DR KAUSAR SADIA FAKHRUDDIN (Orcid ID : 0000-0003-0135-6597)

Accepted Article
Article type : Review Article

Title:

Cariogenic microbiome and microbiota of the early primary dentition:

a contemporary overview
Running Title:

Cariogenic microbiome in primary dentition

Keywords:

Primary dentition; microbiome; microbiota; early childhood caries; Next-Generation Sequencing

(NGS)

Authors:

Kausar Sadia Fakhruddin1

Hien Chi Ngo1

Lakshman Perera Samaranayake1, 2

1Department of Preventive and Restorative Dentistry, University of Sharjah, UAE

2 The University of Hong Kong, Hong Kong Special Administrative Region, China

Corresponding author:

Professor Lakshman Samaranayake

M28-125, College of Dental Medicine,

University of Sharjah, 27272

United Arab Emirates

Email: lsamaranayake@sharjah.ac.ae

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/odi.12932
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 Telephone: 971-06 5057317

Fax: 971 -06 558 5641

Accepted Article
Abstract

Recent advances in the field of molecular microbiology provide an unprecedented

opportunity to decipher the vast diversity of the oral microbiome in health and disease. Here,
we provide a contemporary overview of the oral microbiome and the microbiota of Early
Childhood Caries (ECC) with particular reference to newer analytical techniques.

A MEDLINE search revealed a total of 20 metagenomic studies describing cariogenic-

microbiomes of ECC, ten of which also detailed the healthy microbiomes. Additionally,
seven studies on site-specific-microbiomes, focusing on acidogenic and aciduric-microbiota
of deep-dentinal lesions were also reviewed. These studies evaluated plaque and saliva of
children aged-1.5 - 11-years, in cohorts of 12 to 485 individuals.

These studies reveal a very rich and diverse microbial communities, with hundreds of
different phylotypes and microbial-species, including novel species and phyla such as
Scardovia wiggsiae, Slackia exigua, Granulicatella elegans, Firmicutes in the plaque
biofilms of children with ECC. On the contrary, bacteria such as Streptococcus cristatus, S.
gordonii, S. sanguinis, Corynebacterium matruchotii, and Neisseria flavescens, were
common in plaque biofilm of non-carious, healthy, tooth surfaces in subjects with caries.

The review illustrates the immense complexity and the diversity of the human oral-
microbiota of the cariogenic plaque microbiome in ECC, and the daunting prospect of its
demystification.

Introduction

The oral microbiome comprises a vast assortment of cohabiting plaque microbiota of more
than 700 phylotypes of which approximately one half can be present in any individual at any
one time (Kuramitsu et al., 2007, He & Shi, 2009, Palmer, 2014). The supragingival-plaque
biofilm, in particular, comprises a sub-micro-ecosystem of this polymicrobial community

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 exhibiting a variety of patho-physiological characteristics (Takahashi & Nyvad, 2008,
Takahashi & Nyvad, 2011).
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In health, the oral microbiome has a symbiotic or a eubiotic relationship with the host but
when a disease such as dental caries supervenes, this balance is perturbed and a dysbiotic
relationship ensues. The dysbiosis is essentially due to the proliferation or overgrowth of
cariogenic microbes such as mutans-group streptococci, which metabolizes dietary sugars,
leading to a low pH eco-niche, demineralization of tooth structure and dental caries (Marsh
et al., 2008). Conversely, some biofilm microbes through their arginine-deiminase system can
also produce alkali, resulting in a neutralizing micro-niche and a raised pH leading to
remineralization (Takahashi & Nyvad, 2011, Burne & Marquis, 2000). The dampening of the
demineralization process which retards the velocity of cariogenesis is one of the core
regulators of dental caries activity (Kleinberg, 2002). In essence, dental caries is an
endogenous infection caused by overgrowth of specific groups of `cariogenic` normal
commensals due to an ecological imbalance (Seneviratne et al., 2011, Pereira et al., 2017,
Marsh, 2006).

Beyond Streptococcus mutans

There is a voluminous literature on the link between S. mutans and dental caries (Klein et al.,
2015, Aas et al., 2005, Aas et al., 2008). It is widely accepted that the cariogencity of
S.mutans is due to its superior acidogenic and aciduric potential. The ability of mutans-group
streptococci to produce water-insoluble glucan and surface antigens I/II proteins helps their
persistence in saliva as well as in the extracellular matrix of biofilms, in particular. Moreover,
antigen I/II proteins are associated with tubular invasion and binding to Collagen type-I of
dentine, which is a crucial factor for invasion of dentinal tubules during the caries process
(Petersen, 2002, Love, 1997).

As acidogenicity is an essential pre-requisite to be classsified as a cariogenic organism, there

has been a search for similar candidate oral microbiota, and recently spotlight has fallen on
the opportunistic eukaryote, Candida species, that are frequenty isolated from caries lesions
both in children and adults (Beighton et al., 2004, Xiao et al., 2016). Candida species are
prevalent in 40-60 per cent of the human oral cavities (Samaranayake and MacFarlane, 1990)
and available data tend to imply this opportunist yeast to be a strong contender plausibly

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 as a secondary perpetrator of the caries process (Pereira et al., 2017). Alike mutans
streptococci (MS), Candida are aciduric and acidogenic, mainly producing short chain
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carboxylic acids such as acetic and, pyrvic acid in such milieus (Samaranayake et al., 1982).
Other virulence attributes of this yeast including their adhesion potential to abiotic surfaces
such as hydroxyapatite and its crystals (Hemadi et al., 2017), and prolific biofilm forming
potential (Samaranayake & Ellepola, 2000), and the extensive array of proteinases which
facilitates collagenolysis and dentinal tubular invasions makes this proposition eminently
viable (Pereira et al., 2017, Xiao et al., 2018).

Lactobacilli are the third group of organisms often isolated, particularly from the advancing
front of the carious lesions. Indeed, lactobacilli were considered as prime movers of the caries
process in 1960`s after which this notion fell into disrepute when mutans streptococci (MS)
came to the fore. The current understanding is that mutans streptococci (MS) play a crucial
role of paving an initial pathway and cavitation necessary for secondary colonization by
aciduric organisms such as lactobacilli and Candida (Caufield et al., 2015, Badet et al.,
2004). Nevertheless, this notion remains a hypothesis as convincing data are not yet available
with proper longitudinal studies tracking the microbiota in early and late caries.

On the contrary, others have questioned the role of mutans streptococci (MS) in caries, as
10-20 per cent of subjects with caries do not have detectable levels of mutans streptococci
(MS) (Aas et al., 2008) and converesly, significant numbers of mutans streptococci (MS)
have been isolated from the tooth surface biofilms devoid of caries (Innes & Robertson,
2018, Philip et al., 2018). Interestingly, novel molecular methods for bacterial identification
and enumeration used in recent studies indicate a diverse microflora comprising novel
phenotypes that are associated with caries in both adults and children (Aas et al., 2008,
Becker et al., 2002). This implies that the microbiota of caries is complex and hitherto
unknown organisms play a decisive role in the pathologic process. What follows is an
attempt to contextualise these new findings with reference to the traditional cariogenic
microbiome, focusing on childhood caries in particular. Initally, we provide a thumb sketch
of the recent advances in microbial identifications systems that has paved the way for the
demystification of the oral microbiome.

Recent technical advances in identification of oral microbiota

As mentioned, understanding the etiology and pathogenesis of dental caries is a challenging

prospect due to its multifaceted microbiome. Moreover, due to the multifactorial nature of the

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 disease it is difficult to find a causal relationship at individual levels. The traditional methods
of cultivating plaque biofilms from carious lesions using laboratory cultures of selective
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media, and identification of the resultant harvest through either phenotypic or first generation
genotypic analyses are falling into disrepute due to the advent of more efficient and effective
ribosomal RNA (rRNA) gene sequencing and next-generation sequencing (NGS) technology.
For instance, traditional cloning and sequencing techniques can detect but only the
predominant members of the microbial community within a complex microbial habitat (Ling
et al., 2010). Subsequent development of broad-range-PCR assays and reverse-capture
checkerboard hybridization methods that used a limited number of primers were a marginal
improvement and provided only a relatively broad-brush view of the composition of the
plaque biofilm microbiota.

A comparatively robust understanding of the complex bacterial diversity in varying

microhabitats is now possible through `big data` generated through NGS technology (Tanner
et al., 2011, Xu et al., 2014). 16S rRNA gene, present in all bacteria, is a highly conserved
component of the transcriptional machinery. 16S rRNA gene sequencing enables accurate
identification, classification and quantification of microbes in complex biological samples
such as polymicrobial plaque biofilms with hundreds of different phylotypes and microbial
species. Currently, various equipment for NGS, with differing technologies is available, that
work principally on four main DNA sequencing methods. These include i) pyrosequencing,
ii) sequence by synthesis, iii) sequence by ligation and iv) Ion semiconductor sequencing
(Mardis, 2008, Buermans & den Dunnen, 2014). The latter DNA sequencing method, based
on the detection of hydrogen ions that are released during the polymerization of DNA, is
gaining favor amongst microbiologists due to its rapid sequencing speed, low operating costs
and scalable nature with the ability to evaluate microbiota and the metagenome of many
samples within hours (Libertini et al., 2016). Currently available data on early childhood
caries (ECC) from NGS and comparative technology are reviewed in some detail below.

Oral microbiota in childhood in health and disease: Microbiomes associated with caries
and health in primary dentition

There are now a number of studies in the literature that utilizes novel analytical tools
described above to evaluate the oral microbiome in childhood (Tables 1 and 2). At the time of
writing a MEDLINE search revealed a total of 20 -studies on the microbiomes related to
ECC, of which ten manuscripts describing the healthy microbiome, and further seven
describing microbiomes of deep-dentinal lesions. The age range of children evaluated in
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 these studies ranged from 1.5 to 11- years with cohort size ranging from 12 to 485
individuals.
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The first of such contemporaneous study was by Kansai et al., (2010) who used 16sRNA
gene cloning and sequencing to evaluate ` cariogenic` plaque biofilm of 80 children that were
2-6 years old. They described 139 taxa and significant increased levels of S. mutans,
Veillonella sp., Bifidobacterium species and Granulicatella elgans in the microbiome of the
caries-affected group. Ling et al., (2010) subsequently compared phylotypes of oral
microbiome in saliva as well as plaque biofilms, based on nucleic acid sequences. Using
high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis
(DGGE), they examined bacterial diversity in pre-school children with and without caries.
PCR-DGGE can detect changes of predominant microbiota in specific microhabitats while
pyrosequencing provides a high-throughput, high-depth approach to analyze 16S rRNA gene
sequences. The latter technique facilitates the exploration of bacterial diversity in different
microhabitats and permits the detection of minor variations in microbial populations. Study
by Ling and co-workers reported of a very rich microbial communities with over 200
genera belonging to 10 phyla, in saliva and plaque biofilm samples of children with dental
caries.

Luo et al., (2012) observed an abundance of Bacteroidetes species, Capnocytophaga

sputigena, Tannerella, Campylobacter showae, Selenomonas and Parvimonas in a caries-
active (DMFS⦣8) group of children in their mixed dentition. However, Ma et al., (2015)
could not verify these findings in saliva and plaque biofilm samples of pre-school children,
independent of their caries-or caries-free status (Luo et al., 2012, Ma et al., 2015).

Following Ling’s study, Xu et al., in 2014, analyzed supragingival plaque microbial diversity
in children less than 30-months of age. Using 16S rRNA genes, subjected to 454-
pyrosequencing, they compared the microbial profile of plaque samples from children with
severe ECC and without. They reported, using metagenomics analyses, seven-novel bacterial-
phylotypes from supragingival plaque biofilm of children less than three-years of age. The
new phylotypes were Schlegelella thermodepolymerans, Actinomyces timonensis, Ottowia
thiooxydans, Selenomonas bovis, Streptobacillus moniliformis, Eubacterium saburreum, and
Dechloromonas agitate. Interestingly, these strains were neither listed in the Human Oral
Microbiome databases (HOMD) nor in phylogenetically curated 16S rDNA database of the
Core Human Oral Microbiome (CORE), at the time (Chen et al., 2010, Griffen et al., 2011) .

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 The most extensive series of recent studies on childhood caries using NGS technology has
been conducted by Jiang and co-workers in (2013 and 2014). They evaluated, employing a
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pyrosequencing method based on 16S rRNA and a NGS Illumina platform, the microbial
profile and dynamic characteristics of cariogenic bacteria during disease progression. By
analyzing plaque biofilm and saliva samples of 50, pre-school children with caries and a
control cohort of 50 children without caries they reported that the oral microbiomes tend to
be stage-specific during the progress of a carious lesion (Jiang et al., 2014, Jiang et al., 2013).
One year later, Teng et al., (2015) studied changes of microbiota in plaque biofilm and saliva
samples of careis –free, healthy preschoolers, over a period of two-years. Presenting a
predictive model of ECC, they documented a temporal transition of the pre-cariogenic
microbiome to a complex community structure, associated with caries progression in caries-
active children over the period. They also observed, through a pyrosequencing analytical
platform, an abundance of Prevotella spp.in both saliva and plaque samples of children with
ECC, in addition to 20 further caries-related taxa which included S. mutans, Veillonella
dispar, V. atypical, V. parvula (Teng et al., 2015). Subsequently Jiang et al., (2016) also
studied microbial profile of cariogenic bacteria from n=40, saliva samples of pre-school
children aged-3-4-years with and without severe ECC, and noted 151 genera belonging to 17
phyla and 310 bacterial species in the saliva sample (Jiang et al., 2016).

In an interesting, recent metagenomic study of 5-11-years-old mono and dizygotic twins,

Gomez et al., (2017) reported similar composition of the oral microbiome in the cohorts
studied despite the host genotype. They concluded that the phenotype and the biotype of the
cariogenic microbiota are unlikely to be influenced by host genetics (Gomez et al., 2017).

Oral Microbiota in childhood in health and disease: Microbiota of deep-dentinal lesions

of primary dentition:

Ecological plaque theory suggests decreasing microbial diversity in progressive carious

lesions. Dental plaque biofilm is challenged by frequent changes in environmental conditions
such as food intake, temperature, pH, salivary flow (Marsh, 1994, Marsh, 2006, Takahashi &
Nyvad, 2008, Takahashi & Nyvad, 2011). Constant availability of fermentable carbohydrate
and prolonged acidic conditions (pH≤5) within cavities due to non-clearance of acids and
death of acid-sensitive microbiomes results in a predominance of aciduric species.
Consequently, it has been argued that the microbial diversity is relatively lower in deep-

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 dentinal lesions compared with superficial lesions. Furthermore, the dominant aciduric-
microbes in the plaque-microbial community of cavitated carious lesions promote further
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lesion progression (Figure 1). The so called extended ecological plaque hypothesis and the
predominance of aciduric-microbes in deep-dentinal lesions are amply corroborated in recent
studies using 16S rRNA gene analysis, (Becker et al., 2002, Corby et al., 2005)

With 16S rRNA gene sequencing, Aas et al., (2008), examined changes in bacterial profiles
associated with different stages of carious lesions and between primary and permanent
dentition. They observed changes in microbial profile as the lesion progresses. Their team
found a clear dominance of potential acid-producing species other than S. mutans involved in
deep-dentinal lesions. Aciduric species was characterized by Lactobacillus spp.,
Propionibacterium sp. strain FMA5, and Bifidobacterium in primary teeth and Atopobium
genomospecies C1, in permanent teeth.The most recent observation of microbiomes of
dentine carious lesions using NGS approach, also reported site specific dominance of
acidogenic and aciduric species. Richards et al., (2017) for instance, found Scardovia
wiggsiae, Lactobacillus salivarius, Streptococcus mutans and Parascardovia denticolens
species which are all acidogenic in caries -active dentine lesions of children aged 2-7-years
old. The foregoing clearly suggests substantial diversity and differentiation in microbial
composition between specific sites, and stages of caries progression, in addition to inter-
individual, intrinsic variations in microbiota (Xu et al., 2014).

Microbiota of ECC: Insights into novel species and phylotypes

There is a voluminous literature on the traditionally recognized microbiota such as

Streptococcus sanguis, S. gordonii, S. mitis, and S. oralis isolated from biofilms of healthy
enamel surfaces (Kreth et al., 2009, Facklam, 2002, Wang & Kuramitsu, 2005). However, the
new technology such as NGS has provided a whole new panorama of phylotypes associated
with both healthy and affected enamel surfaces in childhood caries. A picture appears to be
now emerging of the key discernible phylotypes in health and disease, and these are reviewed
next. As will be seen, it is clear that a single universal model for different types of caries
such as surface vs. deep, enamel vs. dentine, and initial vs. cavitated lesions may not be
viable proposition given the new findings in the literature.

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 Scardovia species: In 2011, Tanner et al., reported of an hitherto unknown species,
Scardovia wiggsiae in the plaque biofilm samples of children with S-ECC. Even in the
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absence of S. mutans detection, Tanner and team observed an association of this novel
phylotype with early-white-spot and advanced dentine-carious lesions. Interpreting this
intriguing observation, they hinted at the possibility that the newly recognized pathogen
may be independently associated with caries or involved at an advanced stage of lesions
where S. mutans is not the predominant species (Gross et al., 2010). Confirmatory data are
now available from recent NGS studies that Scardovia wiggsiae may indeed be highly
prevalent in caries -active dentinal lesions of children (Richards et al., 2017). This species is
acidogenic and have acid-tolerant potential at pH 5 (Downes et al., 2011, Tanner et al., 2011)
yet, further studies examining their cariogenic attributes are required and longitudinal clinical
trials can also help to determine whether S. wiggsiae is a sentinel organism of advanced
caries lesion and a risk indicator for caries.

Slackia species: Tanner et al., (2011) also observed a strong association between a novel
species, Slackia exigua, with severe early childhood caries. They also reported a positive
relationship between S. exigua, a fastidious anaerobic-Gram-positive rod and S. wiggsiae in
the eco-niche of advance carious lesions. A very recent study evaluating site-specific
microbiome also confirmed presence of species S. wiggsiae, exclusively in dentinal carious
lesions (Richards et al., 2017).

Firmicutes species: Agnello et al., (2017), studying oral-microbiomes in young indigenous

children of North America, observed an abundance of phyla Firmicutes in the caries-affected
group. Though, their team noticed the multitude of phyla Actinobacteria and Fusobacteria
among children with no-caries from the indigenous population. This corroborates the finding
of Gross et al., (2012) longitudinal study, which found a decrease in Actinobacteria, and an
increase of Firmicutes as the lesion progresses. Likewise, Jiang et al., (2016), have also
reported a significantly higher abundance of Firmicutes in children affected with caries
corroborating the observations of Agnello et al., (2017).

Granulicatella species: On clonal analysis of the oral microbiome Kanasi et al., (2010),
reported a higher mean proportion of Granulicatella elegans, in plaque biofilm sample of
children with S-ECC. A recent report by Agnello and team (2017) also confirmed significant
abundance of these phyla in their study group of children with S-ECC. It is noteworthy that
G. elegans was previously classified as a nutritionally variant Streptococcus, and it has been

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 reclassified as a Granulicatella species (Samaranayake 2018). The clinical significance of
this species is yet to be established.
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Bifidobacterium species: This organism, a known lactate producer, has been significantly
associated with caries in both children as well as adults (Becker et al., 2002, Aas et al., 2008,
Ventura et al., 2009). Molecular analysis by Becker and team observed Bifidobacterium as
the most numerous species after Actinomyces gerencseriae in cavitated and deep-dentinal
lesions, suggesting their role in caries progression. According to Becker et al., (2002) their
numbers far exceed S. mutans and even surpassed Lactobacillus fermentum in deep caries
lesions level ranges from 5x107 to 4x109 cell number.

Corynebacterium species: Several studies assessing health-related bacterial species in

children with no caries, have noted numerous species that are `protective` against caries onset
and progression. Such a key emerging `eco-friendly` microbe is C. matruchotii (Gross et al.,
2012, Kanasi et al., 2010a, Becker et al., 2002, Xu et al., 2014). These are Gram positive
bacilli with long filaments and short, thick terminal ends. Within the plaque biofilm they
provide a central core axis and a structural skeleton for other bacteria such as streptococci to
aggregate leading to the formation of polymicrobial complexes, traditionally known as the
"corn-cob” arrangement (Samaranayake, 2018). Becker et al., (2002), for instance, found
high levels of C. matruchotii in plaque biofilm samples from intact enamel. C. matruchotii
also present in supra-gingival plaque, can help raise biofilm-pH, by utilizing lactate produced
by other co-habiting plaque microbes (Gross et al., 2012, Mark Welch et al., 2016).

Streptococcus cristatus: Several studies also reported the presence of S. cristatus in plaque
biofilm from healthy subjects and on intact enamel in subjects with caries. S. cristatus and
another species, S. gordonii are highly arginolytic species. Through their arginine-deiminase-
system (ADS) they produce ammonia, thus raising plaque-pH. This may ultimately contribute
to retarding the initiation and progression of caries. (Corby et al., 2007, Kanasi et al., 2010b,
Aas et al., 2005).

Streptococcus sanguinis and Streptococcus gordonii: These two species when grown under
aerobic conditions can inhibit S. mutans. Under these conditions they produce hydrogen
peroxide (H2O2), thus inhibiting biofilm formation and bacteriocin production by S. mutans.
These inhibitory effects on the competing species of is thought to keep the S. mutans
numbers at bay (Kreth et al., 2008).

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 Neisseria species: Additionally, several studies employing advanced molecular methods
noted association of Neisseria flavescens-an asaccharolytic, N. mucosa and N. pharynges
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with health (Gross et al., 2012, Crielaard et al., 2011). It has been traditionally known that
representative organisms of the Genus Neisseiraceae are commonly isolated from plaque and
saliva samples and the emerging data points to their importance in maintaining the
homeostasis of the healthy plaque microbiome.

Some unanswered questions and insight for future research

As noted the plaque microbiome is an immensely complex microbial system which

maintains its exquisite homeostasis with multifarious interactions amongst the resident
microbes, most of which remains to be deciphered (Kuramitsu et al., 2007, Kaidonis &
Townsend, 2016, Marsh, 1994, Marsh, 2006). Thus far, very few investigators have ventured
to study in vitro, dental plaque from an ecological perspective. Most of them have evaluated
only mono- or dual-species or at most quadruple microbial consortia, essentially using the
artificial mouth model systems (Tang et al., 2003). This is not surprising as there are many
issues that constrain these studies such as the maintenance of pH, Eh and temperature within
in vitro systems leaving apart the differential growth rates of the cohabiting microbes that
may overwhelm the other organisms in the polymicrobial habitat.

Another area of study that is direly wanting of information is the quorum sensing studies of
the caries microbiota. Quorum sensing is a chemical messenger system that is universally
employed by microbiota to coordinate biofilm metabolism, progeny and populations
(Bandara et al., 2012). Quorum-sensing- compounds also have a major impact on the
expression of bacterial phenotypes of the community. Once the quorum sensing molecules of
a given habitat are known these may be exploited as targets for drug design which can be
promising in preventive and therapeutic aspects of dental caries.

To date, very few molecular studies employing longitudinal or pre-post study designs have
investigated dynamics of microbial community profile during the initial demineralization and
progression and demineralization of caries in primary dentition. Further, longitudinal
monitoring can help not only to identify but also to understand the impact of cariogenic
microbes on recurrent or new carious lesions particularly after management interventions.

Almost all studies using advanced gene-sequencing platfors have assessed oral-microbial
profiles related to childhood caries using pooled-plaque samples. (Xu et al., 2014, Tanner et

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 al., 2011, Gross et al., 2012, Agnello et al., 2017, Jiang et al., 2013). Due to the documented
variation of intra-individual, site and surface-specific microbes in the oral cavity, pooled
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plaque sample may not be a justifiable surrogate for identifying these specific microbiomes.
Hence, unresolved complexity exists, regarding microbes associated with particular site or
surfaces in the oral cavity.

Finally, phylotypes identified in saliva and supra-gingival plaque biofilm are not comparable
(Ling et al., 2010). Given that, use of saliva as a proxy for microbial composition at different
tooth sites and surfaces may not provide valid tie-in between microbial composition and
disease stages, crucial for etiological studies.

NGS technology now offers an unprecedented window of opportunity to evaluate and

comprehend the vast diversity of the caries-microbiome in vivo. It is hoped that once such an
in-depth understanding of the microbial profile and physiological characteristics of caries-
associated and caries-deterrent microbiota are discerned then newer targets for disease
intervention could be further explored in future.

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Accepted Article Legend for Figure

Figure1. Schematic diagram of the dynamics of dental plaque-microbiome during ECC

Funding:

This review was not supported by any specific funding agency.

Conflict of Interest:

None.

Author’s contribution:

K.S.F. was involved in manuscript’s conception and drafted the tentative manuscript. L.P.S.
critically examined and revised the manuscript and gave approval of the final version to be
published. H.C.G. critically examined the manuscript and gave final approval of the version
to be published.

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 Table 1. Contemporary molecular data on microbiota of caries and healthy plaque biofilm in
primary dentition (from 2010 -2017)
Accepted Article
Study Sample Type of Method of No. of Microbiomes- caries Microbiomes-
Year size sample analysis taxa/phyla affected group caries-free
Age acc.to HOMD group
(years)
Kanasi et N=80 Plaque 16S rRNA gene 139 taxa Granulicatella elegans Capnocytophaga
al., (2010) 2-6-yrs- biofilm cloning and (p≤0.01) gingivalis (p≤0.01)
old sequencing Veillonella sp. HOT-780 Abiotrophia
(p≤0.01) defectiva (p≤0.01)
Lachnospiraceae
PCR amplification sp. HOT-100
for S. mutans and S. mutans (p≤0.005) (p≤0.05)
Bifidobacteriaceae Bifidobacteriaceae spp. Streptococcus
species (p≤0.001) sanguinis (p≤0.05)
Streptococcus
cristatus (p≤0.05)
Ling et al., N=60 Unstimulated High-throughput 200 genera of The phylotypes of saliva and
(2010) 3-6-yrs- saliva and barcoded ten phyla. plaque were significantly
old plaque pyrosequencing different. Following genera
biofilm and PCR-DGGE were significantly associated
(denaturing with caries:
gradient gel Streptococcus
electrophoresis- Leptotrichia
DGGE) for Veillonella
bacterial diversity Actinomyces
Granulicatella
Thiomonas

Tanner et N=82 Plaque Partial sequences 198 HOMD taxa; Streptococcus mutans
al., (2011) 2-6-yrs- biofilm for 16S rRNA 29 extended Scardovia wiggsiae
old gene. Isolate HOMD taxa; 45 Veillonella parvula
sequence potential novel Streptococcus cristatus
compared with group Scardovia wiggsiae
taxon sequences Actinomyces gerensceriae
in HOMD (significantly associated
with severe caries)

Tanner et N=85 Plaque 16S rRNA-based Slackia exigua (p≤0.002)

al., (2011) 2-6-yrs- biofilm microarray Streptococcus
old parasanguinis (p≤0.013)
Children Prevotella species (p≤0.02)
monitored
for 12- PCR amplification Bifidobacteriaceae species
months for S. mutans, (p≤0.001)
post Bifidobacteriaceae Scardovia wiggsiae
therapy specie, and (p≤0.003)
Scardovia S. mutans with S.wiggsiae
wiggsiae (p≤0.001)
S. mutans with
Bifidobacteria (p≤0.001)

Gross et N=72 Plaque 16S rRNA gene 134 species (two S. mutans
al., (2012) 12-36- biofilm sequencing for novel taxa); S. vestibularis
months bacterial S. salivarius
community S. sobrinus
analysis S. parasanguinis
Veillonella

In advanced caries diversity

decreases as following
species were found at lower
level or not at all:
S. mitis; S. infantis; S. oralis;
S. pneumoniae; S. sanguinis
group. N. flava, N. mucosa;
N. pharynges group

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 Luo et al., N=50, 6 to Saliva sample 16S rRNA genes, 94 bacterial Eight-species were detected Six- different
(2012) 8years- sampled were species more frequently in caries species were
old assayed using representing six group detected more
Accepted Article HOMIM bacterial phyla frequently in
microarray and 30 genera caries-free group

Tao et al., N=12 at 8, Dental Total microbial A total of 21 onset of S-ECC is number of bands
(2013) 14, 20, 26 plaque genomic DNA genera were accompanied by a decrease was significantly
and 32 sample PCR-denaturing identified in all in microbial diversity higher in the CF
months of gradient gel subjects group (18.17±4.91
age - electrophoresis bands)
follow-up (DGGE) analyses
Jiang et N=40, 3 to Dental 16S rRNA genes 14 phyla, and 63 Following genera were
al., (2013) 5years old plaque subjected to genera dominant in caries group:
biofilm pyrosequencing Streptococcus, Actinomyces
Granulicatella,

Xu et al., N=19 Plaque DNA amplicons of 8 phyla; 15 Following genera are Following genera
(2014) Children biofilm the V1-V3 classes; 21 predominant in caries are predominant in
prior to hypervariable orders; 30 group: caries-free group:
eruption region of bacterial families; 41 Streptococcus Leptotrichia
of 16S rRNA gene genera; and 99 Veillonella Selenomonas
deciduous subjected to 454- species Fusobacterium
second pyrosequencing Capnocytophaga
molars Porphyromonas

Ma et al., N=40 Saliva and Samples were 379 bacterial Following genera are
(2015) 3-4-yrs- plaque assayed using the species detected strongly associated with
old biofilm Human Oral in both plaque caries:
Microbe and saliva Streptococcus
Identification samples Porphyromonas
Microarray Actinomyces
(HOMIM)
Teng et al., N=50 Dental 16S rRNA genes Streptococcus spp.,
(2015) 4-yrs-old plaque (V1-V3 Prevotella spp.,
children biofilm hypervariable Veillonella spp.,
followed regions) subjected
for 2 years to pyrosequencing
Jiang et N=40 Unstimulated DNA amplicons of 17 phyla; 26 (Dominant genera but no Fusobacterium
al., (2016) 3-4-yrs- saliva V3-V4 classes; 40 statistical difference) periodonticum
old hypervariable orders; 80 Rothia dentocariosa
region of bacterial families; 151 Actinomyces graevenitzii Leptotrichia sp.
16S rRNA gene genera; 310 Veillonella sp.oral taxon Oral clone FP036
subjected to species 780
Illumina Miseq Prevotella salivae
sequencing Streptococcus mutans

Agnello et N=50 Plaque 16S rRNA gene 28-species-level Streptococcus mutans Streptococcus
al., (2017) Less than biofilm sequence taxonomic units (extremely high levels) gordonii (5 fold
72 clustered into significantly Veillonella sp. HOT-780 (4.6 higher)
months operational different fold higher) Streptococcus
taxonomic units between groups Porphyromonas HOT 284 (9 sanguinis (2 fold
via HOMD fold higher) higher)

Table 1 (continued)

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 Table 2. Microbiota of deep-dentinal lesions of caries- affected primary dentition
Accepted Article Study Sample Type of Method of No. of Dentinal Microbiomes-
Year size sample analysis taxa/phyla microbiomes -caries caries-free group
Age acc.to HOMD affected group
(years)
Marchant N=14; 3-5- Carious Culture method Predominant microflora
et al., years-old dentine (infected dentin) at pH 5.2
(2001) from S. oralis (46%)
caries S.mutans (38%)
group S. sanguis (13%)
S. parasanguis (5.7%)
Plaque S. intermedius (7.6%)
sample S. mitis (1.9%)
from S. anginosus (1.9%)
caries-free S. sobrinus (Not detected)
group S. salivarius (1.9%)
S. gordonii (Not detected)
A. israelii (27%)
A. naeslundii (3.8%)
A. odontolyticus (1.9%)
A. gerencseriae (8%)
Lactobacillus spp. (13%)
H. parainfluenzae (1.9%)

*33 genotypes of S.
mutans were isolated
from only one teeth
Molecular method *Up to four genotypes
were recovered of
Repetitive extragenic Lactobacillus spp. From a
palindromic PCR single nursing carious
(REP-PCR) lesion
Becker et N=60; 2-8- Plaque 16S rRNA gene Species from dentin S. sanguinis
al., (2002) years-old biofilm sequence and lesion: Abiotrophia defective
reverse-capture Streptococcus mutans S. mitis or S. oralis
checkerboard Veillonella dispar Neisseria mucosa
hybridization Bifidobacterium sp.cl S. mitis biovar II
CX010
Actinomyces sp.cl AP064
S. mitis or S. oralis
Lactobacillus fermentum
Selenomonas sputigena

Corby et N=204 Plaque 16S rRNA gene Actinomyces sp. strain S. mitis
al., (2005) twins; 1.5- biofilm sequence and B195C S. oralis
7-years- reverse-capture Streptococcus mutans, S. cristatus
old checkerboard Lactobacillus spp. S. sanguinis
hybridization Fusobacterium S. parasanguinis
Cardiobacterium sp. A. C. A. defective
hominis G. hemolysans
Selemonas sp. Clone
EY047
Porphyromonas sp. clone
DS033
Atopobium sp. clone
GW027
Haemophilus
parainfluenzae
Bacteroidetes sp. clone
AU126

Aas et al. N=90; 2- Plaque 16S rRNA gene were 22-new Dentinal lesion Leptotrichia all
(2008) 21-years- biofilm PCR amplified phylotypes; Veillonella all Veillonella all
old 197 species, of Streptococcus mutans S. anginosus
this 50% have not Actinomyces sp. cl. GU067 Actinomyces sp. cl.
been cultivated Streptococcus sanguinis GU067
Streptococcus salivarius* Capnocytophaga
Veillonella sp. cl. BU083 granulosa
Streptococcus mitis Fusobacterium all
Selenomonas sp. cl. S. sanguinis

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 EY047 S. cristatus
Leptotrichia all Capnocytophaga
Actinomyces gerencseriae sputigena
Accepted Article (all, all species of the Campylobacter
genus) gracilis

Deep dentinal lesions (all, all species of the

Streptococcus mutans genus)
Veillonella all
Actinomyces sp. cl. GU067
Lactobacillus all
Streptococcus salivarius
Streptococcus sanguinis
Streptococcus mitis
Veillonella sp. cl. BU083
Actinomyces sp. strain
B19SC
Selenomonas sp. cl.
EY047
(all, all species of the
genus)
Jiang et N=60, 3-7- Dental 16S rRNA genes (V1- Identified 25,444 Following genera were Following health
al., (2014) year-old plaque V3 hypervariable unique dominant in cavitated related genera in
biofilm regions) subjected to phylotypes lesions: caries-free group:
454 pyrosequencing representing 18 Streptococcus Porphyromonas,
phyla and 145 Megasphaera, Olsenella, Abiotrophia,
genera Cryptobacterium, Capnocytophaga,
Lactobacillus, Scardovia, Fusobacterium,
Shuttleworthia, Comamonas,
Cryptobacterium, Tannerella,
Actinobaculum,
Stenotrophomonas,
Eikenella, and
Paludibacter
Richards N=55, 2-7- Dental 16S rRNA genes (V3- Following species were Following health
et al., year-old plaque V4 hypervariable dominant in dentine related species in
(2017) biofilm regions) was carious lesions: Scardovia caries-free group
sequenced by the wiggsiae Streptococcus
Illumina MiSeq Lactobacillus salivarius sanguinis
Parascardovia denticolens
Streptococcus mutans
Gomez et N=485 Dental 16S rRNA gene Enamel: Prevotella .pallens
al., (2017) 5-11-yrs- plaque subjected to Illumina Neisseriaceae Veillonella-oral taxon
old (twins) biofilm Miseq sequencing Prevotella oulorum Streptococcus
clustered into Tannerella S. anginosus
operational Rothia Cardiobacterium
taxonomic units Aggregatibacter Actinomycetaceae
Corynebacterium
Dentin: Rothia.100
Selenomonas.100
Cardiobacterium.99
Table 2 (continued)

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Accepted Article

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