ-
ments and conditions. Nowadays, we are able to obtain information
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on the expression profile of genes in the community by shotgun
metagenomic and metatranscriptomic analyses.36,91 Interestingly,
we can assess the expression of any gene in a specific species and
also determine the functional activities expressed by the whole com-
munity, acting as a meta-organism. As stated before, not only does
the oral environment or niche shape the diversity of the microbiome,
it also modulates the biological pathways within the microbiome.
For instance, gamma-aminobutyric acid biosynthesis is increased on
the tongue dorsum. Gamma-aminobutyric acid is a suppressor neu-
rotransmitter known to counterbalance the action of the excitatory
neurotransmitter glutamate. To date, the role of gamma-aminobu-
tyric acid produced in the oral cavity is still unknown. The supragin-
gival plaque is also enriched in metabolic functions associated with
environmental information processing (eg, secretion systems, signal
transduction, and molecules involved in signaling) interaction), and
energy metabolism (eg, oxidative phosphorylation, methane metab-
olism, and nitrogen and sulfur metabolism).
To have a baseline with which the metatranscriptome can be
compared is crucial in order to characterize the transcriptome pro-
file under healthy conditions. Interest in the expression profile of
health-associated genes is increasing; however, the number of stud-
ies on patterns of gene expression in vivo remains limited. Benitez-
Páez et al92 published 1 of the few studies on characterization of the
metatranscriptome during plaque formation.
Interestingly, the biofilm-formation process is also determined
by person-specific factors after digestion of food, as determined in
a recent metatranscriptomic analysis.92 In some people, more than
80% of active bacteria belonged to only 3 genera (Actinomyces,
Corynebacterium, and Rothia), whereas in others no predominant
genera were detected in their active microbial community.
In some individuals, there were virtually no changes in the active
bacterial population after ingestion of food, suggesting that their mi-
crobiota is not affected by food ingestion, potentially reducing the
risk of acidic pH and promoting dental health. Benitez-Páez et al92
also showed that expression of genes linked to translation machin-
ery is higher in early stages of biofilm formation, whereas in mature
biofilm, genes involved in competence, quorum sensing, mutacin
production, and DNA uptake were over-expressed, indicating more
sophisticated levels of interactions in mature biofilm than during
earlier stages of biofilm formation.
In the majority of cases, active members of the microbial
community were from the genera Streptococcus (12%-19%) and
Actinomyces (3%-12%). Bacteria from the genera Actinomyces
were present at higher frequencies in early plaque samples, con-
sistent with its known role as an early colonizer. Other phyla from
which active members of the microbial community were fre-
quently identified were Actinobacteria (Rothia, Angustibacter, and
Kineococcus) Proteobacteria (Neisseria, Kingella, and Alysiella), the
Firmicutes (Gemella, Paenibacillus, and Veillonella), Bacteroidetes
(Capnocytophaga), and Fusobacteria (Fusobacterium).93 In the early
stages of supragingival plaque development, genes involved in the
metabolism of carbohydrates, energy, amino acids, cofactor/vita-
mins, and xenobiotic degradation were predominantly upregulated.
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34       DURAN-PINEDO
Moreover, in the late stages of supragingival plaque formation, the
upregulation of genes involved in the quorum sensing response, in
particular genes identified as belonging to type II secretion systems,
was reported. As type II secretion systems promote the secretion of
folded periplasmic proteins that typically play a role in survival, this
finding indicates that the community is adapting as it develops.
The influence of oral microbes goes beyond the oral cavity.
There is a strong association between the oral microbiome and sys-
temic health. The oral microbiome has been implicated in the main-
tenance of vascular health through the reduction of nitrate to nitric
94-96
oxide. Saliva contains high levels of dietary nitrate that may be
reduced by oral nitrate-reducing bacteria.97 The contribution of the
oral microbiome to the reduction of nitrate remains to be explored.98
We are just at the dawn of functional studies of the oral micro-
biome. In the years to come, we will have an excellent understand-
ing of how biological activities maintain a healthy microbiome, and
we are likely to obtain a clearer picture of the transition to disease.
Changes in microbial diversity and gene-expression profiles result-
ing from that transition could be used as microbial biomarkers for
oral infectious diseases.
4 |   M I C RO B I A L M E TATR A N S C R I P TO M E
I N PRO G R E S S I O N O F G I N G I V ITI S A N D
PE R I O D O NTITI S
Gingivitis is a reversible stage of periodontal disease affecting supra-
and subgingival sulci. Like periodontitis, gingivitis is characterized by
inflammation induced by the oral microbiota. However, unlike perio-
dontitis, gingivitis is initiated at the apical gingival border, namely the
upper limit between the supra- and subgingival areas.90 Classically,
gingivitis has been correlated with a reduction in, or absence of, oral
hygiene for a short period of time and resolves typically after reini-
99,100
tiation of a dental prophylaxis routine. New studies have re-
vealed a stronger correlation between microbial abundance and the
clinical severity of gingivitis than the oral hygiene status, suggesting
that brushing affects the relative abundance of microbes more than
33
the diversity of different taxa. The oral biofilm established during
gingivitis, can lead to the development of periodontitis.
Molecular analyses of dental plaque communities in volunteers
with experimentally induced gingivitis have revealed a unique co-
hort of microbes exclusively associated with gingivitis, which include
Fusobacterium nucleatum spp. polymorphum, Lachnospiraceae spp.,
Lautropia spp., and Prevotella oulorum.100 Recently, Nowicki et al also
found that the genera Oribacterium, Leptotrichia, Tannerella, and
Lachnoanaerobaculum are significantly more abundant during gingi-
33
vitis. This finding reveals involvement of new players during the in-
duction of dysbiosis of the oral microbiome, which occurs at the very
early transition to periodontal disease. Nowicki et al also focused on
33
the expression of virulence factors by those species.
Metatranscriptomic analysis of gingivitis samples revealed a
mixed picture of expression profiles of specific genes and virulence
factors in both health and disease, suggesting the existence of a
transitional state during progression from gingivitis to periodonti-
tis. Interestingly, during gingivitis, the oral microbiota expresses not
only nonspecific peptidases, proteases, and stress response proteins
associated with colonization, but also putative virulence factors,
such as collagenases, gingipains, and hemagglutinins.82 Virulence-
related genes of Leptotrichia buccalis were upregulated, including an-
tibiotic resistance and nonspecific proteases, as well as the response
regulator, MprA (which is strongly associated with pathogenesis in
Mycobacterium tuberculosis, suggesting that it may also be involved
in L buccalis pathogenicity).101 During gingivitis, the expression of
genes involved in antibiotic resistance, proteolysis, breakdown of
collagen, and iron intake were increased in both F nucleatum and
Prevotella nigrescens. Prevotella nigrescens also expressed endothe-
lin-converting enzyme 1 and a gingipain, and several arginine/ly-
sine-specific proteases. The expression of those enzymes, which are
related to virulence, could moderate the immune response as they
are able to degrade host cytokines.102,103
Nowicki et al also described high homology of the expression
of collagenase with one of the Xaa-Pro aminopeptidases on the
T forsythia genome (BFOR_2955).33 Expression of this protein could
link T forsythia to the initial steps of collagen damage present in
periodontitis.
There is no doubt that our current knowledge of the communi-
ty-level shifts occurring during the transition from health to gingivi-
tis, and later to periodontitis, has increased considerably in the last
decades. However, to unveil the exact process of transition to dis-
ease, further exploration in longitudinal studies is needed, involving
a larger number of samples. This exploration should include analysis
of unpooled samples, thus allowing an independent study of each
site to be performed.
The question of why some untreated teeth with clinical manifes-
tation progress to periodontal disease and others remain stable is
still unanswered.104 To address this question, research has been fo-
cused on identifying markers that might distinguish between active
and nonactive sites. Those biomarkers include protein activities,105
bacterial profiles,106,107 cytokine levels,108 genetic markers,109,110
and clinical markers.107 Clinically, the progression of periodontal
disease follows cycles of periods of exacerbation (activity) and re-
mission.104,111-114 Some studies have suggested that changes in the
composition of subgingival biofilm could explain the transition to
disease. However, this hypothesis is invalid because of considerable
overlap in the composition of the microbial communities in progress-
ing and nonprogressing sites.
To elucidate the biological processes linked to the progres-
sion of periodontal disease Yost et al applied metatranscriptomic
approaches.32 The authors assessed gene-expression profiles in
plaque samples isolated from the teeth of patients in whom clinical
progression of periodontal disease was observed. That longitudinal
metatranscriptomic study was able to provide us with a snapshot on
long-term healthy subgingival sites, unveiling the changes leading to
the progression of periodontitis. Interestingly, sites that remained
healthy between the first visit and the time when subsequent
samples were taken showed an unaltered gene-expression profile.
DURAN-PINEDO
Changes in expression were observed on sites that progressed to
disease.
It is noteworthy that differences in the gene-expression profiles
of baseline (or initial samples) of progressing and nonprogressing
sites were found. In the baseline of progressing sites, Gene Ontology
Term Enrichment analysis showed overrepresentation of functions
related to iron, potassium, chloride, citrate, amino acid transport,
lipid A and peptidoglycan biosynthesis, cell motility, and protein
kinase C-activating and G protein-coupled receptor signaling path-
ways. By contrast, in baseline samples from nonprogressing lesions,
the gene ontology terms that were overrepresented were related
to metal ion transport, the phosphoenolpyruvate-dependent sugar
phosphotransferase system, tricarboxylic acid, and protein secre-
32
tion. Interestingly, Yost et al described overexpression of histidine
biosynthesis in baseline samples of progressing lesions,, while Jorth
et al82 found upregulation of histidine catabolism in disease sites. It
is tempting to think that the availability of histidine might represent
a signal to disease.
As a general picture, during health, genes involved in ascorbate
and aldarate metabolism,115,116 vitamin B6 metabolism and glycoly-
sis, and gluconeogenesis, as well as the pentose phosphate pathway,
pyruvate metabolism, and propanoate and butanoate metabo-
lism,82,117,118 were upregulated by the oral microbiota.
On sites that progressed to disease, the known periodontal
pathogens Treponema denticola and T forsythia were not significantly
active, while P gingivalis showed increased activity, supporting the
keystone-pathogen hypothesis.32 According to the keystone-patho-
gen hypothesis, specific low-abundance bacteria can transform an
ordinarily healthy microbiome into a dysbiotic state, causing peri-
32,119
odontal inflammation and tissue destruction. The increased
activity of P gingivalis in initial samples of active sites supports the
hypothesis that P gingivalis leads to the dysbiotic process which initi-
ates the disease.120,121 It is tempting to think that particular proteins
expressed by P gingivalis could be a bacterial biomarker for periodon-
titis. In this regard, more extensive longitudinal studies are neces-
sary to identify the expression of hallmark genes that will allow us
to track the progression of periodontal disease at a particular site.
A less studied aspect of the survival and physiological adapta-
tion of microorganisms in the oral cavity is the regulatory functions
of bacterial small noncoding RNAs. Those elements are rapidly syn-
thesized and capable of modulating the microbial metabolism faster
than the 2-component systems or any regulatory mechanisms for
which protein synthesis is required.122,123 Small noncoding RNAs
comprise a diverse group of RNA molecules with high heterogene-
ity in size and structure, which do not become translated to pro-
teins. This is especially crucial in the oral cavity, where conditions
change constantly, and over short periods of time, because of in-
gestion of nutrients, talking, and the fact that the oral cavity is an
open entrance in contact with the external environment. It is now
well established that small noncoding RNAs could regulate physio-
logical roles, including adaptation to new environmental conditions
124
through quorum-sensing systems or the development of biofilms.
Some reports have shown that small noncoding RNAs also play
|
      35
important roles in microbial virulence and infection.125,126 Small non-
coding RNAs repress translation of mRNA by attaching to the ribo-
some-binding site, competing with the ribosome and leading to the
rapid degradation of the mRNA.127 Other noncanonical mechanisms
of translation repression have been described, such as the binding
of cis-acting antisense RNA to a ribosome standby site upstream of
the ribosome-binding site.128 Besides, small noncoding RNAs can
activate the translation of mRNAs, or can modulate gene expression
by varying the level of transcript stability.129 Small noncoding RNAs
can also modulate protein activity by mimicking the structures of
other nucleic acids and sequestering proteins that otherwise will act
on their real target.130 Diverse experimental approaches have been
applied to identify bacterial small noncoding RNAs.131,132 Those ap-
proaches include direct labeling of RNA, and sequencing or collect-
ing small noncoding RNA genes by shotgun cloning of their cDNA
and use of microarrays and next-generation sequencing technolo-
gies. However, those methods are laborious and expensive, espe-
cially if a large number of elements are analyzed. Global detection
of small noncoding RNAs has been facilitated by the development
of microarrays and next-generation sequencing techniques. Next-
generation sequencing has allowed the discovery of numerous
novel small noncoding RNA transcripts in different bacteria, such as
Salmonella spp.,133 Vibrio cholerae,134 and Bacillus subtilis.135
Our knowledge of the small noncoding RNA metatranscriptome
in complex microbial communities is even more limited than our un-
derstanding of the role of small noncoding RNAs produced by spe-
cific bacteria. Community-wide studies have been mainly limited
to controlled environments.136,137 The first community-wide meta-
transcriptomic analysis of small noncoding RNAs by Shi et al136 was
reported in samples from the ocean. In that study, the researchers
found a large number of previously unknown, putative small non-
coding RNAs, as appropriate. Gosalbes et al reported the presence
of small noncoding RNAs in the metatranscriptome of human fecal
microbiota, although these researchers did not identify the possible
functions of the small noncoding RNAs.138 Research on small non-
coding RNAs in the oral microbiome is very limited. Based on their
study of expression profiles in periodontitis and in health, Duran-
Pinedo et al identified small noncoding RNAs by mapping transcripts
to an Intergenic region (IGR) database generated from the genomes
used in that analysis.84 The authors reported a high correlation be-
tween expression profiles of mRNAs and several clusters of small
noncoding RNAs. Moreover, the functions of those clusters were
surprisingly similar to signature functions described on the mRNA
metatranscriptome during periodontitis progression,32 highlighting
the hypothesis that the differentially expressed small noncoding
RNAs identified participate in the regulation of functions. The small
noncoding RNA clusters found were associated with ferrous iron
transport, proteolysis, potassium ion transport, beta-lactam catab-
olism, cell adhesion, protein secretion and import, and cobalamin
biosynthesis, activities that have all been identified in progressing
and severe periodontitis.84
Additionally, gene ontology terms associated with pathogene-
sis were reported, supporting the hypothesis that small noncoding
 |
36       DURAN-PINEDO
RNAs play a key role in the regulation of bacterial activity during
periodontitis progression. The association of small noncoding RNAs
with the regulation of bacterial virulence has been described for in-
dividual species such as Staphylococcus aureus,139-141 Pseudomonas
aeruginosa,142 and V cholerae.134,143
Small noncoding RNAs have been associated with the regulation
of conjugative transposons in Bacteroides spp.144 High levels of ex-
pression of transposases have been reported in F nucleatum and T
denticola145,146; furthermore, in P gingivalis, small noncoding RNAs
147
have been found within putative transposons.
These results suggest that small noncoding RNAs might play a
key role in modulation of the oral microbiome from a commensal
state to a dysbiotic one by controlling survival, response to the host
immune system, and virulence activities.
5 |   M I C RO B I A L M E TATR A N S C R I P TO M E I N
PE R I O D O NTITI S
Periodontitis is a progressive and chronic disease induced by the
overgrowth of oral microbiota within the gingival sulcus, resulting
in the formation of an inflammatory infiltrate that leads to the de-
struction of the periodontium and alveolar bone, and may eventu-
ally result in tooth loss.148,149 During this process, oral homeostasis
is disrupted, and the local microbiota changes from symbiosis to a
dysbiotic state, leading to disease. The transition of the oral micro-
biome from commensal to pathogenic is a dynamic process between
the complete oral endogenous microbiota and the host response.119
Classically, periodontal disease has been associated with bac-
teria of the so-called red complex (P gingivalis, T forsythia, and
T denticola). Currently, these microorganisms are known as patho-
bionts rather than pathogens because they are present at low
numbers in microbiomes of patients with no clinical signs of peri-
150
odontal disease. The pathobionts group now includes anaerobes
of the genera Parvimonas, Fusobacterium, and Prevotella as well as
gram-positive bacteria such as Filifacter alocis.35,148,151 Periodontitis
is characterized by changes in the structure of the microbial commu-
nity; namely an increase in the number of bacterial taxons and in the
relative abundance of pathobionts.148,152,153 As in other infectious
diseases, the microbial diversity has been studied in detail, in con-
trast to the bacterial gene-expression profiles in vivo, which remain
less explored. Fortunately, the application of metatranscriptomics to
study the functional activities associated with periodontal disease
has increased in the last few years. Despite high interpatient vari-
ability in the oral microbiome, those studies suggest that we should
focus more on the functional changes of the whole community than
on specific pathogens or the microbial composition.32,75,82
Recently, the phylogeny and diversity of the oral microbiota have
been characterized at unprecedented levels using high-throughput
sequencing. Early metabolic activities in the dysbiotic microbiome
revealed a functional signature that distinguishes periodontal sites
from healthy ones, supporting the idea that microbial commu-
nities as a whole might drive disease progression.75 In that study,
Duran-Pinedo et al31 reported an increase in metabolic activities
related to iron acquisition, lipopolysaccharide synthesis, flagella
synthesis, and expression of genes related to beta-lactam antibi-
otic degradation processes, as the significant activities defining se-
vere periodontitis. Iron is an essential enzyme cofactor and many
microbial pathogens have developed a rage of strategies to acquire
it.154,155 The presence of high levels of bacterial lipopolysaccharide
has been described as an essential element in periodontitis.156,157 In
P gingivalis, high levels of lipopolysaccharide and lipid A have been
described as a mechanism to modulate the inflammation in peri-
odontal tissues by delaying neutrophil apoptosis.158-160
Isolation of beta-lactamase-producing bacteria from the oral
cavity has previously been reported.161 Moreover, beta-lactamase
activity has been observed at a low level, but with high prevalence,
in patients with periodontitis.162 This raises the question of the
role of beta-lactamase in periodontitis as none of the patients was
being treated with beta-lactam antibiotics at the time of sampling.
Duran-Pinedo et al31 also analyzed the expression of virulence fac-
tors during periodontal disease, and demonstrated upregulation of
metalloproteases and peptidases in bacteria of the red complex.
Red complex bacteria also expressed proteins involved in iron me-
tabolism, including ferrous iron transport protein B, iron compound
ABC transporter ATP-binding proteins, ferric enterobactin transport
ATP-binding protein FepC, and iron chelate uptake ABC transporter,
FeCT family, permease protein. Porphyromonas gingivalis upregu-
lated expression of hemolysin genes, and P gingivalis and T denticola
showed upregulated expression of genes involved in vitamin B12
synthesis (for example, vitamin B12 ABC transporter btuFCD sys-
tem). Among the proteins not related directly to iron metabolism
were ATP-dependent Clp protease ATP-binding subunit ClpA and
chaperone protein ClpB, described as necessary in cellular adhesion
and invasion of P gingivalis. Treponema denticola overexpressed genes
related to flagella synthesis, such as flgC, flgF, and flgG, the flagellar
hook proteins FlgE and FlgK, the flagellar motor rotation proteins
MotA and MotB, the flagellar motor switch proteins FliG and FliM,
flagellin protein FlaA, flagellar M-ring protein FliF, flagellum-specific
ATP synthase FliI, and proteins involved in flagellum biosynthesis,
supporting the advance of periodontal disease by continued inva-
sion of the tissue. Tannerella forsythia and T denticola were found
to upregulate homologs to internalins, surface proteins that are in-
volved in the invasion of mammalian cells by L monocytogenes163,164
and which could play a similar role in those periodontal pathogens.
These observations strongly support the idea that in severe peri-
odontitis, members of the ‘red complex' actively invade host cells.
There is no doubt that the most intriguing result of that work was
the upregulation of a vast majority of virulence factors by organ-
isms not considered as pathogens. Surprisingly, the gram-positive
bacteria Corynebacterium matruchotii, Rothia dentocariosa, Veillonella
parvula, and Neisseria sicca, classically associated with the health of
the periodontium, expressed a large variety of putative virulence
factors. Interestingly, candidate division TM7 (Saccharibacteria), not
cultivated at the time of that publication and with only a partially
sequenced genome, showed overexpression of several homologs
DURAN-PINEDO |
      37

to virulence factors in the diseased community.31 These included:
the oligopeptide ABC transporter periplasmic oligopeptide-bind-
ing protein, previously described as a virulence factor in group A
Streptococci165; a hemagglutinin-related protein; exoproteins in-
volved in heme utilization or adhesion; and a homolog of LemA, a
2-component regulatory system identified as responsible for regu-
lating virulence factors and toxin production in Pseudomonas syrin-
gae.73 Those results support the proposed idea of the “pathogenic
microbial community”166 or “the community as pathogen”,167 which
postulates that the integrated actions of the components of the mi-
crobial community would result in disease (Figure 4).
Later on, Yost et al32 showed a general picture of the bacterial
pathways associated with progression to periodontitis. The authors
analyzed the metatranscriptome in subgingival samples from sta-
ble and progressing sites. Disease was considered to be progress-
ing if an increase in clinical attachment loss, of ≥2  mm compared
with baseline, was recorded at any follow-up visit. Stable sites were
defined as demonstrating clinical attachment loss no greater than
1 mm from the measuremnet made at baseline. The authors ana-
lyzed the gene-expression profile of the community by identifying
the gene ontology terms that were enriched. Changes took place in
the gene-expression profiles within all members of the red complex
during periodontal disease progression. Specifically, genes with gene
ontology terms associated with transport (iron, lactate, citrate, so-
dium, and phosphate), proteolysis, the protein kinase C-activating
G-protein coupled receptor signaling pathway, and response to
antibiotic were upregulated; and genes with gene ontology terms
associated with cobalamin (vitamin B12) biosynthesis were down-
regulated. Motility of T denticola was indicated by the upregulation
of genes related to flagella biosynthesis (flaN, flaG, fliQ, and fliW).
Tannerella forsythia and P gingivalis both upregulated different Ton
B-dependent receptors, genes involved in iron transport (ferric up-
take siderophores and ferrous iron transport protein B), and a large
number of peptidases and proteases, including ClpB. They also upreg-
ulated genes associated with aerotolerance, and clustered regularly
interspaced short palindromic repeat-associated genes (csp, csm, and
cas).32 Finally, P gingivalis specifically upregulated genes related on
biotin synthesis (biotin synthase, bioC, and bioG), proteins involved
in the biosynthesis of capsular polysaccharide, a large number of
proteins of conjugative transposons (TraA, TraB, TraE, TraF, TraG,
TraI, TraJ, TraK, TraL, TraM, TraN, TraO, Trap, and TraQ), transposases
(ISPg2, ISPg3, ISPg4, ISPg5, and ISPg6), and the 2 clustered regu-
larly interspaced short palindromic repeat-Cas operons (CRISPR-cas)
found in P gingivalis ATCC 33277. Porphyromonas gingivalis showed

A B

F I G U R E 4   Species ranked according to the number of upregulated putative virulence factors in the metatranscriptome. Putative
virulence factors were identified by aligning the protein sequences from the different genomes against the Virulence Factors of Pathogenic
Bacteria Database. The numbers in the bar chart are the absolute number of hits, for each bacterial species, of the upregulated putative
virulence factors identified. Members of the red complex are shown in red, members of the orange complex are shown in orange, and
members of the yellow complex are shown in blue. (A) Comparison of health with severe periodontitis (adapted from Duran-Pinedo et al75).
(B) Comparison of baseline with sites showing progression of periodontitis (adapted from Yost et al32).
 |
38       DURAN-PINEDO
upregulation of a large number of genes associated with virulence in
healthy sites that later progressed to periodontitis, while T forsythia
and T denticola upregulated only one of a few virulence genes until
later time points in this study.Individually, T denticola upregulated
genes related to flagella biosynthesis (flaA, flaG, fliQ, and fliW), oligo-
peptide ABC transporters, and a large number of hypothetical pro-
teins. Tannerella forsythia and P gingivalis both upregulated different
TonB-dependent receptors, genes involved in iron transport (ferric
uptake siderophores and ferrous iron transport protein B), a large
number of proteases such as ClpB, and clustered regularly inter-
spaced short palindromic repeat-associated genes (csp, csm, and cas).
Tannerella forsythia specifically upregulated transposases (IS116,
IS110, IS902, and IS4 families) and large numbers of homologs of
32
SusC and SusD family proteins involved in polysaccharide binding.
In addition, T forsythia significantly upregulated the expres-
sion of genes related to the destruction of the periodontium. The
bfor_c_1_1659 open reading frame was highly expressed in sites that
31,32
progressed to periodontitis. BFOR_c_1_1659 has homology to
dipeptidyl aminopeptidase IV, a serine protease that cleaves X-Pro
or X-Ala dipeptide at the penultimate position from the N-terminal
ends of polypeptide chains. Collagen, rich in X-Pro or X-Ala dipep-
tide, is the main supporting structure in connective tissues, which
168
are present on all structures of the periodontium. The ability
of BFOR_c_1_1659 to degrade collagen was recently assessed.
Expression of this putative collagenase was only found in sites with
169
severe periodontitis.
By contrast, during gingivitis, T forsythia downregulated BFOR-
1659 and upregulated another Xaa-dipeptidase (BFOR-2955), as de-
scribed by Nowicki et al33. Those results suggest that the ability of
T forsythia to contribute to the destruction of collagen could be a
result of selective expression of proteins triggered by still unknown
mechanisms. The over-representation of gene ontology terms re-
lated to potassium ion transport is also an essential signature of
31,32
periodontal disease. Potassium transport systems have been
linked to the pathogenesis of S aureus170 and Salmonella171, but inter-
estingly have not been described in oral bacteria. The link between
potassium transport and bacterial pathogenesis is correlated to the
significant increase in levels of potassium in both saliva and ginival
172
crevicular fluid (GCF) during periodontitis. It is tempting to hy-
pothesize that higher levels of potassium could increase the viru-
lence of the oral community. At the same time, microbial imbalance
can alter the immune response of the gingival epithelium, increasing
the production of tumor necrosis factor-alpha and reducing the ex-
pression of interleukin-6 and the antimicrobial peptide human be-
ta-defensin 3.173
The gene-expression profiles in bacteria of the orange com-
plex are very similar to those in bacteria of the red complex. Both
upregulate different TonB-dependent receptors, a large number
of peptidases and proteases (including ClpB), genes associated
with aerotolerance (Bacteroides aerotolerance operon batA-E i and
moxR-like ATPase of the aerotolerance operon in P intermedia and
P nigrescens), genes involved in iron transport (ferric uptake sidero-
phores and ferrous iron transport protein B), hemolysins, clustered
regularly interspaced short palindromic repeat-associated genes (in
Centruroides gracilis, Campylobacter rectus, Campylobacter showae,
P nigrescens, and Streptococcus constellatus), and the chaperone pro-
teins GroEL, GroES, and GrpE. As in the case of P gingivalis, both P in-
termedia and P nigrescens upregulated a large number of mobilization
genes (traA, traB, traD, traE, traF, traG, traI, traJ, traK, traL traM, traN,
traO, and traQ) from conjugative transposons.32
Interestingly, organisms not associated with periodontal diseases,
such as Streptococcus oralis, Streptococcus mutans, Staphylococcus in-
termedius, Streptococcus mitis, Veillonella parvula, and Pseudomonas
fluorescens, were particularly active, transcribing genes related to
virulence. All of the above-cited organisms upregulated genes en-
coding different oligopeptide transport systems (oppA, oppD, oppF,
and oppB), several hemolysins, manganese ABC transporters, man-
ganese superoxide dismutase, and a protein serine-threonine phos-
phatase (PrpC) involved in regulating the stationary phase of the cell
cycle. Streptococcus oralis, S mitis, and V parvula upregulated vitamin
B12 ABC transporters. Streptococcus oralis, S mutans, S intermedius,
and S mitis all upregulated the Clp protease system and the tran-
scription attenuator LytR. Pseudomonas fluorescens upregulated the
complete set of genes associated with flagellar synthesis (flaA-S and
motB) and genes related to chemotaxis.32 These findings suggest
that the whole community, not just a handful of oral pathogens, is
responsible for the increase in virulence that leads to progression of
periodontal disease.
Tannerella forsythia also specifically upregulated several trans-
posases (IS116, IS110, IS902, and IS4 families), and large numbers
of different homologs of SusC and SusD family proteins that are in-
volved in polysaccharide binding. Regarding proteins downregulated
by red complex bacteria, mostsuch proteins were hypothetical.31
Archaea have not been as widely studied as Bacteria although
they might have a direct effect on the development of periodonti-
tis. Archaea are found in 36% of patients with periodontitis, with
the Methanobrevibacter oralis-like phylotype being the predominant
archaeal community at periodontally diseased sites; moreover, a dis-
tinct Methanobrevibacter subpopulation is mainly found in the gut of
mammals. Methanobrevibacter oralis has been found in periodontal
pockets and in dental root canals.174 The two main species of Archaea
identified so far in humans are M oralis and Methanobrevibacter
smithii. They can colonize the intestine, oral cavity, and skin, and
can favor the growth of fermenting bacteria through interspecies
hydrogen transfer. There is very little information on the relationship
between Archaea and periodontitis or on the possible mechanisms
of interaction between Archaea and bacteria.175
6 | TH E M I C RO B I A L
M E TATR A N S C R I P TO M E I N D E NTA L C A R I E S
The prevalence of caries in permanent teeth has been calculated to
be 34%; thus, caries affects 2.5 billion people worldwide. 20 Caries
is characterized by lesions on tooth enamel and dentin induced by
an sugar rich diet. Saccharolytic bacteria in the oral microbiome
DURAN-PINEDO
ferment sucrose and other sugars to produce ATP and lactic acid as
a waste product. The accumulation of lactate is responsible for the
local acidification.176 Lowering the pH (to below 5.5) within the bio-
film177 for extended periods of time favors the growth of acidogenic
and acid-tolerant (aciduric) bacteria.178
Historically, only acidogenic species of the genus Streptococcus,
mainly S mutans, have been considered as the etiological agents of
dental caries. Recent studies using metatranscriptomics and metabo-
lomics approaches found a much higher diversity of bacteria, than pre-
viously considered, in alkali-generating pathways within complex oral
biofilms, thus linking caries to a more diverse microbiota. Interestingly,
the composition of microbes in the oral biofilm changes at different
stages of caries development.179,180 In contrast to what is observed
in periodontal diseases, the initial stage of caries is characterized by
higher microbial diversity. Caries progression is accompanied by a loss
of diversity and a reduction in the levels and activity of commensal
bacteria.181,182 Once the caries lesion reaches the dentin, diversity
may increase again, highlighting the influence of the oral niches on
the activity of the oral microbiome. For instance, in dentin, the rela-
tive proportion of S mutans increases from 0.12% in healthy dental
plaque to 0.72% in enamel lesions.34,183 By metatranscriptomic anal-
34
ysis, Simón-Soro et al identified the metabolically active bacterial
fraction in caries lesions at different stages of disease progression to
reveal a big picture of the bacterial community causing tooth decay.
They analyzed active lesions (identified by their color and texture)
and active white spot lesions or noncavitated enamel caries (iden-
tified based on their chalky white, opaque, and rough texture), on
which the carious lesion might extend into dentin without a clinically
visible crack at the enamel surface.184 Their data showed variation
in bacterial consortia between caries lesions of the same individuals
and among individuals. Lactobacilli were found almost exclusively in
cavities in dentin. Streptococci accounted for 40% of the entire active
community in enamel caries and 20% in dentin caries. As anticipated,
white spot lesions appeared to be a restrictive environment, whereas
open dentin cavities had the most diverse bacterial communities.
Open dentin lesions also showed the highest variability of bacteria be-
cause of their exposure to saliva. Streptococci, Rothia, Leptotrichia, and
Veillonella, for example, were more abundant in carious enamel lesions,
whereas Lactobacillus, Shlegelella, Pseudoramibacter, and Atopobium
were associated with dentin lesions. The minority species that were
only present in enamel lesions included Tannerella, Olsenella, Filifactor,
and Treponema, all of which are strongly associated with periodontal
disease.
Interestingly, the level of Streptococcus sanguinis was markedly
increased in dentin cavities, whereas S mitis was more abundant in
enamel lesions. Regarding S mutans (the most studied caries-associ-
ated species), the authors reported a low prevalence in all samples:
0.73% in enamel lesions, 0.48% in open dentin lesions, and 0.02% in
hidden dentin lesions.34 Those data suggest that the etiology of den-
tal caries is a very dynamic process shaped by the tissue, and it has
a polymicrobial origin. The cariogenic properties of the bacteria re-
cently identified to be associated with caries should be investigated
in future experimental work. Simón-Soro et al34 also reported that
|
      39
each sample expressed between 70 and 100 metabolically active
species associated with oxidative stress. They reported high expres-
sion of proteins that metabolize superoxides and peroxides present
in enamel/coronal caries.
A number of studies on dental caries have shown the presence of
many activities associated with oxidative stress and that high levels
of proteins which metabolize superoxides and peroxides are pres-
ent in enamel/coronal caries.180,185 The functional profiles of car-
ies-associated bacterial communities indicate that genes involved in
production of proteins implicated in oxidative stress and metabolism
of superoxides and peroxides are over-represented only at the ini-
tial stage (enamel caries). During later stages of caries, expression
of genes coding for osmotic stress tolerance proteins as well as pu-
tative collagenases, arginine deaminase, and urease counterbalance
the acidic pH, enabling degradation of dentin in dentin cavities.168
The classical mechanism against acidification of the ecosystem was
thought to be through the production of ammonia (an alkali) from
arginine and urea.186 However, Edlund et al found that glutamate de-
hydrogenase, threonine, and serine deaminase, and upregulation of
membrane proteins involved in ammonia gas conduction, acted as a
way to control pH besides the urease activity and arginine deaminase
system.187 It is becoming clear that caries is a more dynamic process
than originally thought, being characterized by a shift in composition
of the supragingival microbial community that induces the caries le-
sion on enamel. Numerous metagenomic studies support the strong
correlation between S mutans and Lactobacillus spp. and active car-
ies.181,182,188 At low concentrations of fermentable carbohydrates,
S mitis bind, with higher avidity than S mutans, to teeth coated with
saliva. Then, S mitis rapidly increase in number, and by secreting
hydrogen peroxide antagonize competitors.119,189 The presence
of acidogenic and aciduric oral microbes, in conjunction with fre-
quent consumption of dietary sugars, results in caries lessons.
Recent studies present evidence that other species than S mutans
and S mitis are likely to play a roles in caries development.188,190,191
They include: Actinomyces, Fusobacterium, Porphyromonas, Rothia,
Granulicatella, Gemella, Selenomonas, Bifidobacteria, Scardovia, and
Haemophilus, well as nonaciduric genera including Corynebacterium
spp, Granulicatella spp, Propionibacterium spp., Bifidobacterium spp.,
and Scardovia spp. Actinomyces and Candida albicans.190 Actinomyces
and Candida albicans are strongly associated with caries progression
in vivo, especially in the case of early childhood caries.192 Similarly
to periodontitis, an extended group of species becomes more
pathogenic.
Supporting those results, Kressirer et al found in 2018, that
genes expressed in caries mapped to 108 named species.190 Simón-
Soro et al34 also found that Streptococcus and Veillonella were highly
active in all types of caries, whereas Lactobacillus spp. were only
highly active in the enamel-dentin caries sites. In a parallel study on
caries lesions in children, Kressirer et al190 reported that Lactobacillus
spp. are highly active in coronal caries, while in dentin caries,
the most representative members of the active community are
Neisseriales, Prevotellaceae, and Actinobacteria. Scardovia wiggsiae
is strongly associated with dentin caries, suggesting a role in caries
 |
40       DURAN-PINEDO
development.183 By contrast, in a previous study, when looking at
community-wide expression profiles of dental plaque, Peterson
et al193 found that a small fraction of the community is responsible
for the majority of transcripts in the community. Streptococci rep-
resent the majority of the active members in caries. Streptococcus
sanguinis was found to be the most active member of the commu-
nity, with 16% of the transcripts, followed by S mitis (10%), V parvula
(9%), Capnocytophaga spp. (9%), S oralis (8%), Streptococcus spp. (7%),
Gemella haemolysans (5%), Streptococcus gordonii (4%), and Neisseria
185
spp. (3%).
The gene-expression profile of caries lesions reported by
Kressirer et al190 showed a higher diversity of genes in dentin than in
caries-free samples or in caries samples from coronal sites. In den-
tin lesions, larger number of enzymes were detected. This finding
is consistent with other observations of acidogenic and proteolytic
activities in dentin caries.92
Caries lesions showed upregulation of genes involved in the
metabolism of fructose and mannose, sugar phosphotransferases
involved in the metabolism of beta-glucoside, as well as the use
of sorbitol as an additional source of carbon in caries lesions. May
et al194 confirmed this observation on their analysis of already ex-
isting metatranscriptomic data. In S mutans, the authors found a
strong association of the pathway that converts sorbitol to fructose
6-phosphate with caries lesions. This Kegg Orthology (KO) group
converts fructose to fructose 6-phosphate, suggesting that in den-
tal caries sorbitol might be used as an additional source of carbon.
They also described 9 KEGG Orthology groups from pathways of the
phosphotransferase system and mannose metabolism that were as-
sociated with caries lesions. Those genes have also been associated
195,196
with the colonization process.
By contrast, in caries-free samples or healthy sites, alcohol dehy-
drogenase and methylenetetrahydrofolate reductase genes were sig-
nificantly upregulated. Alcohol dehydrogenase has been described
in the neutralization of acid through the reduction of nicotinamide
adenine dinucleotide (NAD+ to NADH). Alcohol dehydrogenase is
mainly produced by N sicca.197 Genes expressing methylenetetrahy-
drofolate reductase are involved in methionine production and are
190
mainly expressed by S sanguinis.
7 |  CO N C LU S I O N S
Current concepts of caries and periodontal disease highlight the
importance of oral biofilms as and its participation as etiological
agents of those diseases. There has been a shift in focus from the
role of individual species within the biofilm to a more complex
view, in which biofilms as a whole cause and perpetuate these
chronic infections.
Despite the relevance of the biofilm to the infectious oral dis-
ease, it is clear that not all compositions of the oral microbiota re-
sult in tissue destruction, and that not all elements of a pathogenic
biofilm have the same significance in the disease process. Although
some organisms are highly associated with disease, changes in bio-
film composition alone do not explain the onset or development of
disease.
It is now clear that the functions of distinct species within the
subgingival microbiota are intimately intertwined with the rest of
the microbial community. This point highlights the relevance of ex-
amining the gene expression profile of the subgingival microbiota in
periodontal disease or caries lesions in the context of other mem-
bers of the biofilm in vivo.
Metatranscriptomic analysis of the oral community has allowed
us to begin answering questions of how homeostasis is maintained.
These analyses are the starting point for identifying environmental
signals that modulate the metabolic patterns of the community as it
shifts from commensal to dysbiotic. This wealth of information was
unimaginable just a decade ago.
These studies give a shot of the gene expression patterns of
microbial communities and also allow us to determine triggers to
diseases. In the case of caries, new observations on sugar metabo-
lism (classically identified as an essential element that distinguishes
health from disease), were made, such as the potential use of sorbitol
as an additional source of carbon by S mutans. In the case of peri-
odontal disease, high levels of extracellular potassium could be a sig-
nal of disease. These are just some examples, among other dysbiotic
signals identified by metatranscriptomic analysis.
Thus, in the near future, the application of metatranscriptomic
analysis along with other omics approaches will continue to pro-
vide the opportunity to identify the group of metabolic pathways
that trigger the development of periodontal disease and caries.
Longitudinal studies are needed to identify the real markers of the
initial stages of oral diseases. Of note, the metatranscriptome does
not necessarily represent the final metabolic products generated by
the microbial community. Improvements in the integration of omics
approaches should allow us to determine the origin of proteins and
metabolites produced by the microbiome in health and disease.
Finally, we need more information on the gene-expression profiles
of the host, along with the patterns from the microbiome, to obtain a
better view of the modulation of health and disease. Understanding
the communication between the host and the microbiome is cru-
cial to complete the picture between oral health and disease. Finally,
there is a missing piece on the transcriptome, namely the fungal frac-
tion of the oral microbiome, which could influence the dynamics of
the whole microbial community.
AC K N OW L E D G E M E N T S
I thank Jorge Frias-Lopez and Susan Yost for their helpful com-
ments on this manuscript. This work was supported by the National
Institute of Dental and Craniofacial Research of the National
Institutes of Health (2R01DE021553).
C O N FL I C T S O F I N T E R E S T
The author declares no potential conflicts of interest with respect to
the authorship and/or publication of this article.
DURAN-PINEDO |
      41

REFERENCES 23. McCarren J, Becker JW, Repeta DJ, et al. Microbial community
transcriptomes reveal microbes and metabolic pathways associ-
1. Sender R, Fuchs S, Milo R. Revised estimates for the num-
ated with dissolved organic matter turnover in the sea. Proc Natl
ber of human and bacteria cells in the body. PLoS Biol.
Acad Sci U S A. 2010;107(38):16420-16427.
2016;14(8):e1002533.
24. Carlisle EM, Poroyko V, Caplan MS, Alverdy J, Morowitz MJ, Liu D.
2. Lederberg J, McCray A. Ome sweet ’omics: – A genealogical trea-
Murine gut microbiota and transcriptome are diet dependent. Ann
sury of words. Scientist. 2001;15:8.
Surg. 2013;257(2):287-294.
3. Wade WG. The oral microbiome in health and disease. Pharmacol
25. Giannoukos G, Ciulla DM, Huang K, et al. Efficient and robust
Res Off J Ital Pharmacol Soc. 2013;69(1):137-143.
RNA-seq process for cultured bacteria and complex community
4. Abeles SR, Robles-Sikisaka R, Ly M, et al. Human oral vi-
transcriptomes. Genome Biol. 2012;13(3):R23.
ruses are personal, persistent and gender-consistent. ISME J.
26. Xiong X, Frank DN, Robertson CE, et al. Generation and analysis
2014;8(9):1753-1767.
of a mouse intestinal metatranscriptome through illumina based
5. Ly M, Abeles SR, Boehm TK, et al. Altered oral viral ecology in as-
RNA-sequencing. PLoS One. 2012;7(4):e36009.
sociation with periodontal disease. MBio. 2014;5(3):e01133-1114.
27. Booijink CCGM, Boekhorst J, Zoetendal EG, Smidt H, Kleerebezem
6. Wade WG. The oral microbiome in health and disease.
M, de Vos WM. Metatranscriptome analysis of the human
Pharmacological Research. 2013;69 (1):137-143. http://dx.doi.
fecal microbiota reveals subject-specific expression profiles,
org/10.1016/j.phrs.2012.11.006
with genes encoding proteins involved in carbohydrate me-
7. Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC, Relman
tabolism being dominantly expressed. Appl Environ Microbiol.
DA. Methanogenic Archaea and human periodontal disease.
2010;76(16):5533-5540.
Proc Natl Acad Sci U S A. 2004;101(16):6176-6181. https://doi.
28. Solbiati J, Frias-Lopez J. Metatranscriptome of the oral microbi-
org/10.1073/pnas.03087​66101
ome in health and disease. J Dent Res. 2018;97(5):492-500.
8. Jenkinson HF. Beyond the oral microbiome. Environ Microbiol.
29. Belstrøm D, Fiehn N-E, Nielsen CH, et al. Differences in bacterial
2011;13(12):3077-3087.
saliva profile between periodontitis patients and a control cohort.
9. Paster BJ, Boches SK, Galvin JL, et al. Bacterial diversity in human
J Clin Periodontol. 2014;41(2):104-112.
subgingival plaque. J Bacteriol. 2001;183(12):3770-3783.
30. Belstrøm D, Fiehn N-E, Nielsen CH, et al. Altered bacterial profiles
10. Peterson J, Garges S, Giovanni M, et al. The NIH human microbi-
in saliva from adults with caries lesions: a case-cohort study. Caries
ome project. Genome Res. 2009;19(12):2317-2323.
Res. 2014;48(5):368-375.
11. Dahlén G. Bacterial infections of the oral mucosa. Periodontol.
31. Duran-Pinedo AE, Chen T, Teles R, et al. Community-wide tran-
2000;2009(49):13-38.
scriptome of the oral microbiome in subjects with and without
12. Dewhirst FE, Chen T, Izard J, et al. The human oral microbiome. J
periodontitis. ISME J. 2014;8(8):1659-1672.
Bacteriol. 2010;192(19):5002-5017.
32. Yost S, Duran-Pinedo AE, Teles R, Krishnan K, Frias-Lopez J.
13. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining
Functional signatures of oral dysbiosis during periodontitis pro-
the normal bacterial flora of the oral cavity. J Clin Microbiol.
gression revealed by microbial metatranscriptome analysis.
2005;43(11):5721-5732.
Genome Med. 2015;7(1):27.
14. Dewhirst FE, Paster BJ, Tzellas N, et al. Characterization of novel
33. Nowicki EM, Shroff R, Singleton JA, et al. Microbiota and meta-
human oral isolates and cloned 16S rDNA sequences that fall in
transcriptome changes accompanying the onset of gingivitis. MBio.
the family Coriobacteriaceae: description of olsenella gen. nov.,
2018;9(2):e00575.
reclassification of Lactobacillus uli as Olsenella uli comb. nov. and
34. Simón-Soro A, Guillen-Navarro M, Mira A. Metatranscriptomics
description of Olsenella profusa sp. nov. Int J Syst Evol Microbiol.
reveals overall active bacterial composition in caries lesions. J Oral
2001;51(Pt 5):1797-1804.
Microbiol. 2014;6:25443.
15. Haffajee AD, Socransky SS. Introduction to microbial aspects of
35. Griffen AL, Beall CJ, Campbell JH, et al. Distinct and complex bac-
periodontal biofilm communities, development and treatment.
terial profiles in human periodontitis and health revealed by 16S
Periodontol. 2000;2006(42):7-12.
pyrosequencing. ISME J. 2012;6(6):1176-1185.
16. Haffajee AD, Socransky SS, Patel MR, Song X. Microbial
36. Segata N, Haake SK, Mannon P, et al. Composition of the adult di-
complexes in supragingival plaque. Oral Microbiol Immunol.
gestive tract bacterial microbiome based on seven mouth surfaces,
2008;23(3):196-205.
tonsils, throat and stool samples. Genome Biol. 2012;13(6):R42.
17. Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial
37. Zhou Y, Gao H, Mihindukulasuriya KA, et al. Biogeography
diversity in the human periodontal pocket and other oral sites.
of the ecosystems of the healthy human body. Genome Biol.
Periodontol. 2000;2006(42):80-87.
2013;14(1):R1.
18. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin
38. Li K, Bihan M, Yooseph S, Methé BA. Analyses of the microbial di-
AE. Checkerboard DNA-DNA hybridization. Biotechniques.
versity across the human microbiome. PLoS One. 2012;7(6):e32118.
1994;17(4):788-792.
39. Smith QT, Geegan SJ. Repeated measurement of crevicular fluid
19. Eke PI, Thornton-Evans GO, Wei L, Borgnakke WS, Dye BA, Genco RJ.
parameters at different sites. J Clin Periodontol. 1991;18(3):171-176.
Periodontitis in US Adults: National health and nutrition examination
40. Xu X, He J, Xue J, et al. Oral cavity contains distinct niches
survey 2009–2014. J Am Dent Assoc 1939. 2018;149(7):576-588.e6.
with dynamic microbial communities. Environ Microbiol.
20. Kassebaum NJ, Smith AGC, Bernabé E, et al. Global, regional, and
2015;17(3):699-710.
national prevalence, incidence, and disability-adjusted life years
41. Kenney EB, Ash MM. Oxidation Reduction Potential of Developing
for oral conditions for 195 countries, 1990–2015: A systematic
Plaque, Periodontal Pockets and Gingival Sulci. J Periodontol-
analysis for the global burden of diseases, injuries, and risk factors.
Periodontics. 1969;40(11):630-633.
J Dent Res. 2017;96(4):380-387.
42. Diaz PI, Dupuy AK, Abusleme L, et al. Using high throughput se-
21. Frias-Lopez J, Duran-Pinedo A. Effect of periodontal pathogens on
quencing to explore the biodiversity in oral bacterial communities.
the metatranscriptome of a healthy multispecies biofilm model. J
Mol Oral Microbiol. 2012;27(3):182-201.
Bacteriol. 2012;194(8):2082-2095.
43. Kumar PS, Mason MR. Mouthguards: does the indigenous mi-
22. Frias-Lopez J, Shi Y, Tyson GW, et al. Microbial community gene
crobiome play a role in maintaining oral health? Front Cell Infect
expression in ocean surface waters. Proc Natl Acad Sci U S A.
Microbiol. 2015;5:35.
2008;105(10):3805-3810.
 |
42       DURAN-PINEDO
44. Mager DL, Ximenez-Fyvie LA, Haffajee AD, Socransky SS.
Distribution of selected bacterial species on intraoral surfaces. J
Clin Periodontol. 2003;30(7):644-654.
45. Proctor DM, Relman DA. The landscape ecology and microbi-
ota of the human nose, mouth, and throat. Cell Host Microbe.
2017;21(4):421-432.
46. Ximenez-Fyvie LA, Haffajee AD, Socransky SS. Microbial compo-
sition of supra- and subgingival plaque in subjects with adult peri-
odontitis. J Clin Periodontol. 2000;27(10):722-732.
47. Mark Welch JL, Dewhirst FE, Borisy GG. Biogeography of the oral
microbiome: the site-specialist hypothesis. Annu Rev Microbiol.
2019;73(1):335-358.
48. Valm AM. The structure of dental plaque microbial communities in
the transition from health to dental caries and periodontal disease.
J Mol Biol. 2019;431(16):2957-2969.
49. Listgarten MA. Structure of the microbial flora associated with
periodontal health and disease in man. A light and electron micro-
scopic study. J Periodontol. 1976;47(1):1-18.
50. Nyvad B, Fejerskov O. Scanning electron microscopy of early mi-
crobial colonization of human enamel and root surfaces in vivo.
Scand J Dent Res. 1987;95(4):287-296.
51. Listgarten MA, Mayo HE, Tremblay R. Development of dental
plaque on epoxy resin crowns in man. A light and electron micro-
scopic study. J Periodontol. 1975;46(1):10-26.
52. Palmer RJ, Shah N, Valm A, et al. Interbacterial adhesion networks
within early oral biofilms of single human hosts. Appl Environ
Microbiol. 2017;83(11):e00407-17.
53. Periasamy S, Chalmers NI, Du-Thumm L, Kolenbrander PE.
Fusobacterium nucleatum ATCC 10953 requires Actinomyces
naeslundii ATCC 43146 for growth on saliva in a three-species
community that includes Streptococcus oralis 34. Appl Environ
Microbiol. 2009;75(10):3250-3257.
54. Egland PG, Palmer RJ, Kolenbrander PE. Interspecies communica-
tion in Streptococcus gordonii-Veillonella atypica biofilms: signal-
ing in flow conditions requires juxtaposition. Proc Natl Acad Sci U S
A. 2004;101(48):16917-16922.
55. Periasamy S, Kolenbrander PE. Aggregatibacter actinomy-
cetemcomitans builds mutualistic biofilm communities with
Fusobacterium nucleatum and Veillonella species in saliva. Infect
Immun. 2009;77(9):3542-3551.
56. Mark Welch JL, Rossetti BJ, Rieken CW, Dewhirst FE, Borisy GG.
Biogeography of a human oral microbiome at the micron scale.
Proc Natl Acad Sci U S A. 2016;113(6):E791-800.
57. Wecke J, Kersten T, Madela K, et al. A novel technique for moni-
toring the development of bacterial biofilms in human periodontal
pockets. FEMS Microbiol Lett. 2000;191(1):95-101.
58. Drescher J, Schlafer S, Schaudinn C, et al. Molecular epidemiology
and spatial distribution of Selenomonas spp. in subgingival bio-
films. Eur J Oral Sci. 2010;118(5):466-474.
59. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communi-
ties, conflict and control. Periodontol 2000. 2011;55(1):16-35.
60. Noiri Y, Ozaki K, Nakae H, Matsuo T, Ebisu S. An immunohisto-
chemical study on the localization of Porphyromonas gingivalis,
Campylobacter rectus and Actinomyces viscosus in human peri-
odontal pockets. J Periodontal Res. 1997;32(7):598-607.
61. Noiri Y, Li L, Ebisu S. The localization of periodontal-disease-as-
sociated bacteria in human periodontal pockets. J Dent Res.
2001;80(10):1930-1934.
62. Noiri Y, Li L, Yoshimura F, Ebisu S. Localization of Porphyromonas
gingivalis-carrying fimbriae in situ in human periodontal pockets. J
Dent Res. 2004;83(12):941-945.
63. Vartoukian SR. Cultivation strategies for growth of uncultivated
bacteria. J Oral Biosci. 2016;58(4):142-149.
64. Beall CJ, Campbell AG, Dayeh DM, Griffen AL, Podar M, Leys EJ.
Single cell genomics of uncultured, health-associated Tannerella
BU063 (Oral Taxon 286) and comparison to the closely related
pathogen Tannerella forsythia. PLoS One. 2014;9(2):e89398.
65. Stewart EJ. Growing unculturable bacteria. J Bacteriol.
2012;194(16):4151-4160.
66. Kroes I, Lepp PW, Relman DA. Bacterial diversity within
the human subgingival crevice. Proc Natl Acad Sci U S A.
1999;96(25):14547-14552.
67. Sakamoto M, Huang Y, Umeda M, Ishikawa I, Benno Y. Detection
of novel oral phylotypes associated with periodontitis. FEMS
Microbiol Lett. 2002;217(1):65-69.
68. Wade W. Unculturable bacteria—the uncharacterized organisms
that cause oral infections. J R Soc Med. 2002;95(2):81-83.
69. de Lillo A, Booth V, Kyriacou L, Weightman AJ, Wade WG.
Culture-independent identification of periodontitis-associated
Porphyromonas and Tannerella populations by targeted molecular
analysis. J Clin Microbiol. 2004;42(12):5523-5527.
70. Vartoukian SR, Moazzez RV, Paster BJ, Dewhirst FE, Wade WG.
First cultivation of health-associated Tannerella sp. HOT-286
(BU063). J Dent Res. 2016;95(11):1308-1313.
71. Beall CJ, Campbell AG, Griffen AL, Podar M, Leys EJ. Genomics
of the uncultivated, periodontitis-associated bacterium Tannerella
sp. BU045 (Oral Taxon 808). mSystems. 2018;3(3):e00018-18.
72. Ferrari B, Winsley T, Ji M, Neilan B. Insights into the distribution
and abundance of the ubiquitous Candidatus Saccharibacteria
phylum following tag pyrosequencing. Sci Rep. 2015;4(1):3957.
73. Kitten T, Kinscherf TG, McEvoy JL, Willis DK. A newly identi-
fied regulator is required for virulence and toxin production in
Pseudomonas syringae. Mol Microbiol. 1998;28(5):917-929.
74. Marcy Y, Ouverney C, Bik EM, et al. Dissecting biological “dark
matter” with single-cell genetic analysis of rare and uncultivated
TM7 microbes from the human mouth. Proc Natl Acad Sci U S A.
2007;104(29):11889-11894.
75. Duran-Pinedo AE, Baker VD, Frias-Lopez J. The periodontal
pathogen Porphyromonas gingivalis induces expression of trans-
posases and cell death of Streptococcus mitis in a biofilm model.
Infect Immun. 2014;82(8):3374-3382.
76. Liu B, Faller LL, Klitgord N, et al. Deep sequencing of the oral
microbiome reveals signatures of periodontal disease. PLoS One.
2012;7(6):e37919.
77. Brinig MM, Lepp PW, Ouverney CC, Armitage GC, Relman DA.
Prevalence of bacteria of division TM7 in human subgingival
plaque and their association with disease. Appl Environ Microbiol.
2003;69(3):1687-1694.
78. Podar M, Abulencia CB, Walcher M, et al. Targeted access to the
genomes of low-abundance organisms in complex microbial com-
munities. Appl Environ Microbiol. 2007;73(10):3205-3214.
79. He X, McLean JS, Edlund A, et al. Cultivation of a human-associ-
ated TM7 phylotype reveals a reduced genome and epibiotic para-
sitic lifestyle. Proc Natl Acad Sci U S A. 2015;112(1):244-249.
80. Wang B, Wu J, Lamont RJ, Lin X, Xie H. Negative correlation of dis-
tributions of Streptococcus cristatus and Porphyromonas gingiva-
lis in subgingival plaque. J Clin Microbiol. 2009;47(12):3902-3906.
81. Larsen MH, Leisner JJ, Ingmer H. The chitinolytic activity of
listeria monocytogenes EGD is regulated by carbohydrates but
also by the virulence regulator PrfA. Appl Environ Microbiol.
2010;76(19):6470-6476.
82. Jorth P, Turner KH, Gumus P, Nizam N, Buduneli N, Whiteley M.
Metatranscriptomics of the human oral microbiome during health
and disease. MBio. 2014;5(2):e01012–14.
83. Hoeijmakers WAM, Bártfai R, Françoijs K-J, Stunnenberg
HG. Linear amplification for deep sequencing. Nat Protoc.
2011;6(7):1026-1036.
84. Duran-Pinedo AE, Yost S, Frias-Lopez J. Small RNA transcriptome
of the oral microbiome during periodontitis progression. Appl
Environ Microbiol. 2015;81(19):6688-6699.
DURAN-PINEDO
85. Hart SN, Therneau TM, Zhang Y, Poland GA, Kocher J-P.
Calculating sample size estimates for RNA sequencing data. J
Comput Biol. 2013;20(12):970-978.
86. Ching T, Huang S, Garmire LX. Power analysis and sample size
estimation for RNA-Seq differential expression. RNA N Y N.
2014;20(11):1684-1696.
87. Liu Y, Zhou J, White KP. RNA-seq differential expression studies: more
sequence or more replication? Bioinformatics. 2014;30(3):301-304.
88. Beasley DE, Koltz AM, Lambert JE, Fierer N, Dunn RR. The evo-
lution of stomach acidity and its relevance to the human microbi-
ome. PLoS One. 2015;10(7):e0134116.
89. He X, McLean JS, Guo L, Lux R, Shi W. The social structure of
microbial community involved in colonization resistance. ISME J.
2014;8(3):564-574.
90. Kilian M, Chapple ILC, Hannig M, et al. The oral microbi-
ome - an update for oral healthcare professionals. Br Dent J.
2016;221(10):657-666.
91. Abubucker S, Segata N, Goll J, et al. Metabolic reconstruction for
metagenomic data and its application to the human microbiome.
PLoS Comput Biol. 2012;8(6):e1002358.
92. Benítez-Páez A, Belda-Ferre P, Simón-Soro A, Mira A. Microbiota
diversity and gene expression dynamics in human oral biofilms.
BMC Genom. 2014;15:311.
93. Kraal L, Abubucker S, Kota K, Fischbach MA, Mitreva M. The prev-
alence of species and strains in the human microbiome: a resource
for experimental efforts. PLoS One. 2014;9(5):e97279.
94. Marsh PD. In sickness and in health-what does the oral micro-
biome mean to us? An ecological perspective. Adv Dent Res.
2018;29(1):60-65.
95. Raizada MK, Joe B, Bryan NS, et al. Report of the national heart,
lung, and blood institute working group on the role of microbiota
in blood pressure regulation: current status and future directions.
Hypertension. 2017;70(3):479-485.
96. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-ni-
tric oxide pathway in physiology and therapeutics. Nat Rev Drug
Discov. 2008;7(2):156-167.
97. Hyde ER, Andrade F, Vaksman Z, et al. Metagenomic analysis of
nitrate-reducing bacteria in the oral cavity: implications for nitric
oxide homeostasis. PLoS One. 2014;9(3):e88645.
98. Desvarieux M, Demmer RT, Jacobs DR, et al. Periodontal bacteria
and hypertension: the oral infections and vascular disease epide-
miology study (INVEST). J Hypertens. 2010;28(7):1413-1421.
99. Kurgan S, Kantarci A. Molecular basis for immunohistochemical
and inflammatory changes during progression of gingivitis to peri-
odontitis. Periodontol 2000. 2018;76(1):51-67.
100. Kistler JO, Booth V, Bradshaw DJ, Wade WG. Bacterial com-
munity development in experimental gingivitis. PLoS One.
2013;8(8):e71227.
101. Smith I. Mycobacterium tuberculosis pathogenesis and molecular
determinants of virulence. Clin Microbiol Rev. 2003;16(3):463-496.
102. Kostic AD, Gevers D, Pedamallu CS, et al. Genomic analysis iden-
tifies association of Fusobacterium with colorectal carcinoma.
Genome Res. 2012;22(2):292-298.
103. Gur C, Ibrahim Y, Isaacson B, et al. Binding of the Fap2 pro-
tein of Fusobacterium nucleatum to human inhibitory recep-
tor TIGIT protects tumors from immune cell attack. Immunity.
2015;42(2):344-355.
104. Goodson JM, Tanner AC, Haffajee AD, Sornberger GC, Socransky
SS. Patterns of progression and regression of advanced destruc-
tive periodontal disease. J Clin Periodontol. 1982;9(6):472-481.
105. Eley BM, Cox SW. A 2-year longitudinal study of elastase in human
gingival crevicular fluid and periodontal attachment loss. J Clin
Periodontol. 1996;23(7):681-692.
106. Byrne SJ, Dashper SG, Darby IB, Adams GG, Hoffmann B,
Reynolds EC. Progression of chronic periodontitis can be
|
      43
predicted by the levels of Porphyromonas gingivalis and
Treponema denticola in subgingival plaque. Oral Microbiol
Immunol. 2009;24(6):469-477.
107. Charalampakis G, Dahlén G, Carlén A, Leonhardt A. Bacterial
markers vs. clinical markers to predict progression of chronic peri-
odontitis: a 2-yr prospective observational study. Eur J Oral Sci.
2013;121(5):394-402.
108. Khalaf H, Lönn J, Bengtsson T. Cytokines and chemokines are
differentially expressed in patients with periodontitis: possible
role for TGF-β1 as a marker for disease progression. Cytokine.
2014;67(1):29-35.
109. Heitz-Mayfield LJA. Disease progression: identification of high-
risk groups and individuals for periodontitis. J Clin Periodontol.
2005;32(Suppl 6):196-209.
110. Ricci M, Garoia F, Tabarroni C, et al. Association between genetic
risk score and periodontitis onset and progression: a pilot study.
Arch Oral Biol. 2011;56(12):1499-1505.
111. Socransky SS, Haffajee AD, Goodson JM, Lindhe J. New con-
cepts of destructive periodontal disease. J Clin Periodontol.
1984;11(1):21-32.
112. Haffajee AD, Socransky SS. Attachment level changes in destruc-
tive periodontal diseases. J Clin Periodontol. 1986;13(5):461-475.
113. Jeffcoat MK, Reddy MS. Progression of probing attachment loss in
adult periodontitis. J Periodontol. 1991;62(3):185-189.
114. Clerehugh V, Worthington HV, Lennon MA, Chandler R. Site pro-
gression of loss of attachment over 5 years in 14- to 19-year-old
adolescents. J Clin Periodontol. 1995;22(1):15-21.
115. Sculley DV, Langley-Evans SC. Periodontal disease is associ-
ated with lower antioxidant capacity in whole saliva and evi-
dence of increased protein oxidation. Clin Sci Lond Engl 1979.
2003;105(2):167-172.
116. Leggott PJ, Robertson PB, Rothman DL, Murray PA, Jacob RA. The
effect of controlled ascorbic acid depletion and supplementation
on periodontal health. J Periodontol. 1986;57(8):480-485.
117. Singer RE, Buckner BA. Butyrate and propionate: import-
ant components of toxic dental plaque extracts. Infect Immun.
1981;32(2):458-463.
118. Szafranski SP, Wos-Oxley ML, Vilchez-Vargas R, et al. High-
resolution taxonomic profiling of the subgingival microbiome for
biomarker discovery and periodontitis diagnosis. Appl Environ
Microbiol. 2015;81(3):1047-1058.
119. Lamont RJ, Koo H, Hajishengallis G. The oral microbiota: dy-
namic communities and host interactions. Nat Rev Microbiol.
2018;16(12):745-759.
120. Hajishengallis G, Liang S, Payne MA, et al. Low-abundance biofilm
species orchestrates inflammatory periodontal disease through
the commensal microbiota and complement. Cell Host Microbe.
2011;10(5):497-506.
121. Hajishengallis G, Darveau RP, Curtis MA. The keystone-pathogen
hypothesis. Nat Rev Microbiol. 2012;10(10):717-725.
122. Beisel CL, Storz G. Base pairing small RNAs and their roles in global
regulatory networks. FEMS Microbiol Rev. 2010;34(5):866-882.
123. Storz G, Vogel J, Wassarman KM. Regulation by small RNAs in bac-
teria: expanding frontiers. Mol Cell. 2011;43(6):880-891.
124. Michaux C, Verneuil N, Hartke A, Giard J-C. Physiological roles
of small RNA molecules. Microbiol Read Engl. 2014;160(Pt
6):1007-1019.
125. Gong H, Vu G-P, Bai Y, et al. A Salmonella small non-coding
RNA facilitates bacterial invasion and intracellular replication
by modulating the expression of virulence factors. PLoS Pathog.
2011;7(9):e1002120.
126. Koo JT, Alleyne TM, Schiano CA, Jafari N, Lathem WW. Global
discovery of small RNAs in Yersinia pseudotuberculosis identifies
Yersinia-specific small, noncoding RNAs required for virulence.
Proc Natl Acad Sci. 2011;108(37):E709-E717.
 |
44       DURAN-PINEDO
127. Tsai C-H, Liao R, Chou B, Palumbo M, Contreras LM. Genome-
wide analyses in bacteria show small-RNA enrichment for long and
conserved intergenic regions. J Bacteriol. 2015;197(1):40-50.
128. Urban JH, Vogel J. Translational control and target recogni-
tion by Escherichia coli small RNAs in vivo. Nucleic Acids Res.
2007;35(3):1018-1037.
129. Caron M-P, Lafontaine DA, Massé E. Small RNA-mediated regula-
tion at the level of transcript stability. RNA Biol. 2010;7(2):140-144.
130. Waters LS, Storz G. Regulatory RNAs in bacteria. Cell.
2009;136(4):615-628.
131. Sharma CM, Vogel J. Experimental approaches for the discovery
and characterization of regulatory small RNA. Curr Opin Microbiol.
2009;12(5):536-546.
132. Altuvia S. Identification of bacterial small non-coding RNAs: ex-
perimental approaches. Curr Opin Microbiol. 2007;10(3):257-261.
133. Sittka A, Sharma CM, Rolle K, Vogel J. Deep sequencing of
Salmonella RNA associated with heterologous Hfq proteins in vivo
reveals small RNAs as a major target class and identifies RNA pro-
cessing phenotypes. RNA Biol. 2009;6(3):266-275.
134. Liu JM, Livny J, Lawrence MS, Kimball MD, Waldor MK, Camilli
A. Experimental discovery of sRNAs in Vibrio cholerae by direct
cloning, 5S/tRNA depletion and parallel sequencing. Nucleic Acids
Res. 2009;37(6):e46.
135. Irnov I, Sharma CM, Vogel J, Winkler WC. Identification
of regulatory RNAs in Bacillus subtilis. Nucleic Acids Res.
2010;38(19):6637-6651.
136. Shi Y, Tyson GW, DeLong EF. Metatranscriptomics reveals
unique microbial small RNAs in the ocean’s water column. Nature.
2009;459(7244):266-269.
137. Murakami S, Fujishima K, Tomita M, Kanai A. Metatranscriptomic
analysis of microbes in an Oceanfront deep-subsurface hot spring
reveals novel small RNAs and type-specific tRNA degradation.
Appl Environ Microbiol. 2012;78(4):1015-1022.
138. Gosalbes MJ, Durbán A, Pignatelli M, et al. Metatranscriptomic ap-
proach to analyze the functional human gut microbiota. PLoS One.
2011;6(3):e17447.
139. Romby P, Vandenesch F, Wagner EGH. The role of RNAs in the
regulation of virulence-gene expression. Curr Opin Microbiol.
2006;9(2):229-236.
140. Soutourina OA, Monot M, Boudry P, et al. Genome-wide identi-
fication of regulatory RNAs in the human pathogen Clostridium
difficile. PLoS Genet. 2013;9(5):e1003493.
141. Pichon C, Felden B. Small RNA gene identification and
mRNA target predictions in bacteria. Bioinforma Oxf Engl.
2008;24(24):2807-2813.
142. Wilderman PJ, Sowa NA, FitzGerald DJ, et al. Identification of
tandem duplicate regulatory small RNAs in Pseudomonas aeru-
ginosa involved in iron homeostasis. Proc Natl Acad Sci U S A.
2004;101(26):9792-9797.
143. Bardill JP, Hammer BK. Non-coding sRNAs regulate virulence in the
bacterial pathogen Vibrio cholerae. RNA Biol. 2012;9(4):392-401.
144. Waters JL, Salyers AA. The small RNA RteR inhibits transfer of
the bacteroides conjugative transposon CTnDOT. J Bacteriol.
2012;194(19):5228-5236.
145. Lee H-R, Rhyu I-C, Kim H-D, et al. In-vivo-induced antigenic deter-
minants of Fusobacterium nucleatum subsp. nucleatum. Mol Oral
Microbiol. 2011;26(2):164-172.
146. Mitchell HL, Dashper SG, Catmull DV, et al. Treponema denti-
cola biofilm-induced expression of a bacteriophage, toxin-an-
titoxin systems and transposases. Microbiology. 2010;156(Pt
3):774-788.
147. Phillips PL, Progulske-Fox A, Grieshaber NA. Expression of
Porphyromonas gingivalis small RNA in response to hemin avail-
ability identified using microarray and RNA-seq Analysis. FEMS
Microbiol Lett. 2014;351(2):202-208.
148. Abusleme L, Dupuy AK, Dutzan N, et al. The subgingival microbi-
ome in health and periodontitis and its relationship with commu-
nity biomass and inflammation. ISME J. 2013;7(5):1016-1025.
149. Hajishengallis G. Periodontitis: from microbial immune subversion
to systemic inflammation. Nat Rev Immunol. 2015;15(1):30-44.
150. Chow J, Tang H, Mazmanian SK. Pathobionts of the gastrointes-
tinal microbiota and inflammatory disease. Curr Opin Immunol.
2011;23(4):473-480.
151. Dabdoub SM, Ganesan SM, Kumar PS. Comparative metagenom-
ics reveals taxonomically idiosyncratic yet functionally congruent
communities in periodontitis. Sci Rep. 2016;6:38993.
152. Naik S, Bouladoux N, Wilhelm C, et al. Compartmentalized
control of skin immunity by resident commensals. Science.
2012;337(6098):1115-1119.
153. Dalal SR, Chang EB. The microbial basis of inflammatory bowel dis-
eases. J Clin Invest. 2014;124(10):4190-4196.
154. PubMed entry. http://www.ncbi.nlm.nih.gov/pubme​d/11240860.
Accessed May 7, 2013.
155. Nairz M, Schroll A, Sonnweber T, Weiss G. The struggle for
iron - a metal at the host-pathogen interface. Cell Microbiol.
2010;12(12):1691-1702.
156. Fives-Taylor PM, Meyer DH, Mintz KP, Brissette C. Virulence
factors of Actinobacillus actinomycetemcomitans. Periodontol.
2000;1999(20):136-167.
157. Jain S, Darveau RP. Contribution of Porphyromonas gingi-
valis lipopolysaccharide to periodontitis. Periodontol 2000.
2010;54(1):53-70.
158. Hiroi M, Shimojima T, Kashimata M, et al. Inhibition by
Porphyromonas gingivalis LPS of apoptosis induction in human
peripheral blood polymorphonuclear leukocytes. Anticancer Res.
1998;18(5A):3475-3479.
159. Preshaw PM, Lauffart B, Zak E, Jeffcoat MK, Barton I, Heasman
PA. Progression and treatment of chronic adult periodontitis. J
Periodontol. 1999;70(10):1209-1220.
160. Murray DA, Wilton JMA. Lipopolysaccharide from the periodon-
tal pathogen Porphyromonas gingivalis prevents apoptosis of
HL60-derived neutrophils in vitro. Infect Immun. 2003;71(12):
7232-7235.
161. Handal T, Olsen I, Walker CB, Caugant DA. Beta-lactamase
production and antimicrobial susceptibility of subgingival bac-
teria from refractory periodontitis. Oral Microbiol Immunol.
2004;19(5):303-308.
162. van Winkelhoff AJ, Winkel EG, Barendregt D, Dellemijn-Kippuw
N, Stijne A, van der Velden U. beta-Lactamase producing bacteria
in adult periodontitis. J Clin Periodontol. 1997;24(8):538-543.
163. Cossart P, Pizarro-Cerdá J, Lecuit M. Invasion of mammalian cells
by Listeria monocytogenes: functional mimicry to subvert cellular
functions. Trends Cell Biol. 2003;13(1):23-31.
164. Lecuit M, Ohayon H, Braun L, Mengaud J, Cossart P. Internalin
of Listeria monocytogenes with an intact leucine-rich repeat
region is sufficient to promote internalization. Infect Immun.
1997;65(12):5309-5319.
165. Wang C-H, Lin C-Y, Luo Y-H, et al. Effects of oligopeptide
permease in group a streptococcal infection. Infect Immun.
2005;73(5):2881-2890.
166. Berezow AB, Darveau RP. Microbial shift and periodontitis.
Periodontol 2000. 2011;55(1):36-47.
167. Relman DA. The human microbiome: ecosystem resilience and
health. Nutr Rev. 2012;70(Suppl 1):S2-9.
168. Uitto VJ, Suomalainen K, Sorsa T. Salivary collagenase. Origin,
characteristics and relationship to periodontal health. J Periodontal
Res. 1990;25(3):135-142.
169. Yost S, Duran-Pinedo AE. The contribution of Tannerella forsythia
dipeptidyl aminopeptidase IV in the breakdown of collagen. Mol
Oral Microbiol. 2018;33(6):407-419.
DURAN-PINEDO
170. Moscoso JA, Schramke H, Zhang Y, et al. Binding of Cyclic Di-AMP
to the Staphylococcus aureus Sensor Kinase KdpD occurs via the
universal stress protein domain and downregulates the expression
of the Kdp potassium transporter. J Bacteriol. 2016;198(1):98-110.
171. Su J, Gong H, Lai J, Main A, Lu S. The potassium transporter Trk
and external potassium modulate salmonella enterica protein se-
cretion and virulence. Infect Immun. 2009;77(2):667-675.
172. Tenenbaum H, Jehl F, Gallion C, Dahan M. Amoxicillin and
clavulanic acid concentrations in gingival crevicular fluid. J Clin
Periodontol. 1997;24(11):804-807.
173. Yost S, Duran-Pinedo AE, Krishnan K, Frias-Lopez J. Potassium is
a key signal in host-microbiome dysbiosis in periodontitis. PLOS
Pathog. 2017;13(6):e1006457.
174. Huynh HTT, Nkamga VD, Drancourt M, Aboudharam G.
Genetic variants of dental plaque Methanobrevibacter ora-
lis. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol.
2015;34(6):1097-1101.
175. Huynh HTT, Pignoly M, Drancourt M, Aboudharam G. A new
methanogen “Methanobrevibacter massiliense” isolated in a case
of severe periodontitis. BMC Res Notes. 2017;10(1):657.
176. Guo L, Hu W, He X, Lux R, McLean J, Shi W. investigating acid pro-
duction by Streptococcus mutans with a surface-displayed pH-sen-
sitive green fluorescent protein. PLoS One. 2013;8(2):e57182.
177. Zeng L, Burne RA. Sucrose- and fructose-specific effects on the
transcriptome of Streptococcus mutans, as determined by RNA
sequencing. Appl Environ Microbiol. 2016;82(1):146-156.
178. Marsh PD. Microbial ecology of dental plaque and its significance
in health and disease. Adv Dent Res. 1994;8(2):263-271.
179. Simón-Soro A, Tomás I, Cabrera-Rubio R, Catalan MD, Nyvad
B, Mira A. Microbial geography of the oral cavity. J Dent Res.
2013;92(7):616-621.
180. Simón-Soro A, Belda-Ferre P, Cabrera-Rubio R, Alcaraz LD, Mira
A. A tissue-dependent hypothesis of dental caries. Caries Res.
2013;47(6):591-600.
181. Gross EL, Leys EJ, Gasparovich SR, et al. Bacterial 16S sequence
analysis of severe caries in young permanent teeth. J Clin Microbiol.
2010;48(11):4121-4128.
182. Johansson I, Witkowska E, Kaveh B, Lif Holgerson P, Tanner ACR.
The microbiome in populations with a low and high prevalence of
caries. J Dent Res. 2016;95(1):80-86.
183. Simón-Soro A, Mira A. Solving the etiology of dental caries. Trends
Microbiol. 2015;23(2):76-82.
184. Nyvad B, Machiulskiene V, Baelum V. Reliability of a new caries di-
agnostic system differentiating between active and inactive caries
lesions. Caries Res. 1999;33(4):252-260.
185. Peterson SN, Snesrud E, Liu J, et al. The dental plaque microbiome
in health and disease. PLoS One. 2013;8(3):e58487.
|
      45
186. Burne RA, Marquis RE. Biofilm acid/base physiology and gene ex-
pression in oral bacteria. Methods Enzymol. 2001;337:403-415.
187. Edlund A, Yang Y, Yooseph S, et al. Meta-omics uncover temporal
regulation of pathways across oral microbiome genera during in
vitro sugar metabolism. ISME J. 2015;9(12):2605-2619.
188. Tanner ACR, Kent RL, Holgerson PL, et al. Microbiota of se-
vere early childhood caries before and after therapy. J Dent Res.
2011;90(11):1298-1305.
189. Jakubovics NS. Intermicrobial interactions as a driver for com-
munity composition and stratification of oral biofilms. J Mol Biol.
2015;427(23):3662-3675.
190. Kressirer CA, Chen T, Lake Harriman K, et al. Functional pro-
files of coronal and dentin caries in children. J Oral Microbiol.
2018;10(1):1495976.
191. Tanner ACR, Mathney JMJ, Kent RL, et al. Cultivable anaero-
bic microbiota of severe early childhood caries. J Clin Microbiol.
2011;49(4):1464-1474.
192. Tanner ACR, Kressirer CA, Rothmiller S, Johansson I, Chalmers NI.
The caries microbiome: implications for reversing dysbiosis. Adv
Dent Res. 2018;29(1):78-85.
193. Peterson SN, Meissner T, Su AI, et al. Functional expression of
dental plaque microbiota. Front Cell Infect Microbiol. 2014;4:108.
194. May A, Brandt BW, El-Kebir M, et al. metaModules identifies
key functional subnetworks in microbiome-related disease.
Bioinformatics. 2016;32(11):1678-1685. https://doi.org/10.1093/
bioinformatics/btv526. Epub 2015 Sep 5.PMID: 26342232
195. Kilian M, Poulsen K, Blomqvist T, et al. Evolution of Streptococcus
pneumoniae and its close commensal relatives. PLoS One.
2008;3(7):e2683.
196. Loo CY, Mitrakul K, Voss IB, Hughes CV, Ganeshkumar N.
Involvement of an inducible fructose phosphotransferase op-
eron in Streptococcus gordonii biofilm formation. J Bacteriol.
2003;185(21):6241-6254.
197. Muto M, Hitomi Y, Ohtsu A, Ebihara S, Yoshida S, Esumi H.
Association of aldehyde dehydrogenase 2 gene polymorphism
with multiple oesophageal dysplasia in head and neck cancer pa-
tients. Gut. 2000;47(2):256-261.
How to cite this article: Duran-Pinedo AE. Metatranscriptomic
analyses of the oral microbiome. Periodontol 2000.
2021;85:28–45. https://doi.org/10.1111/prd.12350