Microorganisms 12 00121 v2

microorganisms
Review
The Evolving Microbiome of Dental Caries
Grace Spatafora 1, * , Yihong Li 2 , Xuesong He 3 , Annie Cowan 4 and Anne C. R. Tanner 3, *
1 Biology and Program in Molecular Biology and Biochemistry, Middlebury College,

Middlebury, VT 05753, USA
2 Department of Public and Ecosystem Health, Cornell University, Ithaca, NY 14853, USA;
yihong.li@cornell.edu
3 ADA-Forsyth Institute, Cambridge, MA 02142, USA; xhe@forsyth.org
4 The Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA; anniencowan@gmail.com
* Correspondence: spatafor@middlebury.edu (G.S.); annetanner@forsyth.org (A.C.R.T.)
Abstract: Dental caries is a significant oral and public health problem worldwide, especially in low-
income populations. The risk of dental caries increases with frequent intake of dietary carbohydrates,
including sugars, leading to increased acidity and disruption of the symbiotic diverse and complex
microbial community of health. Excess acid production leads to a dysbiotic shift in the bacterial
biofilm composition, demineralization of tooth structure, and cavities. Highly acidic and acid-tolerant
species associated with caries include Streptococcus mutans, Lactobacillus, Actinomyces, Bifidobacterium,
and Scardovia species. The differences in microbiotas depend on tooth site, extent of carious lesions,
and rate of disease progression. Metagenomics and metatranscriptomics not only reveal the structure
and genetic potential of the caries-associated microbiome, but, more importantly, capture the genetic
makeup of the metabolically active microbiome in lesion sites. Due to its multifactorial nature, caries
has been difficult to prevent. The use of topical fluoride has had a significant impact on reducing
caries in clinical settings, but the approach is costly; the results are less sustainable for high-caries-risk
individuals, especially children. Developing treatment regimens that specifically target S. mutans
and other acidogenic bacteria, such as using nanoparticles, show promise in altering the cariogenic
microbiome, thereby combatting the disease.
Keywords: caries; microbiology; ecology; treatment; Streptococcus mutans

Citation: Spatafora, G.; Li, Y.; He, X.;
Cowan, A.; Tanner, A.C.R. The
Evolving Microbiome of Dental
Caries. Microorganisms 2024, 12, 121.
1. Introduction
https://doi.org/10.3390/
microorganisms12010121
Dental caries is a multifactorial disease associated with the structure of deciduous and
permanent teeth, the pathogenicity of oral microbial composition, and micro-environments
Academic Editor: Todd
of the oral cavity influenced by sugar, salivary, and genetic factors. While the initial signs
Riley Callaway
of caries affect the outer enamel surfaces of teeth first, in older adults the exposed dentin
Received: 3 December 2023 of root surfaces can be the main risk site for caries initiation [1,2]. Untreated caries can
Revised: 28 December 2023 progress into the tooth root canal and produce abscesses. Bacteria produce acids as a
Accepted: 4 January 2024 byproduct of their metabolism of dietary carbohydrates. Since bacterial metabolism is
Published: 7 January 2024 the source of acid that precedes caries, the systemic host response to bacterial infection
plays a significant role in dental caries risk. Host-based factors may be genetic or related
to medication side effects, other diseases, or exposure to environmental toxins that can
weaken tooth structure. Thus, bacterial composition, diet, and the host defense system are
Copyright: © 2024 by the authors. critical factors that can directly or indirectly influence the clinical outcome of the destruction
Licensee MDPI, Basel, Switzerland.
of tooth substance.
This article is an open access article
Decades of scientific investigations have indicated that sugar consumption is the most
distributed under the terms and
significant contributing factor to disease initiation and progression. Unhealthy behaviors,
conditions of the Creative Commons
poor oral hygiene, and lack of fluoride exposure are substantial contributing factors to
Attribution (CC BY) license (https://
the disease process [2]. The global burden of sugar-related dental caries based on data
creativecommons.org/licenses/by/
4.0/).
from 168 countries has provided new evidence that confirms the correlation between the
Microorganisms 2024, 12, 121. https://doi.org/10.3390/microorganisms12010121 https://www.mdpi.com/journal/microorganisms

Microorganisms 2024, 12, 121 2 of 45
excessive consumption of sugar and dental caries and documents the many dimensions of
disparities and financial burdens in dental caries [3].
Furthermore, the global burden of disease suggests that dental caries and untreated
caries are responsible for more than 26% of the total dental disease burden and 2.7 million
disability-adjusted life years which cost over 172 billion U.S. dollars to treat [4]. On a
national level, the U.S. spent over $110 billion in 2016 on dental visits [5], of which over
60% was directed toward treating dental caries. Many high-income countries have a lower
disease burden caused by fermentable mono- and disaccharide [MDS]-related dental caries
than most middle- and low-income countries [6] and disadvantaged populations and
nations with limited access to caries prevention and treatment. Untreated caries can cause
pain and infection, impairment of dental function, tooth mortality, missing school hours,
and productivity losses. Thus, dental caries remains a serious public health issue with
substantial long-term health, economic, and societal impact due to the persistently high
prevalence and excessive treatment costs. The disease negatively affects general health and
oral health-related quality of life for all age groups [7,8], including infection, impairment of
dental function, tooth mortality, missing school hours, and productivity losses. Overall,
dental caries remains a serious challenge with substantial long-term health, economic, and
societal impact due to its persistently high prevalence and excessive treatment costs [7,8].
In this review, we probe the epidemiology of dental caries and its association with
diet. We delve into the history of caries, ecology-based theories about the role of bacteria
in caries, and their association with the development of the oral microbiome as a biofilm.
We examine the microbiotas associated with enamel and dentin caries and aggressive
diseases with the functional activity of bacterial communities. Using an advanced map of
the caries microbiome, we explore new control and treatment strategies for modulating
disease-associated microbial dysbiosis and the role of therapeutics in alleviating caries and
re-establishing a healthy microbiota in the human mouth. The review is based on literature
searches in the categories of caries microbiology, epidemiology, etiology, and treatment.
2. Background
2.1. Epidemiology of Dental Caries
As a bacteria-mediated, sugar-driven, and oral-hygiene-modified dynamic disease,
dental caries persists as one of the most common chronic diseases affecting the general
population of all age groups. Worldwide, the estimated average prevalence of dental
caries of deciduous teeth in 2019 was 43% (38% in high-income countries and 46% in low-
income counties) [9]. It is estimated that 514 million children suffer from untreated caries
in deciduous teeth worldwide. The estimated age-standardized prevalence of dental caries
in permanent teeth was 29.4%, and 2.3 billion adults had untreated caries in permanent
teeth [4,10,11].
There has been a decrease in caries in younger age groups in selected regions over the
last few decades due to community water fluoridation [12–15], the widespread availability
of fluoride toothpaste [16], reduced sugar intake [17], and increased access to oral health
services [18]. In the United States, caries rates slightly decreased in the primary teeth of
2- to 5-year-old children but not in the permanent teeth of 6- to 11-year-old adolescents
between 1988 and 2016 (Figure 1A) [19–21]. These downward trends, however, were not
observed in many low- or middle-income nations when associated with increased sugar
consumption during that time period [22].
There were no significant changes in overall caries status in adults aged 20 to 64 years,
but a notable increase in older American adults over 65 years [19–21] (Figure 1B). World-
wide, billions of people with untreated caries have significantly and dramatically increased
the burden on healthcare systems [23]. One contributing factor is an increase in life ex-
pectancy, leading to more older people maintaining their teeth, which has an associated
caries burden [19–21,24,25]. This challenges the healthcare system everywhere, particularly
in low- and middle-income countries.
Microorganisms 2024, 12, 121 sociated caries burden [19–21,24,25]. This challenges the healthcare system everywhere,
3 of 45
particularly in low- and middle-income countries.
Figure
Figure1.1.TheThedistribution
distributionofofdental
dentalcaries
cariesby
byage,
age,race,
race,and
andethnicity,
ethnicity,and
andfamily
familyincome
incomeininthe
theU.S.
U.S.
population.
population.(A) (A)Prevalence
Prevalenceof ofcaries
cariesininprimary
primaryteeth.
teeth. Compared
Comparedwith withNHANES
NHANES1988–1994,
1988–1994,caries
caries
experience
experience slightly decreasedfor
slightly decreased forchildren
children2-2-
to to 5-year-olds.
5-year-olds. However,
However, the the decrease
decrease was was not ob-
not observed
served
for 6- to 8-year-old children between 1988 and 2016. (B) Trend in dental caries. There was awas
for 6- to 8-year-old children between 1988 and 2016. (B) Trend in dental caries. There a
slight
slight decrease in caries experience in the permanent dentition of young children and adolescents
decrease in caries experience in the permanent dentition of young children and adolescents between
between 1988 and 2016. However, there are no significant changes in overall caries status in adults
1988 and 2016. However, there are no significant changes in overall caries status in adults aged
aged 20 to 64 years old. Caries prevalence significantly increased for the elderly aged 64 years and
20 to(C–E)
over. 64 years old. Caries
Prevalence prevalence
of caries significantly
in primary increasedteeth
and permanent for the elderly aged
in 2011–2016 by 64
ageyears and over.
(C), race and
ethnicity (D), and family poverty status, FPL = Federal Poverty Level (E). (F–H) Prevalenceethnicity
(C–E) Prevalence of caries in primary and permanent teeth in 2011–2016 by age (C), race and of un-
(D), andcaries
treated family bypoverty
age (F),status,
race andFPLethnicity,
= FederalNHisp
Poverty= Level (E). (F–H)(G),
Non Hispanic Prevalence of untreated
and family poverty caries
level
(FPL)
by age(H)(F),inrace
the U.S. population.
and ethnicity, NHisp = Non Hispanic (G), and family poverty level (FPL) (H) in the
U.S. population.
The National Health and Nutrition Examination Survey 2015–2016 data reported
Theprevalence
that the National Health
of dental and Nutrition
caries Examination
in permanent Survey 2015–2016
teeth increased with age data
fromreported
9.5% to
that the prevalence of dental caries in permanent teeth increased
96% (Figure 1C) [9]. Significant socioeconomic inequalities exist in caries experience with age from 9.5%
and
to 96% (Figure
untreated caries1C)
in [9]. Significant
children socioeconomic
and adults. Hispanicinequalities
youth andexist adultsin caries experience
experienced the
and untreated
highest caries
prevalence of in children
total and adults.
caries (32.9% at 2–5Hispanic
years old,youth
72.8% and adults
at 6–8 experienced
years old, 69.7%the
at
highest prevalence of total caries (32.9% at 2–5 years old, 72.8%
12–19 years old, and 86.6% at 20–64 years old) compared with other ethnic groups [9]at 6–8 years old, 69.7%
at 12–191D).
(Figure years
Theold, and 86.6%
prevalence at 20–64
was higheryears
among old) compared
poor with other
and near-poor ethnicand
children groups [9]
adults
(Figure 1D). The prevalence was higher among poor and near-poor
than in non-poor counterparts (Figure 1E). Among millions of American children and children and adults
than in16.4%
adults, non-poor
of 6- counterparts (Figure 1E).
to 8-year-old children andAmong
more than millions
25% ofof20-American children
to 74-year-old and
adults
adults, 16.4% of 6- to 8-year-old children and more than 25% of 20-
had at least one untreated decayed tooth (Figure 1F). Non-Hispanic black populations to 74-year-old adults
had the
had at least one untreated
highest prevalencedecayed tooth (Figure
of untreated caries in1F). Non-Hispanic
primary black populations
teeth (22.4%) and permanent had
the highest prevalence of untreated caries in primary teeth (22.4%)
teeth (40.2%) (Figure 1G). The prevalence increased for both total caries and untreatedand permanent teeth
(40.2%)
caries as(Figure 1G). Thelevels
family income prevalence
decreasedincreased for both
[9,19,26–28] total caries
(Figure 1H). and untreated caries as
family income levels decreased [9,19,26–28] (Figure 1H).
For a long time, the caries diagnosis and risk assessment methods used in epidemi-
For a long time, the caries diagnosis and risk assessment methods used in epidemi-
ologic studies varied worldwide, resulting in different outcomes for reporting, evaluat-
ologic studies varied worldwide, resulting in different outcomes for reporting, evaluat-
ing, and monitoring disease progression. Many government-funded water fluoridation
programs or caries risk assessment programs measured caries management using a risk
assessment (CAMBRA® v.4.0) model (cda.org/CAMBRA4) [29] or the Cariogram software

program, v.1.0 [30]. Meanwhile caries diagnosis and measurement for population-level stud-
ies relied on the DMFT/DMFS (decayed, missing due to caries, and filled tooth/surface)
Index [31]. This methodology has been challenged for its lack of sensitivity and discrimina-
tory validity, resulting in substantial underestimation or biased estimation of caries rates
and severity. As such, it has had a limited impact on the evaluation of caries experience
for high-risk populations [32,33]. In response to these concerns, the International Caries
Detection and Assessment System (ICDAS) was developed by an interdisciplinary team
of caries researchers, epidemiologists, and clinicians [34,35]. This integrated system has
been tested and proved to be easy to use with clearly defined clinical visual criteria for
caries detection, a seven-point ordinal scale ranging from sound to extensive cavitation to
measure caries severity, and codes to monitor caries activity over time [36]. Along with
simplified merged-code options, ICDAS has been increasingly adopted by many countries
for caries surveillance and caries risk assessment and management.
In the future, new scientific-evidence-based approaches, such as using new biomarkers
for caries pathogens or disease activity in caries epidemiological studies, can evaluate
disease incidence and progression and support decision-making at both the individual and
public health levels.
2.2. Diet and Dental Caries

While it is generally appreciated that the consumption of sugar-based foods and
drinks is “bad for your teeth”, the history of dental caries and its relationship with diet
goes back to the origins of agriculture. As hunter-gatherers of the European Mesolithic age
(9600–4000 BC), humans had a low prevalence of caries impacting ~0–2% of the popula-
tion [37]. In post-agricultural societies, however, computer modeling analysis indicates that
caries incidence has increased to 5–50%, in direct correlation with a concomitant increase
in dietary carbohydrate consumption [37] and the availability of processed foods in the
Western marketplace in the mid-20th century [38]. This historical sequence of dental caries
emergence has been recapitulated in populations under dietary stress. During World War
II (1939–45), a decrease in caries was documented in European countries (Czechoslovakia,
Denmark, England, Finland, Germany, Holland, Norway, Scotland, and Sweden) where
there were reductions in refined carbohydrates including sugar and calorie-restricted di-
ets [39,40]. When the period of diet deprivation ended in the post-war era, caries increased
to pre-war levels. The late 1940s post-war caries increase was so alarming that it led
to a study to better understand the link between carbohydrates, including sugars, and
their frequency of intake over time with caries development in Sweden. This “Vipeholm”
study conclusively demonstrated the role of frequency of carbohydrate intake in caries
susceptibility [41].
More recently, populations with rapid “Westernization” showed a sharp increase
in dental caries experience directly related to changes from eating primarily meat-based
diets to those with increased proportions of carbohydrates, as in American Indian and
First Nation populations in the US and Canada [42]. Caries-associated carbohydrates go
beyond desserts and sugary drinks to include beverages and foods like beer, potato chips,
and sweetened yogurts. Individuals who subsist on a Western diet, regardless of their
consumption of standard sugar-based foods such as dessert items, are significantly more
likely to score higher on the standard decayed, missing, and filled teeth (DMFT) index than
those who do not [43].
2.3. Plaque pH and Host Factors

In 1944, Stephan suggested that the link between diet and caries was the development
of a low plaque pH [44]. His experiments included measuring the pH of plaque over time
following a glucose rinse in human volunteers. After an initial drop in pH following the
rinse, the acid pH readings gradually returned to baseline levels and plots of the change in
pH readings became known as a “Stephan curve”. Of particular interest was the lowering
of plaque pH to below 5.5, as this was considered the level of acidity needed for enamel
demineralization. Marked differences in plaque acid responses were observed based on
caries clinical status including caries-free, slight caries, obvious lesions, and aggressive,
advanced lesions. In caries-free subjects, post-glucose rinse acidity remained above pH 5.5,
suggesting no enamel demineralization. By contrast, in aggressive caries, the initial resting
plaque acidity was below that needed for demineralization, with further lowering after the
glucose challenge to under a pH of 4.
But how do sugars, particularly sucrose and fructose, impact the rate of caries de-
velopment? As a metabolite for caries-associated pathogens, sucrose is easily converted
into its monosaccharide constituents, glucose and fructose, each of which can serve as sub-
strates for bacterial fermentation to produce lactic acid byproducts [45]. The combination
of acid-producing bacteria and fermentable carbohydrates, some of which can be stored as
intracellular polysaccharide, fosters continuous exposure of the host dentition to organic
acids for extended periods, including non-meal times [46]. Thus, to understand the impact
of the cariogenic process on caries development and treatment, we must understand these
and other players that contribute to the caries phenotype.
Host-based caries risk factors, including genetics, stress, and access to fluoridated
water, have evolved over time and/or remain disparate for individuals of different socio-
economic strata [32]. Populations that cannot afford or have limited access to healthy
foods are at higher risk for dental caries. Increasing disease incidence among the socioe-
conomically disadvantaged derives primarily from host genetics and individual dietary
intake [47]. While genetic host-based risk factors can impact the expression of salivary
proteins in the tooth pellicle [48,49], dietary risk factors can be related to medications,
exposure to environmental toxins, or systemic disease that can weaken tooth structure
and resistance to bacterial acid attack. The mechanisms that underlie these risk factors are
frequently related to the ability of bacteria to attach to teeth or the resistance of teeth to
acidity and demineralization. Bacterial attachment to teeth via the pellicle is a critical step
in caries formation, as it encourages subsequent biofilm development. This attachment
can be greatly facilitated on the rough surfaces of poorly formed enamel, for example in
individuals with genetically determined amelogenesis imperfecta and other conditions
leading to tooth malformations.
3. Microbiome of Dental Caries

The oral microbiome is at the entryway to the gastrointestinal tract [50]. The evolution
of the human body is coincident with, and dependent on, the evolution of its accompanying
microbiota [51]. The most important and persistently variable relationships between host
and microbe occur within the mouth, esophagus, stomach, small and large intestine, and
anus of the gastrointestinal (GI) tract. Every part of this tubular structure, which collectively
comprises “the gut”, serves as home to a coalition of microbial species that can co-inhabit
its many inherent niches. Research on the microbiomes of the GI tract has focused on the
gastric microbiota and the microbiota of the ileum and proximal colon where a dysbiotic
microbiome may contribute to autoimmune disorders such as inflammatory bowel disease
(IBD) and GI cancers [52,53]. The oral cavity, as the entryway to the digestive tract, plays
a particularly crucial role in mediating overall human health, in large part owing to its
exposure to diet and stressors in the external environment.
While good oral hygiene marks the cornerstone of a healthy mouth and, for those
fortunate enough, a beautiful smile, caries prevention extends well beyond routine practices
such as brushing and flossing. The delicate balance of bacterial species interactions in the
human oral cavity can become dysbiotic under conditions of oxidative stress, acidic pH,
and/or fluctuations in nutrient availability, especially when combined with poor dental
hygiene practices and unfavorable host genetics. The oral microbiome, which includes
microorganisms on the tongue, palate, and dentition, comprises about 700 different species
of bacteria, many of which have not yet been cultured in the laboratory [51]. Ultimately, the
ability to study and characterize the healthy oral microbiome is essential for understanding,
diagnosing, and treating diseases of the human oral cavity, including dental caries.
Since the first publication of a human metagenomic study in 2006 [54], the field of
human microbiome research has exploded. Microbiome research in the 21st century has
incorporated research into the dynamic relationship between the oral microbiome and the
host environment in health and disease. The total number of manuscripts published in the
field of oral health versus the caries-associated microbiome by international researchers has
increased markedly in recent years. A substantial “core model” comprised of approximately
60% of the oral taxa has been suggested to contribute to the deleterious shift in bacterial
diversity to acidic (cariogenic) from neutral (healthy) pH environments [55].
3.1. History of Caries Microbiology

Our understanding of the caries microbiome builds on over 100 years of observa-
tions and research studies. The late 1800s and early 1900s marked the “Golden Age of
Microbiology” based on the germ theory of disease as enunciated by Louis Pasteur and
demonstrated by Robert Koch. Germ theory was expanded to caries by GV Black [56] based
on bacterial acid production that could demineralize teeth. A chemo-parasitic theory was
proposed by Willoughby Dayton Miller in 1890, based on his experiments that involved
soaking teeth in saliva, as a source of bacteria, and bread, as a food source, and observing
tooth demineralization [57]. Miller determined that the demineralization he observed came
from lactic acid that was produced by bacteria that we now recognize as Lactobacillus and
Bifidobacterium. These bacteria were later cultured from caries by Percy Howe, the first
research director at the Forsyth Dental Infirmary for Children founded in 1910 [58]. Howe
repeated Miller’s experiments (soaking teeth in saliva with bread) but did not consider the
demineralization he observed to be caries based on his observations of children in the clinic
with advanced caries. Further, Howe was unable to produce caries in experimental animals
using plaque from carious lesions, which led him to question the role of individual species
in dental caries. In the 1930s, Theodore Rosebury, “the grandfather of oral microbiology”,
reported on the acidogenic properties and acid tolerance of Lactobacillus acidophilus of oral
and gastrointestinal tract origin [59]. Rosebury, like Howe, did not advocate for specific
species in dental infections. Thus, these early investigators recognized the role of bacterial
acid production in caries without implicating individual species as had been the Pasteur
and Koch tradition.
In the 1920s, Kilian Clark in the UK was interested in whether the same Bacillus
(Lactobacillus) acidophilus bacteria that Howe had cultured from advanced lesions could
initiate caries. Clarke isolated a streptococcus from many initial carious lesions that was
capable of demineralizing tooth sections [60]. This new streptococcus was a strong acid
producer and was very adherent to the tooth sections so it could resist being washed away
by saliva, characteristics that he reported were important for caries pathogens. Clarke
named this new species Streptococcus mutans, and he considered it capable of causing
caries [60]. In studies that involved isolating S. mutans during the early stages of caries on
enamel surfaces, Clarke was able to isolate lactobacilli from deeper, more advanced lesions,
suggesting that different bacteria were involved in the progression of cavities. Clarke’s
paper was published in a British journal and, before the era of easy access to international
publications, it seems his work was overlooked for several decades.
The essential role of bacteria in caries was demonstrated in the 1940s to 1960s in
studies using experimental animals. The association of Streptococcus mutans with caries was
observed in a series of studies performed mainly at the National Institutes of Health (NIH)
in Bethesda MD, USA. The dental wing of the NIH, later the National Institute of Dental
Research, then the National Institute of Dental and Craniofacial Research, had its origins in
dental caries and was founded in large part to understand the reason why many recruits
for World War II in Europe had many carious teeth and too few teeth to serve in the war.
The critical importance of bacteria as compared with diet had been demonstrated by the
finding of a complete absence of caries in germ-free animals despite maintaining them on
a highly cariogenic high-sucrose diet [61]. Paul Keyes then expanded the importance of
bacteria in caries in a series of studies using Syrian hamsters and other animals. Keyes
summarized many of his experiments in a 1960s publication [62], where he reported that
caries could be transferred from animals with caries to those with no lesions. Keyes also
noted that, in young weanlings, the source of the caries bacteria was transmitted from
the mother. Furthermore, Keyes observed that taking plaque from caries in one animal
could induce caries in non-diseased animals, and that antibiotic administration inhibited
lesions from developing. Caries was also suppressed in pups from mothers with lower
caries experience [62]. In collaboration with Fitzgerald, Keyes reported that the bacterium
responsible for experimental caries was an acidogenic streptococcus [63], which Keyes
observed could by itself induce caries in germ-free animals. It was not until the late 1960s
that Edwardsson made the discovery that the S. mutans he isolated from humans was the
same species that Keyes described from carious lesions in experimental animals, and also
the same species that was described by Clarke in 1924 [64]. Numerous clinical studies have
demonstrated the strong association between S. mutans and dental caries [65–68]. Hence,
the concept of a key role for S. mutans in dental caries was established.
The development of a caries-associated microbiome was subsequently described using
an ecological plaque hypothesis [69]. This concept was based, in part, on chemostat studies
and the growth of a mixture of nine oral bacteria pulsed with glucose at different pHs
over time [70]. The experimental findings indicated that when a healthy resting oral
pH of 7.0 was maintained, Streptococcus gordonii, which is acidogenic but not very acid-
tolerant, dominated the community. When the acidity of the medium was not controlled in
the experiment, however, the pH dropped to 4.5 and the acid-tolerant species S. mutans,
Lactobacillus casei, and Veillonella dispar dominated the community [70]. According to this
model, the metabolism of all acid-producing species in the nine-species cocktail lowered the
environmental pH, but only the acid-tolerant species could survive. Thus, the oral biofilm
under acid stress undergoes a selective succession that favors acid-tolerant species. In
accordance with the ecological plaque hypothesis, it is dietary carbohydrates, particularly
sucrose, that are the drivers of acid production in tooth-associated microbial communities.
Consistent with this hypothesis are the noticeable shifts in biofilm architecture and microbial
population dynamics that were observed after the consumption of a meal [71].
3.2. Development of the Tooth-Associated Microbiome Leading to Caries

The tooth-associated microbiome develops in stages that ultimately either defines
health (caries-free) or disease (caries-associated). An association between clinical outcomes
and microbial changes in oral biofilms under dietary stress was described in 2011 as an
extension of the caries ecological hypothesis as proposed by Takahashi and Nyvad [72,73].
The authors described the earlier stages of microbiome change as reversible, depending on
the availability and duration of dietary carbohydrates in the mouth.
3.2.1. Pioneer Species

The dynamic stability stage of the extended ecological plaque hypothesis represents
health, as dietary carbohydrate challenges are counterbalanced by microbial community
activity to rebalance any acid produced. This stage encompasses colonization of the
dentition by pioneer species in the tooth pellicle. As described below, microorganisms in
the pellicle are comprised mainly of non-mutans streptococci and Actinomyces species.
Bacteria can be detected on teeth within two hours after cleaning, and colonization is
initiated after the formation of a dental pellicle, which is induced by healthy host salivary
secretions [74,75]. As the only non-shedding surface in the human body, the teeth are
protected by a hard outer coating of hydroxyapatite, or calcium phosphate, which resists
force and wear that is inherent to chewing and swallowing. Included in the pellicle are
proteins, often negatively charged glycoproteins, that are produced in the saliva and settle
on teeth as biopolymers [75]. The resulting saliva-based film is deposited immediately on
the enamel surface shortly after tooth eruption, within minutes after routine brushing, and
vary secretions [74,75]. As the only non-shedding surface in the human body, the teeth
are protected by a hard outer coating of hydroxyapatite, or calcium phosphate, which
resists force and wear that is inherent to chewing and swallowing. Included in the pelli-
cle are proteins, often negatively charged glycoproteins, that are produced in the saliva
and settle on teeth as biopolymers [75]. The resulting saliva-based film is deposited im-
mediately on the enamel surface shortly after tooth eruption, within minutes after rou-
tine brushing, and reaches maturation within 120 min [76]. The tooth pellicle is believed
reaches
to have maturation within 120
evolved to protect the min
tooth[76]. The tooth
surface from pellicle is believed
acids produced bytolocal
havemicroorgan-
evolved to
protect the tooth surface from acids produced by local microorganisms
isms [69]; yet, microbes were able to exploit this host defense mechanism by using [69]; yet, microbes
the
were able to exploit this host defense mechanism by using the pellicle as a substrate for
pellicle as a substrate for attachment, thereby enhancing the bacterial colonization of
attachment, thereby enhancing the bacterial colonization of tooth surfaces. The host-derived
tooth surfaces. The host-derived pellicle, which is both diet-induced and individual-
pellicle, which is both diet-induced and individual-specific, serves as the first stage in the
specific, serves as the first stage in the development of the plaque biofilm.
development of the plaque biofilm.
The next stage involves colonization of the tooth by so-called “pioneer” species.
The next stage involves colonization of the tooth by so-called “pioneer” species. Most
Most pioneer species have evolved cell-surface ligands which interact directly with pro-
pioneer species have evolved cell-surface ligands which interact directly with proteins
teins as receptors inherent in the dental pellicle. These receptors, deemed “cryptitopes”,
as receptors inherent in the dental pellicle. These receptors, deemed “cryptitopes”, often
often do not appear until after proteins in the salivary pellicle have become absorbed on
do not appear until after proteins in the salivary pellicle have become absorbed on the
the hydroxyapatite surface of the tooth, thereby changing protein conformation to foster
hydroxyapatite surface of the tooth, thereby changing protein conformation to foster
subsequent bacterial adhesion [77,78]. The attachment process begins with reversible
subsequent bacterial adhesion [77,78]. The attachment process begins with reversible
adhesion of bacteria to the tooth via physio-chemical interactions, such as hydrogen
adhesion of bacteria to the tooth via physio-chemical interactions, such as hydrogen bonds,
bonds, hydrophobic
hydrophobic interactions,
interactions, calciumcalcium
bridges,bridges,
and vanandder vanWaals
der Waals
forces,forces, that collec-
that collectively
tively strengthen
strengthen ligand-receptor-mediated
ligand-receptor-mediated surfacesurface attachments.
attachments.
Pioneer species whose presence is consistent withwith
Pioneer species whose presence is consistent caries-free
caries-free sitesgingival
sites and and gingival
health
health include Actinomyces naeslundii, Actinomyces oris, Streptococcus
include Actinomyces naeslundii, Actinomyces oris, Streptococcus gordonii, Streptococcusgordonii, Streptococ-
san-
cus sanguinis,
guinis, and Streptococcus
and Streptococcus oralis (Figure
oralis [79,80] [79,80] (Figure
2). These2).species
These undergo
species undergo adhesin-
adhesin-receptor-
receptor-mediated
mediated associations
associations with in
with proteins proteins in the
the tooth tooththus
pellicle, pellicle, thusthe
forming forming the ini-
initial biofilm
tial biofilm
layer, which,layer, which,
in turn, in turn, specific
can provide can provide specific
bindings sitesbindings sites forbacterial
for subsequent subsequent bac-
coloniza-
terialmediated
tion colonization mediated
by stronger by stronger
physical physical
interactions interactions
between between coaggregation
coaggregation receptor polysac- re-
ceptor polysaccharides (RPS) of the group A streptococci, the oral streptococci,
charides (RPS) of the group A streptococci, the oral streptococci, and the fimbriae of most and the
fimbriae of most
Actinomyces Actinomyces
species [81]. species [81].
Figure 2.2.Major
Figure Majorspecies
species identified
identified from
from caries-free
caries-free sites,sites,
enamelenamel
caries,caries, and dentin/root
and dentin/root caries.
caries. Shown
Shown
are crossare cross sections
sections of the dentin,
of the enamel, enamel,and
dentin, and
root of root of
a tooth a tooth
with their with their associated
associated caries-free
caries-free and caries-
and caries-associated
associated microbiota.microbiotas
microbiota. Caries-free Caries-freecolonize
microbiotas colonize
the tooth surfacethe
as tooth
diversesurface as diverse
communities that
communities that respond to dietary carbohydrate challenge by producing acid, favoring enamel
respond to dietary carbohydrate challenge by producing acid, favoring enamel demineralization that
demineralization that is neutralized by microbiome community activity that includes the produc-
is neutralized by microbiome community activity that includes the production of ammonia deiminase
tion of ammonia deiminase and urease activity (remineralization). Enamel caries derives from the
and urease activity
acid-induced (remineralization).
enrichment Enamel species
of highly acidogenic caries derives from the acid-induced
and suppression enrichment
of acid-sensitive species,
of highly acidogenic species and suppression of acid-sensitive species, leading to a
leading to a reduction in community diversity. Caries in dentin, including root caries at the toothreduction in
community diversity. Caries in dentin, including root caries at the tooth surface, generally comprises
a more diverse microbiota than enamel caries with moderately rather than highly acidogenic species
owing to the reduced mineral content of dentin. Caries progression deep in dentin can involve acidic
demineralization in addition to increasing proteolytic activity, leading to higher pH values in deep
lesions. The sequence of caries progression shown is based on Takahashi and Nyvad [73]. Species
based on text references.
Pioneer streptococci species can dominate the supragingival regions of teeth and
appear to be uniquely attuned to the fluctuating conditions of the oral cavity due to their
advanced ability to regulate gene expression and protein production for attachment to
the dental pellicle [82]. For example, the regulatory mechanisms of S. gordonii allow it to
thrive under a certain set of environmental conditions [83]. Similar mechanisms in other
species help define which bacteria can co-inhabit the same oral niche [84]. Of the pioneer
species, S. gordonii is persistently adherent to the tooth enamel, given its high affinity
for proteins in the tooth pellicle. S. gordonii can utilize adhesion proteins (i.e., Has) and
amylase-binding protein A (AbpA) to mediate receptor–ligand attachments to teeth. The
Has adhesion receptors of S. gordonii can bind specifically to the terminal sialic acid of
host sialoglycoconjugates [80], whereas AbpA interacts directly with human α-amylase, a
salivary enzyme that is paramount for food breakdown [85]. Due to the covalent nature of
these interactions, S. gordonii was found to firmly attach to the tooth, comprising up to 70%
of the bacterial community in this early dynamic stability stage of plaque development [85].
3.2.2. Early Colonizers

An important step in biofilm development involves a substantial increase in the
quantity and variety of streptococci in the dental microbiome [86]. Bacterial adhesion
to proteins in the pellicle extends beyond the more typical ligand–receptor interactions
and includes cooperative cross-species relationships like that between S. sanguinis and
S. oralis, other pioneer colonizers of the caries biofilm. S. gordonii, S. sanguinis, and S. oralis
are facultative anerobes, which is crucial to their pioneering status since they can initiate
biofilm formation on the exposed tooth surface and survive oxygen deprivation as the
biofilm develops and matures. Furthermore, S. sanguinis and S. oralis are considered
commensal species of the healthy oral microbiome, and they maintain genetic factors
which sustain their attachment to the dentition, especially by expressing a variety of
Abps [85,87]. More importantly, S. gordonii, S. sanguinis, and S. oralis can co-aggregate
and form an intricate system for quorum sensing as the plaque biofilm develops. The
comCDE operon, which is highly conserved across the streptococci, encodes an essential
competence-stimulating peptide (CSP) in this trio. The peptide is a product of the comC
gene, which increases the cell division rate and regulates a primary set of quorum-sensing
genes [79]. This allows these streptococci to interact directly with one another, so they may
become sensitive to the environmental conditions around them and, most importantly,
regulate the pH of the oral environment. Major mechanisms for buffering local acidity
include the production of ammonia by arginine deaminase from Streptococcus sanguinis
and by urease from Actinomyces naeslundii [88–90]. Thus, the interspecies communication
among pioneer species is what allows them to survive in the developing biofilm, in large
part because of their abilities to regulate and neutralize local acid production.
Veillonella tobetsuensis, a relatively new species closely related to V. dispar and Veillonella
parvula [91] and likely previously recognized as one of these, is another early colonizer of
human dentition. Further, Veillonella species, including V. tobetsuensis, become especially
well established in active carious lesions by selectively allowing species growth in response
to the increasingly acidic environment. The cellular machinery for Veillonella growth
and division can be induced by association with Streptococcus species within the tooth
pellicle [92]. Hence, biofilm architecture, which is dynamic during the later stages of caries
progression, can have a direct impact on the composition and survival of individual species
on the tooth surface, thereby shaping the landscape of the plaque microbiota to favor either
health or disease depending on the rate of environmental acidification.
3.2.3. Early Colonizers and the Acidogenic Stage of Plaque Biofilm Development
While pioneer species are the first to colonize the tooth pellicle, the appearance of early
colonizer microorganisms in succession parallels changes in the local plaque environment
that derive from the amassing pioneer species; such accumulation generates conditions
no longer favorable for survival of the pioneer species owing to oxygen and nutrient
deprivation as well as increased host sugar consumption [69]. The initial horizontal
coaggregation of bacteria over the tooth surface is followed by vertical coaggregation
away from the tooth surface [93], resulting in biofilm build up that selects for persistent
metabolically diverse species that favor growth at low pH. Co-adhesion between the
co-aggregating pioneer and early colonizers often occurs by uni- or bimodal protein lectin–
oligosaccharide receptor interactions, which allow for the development of a structured
biofilm architecture [94].
S. gordonii, S. sanguinis, and S. oralis, as non-mutans streptococci, are often considered
“accessory pathogens” because of their contributions to plaque architecture. Importantly,
their interactions are often species-specific, such that the attachment of an early colonizer is
entirely dependent on the preceding establishment of its partner pioneer colonizer. Thus,
the development of the caries biofilm is not so much a one-way, stepwise progression
as it is an amalgamation that is dependent on species-specific population dynamics that
essentially generate niches for the next stage of colonizers.
The early colonizers produce acidic byproducts from the fermentative metabolism
of dietary carbohydrates. A longer acidic challenge leads to suppression of more acid-
sensitive oral bacteria and an increase in acid-tolerant species. Biofilm acidity is associated
with demineralization of the enamel surface and weakening of the outer protective enamel
layer of the tooth. This leads to surface roughening, which can instigate the colonization
of more aggressive microbial pathogens [72,95]. This is the stage in which there can be an
acid rebalance of the bacterial community so that enamel remineralization and any clinical
signs of caries are reversed.
3.2.4. Secondary Colonizers and the Aciduric Stage of Plaque Biofilm Development
Next in the microbial succession are the secondary colonizers, which shift the mainly
streptococci, actinomyces, and veillonella microbiota to a more diverse constituency of
microorganisms capable of broader metabolism. This stage represents a more acidogenic
stage of the ecological plaque hypothesis [72,96]. Together, the secondary colonizers
S. mutans and Lactobacillus, Bifidobacterium, and Scardovia species contribute to climax
communities that define the environmental conditions of the oral cavity, and which can
enhance the pathogenic potential of the colonizers involved. If the enamel surface collapses,
there is cavity formation, which can progress to involve the underlying dentin.
3.2.5. The Mature Biofilm

The inter- and intra-species relationships that play out on the tooth surface define
the very biofilm communities that generate organic acids, thereby exposing the dentition
to low-pH environments that are conducive to demineralization. This is supported by
literature reports of specific interspecies relationships that are crucial to a community
structure that promotes caries [92,97,98]. Further, community interactions between local
groups of microorganisms establish intricate networks of communication that regulate
pH, oxygen, and nutrient availabilities. The resulting biofilm ultimately has a distinct
architecture comprised of proteins, carbohydrates, and functionally structured communities
with cariogenic bacteria that together define a cooperative microbiome that derives input
from both host and microbe [69].
Biofilms become organized once they are adherent to the tooth surface. First, an
extracellular polysaccharide (EPS) matrix housing small aggregates of bacterial cells called
micro-colonies can be seen, followed by the establishment of a communication network
comprised of fluids and nutrient channels. The EPS and a heterogeneous secretion of mi-
crobial biopolymers that can include proteins, glycoproteins, glycolipids, and extracellular
DNA form a sort of hydrated gel that protects and nourishes the microbial biofilm commu-
nity and renders it adherent [45]. The biopolymer constituents of the plaque biofilm engage
in electrostatic interactions, which largely define the physical structure of the biofilm. Weak-
ening the EPS structure is one approach to suppressing the caries microbiome [45] and is
the basis of recent novel therapies.
Ultimately, the tooth-adherent microbial community reaches maturation when the

secondary colonizers, called “bridging species”, facilitate the formation of interspecies
coaggregation bridges, causing bacterial population dynamics to become re-organized and
biofilm detachment when this reaches a critical mass [99]. It is this process of maturation
that allows microbial communities to form on the surfaces of the teeth. However, it is the
tenacious attachment of the community to the host dentition that ensures the microbial
production of acidic byproducts and demineralization of the tooth enamel. Thus, the stages
of caries development depend upon both host and bacterial contributions.
3.3. Culture and Genetic Analysis of the Cariogenic Microbiome

Approaches to the study of the composition of the oral microbiome have evolved over
time and incorporate both culture- and molecule-based methods. Culture methods were
used in the 1960s and 1970s to demonstrate the bacterial role in dental caries. Different
selective media were used to enrich for the detection of S. mutans [100] and lactobacilli.
Acidic agar [101] or broths [102] facilitated the isolation of the acid-tolerant microbiota from
carious lesions. Methods for increased sensitivity for bacterial detection followed the use
of anerobic methods for strain isolation [103]. The detection sensitivity for culture-based
methods is ~5% of the microbiota if 40 colonies are processed for identification. Thus,
culture-based studies using isolation agar have the most value for studies that focus on
identifying the dominant species.
Most of our current knowledge of the complex human microbiome, including the
oral microbiome, is derived from 16S rRNA gene sequencing [104,105]. 16S rRNA-based
microbial profiling studies in caries date back to the early 2000s. Since this approach has
a higher detection sensitivity for bacteria present in low proportions in the microbiota,
its applications reveal a more diverse ecosystem than culture-based studies [106–108].
Gene-based studies confirmed that S. mutans frequently accounts for a small fraction of
the bacterial community in carious lesions, even less than noted previously in culture-
based analyses [65]. A consistent observation when using16S rRNA gene community
profiling approaches has been a significant reduction in bacterial community diversity
associated with caries and caries progression compared with caries-free sites [109,110].
This deleterious shift within the microbial community lesions likely reflects suppression of
acid-sensitive species in areas of acid-mediated lesion progression.
Side-by-side analysis using genetic and cultural methods has suggested that for dental
caries, culture techniques can detect most of the taxa observed by molecular methods.
Similar microbiotas were observed using contemporary anerobic culture methods when
isolates and clones were sequenced for bacterial identification from dentin lesions [111] and
severe early-childhood caries (S-ECC) [112,113]. An anerobic culture of S-ECC highlighted
the dominance of acidogenic gram-positive rod species in Actinobacteria, particularly in
Scardovia (in the Bifidobacterium group) and Actinomyces [114]. Sample analysis using pyrose-
quencing on a 454 platform, an updated genetic method compared with previous 16s rRNA
sequencing and cloning techniques, has continued to indicate under-representation of Acti-
nobacteria by genetic methods compared with anerobic cultures [115]. Thus, data deriving
from genetic and culture methods are both valuable for studies of the oral microbiome.
Nevertheless, the advent of massively parallel sequencing technology, known as
next-generation sequencing (NGS), has revolutionized the biological sciences [116]. This
NGS-based 16S rRNA sequencing approach has become a well-established, culture-free
and cost-effective method that enables analysis of the entire microbial community within
a sample. It also allows for the comparative study of bacterial phylogeny and taxonomy
from complex microbiomes [117,118].
4. Caries Microbiome at Different Lesion Sites

There is no single microbiota of dental caries; rather, carious lesions differ in bacterial
composition reflecting several factors, based in part on subject age, diet, and systemic
health, as well as on the site of the lesion, the extent of the lesion, and how rapidly
lesions progress. Studies involving sufficient participants to overcome variations based

on individual differences allow description of the caries microbiota at different clinical
states (Figure 2). We first describe the species in various caries lesions and then focus on
the characteristics of selected caries-associated bacteria including S. mutans, Lactobacillus,
Bifidobacterium, and Scardovia species. Considering the complexity of the oral microbiome
and community activities, the role of individual species in disease has been challenged.
Rather, there is consideration of acid production from the microbial complex as a whole,
with a reduced focus on selected putative cariogenic pathogens.
4.1. Initial Caries

Initial caries can present as white-spot enamel lesions (WSL), which, according to
the ecological plaque hypothesis, result from biofilm colonization with more acidogenic
and acid-tolerant bacteria than is otherwise characteristic of health [72]. Initial caries had
been studied in culture by Kilian Clarke with the first description of S. mutans [60]. A
longitudinal study of the development of WSL in schoolchildren using anerobic culture
methods 30 years later revealed detection of S. mutans and lactobacilli in lesions in some
but not all of the children [119]. They observed the ubiquitous presence of Actinomyces,
Streptococcus, Veillonella, and Bacteroides species, and, at lower proportions, Neisseria, Lacto-
bacillus, Haemophilus, Fusobacterium, Rothia, Bacterionema (Corynebacterium), Leptotrichia, and
Eubacterium species.
In college students, associations between S. mutans and caries compared with non-
lesion sites required pooling together plaque from several WSL [120]. Proportions of S.
mutans ranged from 0.0001–10% of the microbiota assayed by culture of lesions. Based on
the ability of plaque to produce a low pH in in vitro assays, van Houte et al. concluded
that several bacteria other than S. mutans and lactobacilli contributed to the cariogenic
microbiome of WSL. Using an approach based on low-pH agars, similar numbers of acid-
tolerant bacteria were present in health and initial caries, including lactobacilli streptococci
(principally S. mutans and S. oralis), with Veillonellae being associated with lesions in
adolescents [101].
The microbiota of WSL has also been examined in an orthodontic fixed-banding model.
In adolescents with poor oral hygiene, plaque collects around the metal bands and can
be associated with rapid enamel demineralization, which appears clinically as whitened
zones. Consistent with the above-cited studies, S. mutans was detected in only a few of
the developing carious lesions [121]. Using a combination of a 16S rRNA gene-based
methods, the association of S. mutans with WSL was confirmed [122]. The lesion-associated
microbiota revealed from a microarray approach included the acidogenic streptococci
(S. mutans, non-mutans streptococci) as well as Atopobium parvulum, Dialister invisus, S.
wiggsiae, and Prevotella species [122,123].
A cross-sectional model of caries using 16S rRNA sequencing and cloning and probe as-
says was used to compare the microbiotas of caries-free children with those of children with
caries-free sites, white-spot lesions, cavities, and advanced deep dentin caries in the same
mouth [106,124]. According to this model, species associated with initial lesions, in addition
to S. mutans, included S. sanguinis, S. salivarius, Actinomyces gerensceriae, Veillonella species,
and a Corynebacterium species, although differences were not all statistically significant.
These were not, however, the only species detected, as almost 200 distinct species/taxa were
revealed in the samples, reflecting the complex microbiota in oral biofilms [124]. Using a
similar disease model, but with longitudinal monitoring to detect new lesion development,
investigators reported several microbiotas associated with initial lesion development [110].
These included a major group characterized by S. mutans and other smaller groups char-
acterized by either Streptococcus sobrinus or Streptococcus vestibularis/salivarius (the assay
could not differentiate these streptococci). Other progressing lesions were not included in
these groups. In this study, Veillonella species were indicators of future caries, reflecting the
presence of an acid-producing microbiota as described above [110].
These findings indicate that the microbiota of initial caries is consistent with the
ecological plaque hypothesis with regards to the sequence of colonization and inclusion
of highly acidogenic and acid-tolerant species, which include S. mutans and S. wiggsiae
(when assayed) and several other streptococci in the caries microbiome. Initial lesions
are clinically reversible, and many will not progress to cavities. Many of the white-spot-
lesion-associated microbiota, particularly the actinomyces, are moderately acidogenic and
acid-tolerant, consistent with the microbiome at the borderline of the dynamic stability
and acidogenic stages (Figure 2) of the expanded ecological plaque hypothesis. Following
the recent observation that S. mutans can be concentrated as “rotunds” over white-spot
lesions [125], it seems likely that S. mutans alone could be the prime species that drives
enamel demineralization. To evaluate how frequently this highly localized, species-specific
demineralization occurs in initial and more advanced caries, highly localized site sampling
of progressing lesions will be required. If found, it would suggest that S. mutans is a
keystone pathogen of caries.
4.2. Dentin Caries

Dentin caries occurs either from an extension of deep cavities in the enamel or directly
when tooth roots become exposed and lack the protective benefits of an overlying enamel
layer. Tooth root exposure can follow gingival recession either associated with aging or
after periodontal surgery. Reduced salivary flow as a side effect of medications, following
head and neck radiation treatment, or diseases like Sjögren’s syndrome can significantly
impact the microenvironment of the root surface, favoring biofilm formation and other
high-risk factors for root caries.
4.2.1. Root Caries

Studies in the 1970s reported a complex microbiota from anerobic culture of root
caries in adults [126]. Loesche, Syed, and co-workers detected S. mutans, S. sanguinis and
other streptococci, Actinomyces viscosus, Lactobacillus, and Veillonella species in caries. Using
culture isolation on acidic media, the bacterial taxa detected in association with root caries
included S. mutans, Actinomyces israelii, Propionibacterium, Bifidobacterium, Lactobacillus,
yeast, and non-mutans Streptococcus species, expanding the acidogenic bacteria in root
caries [127–131]. On a newly formulated Bifidobacterium selective medium, the microbiome
of active lesions was comprised of 8% Bifidobacteria [Bifidobacterium dentium, Parascardovia
denticolens, Scardovia inopinata, S. wiggsiae (Scardovia genomosp. C1), Bifidobacterium breve,
and Bifidobacterium subtile], 4% S. mutans, and 31% lactobacilli, as a proportion of total
anerobic counts [130]. In other studies, yeast species were detected in root caries in over
60% of older Chinese individuals, although yeast levels were low and included Candida
dubliniensis, which had previously been reported in HIV subjects [132]. The dominant
species cultured in root caries in several studies thus were principally acidogenic and
acid-tolerant taxa.
Molecular-based methods including clonal methods combined with 16S rRNA probe
analyses were used to reveal the diverse microbiota in root caries, including S. mutans,
Enterococcus faecalis, Actinomyces, Lactobacillus, Atopobium, Olsenella, Pseudoramibacter, Propi-
onibacterium, Selenomonas, and Prevotella species [133]. The authors noted that the microbial
composition of lesions varied between subjects, and that no defined microbiota was con-
sistently observed. A pyrosequencing study indicated higher levels of several acidogenic
species, including S. mutans, several Lactobacillus species, S. wiggsiae, B. dentium, and P.
denticolens as from the culture analyses, with the addition of several proteolytic Prevotella
species [134].
Although several Actinomyces species have been considered pathogens of root caries
from the above culture-based studies, a recent study described similar levels of Actinomyces
gene expression in both sound and carious root biofilms. This suggests either a role for
these bacteria as commensals on the root surface or their essential function in microbial
metabolic pathways and the formation of an environment on root surfaces that is suitable for
cariogenesis [135]. The study authors concluded that some Actinomyces could be cariogenic,
considering their ability to survive in acidic environments and to ferment carbohydrates.
4.2.2. Deep Dentin Caries

On tooth crowns, caries involving dentin occurs after breaching the overlying enamel,
and the microbiota has been considered to differ by lesion depth. The microbiota from
sequential excavator samples that went deeper and deeper in lesions, however, did not
differ in a study with microbial analysis by 16S rRNA cloning and by anerobic culture [111].
Some differences in the microbiota were observed based on microbiology technique, al-
though S. mutans, Rothia dentocariosa, and Propionibacterium acidifaciens were detected with
both approaches. The major species detected in culture included the gram-positive rods
P. acidifaciens, Olsenella profuse, and Lactobacillus rhamnosus, whereas the dominant species
in the molecular cloning analysis were S. mutans, Lactobacillus gasseri/johnsonii, L. rham-
nosus, Veillonella species (mainly Veillonella dispar), and several Prevotella and Fusobacterium
species. Similarly, no differences in microbiota were observed by lesion depth in a 16S
rRNA probe hybridization study of adolescents [136]. In this latter study, dentin species in-
cluded Fusobacterium nucleatum, Atopobium genomospecies C1, Lactobacillus casei, veillonellae,
S. mutans, bifidobacteria, and Rothia dentocariosa. S. mutans was identified in 44% of lesions,
whereas other streptococci were observed more frequently [136]. Lack of difference by
lesion depth was observed in a more recent study using a shotgun metagenomic sequencing
approach [137]. Overall, many of the species were similar to those detected in root caries,
with the addition of more proteolytic species, including F. nucleatum.
The complexity of dentin caries was clarified, in part, in a report describing differ-
ent microbial combinations: one complex dominated by lactobacilli, another complex
dominated by prevotellae, and a third complex by a combination of Lactobacillus and Pre-
votella species [138]. Other species detected included Selenomonas, Dialister, F. nucleatum,
Eubacterium, Lachnospiraceae, Olsenella, Bifidobacterium/Scardovia, Propionibacterium, and Pseu-
doramibacter. This report suggested that the environment deep in dentin harbors a diverse
microbiota with different dominating taxa, which may or may not be outcompeted by
Lactobacillus species.
The microbiome of dentin caries was subsequently described according to lesion pH,
with a lower pH correlating with increased lesion activity [139]. This is consistent with acid
as a major driving pathogenic mediator in dental caries. In the latter Kuribayashi report,
higher levels of lactobacilli were detected in the more acidic lesions and, in a later study,
lower microbial diversity was associated with increased dentin acidity [55]. In contrast, at
less acidic levels, the microbiota was likely to be more diverse and dominated by Prevotella
species [140]. These observations indicate that the microbiota of aggressive caries is less
diverse than chronic lesions, reflecting suppression of the acid-sensitive bacteria at very
low pH levels.
Microorganisms involved deep in dentin are less likely to be dependent on fermentable
carbohydrates (acidic stress) than in coronal caries, but more sensitive to microenvironmen-
tal stress when at the exposed sites of root surfaces [73]. The presence or absence of specific
nutrients, oxygen, host defenses including secretory immunoglobulin A, lysozymes, lacto-
ferrin and defensins, antimicrobial glycoproteins, peptides, and antibiotics from medication
therapy in saliva and gingival crevicular fluid (GCF) can play significant roles in determin-
ing the composition of microbial colonization and composition on the root surfaces and
susceptibilities for caries [77,141].
Furthermore, it is likely that dentin-caries-associated bacteria may be involved in
caries progression via different mechanisms, either by demineralization by acidogenic
species or proteolytic action deeper in lesions by Prevotella and Fusobacterium species [73]
(Figure 2). Protein-degrading bacteria isolated from root caries included Prevotella, Acti-
nobaculum, and Propionebacterium species [131], which were associated with final pH values
of ~5.0, higher than that of acidogenic species. The proteolytic activity of these taxa likely
explains the higher pH observed in deep carious lesions [140]. The heterogeneity of the
Microorganisms 2024, 12, 121 microbiome of carious dentin compared with crown/coronal tooth sites was observed from
16 of 47
metatranscriptome experiments where a more diverse microbiota was observed when gene
expression profiles were mapped back to species [142] (Figure 3). Moreover, species in the
dentin
ease, as had a wide
reported fromrange of metabolic
16S rRNA capabilities,
gene community reflecting
profiling differences
approaches in the ecology of
in S-ECC com-
pared with
dentin caries-free
caries comparedchildren
with[109].
that on the tooth surface.
Figure 3.3.Microbial
Figure Microbialspecies and taxa,
species including
and taxa, viruses, viruses,
including detected from gene expression
detected from geneprofiles in
expression profiles in
coronal and dentin caries. Bacterial samples were taken from coronal caries and caries-free sites
coronal and dentin caries. Bacterial samples were taken from coronal caries and caries-free
and dentin caries from early childhood caries in children with progressing lesions. Functional pro- sites and
dentin caries
filing was from early
performed childhood
on purified caries
bacterial RNAin children
using HUMAnN with2.0,
progressing
version 0.9.9lesions. Functional profiling
(HMP Unified
was performed on purified bacterial RNA using HUMAnN 2.0, version 0.9.9 (HMP Unified Metabolic
Analysis Network). Species mapped from gene expression profiles showed the greatest diversity from
dentin caries samples. Dentin caries n = 6; coronal caries n = 5; caries n = 4. * difference detection,
Chi-square > 0.05. Data from Kressirer et al. [142].
4.3. Caries Microbiome of Aggressive Lesions

Examining the microbiota of aggressive caries presents an opportunity to identify the
species most likely involved in disease progression. While active caries can occur in adults,
particularly devastating is the rapid destruction of the dentition in young children. Studies
in the 1970s from Walter Loesche’s laboratory using anerobic culture revealed a complex
microbiota in children with lesions extending into the dentin [100]. S. mutans, S. sanguinis,
other streptococci, Actinomyces viscosus, Lactobacillus, and Veillonella species were identified
from caries-associated crown or dentin caries. S. mutans was implicated as a major player
in caries [65]. Another population of children with severe “nursing” caries were found to
harbor a higher diversity of Lactobacillus species, with increased numbers of S. mutans and
lactobacilli detected in association with more sugary diets [143]. Studies since the 1970s
have documented specificity in the acquisition of S. mutans in a child from the mother
where both have dental caries [144–146].
In the U.S., nursing bottle caries is recognized under “severe early childhood caries”
(S-ECC). While generally attributed to S. mutans infection, studies since the 2000s have
assigned greater diversity and differing microbiotas to the active lesions. Using molecular
methods, a seminal study performed in the Griffin laboratory reported the detection of
significantly higher levels of S. mutans in S-ECC than in caries-free children [106]. Other
major caries-associated species included Actinomyces, particularly Actinomyces gerencseriae
in the earlier stages of the disease, an unnamed Bifidobacterium species (now recognized as
Scardovia wiggsiae), veillonellae, and non-mutans streptococci. Anerobic culture analysis
indicated significant associations of S. mutans and S. wiggsiae with S-ECC [113]. Further,
S. wiggsiae was detected in a proportion of caries-affected children without S. mutans, sug-
gesting this Bifidobacterium-like species could be an alternate caries pathogen to S. mutans.
Computer modeling of their microbiota, diet, and clinical characteristics distinguished
S-ECC children with a high frequency of S. mutans, S. sobrinus, and S. wiggsiae and caries
progression from other children, suggesting that these species were associated with more
aggressive caries [147].
Anerobic culture isolation on acidic agar enhanced detection of S. mutans and Scardovia
wiggsiae in S-ECC, but higher proportions of acid-tolerant taxa, including S. thermophilus,
S. intermedius, V. atypica, V. parvula, and V. dispar, were detected in caries-free children,
suggesting that acid tolerance by itself, even in acidogenic species, is not an indicator of a
caries pathogen [113]. Further, there were lower microbial counts on an acidic agar medium
compared with a pH-neutral agar [148], suggesting that, in part, the acidic environment
of carious lesions accounts for the lower microbial diversity in disease, as reported from
16S rRNA gene community profiling approaches in S-ECC compared with caries-free
children [109].
Severe early childhood caries can disproportionately affect American Indian (USA)
and First Nation (Canada) children [149,150]. The caries-associated microbiota includes
a very high prevalence and high levels of S. mutans [151], with differences in the genetic
diversity of S. mutans compared with other children in the local area [152]. To further
investigate the microbiome of aggressive caries, advanced carious lesions observed in
Romanian adolescents who had very limited dental care were compared with a Swedish
population with lower caries experience [153]. The high-caries population demonstrated
a stronger association with S. mutans and S. sobrinus and caries, whereas the low-caries
adolescents harbored a more diverse microbiota that included non-mutans Streptococcus
and Actinomyces species. This provides more evidence for an association between mu-
tans streptococci and aggressive caries. A subsequent report focused on the lower caries
incidence in Swedish children and S. mutans detection in relation to caries. As in the
previous report, adolescents positive for S. mutans had higher caries scores than those with-
out S. mutans [154]. Microbial communities differed between the S. mutans-positive and
-negative groups. Species in the S. mutans-positive groups included Actinomyces sp. HMT
448, S. wiggsiae, Stomatobaculum longum, and Veillonella atypica, most of which are highly
acid-tolerant [114]. In the group with low or no S. mutans, additional taxa were evident,
such as Actinomyces, Dialister, Fusobacterium, Neisseria, Peptostreptococcaceae, Tannerella, and

Treponema, which were associated with low caries activity. The latter less acid-tolerant
group of bacteria, including the proteolytic species involved in caries progression [131], fits
the ecological plaque hypothesis extended to dentin caries [73].
A model of microbial population changes as they might relate to aggressive caries
was assessed by monitoring the demineralization of dentin and enamel sections worn in
the mouths of volunteers for 20 weeks [155]. More notable among the demineralization-
associated species were S. mutans and several Lactobacillus species, including Lactobacillus
gasseri, Scardovia inopinata, and Rothia dentocariosa. Since S. inopinata was the only Scardovia
strain used as a reference, it is possible that the S. inopinata identification could have been
S. wiggsiae. Importantly, these findings indicated that active carious lesions are linked with
more acidogenic and acid-tolerant species in the microbial community, including those
associated with aggressive caries.
Studies centered on the more rapidly progressing forms of dental caries thus revealed
strong involvement of the more acidogenic and acid-tolerant members of the microbiome
compared with less aggressive disease. These observations suggest that the enigma of
caries with and without detection of S. mutans, may, in part, be related to the activity
of the clinical disease. Acidogenic members belonging to the cariogenic microbiotas of
less aggressive disease may not include S. mutans, but rather may include other species
of Streptococcus, Actinomyces, and other gram-positive rods (including Rothia, Atopobium
and Corynebacterium), although the cariogenic potential of many of these taxa singly or in
combination have not been adequately explored.
4.4. Yeasts and Dental Caries

Other oral microorganisms that participate in biofilm formation and cariogenesis
include species of the dimorphic fungus Candida, which, in its yeast form, colonizes
monospecies biofilms comprised of S. mutans in vitro [156]. Candida species are detected in
both health and disease [157], with C. albicans colonizing between 50% and 70% of caries-
free individuals [158]. In the healthy oral microbiome, Candida species have been shown
to co-aggregate with S. gordonii and S. oralis, but not with S. mutans [159]. However, once
the local pH drops below healthy levels and sucrose is introduced into the environment, a
strong adhesive interaction between the Candida species and S. mutans can be facilitated
by S. mutans’ glucosyltransferase (Gtf) exoenzymes, which bind directly to the outermost
protein layer of Candida [156,160]. Hence, the accumulation of acid near the dentition
created by the pioneer colonizers, and the addition of sucrose to the local environment, can
initiate S. mutans colonization and S. mutans–Candida interactions.
The presence of C. albicans in S. mutans biofilms can enhance the ability of both species
to metabolize sucrose [156,161], thereby enhancing the fitness of both organisms within
the biofilm community. In experimental animals, co-infection by C. albicans and S. mutans
led to increased levels of colonization and rampant carious lesions [156]. Co-cultivation of
S. mutans and C. albicans in biofilms revealed changes in the gene expression of S. mutans,
with an increase in carbohydrate metabolism, which could explain the increased severity
of caries observed in dual infections in vivo [161]. A second Candida species, Candida
dubliniensis, previously only isolated from immunocompromised individuals such as those
with HIV/AIDS, was detected in S-ECC [162] and caries-active children [163]. Furthermore,
another S-ECC study revealed C. albicans in association with, in addition to S. mutans,
other acidogenic and acid-tolerant species belonging to Lactobacillus and Scardovia [164].
A meta-analysis focused on evaluating the association of Candida with health and caries
reported that children with oral C. albicans were at a higher risk of caries than those without
the yeast species [157].
Together, these findings point to a role for interkingdom cooperation as a risk factor
for dental caries that goes beyond the observed enhancement of S. mutans-induced caries
by Candida.
5. Principal Caries-Associated Bacteria

5.1. Streptococcus mutans
S. mutans fulfils all of the criteria for species cariogenicity proposed by Van Houte [165].
These criteria include (i) physiological cell traits including acidogenicity and acid tolerance,
(ii) carious lesion production in animal models, and (iii) association with caries in humans.
S. mutans is one of the best-characterized oral symbionts and an important player in dental
caries, whose colonization of the dentition can define an early stage of caries development.
The primary energy source for S. mutans is sucrose, which is obtained by the bacteria
during host mealtimes and is broken down via the homolactic fermentation pathway [166].
However, S. mutans has evolved the ability to metabolize a diverse set of fermentable
carbohydrates, which allows the bacterium to outcompete other secondary colonizers and
reestablish the population dynamics of the developing biofilm in the aciduric stage of
the ecological plaque hypothesis [72,95]. The multiple sugar metabolism system (msm) in
S. mutans is mediated by a cascade of proteins, including ATP-binding cassette transporters,
which allow for the conversion of oligosaccharides into chemical energy [96]. Other com-
pounds, such as the disaccharide sucrose, are processed via the phosphoenolpyruvate:
sugar phosphotransferase system (PTS) [96]. In short, S. mutans has multiple pathways for
sugar metabolism, which, lacking in many other oral pathogens, allow the bacterium to out-
compete its neighbors in the increasingly acidic oral environment. Once S. mutans colonizes
the dentition about 4 h after a mealtime, a subsequent drop in pH can follow within an
hour, thereby establishing an environment that is more conducive to S. mutans survival and
persistence and less welcoming for the original acid-sensitive pioneer species [97]. This pH
drop may be considered a direct consequence of the heightened rate at which S. mutans is
able to metabolize dietary sugars that infiltrate the nutrient channels of the plaque biofilm.
In addition to generating chemical energy, S. mutans can also channel sucrose into the
extracellular polysaccharide (EPS) biosynthetic pathway to promote its tenacious and now
irreversible adherence to the tooth surfaces [96]. The copious quantities of extracellular
polysaccharide typically generated by S. mutans can serve as an extracellular nutrient
repository, as well as a diffusion barrier that traps lactic acid in close proximity to the tooth
surface [95]. This accumulation of acid is crucial to the aciduric stage of the ecological
plaque hypothesis, in which the plaque is dominated by aciduric microorganisms. Thus, it
is no wonder that S. mutans’ obligate biofilm lifestyle allows it to thrive in the human oral
cavity despite the transient environmental conditions and stressors imposed by proximate
bacterial residents and their metabolites. Because the S. mutans biofilm is a more successful
safeguard than those produced by other local microorganisms, the colonization of S. mutans
and secretion of its biofilm metabolites not only appoints the species a leading role in the
mouth but also fosters the survival of additional bacteria.
Research on caries-associated bacterial virulence factors has yielded controversial
results. Comparative genomics of S. mutans from caries-active and caries-free children
revealed genome homogeneity among isolates that ranged from 79.5% to 90.9%, and no spe-
cific genetic loci were identified for either group [167]. Differences in the putative virulence
genes of S. mutans clinical isolates were not found to be associated with caries status [168].
Other studies, however, reported that specific virulence-associated genetic loci, such as
the mutacin-encoding gene mutA, the adhesin-encoding genes for SpaP and Cnm, and
the glycosyltransferase modulating gene SMU.833 for S. mutans biofilm formation could
potentially contribute to S. mutans cariogenicity [169–172]. These variations in S. mutans,
along with other potential cariogenic microorganisms, illustrate the complexity of studying
caries-associated microbiomes and developing effective treatments. Furthermore, while
S. mutans has a solid association with dental caries, this species is not sufficient to explain
all carious lesions, since caries frequently occurs in the absence of S. mutans. A considerable
body of evidence has demonstrated that a range of low-pH non-mutans streptococci bacte-
rial species, in addition to other species, are involved in caries development [165,173,174].
Other caries-associated acidogenic and acid-tolerant species include those in Bifidobacterium,
Lactobacillus, Scardovia, and Actinomyces.
5.2. Lactobacillus Species

The genus Lactobacillus includes over 100 species that are widely distributed in nature,
including in plants, animals, insects, and food, principally dairy products, meat, and
beverages. Lactobacilli are gram-positive, non-sporulating rods, cocci, or coccobacilli that
are highly acidogenic and acid-tolerant. They require a fermentable carbohydrate source
for growth. In humans, Lactobacillus species colonize several body sites, including the
gastrointestinal and urinary tracts and the vagina, although the lactobacilli demonstrate
the highest species diversity in the oral cavity. While over 100 Lactobacillus species have
been identified in different ecological niches [175], many of the lactobacilli are considered
commensals or beneficial species in healthy humans [176,177]. The oral cavity harbors over
20 Lactobacillus species or phylotypes, 50% of which have been associated with active caries
in children or adults [178].
Lactobacilli can be detected in the oral cavity as early as the first two months of
life [179]. The colonization of Lactobacillus species in the mouths of infants and young chil-
dren has been correlated with the mother’s vaginal microflora, breastfeeding, pacifier use,
dietary habits, and the use of antibiotics [179–181]. Teanpaisan et al. reported vertical trans-
mission of Lactobacillus between mothers and their children in a Thai family study [182].
Only 50% of Lactobacillus DNA, however, was a match between mother–child pairs for
individual Lactobacillus strains. Early oral colonization by lactobacilli has been linked to an
increased risk for dental caries in young children [183], and oral Lactobacillus counts are
correlated with a higher prevalence and increased severity of dental caries [184,185]. Con-
versely, Lactobacillus species are infrequently detected in the saliva of caries-free individuals
and are only rarely isolated from dental plaque on sound tooth surfaces or from saliva or
dental plaque lacking S. mutans [185].
The association of Lactobacillus species with caries has varied between investigations
and microbiology methods. Using selective culture-based methods, lactobacilli were
detected at low proportions in the microbiota, with no clear association between individual
species and caries [102]. Several molecular techniques have been developed and introduced
to determine lactobacilli colonization in the oral cavity, including chromosomal DNA
fingerprinting, DNA probes, polymerase chain reaction (PCR) with Lactobacillus genus- and
species-specific primers, matrix-assisted laser desorption/ionization-time of flight mass
spectrometry (MALDI-TOF), and Lactobacillus 16S rDNA gene sequence analysis.
Using gene probes for species detection in less advanced caries revealed several
Lactobacillus species. Still, their associations varied in children with and without caries,
and none of the species predominated in the early carious lesion [186]. In caries-active
adults, on average, 2–8 distinct genotypes of Lactobacillus species were identified in each
individual [187,188]. More recently, multiple Lactobacillus species were detected in children
with S-ECC and in caries-free children (Figure 4A). The abundance and distribution among
those Lactobacillus species, however, differed significantly in the children with S-ECC versus
caries-free children [189] (Figure 4B).
Lactobacillus species were frequently detected in advanced dentin carious lesions [111,190].
As caries progresses, significant increases in Lactobacillus proportions have been observed [106].
Rocas et al. [191] used 16S rRNA gene sequencing to identify the bacteria in the micro-
biome that occupied the deepest layers of carious dentin, 42.3% of which were Lactobacillus
species, followed by Olsenella (13.7%), Pseudoramibacter (10.7%), and then Streptococcus
(5.5%). Half of the advanced caries lesions were dominated by lactobacilli, comprising
63% to 96% of the bacterial sequences in these samples (Figure 5) [191]. Caufield et al. re-
ported that the dominant species in both adult and childhood caries included L. fermentum,
L. rhamnosus, L. gasseri, L. casei/paracasei, L. salivarius, and L. plantarum. The less prevalent
species were L. oris and L. vaginalis [192]. Other clinical studies showed that once carious
lesions were successfully restored, the levels of Lactobacillus colonization were substantially
reduced [193,194].
none of the species predominated in the early carious lesion [186]. In caries-active adults,
on average, 2–8 distinct genotypes of Lactobacillus species were identified in each
individual [187,188]. More recently, multiple Lactobacillus species were detected in chil-
dren with S-ECC and in caries-free children (Figure 4A). The abundance and distribu-
Microorganisms 2024, 12, 121 tion among those Lactobacillus species, however, differed significantly in the children
20 of 45
with S-ECC versus caries-free children [189] (Figure 4B).
The widely divergent species and genotypes that colonize the oral cavity suggest
that the natural sources of Lactobacillus include exogenous and opportunistic colonizers
that reside outside of the human oral cavity, likely originating from food products or
other fermented materials [192]. For example, of the lactobacilli detected in caries, L. fer-
mentum and L. casei are among the species frequently detected in animals, plants, and
fermented
Figure
Figure foods,ofofconsistent
4.4.Diversity
Diversity with species.
oralLactobacillus
oral Lactobacillusaspecies.
dietary
(A) origin.
(A) L. gasseri,
Composition
Composition L. acidophilus,
of Lactobacillus
of Lactobacillus L.isolated
species
species vaginalis,
isolated L.
in
in the
crispatus,
the
oral oral
cavity and
cavity
of L. 5-year-old
3- of
to jensenii
3- are
to 5-year-old ordinary
children (N =colonizers
children (N (B)
74). = 74). of the
(B) healthy
Distribution
Distribution vagina (FigureLactobacillus
of twenty-one
of twenty-one Lactobacillus 6) species
and/or
species
identified identified
the gastrointestinal in with
in children children
tract. L.with
severe severe
salivarius
early early
and
childhood childhood
L.caries
rhamnosus caries
(S-ECC,are (S-ECC,
N =commonly
37) N used
= 37)without
and children as and children
probiotics
caries
without
to suppress
(CF, caries
N = 37). (CF, N = shows
pathogenic
The figure 37).intestinal
Thethat
figure shows that
thebacteria
abundance the
andandsoabundance and
are considered
distribution distribution
of thebeneficial of the Lactobacil-
organisms.
Lactobacillus species were
lus species were significantly different between the two groups of children [189].
significantly different between the two groups of children [189].
Lactobacillus species were frequently detected in advanced dentin carious lesions

[111,190]. As caries progresses, significant increases in Lactobacillus proportions have
been observed [106]. Rocas et al. [191] used 16S rRNA gene sequencing to identify the
bacteria in the microbiome that occupied the deepest layers of carious dentin, 42.3% of
which were Lactobacillus species, followed by Olsenella (13.7%), Pseudoramibacter (10.7%),
and then Streptococcus (5.5%). Half of the advanced caries lesions were dominated by lac-
tobacilli, comprising 63% to 96% of the bacterial sequences in these samples (Figure 5)
[191]. Caufield et al. reported that the dominant species in both adult and childhood car-
ies included L. fermentum, L. rhamnosus, L. gasseri, L. casei/paracasei, L. salivarius, and L.
plantarum. The less prevalent species were L. oris and L. vaginalis [192]. Other clinical
studies showed that once carious lesions were successfully restored, the levels of Lacto-
bacillus colonization were substantially reduced [193,194].
Taken together, these observations suggest that a carious lesion could represent the
primary ecological niche for lactobacilli colonization, furthering the notion that the lac-
tobacilli–caries relationship could be species-specific [179,182,185,186].
Most Lactobacillus species in carious lesions cohabitate with other lactobacilli. Mul-
tiple Lactobacillus species can promote caries progression in dentinal lesions. The reduc-
tion in Lactobacillus colonization once carious lesions were successfully restored is due to
the loss of Lactobacillus’ ecological niche in the cavities [186,187,190–194]. Taken together
with L. fermentum, L. gasseri, L. casei, L. salivarius, L. rhamnosus, and L. plantarum as the
most frequently detected taxa in advanced dentin caries, these species could potentially
be classified
Figure
Figure 5. as caries
5. Relative
Relative pathogens
abundance
abundanceofof [187,195].
thethe
30 30
most prevalent
most bacterial
prevalent genera
bacterial detected
genera in advanced
detected caries
in advanced
[191]. [191].
caries Bacterial samples
Bacterial were taken
samples were from
takendeep
fromocclusal (dentin)(dentin)
deep occlusal caries incaries
permanent molars from
in permanent mo-
10 individuals. DNA was extracted for 16S rRNA gene (V4 variable region) sequence
lars from 10 individuals. DNA was extracted for 16S rRNA gene (V4 variable region) sequence analysis. Ap-
proximately 347,646 partial 16S rRNA gene sequences were obtained. Overall, the Lactobacillus
analysis. Approximately 347,646 partial 16S rRNA gene sequences were obtained. Overall, the ge-
nus accounted for 42.3% of the sequences. The relative abundance per case in five samples ranged
Lactobacillus genus accounted for 42.3% of the sequences. The relative abundance per case in five
from 63% to 96% of the bacterial sequences.
samples ranged from 63% to 96% of the bacterial sequences.
The relative
Taken importance
together, of individual
these observations Lactobacillus
suggest species
that a carious to could
lesion dentalrepresent
caries; their ori-
the pri-
gins, transmission and colonization pathways; their diversity, stability and roles
mary ecological niche for lactobacilli colonization, furthering the notion that the lactobacilli– in caries
ecology;
caries and theircould
relationship interactions with other [179,182,185,186].
be species-specific acid-sensitive or cariogenic bacterial species
needMostfurther investigation and clarification.
Lactobacillus species in carious lesions Clinically,
cohabitatethewith
presence
other of Lactobacillus
lactobacilli. in the
Multiple
oral cavity has been used as an indicator of fermentable carbohydrates
Lactobacillus species can promote caries progression in dentinal lesions. The reduction in and a caries-
inducing oral
Lactobacillus environment.
colonization onceCommercial
carious lesionschairside tests measuring
were successfully salivary
restored is dueLactobacillus
to the loss
counts based on selective isolation have been used in clinical studies
of Lactobacillus’ ecological niche in the cavities [186,187,190–194]. Taken together for caries risk as-
with
L.sessment
fermentum,andL.post-treatment
gasseri, L. casei,evaluations [193].
L. salivarius, Taken together,
L. rhamnosus, and L.this information
plantarum as theshould
most
help scientists and clinicians improve caries management.
frequently detected taxa in advanced dentin caries, these species could potentially be
classified as caries pathogens [187,195].
The widely divergent species and genotypes that colonize the oral cavity suggest that
the natural sources of Lactobacillus include exogenous and opportunistic colonizers that
reside outside of the human oral cavity, likely originating from food products or other
fermented materials [192]. For example, of the lactobacilli detected in caries, L. fermentum
and L. casei are among the species frequently detected in animals, plants, and fermented
foods, consistent with a dietary origin. L. gasseri, L. acidophilus, L. vaginalis, L. crispatus, and L.
Microorganisms 2024, 12, 121 jensenii are ordinary colonizers of the healthy vagina (Figure 6) and/or the gastrointestinal 22 of 47
tract. L. salivarius and L. rhamnosus are commonly used as probiotics to suppress pathogenic
intestinal bacteria and so are considered beneficial organisms.
100%
Vaginal (n=194) Gingival (n=194)
80%
% Subjects
60%
40%
20%
0%
Figure 6. Lactobacillus species detected in vaginal and gingival samples. Oral (gingival) and vaginal
Figure 6.were
samples Lactobacillus species
taken from detected in
194 pregnant vaginalinand
women the gingival samples.
first trimester Oral
[196]. (gingival)
Samples wereand vaginal
analyzed
samples
using DNAwere taken
gene fromin194
probes pregnant women
a checkerboard formatin with
the first
a 10trimester [196].
5 threshold Samples
of species were analyzed
detection. Of the
using DNA gene probes in a checkerboard format with a 105 threshold of species detection. Of the
Lactobacillus species assayed, most were detected in both sample sites, suggesting that the vagina
Lactobacillus species assayed, most were detected in both sample sites, suggesting that the vagina
could be a source of oral lactobacilli in infants. In contrast, other species typical of subgingival
could be a source of oral lactobacilli in infants. In contrast, other species typical of subgingival
sites, including Porphyromonas
sites, including and Prevotella
Porphyromonas and Prevotella species,
species, were
were detected
detected more
more frequently
frequently in
in the
the gingival
gingival
samples. * Difference detection, Chi-square >
samples. * Difference detection, Chi-square > 0.05. 0.05.
The relative importance of individual Lactobacillus species to dental caries; their origins,
5.3. Actinomycetaceae
transmission and colonization pathways; their diversity, stability and roles in caries ecology;
Actinomyces
and their and with
interactions related species
other have been
acid-sensitive recognizedbacterial
or cariogenic as members
speciesofneed
plaque bio-
further
films for many decades. Current taxonomy recognizes several families and
investigation and clarification. Clinically, the presence of Lactobacillus in the oral cavity genera in the
Actinobacteria class that, based on reports in the literature, are relevant
has been used as an indicator of fermentable carbohydrates and a caries-inducing oral to dental caries.
These includeCommercial
environment. species in the generatests
chairside Actinomyces,
measuring Rothia,
salivaryBifidobacterium, Parascardovia,
Lactobacillus counts based
Scardovia,
on Corynebacterium,
selective isolation have Olsenella,
been usedand Atopobium,
in clinical the taxonomy
studies of which
for caries risk can beand
assessment ac-
cessed via the eHOMD
post-treatment evaluations database,
[193]. version 3.1 (https://www.homd.org/taxa/tax_table).
Taken together, this information should help scientists
Only a few
and clinicians species,
improve however,
caries have been examined for cariogenic traits as opposed
management.
to overall disease association. Specifically, Actinomyces were cultured from plaque and
5.3. Actinomycetaceae
dentin samples associated with carious lesions in children [100] and A. viscosus was
identified as theand
Actinomyces dominant
related species in root
species have caries
been in adults
recognized as [126].
members While A. viscosus
of plaque can
biofilms
lower
for thedecades.
many pH belowCurrent
5 in glucose broth,recognizes
taxonomy it was not several
as acidogenic
families orand
acid-tolerant
genera in as
theS.Acti-
mu-
tans isolates
nobacteria at an
class initial
that, basedacidic pH [197].
on reports in theMoreover,
literature,in areexperimental animals,
relevant to dental A. israelii
caries. These
was found
include to beinassociated
species the generawith root caries,
Actinomyces, but not
Rothia, with coronalParascardovia,
Bifidobacterium, caries [198]. Taken col-
Scardovia,
Corynebacterium, Olsenella,
lectively, and despite and Atopobium,
the taxonomy the taxonomy
of Actinomyces beingofupdated
which can be accessed
several via the
times since the
eHOMD
1980s, thedatabase,
actinomyces version 3.1 (https://www.homd.org/taxa/tax_table).
continue to be consistently isolated from carious lesions.
The association of individual Actinomyces species with caries status supports species
specificity in health and disease. Strains of 24 different Actinomyces species were report-
ed as having a broad-based ability to lower pH when grown under neutral (pH7) and
acidic (pH5.5) conditions [114]. When grown in glucose broth, some Actinomyces species,
including Actinomyces johnsonii, Actinomyces graevenitzii, and several unnamed species,
Only a few species, however, have been examined for cariogenic traits as opposed to
overall disease association. Specifically, Actinomyces were cultured from plaque and dentin
samples associated with carious lesions in children [100] and A. viscosus was identified
as the dominant species in root caries in adults [126]. While A. viscosus can lower the pH
below 5 in glucose broth, it was not as acidogenic or acid-tolerant as S. mutans isolates at
an initial acidic pH [197]. Moreover, in experimental animals, A. israelii was found to be
associated with root caries, but not with coronal caries [198]. Taken collectively, and despite
the taxonomy of Actinomyces being updated several times since the 1980s, the actinomyces
continue to be consistently isolated from carious lesions.
The association of individual Actinomyces species with caries status supports species
specificity in health and disease. Strains of 24 different Actinomyces species were reported
as having a broad-based ability to lower pH when grown under neutral (pH7) and acidic
(pH5.5) conditions [114]. When grown in glucose broth, some Actinomyces species, including
Actinomyces johnsonii, Actinomyces graevenitzii, and several unnamed species, were capable
of lowering pH < 4 and were nearly as acid-tolerant as S. mutans and S. sobrinus, consistent
with the characteristics of known cariogenic bacteria. Other Actinomyces species, including
Actinomyces massiliensis, Actinomyces georgiae, and Actinomyces meyeri, were less acidogenic,
reaching final pH values close to pH 5, which is like the acidogenic potential of species
known to reside in caries-free sites. Thus, these differences in acidogenic and acid-tolerant
properties for Actinomyces species support the specificity of Actinomyces in both health and
disease and that a lack of species differentiation will obscure caries associations.
Bifidobacteria have been associated with dental caries since the early 1900s, including
in a study of childhood caries in 1917 [58]. Bifidobacteria isolated from root caries were
highly acidogenic [127], although cariogenicity testing in experimental animals failed to
show significant caries induction, which was attributed to poor colonization in the animals
tested [199,200]. The current taxonomy of the bifidobacterial group recognizes several
species that colonize the oral cavity, including species in the genera Scardovia, Parascardovia,
and Alloscardovia [113,201]. S. wiggsiae was significantly associated with S-ECC in young
children based on molecular analysis of the oral clone CX010 [106] and an anerobic culture
study [113]. A selective medium was used to isolate several Bifidobacterium species from
occlusal lesions in both children and adults, including B. dentium, P. denticolens, Scardovia
inopinata, S. wiggsiae (Scardovia genosp. C1), and Bifidobacterium longum [201]. The results
of selective isolation experiments also revealed a strong association of bifidobacteria with
increasing root caries severity [130].
Bifidobacterium species have been associated with clinical models of disease progression.
Scardovia inopinata was associated with the demineralization of enamel and dentin sections
worn in oral appliances [155]. White-spot initial carious lesions in adolescents with fixed
orthodontic appliances were associated with S. wiggsiae in cross-sectional studies [122]
and with S. wiggsiae and B. dentium in longitudinal monitoring [123]. Further, higher
proportions of bifidobacteria were isolated from caries-active than caries-free children [202].
These findings implicate Bifidobacterium species as playing a larger role in more advanced
carious lesions than in early lesions. Furthermore, higher proportions of children with
advanced caries had Bifidobacterium species and S. wiggsiae in saliva compared with initial
caries and caries-free children [49]. Comparison of bacteria from a metatranscriptomic
analysis of dentin caries detected higher levels of S. wiggsiae and B. dentium compared with
coronal caries and caries-free children [142] (Figure 3).
Clinical and in vitro studies support a symbiotic relationship between Scardovia and
bifidobacteria with S. mutans. For instance, in children’s saliva samples, there was a positive
association between S. wiggsiae and S. mutans [49]. Analysis of adolescents with lower and
higher levels of S. mutans revealed an association of S. wiggsiae with S. mutans in association
with more aggressive caries [154]. Bifidobacterium species did not form biofilms as a single
species when inoculated onto glass slides [203]. This lack of biofilm growth may in part
explain why it was difficult to implant bifidobacterial [199] or S. wiggsiae [200] in animal
models for cariogenicity testing. In vitro biofilms did, however, form when strains were
co-infected with S. mutans [203], and, for P. denticolens and Scardovia inopinata, lower pH
values were achieved in dual-species culture than when either species was cultured alone.
In other in vitro assays, S. wiggsiae strains were found to be as acidogenic and acid-tolerant
as S. mutans [114].
These findings reflect a resurgence in interest in Actinobacteria, especially in Scardovia-
related species. Findings from dual-species analysis indicate the importance of considering
the action of the whole microbial community rather than relying on the activity of individual
species to fully understand their cariogenic potential.
6. Microbiome: Beyond Microbial Composition and the Oral Microbiome:

Functional Genomics
6.1. Metagenome
Although 16S rRNA gene-based microbial profiling can reveal the details of microbial
composition associated with health or disease, this information does not readily translate
into the genetic potential and functional capacity of microbes [204,205]. This is particularly
important for the study of dental caries. It has been shown that the composition of the
microbial consortia associated with health and caries status varies significantly between
individuals and between sites, and that different bacterial consortia may be responsible for
caries pathogenesis [72]. Thus, there are limitations in taxonomy-based studies that can
allow different bacterial consortia to perform similar functions due to the large redundancy
and plasticity of microbes [206]. Meanwhile, accumulating evidence indicates that genomic
makeup and the genetic and metabolic potential of different strains within the same species
(based on 16S rRNA genes) can display high variability at the strain level [169]. Thus,
species-level identifications may not provide adequate information for identifying the
pathogen or understanding the disease process.
Metagenomics is a DNA sequencing approach that allows for the study of the ge-
netic material recovered directly from environmental or host-associated samples. In metage-
nomics, whole-genome shotgun (WGS) sequencing is used. That is, entire DNA samples are
randomly sheared by a “shotgun” method and the resulting short fragments are sequenced
by NGS [207]. The comprehensive sequences can then be analyzed to obtain either bacterial
profiles based on 16S rRNA genes or genomic profiles based on whole genomes [208,209].
Metagenomics not only provides more precise taxonomic diversity and phylogenetic com-
position data associated with specific health/disease status due to the longer 16S RNA
gene sequences obtained, it also offers insight into the genetic potential and patterns of
the metabolic modules/pathways of complex microbial communities, which have been
especially informative in studies of periodontal and caries etiology [99,210,211].
Using metagenomic sequencing analysis, Xie et al. report that the predominant func-
tional categories within a healthy human plaque microbiome include the metabolism of
carbohydrate (11.88% of the assigned reads), amino acids and their derivatives (7.89%), and
proteins (9.34%) [212]. Furthermore, about 2.8% of the total predicted protein-encoding
genes were involved in antibiotic resistance and toxic compound tolerance. Among these
were resistance genes for the major classes of antibiotics, such as β-lactams, aminoglyco-
sides, fluoroquinolones, and the peptide antibiotic bacitracin, as well as genes for general
multidrug or heavy-metal resistance functions, such as efflux pumps.
Comparison of metagenomic DNA sequence data allows the identification of gene
functions or metabolic pathways that are over-represented in, and thus positively associ-
ated with, microbiomes related to health or caries. For example, a direct comparison of
metagenomics DNA sequence data derived from caries-free and caries-active subjects will
likely reveal those genes and functional pathways that promote species adaptation and
strain-level niche exploitation in an acidic microenvironment. The genes or metabolic path-
ways crucial for these adaptations can be revealed as “enriched” in caries-active compared
with caries-free microbiomes.
Using comparative metagenomics, Belda-Ferre and colleagues identified more genes
for mixed-acid fermentation and DNA uptake in microbiomes associated with two subjects
with active caries compared with two healthy individuals [213]. Analysis of the metabolic
potential of the caries metagenome showed that samples from diseased individuals tended
to cluster together, suggesting similar sets of functions were present in their metagenomes.
Interestingly, the oral microbiomes from healthy subjects included significantly enriched
genes involved in the biosynthesis of antibacterial peptides, such as bacteriocins; periplas-
mic stress-response genes like degS and degQ; capsular and extracellular polysaccharides;
and bacitracin stress-response genes; while samples from caries-active subjects had a high
frequency of genes involved in functions such as iron scavenging and oxidative and osmotic
stress response.
The results of these metagenomic studies suggest that specific metabolic genes and
pathways are associated with oral diseases, although more in-depth and functionally
informative analyses are required to confirm these conclusions. Some of these genes may
have the potential to become diagnostic markers.
6.2. Metatranscriptome
Although a metagenomic approach can reveal the total genetic potential of a microbial
community, it cannot elucidate which genes are being actively expressed in real time. The
human oral microbiome exists in a continuously changing environment where pH, organic
carbon, and oxygen levels fluctuate on a hundred-fold or even a thousand-fold scale within
minutes [214,215]. All of these dynamic environmental changes can have varying impacts
on the metabolic activity of different bacterial species in the community and so affect the
genes that are actively expressed by that community. Thus, detecting and analyzing the
gene transcripts of a microbial community offers real-time information that can help detect
the metabolically active microbial members and identify the genes expressed under a given
set of conditions.
Metatranscriptomic analysis characterizes the gene expression profiles of entire mi-
crobial communities based on the sets of transcripts that are synthesized under diverse
environmental conditions. Instead of addressing the question “what are oral microbes
capable of doing?”, as in metagenomics, metatranscriptomics offers answers to “what are
oral microbes actually doing?” [216,217].
Using a metatranscriptomic approach, Simon-Soro et al. identified the RNA-based,
metabolically active bacterial compositions of carious lesions at different stages of disease
progression in an effort to provide a list of potential etiologic agents for tooth decay [218].
They demonstrated that the microbiota associated with dental caries in adults is highly
complex, with each sample containing between 70 and 400 metabolically active species.
The compositions of these bacterial consortia varied among individuals and between caries
lesions in the same individuals. Enamel and dentin lesions also had a different makeup
of metabolically active microbes. In sum, their data confirmed that the etiology of dental
caries is likely tissue-dependent and that the disease has a clear polymicrobial origin. The
relatively low proportion of S. mutans detected in the caries sites examined indicated that
this bacterial species can be present as a minority, leading the authors to question the
importance of S. mutans as the main etiological agent in dental caries. Alternatively, these
observations implicate S. mutans as a keystone pathogen that, despite its low abundance
at the caries site, could remodel the caries microbiota to favor dysbiosis [219] and form
concentrations of S. mutans above localized sites of demineralized enamel [125].
A study of the metatranscriptome of progressing early childhood caries lesions, how-
ever, indicated a clear positive association of genes mapping to S. mutans from coronal
(enamel) and dentin lesions compared with caries-free children [142] (Figure 3). While
there was considerable variability in microbial gene expression within the clinical groups,
similar to that observed in adults [218], total enzyme expression was higher in dentin caries
compared with coronal caries and caries-free samples [142]. Further, there was greater
diversity in the operational taxonomic units (OTUs) that genes from dentin mapped to
than in those from coronal caries, although sample sizes were smaller from dentin than
from coronal sites. Functional profiling revealed higher levels of alcohol dehydrogenase
from Neisseria sicca and choline kinase from several non-mutans streptococci in caries-free
samples; hence, both taxa are associated with healthy plaque. The expressed sequences
in coronal (enamel) caries from S. mutans were mainly derived from DNA ligase genes,
suggesting increased metabolic activity in that species. A wider range of gene expression
activity was observed in dentin than in coronal caries, including expression of uracil DNA
glycosylase from S. wiggsiae and urease from A. naeslundii. Moreover, the higher levels of
gene expression patterns mapping to S. wiggsiae in dentin caries compared with coronal
caries, suggest S. wiggsiae could be a major player in caries progression in dentin. Overall,
metatranscriptomic analysis revealed some differences in the enzyme/metabolic activities
that were expressed in microbiomes associated with dentin caries, coronal caries, and
caries-free samples, respectively [142].
Metatranscriptomic studies thus revealed marked differences in bacterial composition
and metabolism between coronal and dentin caries, suggesting that the microbial commu-
nities in enamel lesions exhibited different functions than those of more advanced, dentin
cavities, where bacteria appear to be specialized in utilizing sugars associated with dentin
tissue and degrading proteins. These data support the hypothesis that dental caries is
not a single disease but a tissue-dependent process with different etiologies, as discussed
above [99] and consistent with the ecological hypothesis for dentin caries proposed by
Takahashi and Nyvad [73].
Using RNA-seq to perform global gene expression analysis of the dental plaque micro-
biota derived from 19 twin pairs that were either concordant (caries-active or caries-free) or
discordant for dental caries [220] revealed similarities in gene expression patterns. Thus,
this twin-pair study allowed an assessment of the relative contributions of human genet-
ics, environmental factors, and caries phenotype on the microbiota’s transcriptome. The
results revealed transcripts encoding functions related to monosaccharide and disaccha-
ride metabolism, accounting for a significant portion of the dental biofilm transcriptome
(around 15% of the total) [220]. Transcripts encoding functions associated with disaccharide
metabolism were more prevalent than those associated with monosaccharide metabolism
by a factor of two. Interestingly, transcripts encoding functions related to antibiotic resis-
tance and tolerance of toxic compounds were also expressed in the oral biofilm, while genes
involved in bacteriocin expression and acid stress represented only a small fraction of all
transcripts across all individuals. Furthermore, correlation analysis of transcription identi-
fied several functional networks, suggesting that inter-personal environmental variables
may co-select for groups of genera and species [220].
By applying a novel computational framework (metaModules) for the automated
identification of key functional differences between health and disease-associated commu-
nities, May et al. [221] were able to identify key functional subnetworks that are relevant
to dental caries, as noted in the metatranscriptomic data obtained by Peterson et al. [220].
Their results showed that the health-associated KEGG Orthology groups (Kos) were mainly
constituents of the pathways associated with amino acid biosynthesis and pyrimidine,
purine, and pyruvate metabolism, as well as glycerophospholipid metabolism. The caries-
associated subnetwork included nine Kos from the pathways of the phosphotransferase
system and fructose/mannose metabolism, supporting the notion that microbial carbohy-
drate acquisition and catabolism are important in dental caries etiology [96].
In sum, metatranscriptomic analysis allows one to gain insight into the genes that are
actively expressed in complex bacterial communities, enabling the elucidation of dynamic
functional changes that govern the microbiome’s functions in given contexts, its interactions
with the host, and the functional alterations that accompany the transition of a healthy
microbiome into a dysbiotic one.
6.3. Multi-Omics
With the advancement of sequencing technologies, protein and small-molecule anal-
yses, and the development of new bioinformatics tools, studies of host-associated micro-
biomes, including the caries microbiome, have entered a new era. Investigators are now
capable of generating information on the functional activity of whole microbial commu-

nities. Metagenomics, which entails sequencing the entirety of the DNA from a given
sample, can provide information not only about which organisms are present, but also
their functional potential, through analysis of metabolic pathway genes and the use of
protein-coding sequence databases. Meanwhile, current investigations in the oral cav-
ity and beyond that combine metatranscriptomics and metagenomics have shown that,
in many cases, the gene sets that are expressed are more important in predicting health
compared with disease than the species that are present [222,223]. Furthermore, meta-
proteomics, metabolomics, and small-molecule analysis have also been applied to study
in vivo [224,225] and in vitro [226,227] multispecies oral microbial communities to com-
plement sequencing data by providing higher-level functional information. Combining
systems-level analyses that generate functional information with knowledge of abundance
and spatial structure can provide invaluable insights into the intricate microbial network of
the oral microbiome, its relationship to the human host, and its role in caries pathogenesis.
7. Treatment of Caries as a Microbial Disease

Treatment approaches for dental caries have evolved, with an increased understanding
of the cariogenic microbiome and the development of new therapies. Since dental caries,
however, has a multifactorial etiology with dietary considerations in addition to bacteria
and host factors, successful and lasting therapy requires attention to all these factors. The
most effective antibacterial therapy is doomed to relapse in the absence of diet manage-
ment. Treatment of dental caries has incorporated suppressing the cariogenic bacteria and
increasing the tooth’s resistance to demineralization. The most frequent approach for caries
prevention, good plaque control through oral hygiene, while effective [228], can be difficult
for individuals to achieve. Thus, additional preventative measures are usually needed,
especially in high-risk populations or once carious lesions have developed.
Earlier approaches to caries treatment related to the microbiota focused on reducing
the colonization and transmission of caries-associated bacteria using antimicrobials and
fluoride-containing formulations. More recent treatment measures have focused on rebal-
ancing the dysbiotic caries microbiome with specific therapies targeting either S. mutans
alone or the acidogenic, acid-tolerant microbiome with the goal of re-establishing a symbi-
otic microbiome compatible with health. Ecological approaches have also been proposed to
avoid the development of a dysbiotic community associated with caries [229]. Highlights
of these approaches are described below.
In studies in the 1970s, topical applications of broad-spectrum antimicrobials, such
as kanamycin and vancomycin, were used with the goal of eliminating S. mutans and
disinfecting carious lesions before restorations were placed [230,231]. Suppression of S.
mutans levels in tooth pits and fissures by antimicrobial agents, however, was short-lived
because agents failed to penetrate the plaque biofilm or tooth demineralized zones. Thus,
this approach was ineffective in reducing counts of target species in carious lesions over
time. In some cases, there were significant increases in the proportions of S. mutans in the
plaque community due to the non-specific bacterial reduction of other species, and caries
progressed twelve months after treatment [230]. Other therapeutic agents evaluated in-
cluded chlorhexidine (CHX), povidone-iodine (PVP-I), casein phosphopeptide-amorphous
calcium phosphate (CPP-ACP), xylitol [232], arginine deiminase system (ADS) [233,234],
tea- and cranberry-derived polyphenols [235], and blue light [236]. In clinical studies, how-
ever, these antimicrobial approaches have had ambiguous results in suppressing cariogenic
bacteria or changing oral microbial ecology [232,237].
7.1. Fluoride and Anticaries Activity

7.1.1. Fluoride as a Preventative Anticaries Agent
Fluoride is the most frequently used agent for caries prevention [238]. The widespread
use of fluoride in water, toothpaste, gels, varnishes, mouthrinses, tablets, drops, milk, and
salt for caries prevention in topical or systemic formulations has been an effective and
significant contributing factor to the remarkable decline in tooth decay in the decades since
it was introduced. Community-based fluoridation in drinking water (≥0.7 parts per million
F) represents a significant public health achievement of the 20th century for its efficacy in
preventing dental caries [12] that has been consistently supported by scientific evidence
worldwide. A study report of 2018 estimated that the preventive fraction for US children
and adolescents was 30% (95% CI 11–48%) for primary teeth and 12% (95% CI 1%, 23%)
for permanent teeth [239]. This cost-effective community-based approach is dependent
on access to drinking water systems and public health policies at national, state, or local
governmental levels [240]. Use of toothpastes or mouthrinses containing sodium fluoride
(NaF), amine fluoride (AmF), acidulated phosphate fluoride (APF), and stannous fluoride
(SnF2 ), among others, reduces plaque accumulation on tooth surfaces [241] and lowers the
cariogenicity of dental plaque [242]. Two mechanisms described as underlying fluoride
anticaries activity—antimicrobial action and promoting enamel hardness via chemical
reaction—will be described in the following two subsections.
7.1.2. Antimicrobial Properties of Fluoride

Fluoride’s anticaries activity as an antimicrobial has been known for several decades [243].
Elemental fluoride can inhibit bacterial carbohydrate metabolism and extracellular polysac-
charide (extracellular polymeric substances, EPS) production and thus reduce bacterial
adherence and the growth of cariogenic bacteria [244]. The targets of action involve (1) sugar
transport via the phosphoenolpyruvate-protein (PEP) phosphotransferase system; (2) sugar
uptake via the protonmotive force (PMF)-associated system; (3) proton-extruding ATPase
(H+ -ATPase); (4) biosynthesis of macromolecules related to bacterial cell division; and
(5) lactate/proton efflux [245]. Fluoride also modifies the plaque ecosystem and influences
bacterial interactions and composition within the plaque community [246].
In a mixed oral species culture model that included S. mutans, S. sanguinis, S. oralis,
Actinomyces naeslundii, Neisseria subflava, Veillonella dispar, Fusobacterium nucleatum, Pre-
votella nigrescens, and Porphyromonas gingivalis, 10 days after exposing to glucose with NaF,
proportions of S. mutans were significantly reduced to <3% of the species mix, compared
with the culture without fluoride (Figure 7). This study suggests that fluoride exerts antimi-
crobial activity against S. mutans by inhibiting critical metabolic processes (direct effect)
and reducing acid production (indirect effect) in biofilms [247]. A later report showed that
NaF inhibited S. mutans more than lactobacilli depending on the Lactobacillus species and
NaF concentration [248].
Clinically, long-term exposure to fluoride reduced the salivary levels of mutans strep-
tococci, lowered caries scores among school children [249,250], and prevented enamel dem-
ineralization [251] and root caries among adults [4]. In other studies, minimal changes in the
microbiota were observed on fixed orthodontic appliances following fluoride mouthrinses
containing 100 ppm AmF and 150 ppm SnF2 [252]. Further, short-term changes in dental
plaque accumulation were observed in pediatric patients after a topical treatment with
0.4% SnF2 or 10% povidone iodine (PVP-I) plus 5% NaF varnish. Further, there was a
minimal antimicrobial effect on the microbial communities of children at high risk for
caries, suggesting that bacteria within dental plaque were resilient to these treaments and
maintained the community structure and ecosystem [253,254].
Fluoride varnish (FV) is a frequently used formulation in clinical practice. FV applied
topically can form globules with fluoride and calcium elements on enamel surfaces [255].
Formulations of FV include those with 5% NaF, 0.9% difluorosilane (SiH2 F2 ), and 6% NaF
with 6% CaF [238]. In three studies, including a review of 22 trials involving 12,455 children
and adolescents worldwide, professionally applied FV (two to four times a year) was
associated with a reduction of 18% to 43% in caries incidence in permanent teeth and a
37% reduction in the primary teeth of children and adolescents compared with placebo
or no intervention [255–258]. Administration of FV containing 5% NaF every three to six
months is the recommended treatment for children with an increased caries risk [259].
For children with early childhood caries, however, clinical studies on FV antimicrobial
molecules related to bacterial cell division; and (5) lactate/proton efflux [245]. Fluoride
also modifies the plaque ecosystem and influences bacterial interactions and composi-
tion within the plaque community [246].
In a mixed oral species culture model that included S. mutans, S. sanguinis, S. oralis,
Microorganisms 2024, 12, 121 Actinomyces naeslundii, Neisseria subflava, Veillonella dispar, Fusobacterium nucleatum, 28 of 45
Prevotella nigrescens, and Porphyromonas gingivalis, 10 days after exposing to glucose with
NaF, proportions of S. mutans were significantly reduced to <3% of the species mix,
efficacy
compared have yielded
with mixedwithout
the culture results. Topical
fluoridefluoride
(Figure application
7). This studyalone, or in combination
suggests that fluoride
with
exertsother antimicrobial
antimicrobial agents,
activity wasS.reported
against mutans byto lead to onlycritical
inhibiting a short-term reduction
metabolic processes in
mutans streptococci
(direct effect) or lactobacilli
and reducing levels in the
acid production saliva and
(indirect dental
effect) plaque [247].
in biofilms [260,261]. Other
A later re-
studies, however,
port showed thatshowed no direct
NaF inhibited S. correlation
mutans more between fluoride release
than lactobacilli and antimicrobial
depending on the Lac-
activity [262,263], and, therefore, more intensive
tobacillus species and NaF concentration [248]. regimens have been proposed [264].
Figure 7. Acid production from a glucose solution with added fluoride on in vitro dental biofilms.
Figure 7. Acid production from a glucose solution with added fluoride on in vitro dental bio-
Effects of a glucose
films. Effects solutionsolution
of a glucose with added
withfluoride on the inon
added fluoride vitro
thepH of dental
in vitro biofilms.
pH of dental pH dropped
biofilms. pH
in 10-day in
dropped biofilms
10-dayafter an overlay
biofilms after anofoverlay
glucoseoforglucose
glucoseor+ glucose
10 ppm +fluoride
10 ppm[247].
fluoride [247].
Silver diamine
Clinically, fluoride
long-term (SDF) tototreat
exposure cariesreduced
fluoride is basedthe
on the activity
salivary of both
levels silver
of mutans
and fluoride. For centuries, elemental silver (Ag) has been known to exhibit antimicrobial
streptococci, lowered caries scores among school children [249,250], and prevented
effects due to its properties as a heavy metal. Silver was the basis of “Howe’s solution”,
used to treat rampant caries in children 100 years ago. The antimicrobial effect of silver
compounds was established for the prevention and treatment of infections in medicine [265].
In dentistry, SDF’s caries-arrest effect was demonstrated in the late 1960s and early 1970s in
Japan. Application of topical SDF, while causing black staining on treated tooth surfaces,
was effective in arresting caries progression in 66% to 75% of primary teeth [266,267], 41%
to 65% of first permanent molars [268,269], and 18% to 71% of root caries in elders [270].
Antimicrobial mechanisms for caries suppression by SDF include, at the cellular
level, interference with bacterial amino and nucleic acid formation, alteration of cell-wall
synthesis and cell division, and disabling of metabolic and reproductive functions, leading
to inhibition of bacterial growth and biofilm formation [271]. An antibacterial effect of SDF
against S. mutans growth with inhibition of dextran-induced agglutination was reported
in the 1970s [272]. Subsequent studies showed that a 3.8% SDF solution could effectively
suppress Enterococcus faecalis biofilm formation [273], which could be valuable for treating
root canal infections. Knight et al. conducted a series of experiments demonstrating that
application of SDF reduced S. mutans viability and inhibited biofilm formation, reducing
the bacteria-induced demineralization of dentin and caries progression [274,275]. Chu and
Mei et al. reported the antimicrobial effect of a 38% SDF solution against multi-species
cariogenic complexes (S. mutans, S. sobrinus, L. acidophilus, L. rhamnosus, and Actinomyces
naeslundii) on dentin carious lesions [276,277]. A formulation of SDF with potassium iodide
(SDF/KI) reduced S. mutans colonization in dentinal tubules compared with chlorhexidine
or other chemo-mechanical products [278]. Furthermore, SDF suppressed S. mutans biofilm
formation on dentin caries models with a superior antibacterial effect against S. mutans
compared with NaF and with SDF + NaF (Figure 8) [279].
zation of the enamel and dentin [286]. In addition to the formation of an impermeable
layer of silver phosphate, calcium fluoride, and fluorohydroxyapatite in SDF-treated car-
ious lesions, highly concentrated silver precipitation zones form around the carious le-
sions, thereby blocking dentin tubules, which could contribute to the physiochemical
Microorganisms 2024, 12, 121 role of silver compounds in caries arrest. 29 of 45
Figure 8. Effects of sodium diamine fluoride (SDF) on S. mutans biofilms cultured on dentin blocks
Figure 8. Effects
in vitro. Shownof sodium diamine
are scanning fluoride
electron (SDF) on S.
micrographs mutans
(SEM, leftbiofilms cultured
panels) of on dentin
the biofilm blocks and
topography
in vitro. Shown are scanning electron micrographs (SEM, left panels) of the biofilm topography
the images from confocal laser scanning microscopy (CSLM, right panels) of the S. mutans biofilm on
dentin caries lesions treated with (a) 38% SDF solution plus 5% NaF varnish; (b) 38% SDF solution
alone; (c) 5% NaF; and (d) a water-treatment control. The green color represents live bacterial cells;
the red color represents dead bacterial cells (magnification ×1000). The green intensity represents the
amount of biofilm formed in the carious lesions. Modified based on Yu et al. 2018 [279].
7.1.3. Chemical Action of Fluoride to Strengthen Teeth

Fluoride affects the enamel structure with the formation of fluorapatite, which resists
demineralization and enhances the remineralization of incipient lesions compared with
hydroxyapatite without fluoride. Fluoride compounds, including sodium fluoride (NaF),
sodium monofluorophosphate (MFP, Na2 PO3 F), stannous fluoride (SnF2 ), amine fluoride
(AmF), acidulated phosphate fluoride (APF), zinc fluoride (ZnF2 ), titanium tetrafluoride
(TiF4 ), and ammonium fluoride (NH4 F) have all been evaluated for cariostatic mecha-
nisms [250,255,280,281]. Clinical studies and in vitro experiments found that NaF, SnF2 ,
and MFP in dentifrice or mouthrinses had better fluoride uptake by enamel lesions,
higher F¯ retention level in saliva and dental plaque, and greater efficacy than other
compounds tested [282]. They, therefore, have been most frequently used in toothpaste and
mouth rinses.
Several chemical reactions have been proposed for interactions between SDF and
tooth structure. A combination of tooth hydroxyapatite, [Ca10 (PO4 )6 (OH)2 ], silver, and
fluoride ions in a 38% SDF solution resulted in the formation of an impermeable layer of
silver phosphate (Ag3 PO4 ) and calcium fluoride (CaF2 ) on the treated tooth surfaces [283].
Four decades later, deep carious lesions treated with SDF were found to have increased cal-
cium, phosphate, and fluoride ions in caries-affected dentin [284]. Mei et al. suggested that
silver particles and fluorohydroxyapatite formation in SDF-treated lesions might contribute
to the increased hardness of treated tooth surfaces [285]. Silver particles were detected on
the surface and in carious lesions treated with SDF, with the extent of silver penetration
and intensity being correlated with the degree of demineralization of the enamel and
dentin [286]. In addition to the formation of an impermeable layer of silver phosphate,
calcium fluoride, and fluorohydroxyapatite in SDF-treated carious lesions, highly con-
centrated silver precipitation zones form around the carious lesions, thereby blocking
dentin tubules, which could contribute to the physiochemical role of silver compounds in
caries arrest.
Overall, topical applications of fluoride have proven to be an effective anti-caries
measure. A review of data from 71 students concluded that the use of fluoride toothpastes
resulted in a 23% to 36% caries reduction depending on the fluoride concentration when
compared with placebo formulations [281]. The use of fluoride mouthrinses in 37 random-
ized controlled trials of over 15,000 children and adolescents resulted in a 27% reduction
in caries increment for permanent tooth surfaces when compared with placebo or no
treatment [250]. Hence, fluoride-containing toothpastes and mouth rinses are effective in
school-based programs for caries prevention in children, particularly in countries with a
high prevalence of caries and limited dental care resources. SDF treatment is minimally
invasive, inexpensive, and time-saving in application. Therefore, it has been considered
as an alternative treatment for at-risk patients when other forms of caries control are not
available or not appropriate [287,288]. Nevertheless, there are limitations in anti-caries
effectiveness for fluoride-containing mouthwashes, FV, and SDF, suggesting that improved
treatment and prevention approaches are needed for dental caries.
7.2. Silver Nanoparticles

Nanoparticles containing active agents represent a new route to deliver antimicrobials
in dentistry, including nano silver fluoride (NSF) and silver nanoparticles (AgNPs). These
silver-containing nanoparticles are aimed at preventing cariogenic bacterial cell adhesion,
attachment to tooth surfaces, and biofilm formation and maturation. These materials have
greater remineralization efficacy to arrest active dentin caries and are more biocompatible
than SDF without producing discoloration of treated tooth surfaces. The approximately
10 nm nano-scaled silver particles can continuously release silver ions, maintain a high
contact surface with microorganisms [289], pass through bacterial cell membranes, disrupt
cell processes, penetrate into dental tubules, and produce a higher antimicrobial effect
against S. mutans and lactobacilli at lower concentrations than SDF, resulting in significantly
decreased toxicity from the amount of silver ions leaching from the materials [290,291].
Acrylic resin fillings containing SF or AgNPs reduced the colonization levels of S. mutans
and C. albicans [292]. Pyrosequencing of 16S rRNA gene studies showed differences in the
composition of early and mature microbial biofilm communities formed on the surfaces
of modified dental polymers incorporated with AgNPs [293]. Therefore, NSF and AgNPs
could be alternatives to SDF as new antimicrobial anticaries agents.
7.3. Approaches for Caries Control Targeting the Microbiome

Development of second- and now third-generation sequencing technologies has al-
lowed investigators to examine the full complexity of the healthy human microbiome. It is
now understood to be highly diverse and contribute significantly to immune modulation,
digestion, colonization resistance against would-be pathogens, and other functions [294].
This insight has led to a major paradigm shift from the concept of “one-germ, one-disease”
to that of dysbiosis and polymicrobial diseases. Despite historical studies of a single
pathogen per disease, the vast majority of therapeutics currently in use to treat maladies
of microbial origin have a wide variety of activities, as discussed above. The adverse
effects of broad-spectrum antimicrobial therapies against many diseases are now well
established [295]. Furthermore, administration of broad-spectrum antibiotics may suppress
not only the pathogen of interest but also wide swaths of the overall microbiota, including
protective species. This leaves the treated body site prone to blooms of antibiotic-resistant
pathogens or recolonization with a less-than-optimal, potentially even harmful, microbial
community. More precise targeting of pathogenic species by treatment modalities that leave
the remainder of the microbiome unharmed is an objective that has inspired a significant
amount of research in recent years.
Historically and contemporarily, there have been several endeavors to combat dental
caries by precisely targeting S. mutans, although none have maintained substantial traction
long-term. Research investigating the feasibility of active or passive immunization against
dental caries has been sporadic. Early investigations on the topic have been excellently
reviewed [296]. More recent studies have explored vaccination using a recombinant P1
adhesin antigen [297], a DNA-based vaccine against glucosyl-transferases and surface pro-
teins [298], and a glycoconjugate vaccine based on rhamnan surface polysaccharides [299].
Unfortunately, past, present, and likely future translational efforts to move anti-caries
vaccine research into clinical trials face significant headwinds because caries, by itself, is
not a life-threatening disease. There are currently no licensed vaccines to prevent dental
caries, and, to our knowledge, only one candidate vaccine has proceeded to phase II clinical
trials [300]. Conceptually, the use of bacteriophages is an attractive approach to combat
S. mutans and dental caries, but this has received relatively little attention. Although the
few phages known to infect S. mutans were lytic and completely eliminated viable counts
from single-species biofilms, the phages demonstrated a highly stringent host specificity,
which was considered a significant disadvantage, particularly considering the high intra-
species diversity exhibited by S. mutans [reviewed in [301]]. No testing in multi-species
communities or further studies has been reported.
Probiotics approaches have sought to displace wild-type S. mutans using S. mutans
strains engineered for reduced pathogenesis or species which either compete with, or
directly antagonize, S. mutans [reviewed in [302,303]]. Although more recently discovered
species, such as Streptococcus dentisani [304] and Streptococcus A12 [90], hold promise com-
pared with older candidates, no probiotic formulations have successfully demonstrated
safety and efficacy in adequately rigorous clinical trials [305,306]. Two small molecules
were recently reported to exhibit the ability to specifically disperse or inhibit S. mutans
biofilms [307,308]. However, disruption of S. mutans biofilm alone, with minimal effect on
the overall dental plaque ecology, is likely to allow rapid reestablishment of the problematic
community and require constant application of the therapy. Recent work has identified
two antimicrobial peptides, ZXR-2 [309] and CLP-4 [310], and a vitamin D derivative, doxer-
calciferol [311], with antimicrobial activities against S. mutans. However, the antimicrobial
specificity of these molecules against S. mutans was not reported in these studies.
Specifically targeted antimicrobial peptides (STAMPs) were developed to address
the need for precision antibiotic therapy. STAMPs are synthetic peptides consisting of a
targeting domain to invoke specificity and a killing domain to exert antimicrobial action
against the intended species [312]. To design a STAMP directed/targeted to S. mutans, a
“G2” killing domain, consisting of a 16-residue segment of the well-characterized potent
broad-spectrum antimicrobial peptide novispirin G10, was chosen [313]. The targeting
domain selected was “C16”, which consisted of the 16 C-terminal residues of the S. mutans
pheromone, a competence-stimulating peptide (CSP). The directed/targeted and killing
moieties of C16G2 were joined by a flexible triglycine linker.
Analyses of C16G2 showed that it was capable of the targeted killing of S. mutans
in either planktonic or biofilm settings and that it could selectively kill S. mutans in a
three-species biofilm [313,314] as well as an in vitro, saliva-derived oral community, which
consisted of over 100 species [315]. This process was accompanied by a significant increase
in the relative abundance of Streptococcus mitis and other Streptococcus species associated
with good dental health [315]. Further work determined that C16G2 exerted its killing
effect through membrane disruption and that the cytotoxic effect of C16G2 was due to rapid
killing of S. mutans in less than one minute of exposure, acceptably swift for application as
an oral care product [316]. This STAMP was also soluble in aqueous solutions for delivery
into the oral cavity in a rinse formulation. A pilot clinical trial showed that, compared with
placebo, C16G2 significantly reduced the number of viable S. mutans in both plaque and
saliva samples, decreased lactic acid production, and increased the resting pH of dental
plaque [314]. Taken together, these results suggest that C16G2 is effective at selectively
suppressing S. mutans from the dental plaque milieu and remodeling the community to one
which is dominated by species that are associated with health. C16G2 is being developed
into a dental product by a biotechnology company (C3J Therapeutics, Inc., Marina del Rey,
CA, USA) and is currently in phase II clinical trials.
Several pH-responsive and acid-activated antimicrobial/anti-biofilm approaches have
been developed to focus on acid-generating bacteria and cariogenic biofilms. Quaternary
pyridinium salt (QPS) was developed by Sun’s group at the American Dental Association
Foundation [317]. Preliminary analysis showed that QPS exhibits pH-controlled antimicro-
bial activity that selectively inhibits the growth of acid-producing bacteria within a multi-
species community. Horev et al. reported pH-activated nanoparticles for controlled topical
delivery of farnesol to disrupt cariogenic biofilms [318]. In this approach, nanoparticles
are formed from diblock copolymers composed of 2-(dimethylamino) ethyl methacrylate
(DMAEMA), butyl methacrylate (BMA), and 2-propylacrylic acid (PAA) (p(DMAEMA)-b-
p(DMAEMA-co-BMA-co-PAA)) that self-assemble into cationic nanoparticles. Due to its
hydrophobic core, nanoparticles could effectively carry farnesol, a hydrophobic antimicro-
bial. The destabilization of nanoparticle cores under acidic pH will trigger the release of
farnesol, thus achieving the killing of cariogenic bacteria. Furthermore, farnesol-loaded
nanoparticles effectively attenuated biofilm virulence in vivo, resulting in a reduced num-
ber and severity of carious lesions using a clinically relevant topical regimen (2×/day) in a
rodent dental caries model.
Similarly, a strategy to control cariogenic biofilms using catalytic nanoparticles (CAT-
NP) was developed by Koo’s team at the University of Pennsylvania [319]. CAT-NP, with
peroxidase-like activity, is pH-responsive and can rapidly catalyze low concentrations of
H2 O2 at an acidic pH to produce free radicals to achieve simultaneous degradation of
the biofilm matrix as well as the killing of matrix-enclosed bacteria. Using 1 min topical
daily treatments, applicable for a clinical setting, they showed that CAT-NP in combination
with H2 O2 effectively suppressed caries development in a rat model. In a recent study, the
same group demonstrated the potential antimicrobial specificity of ferumoxytol iron oxide
nanoparticles (FerIONP) against biofilms harboring S. mutans through preferential binding
that promotes bacterial killing via in situ free-radical generation [320]. While further testing
is needed, these approaches hold the promise of being developed into oral therapeutics
that, instead of targeting one specific pathogen, can achieve the targeted control of a
group of bacteria displaying similar physiological properties that are relevant to disease
pathogenesis, e.g., the acid production and biofilm formation of cariogenic pathogens.
While the targeted approaches discussed above have yet to bear fruit in the form of an
approved therapeutic to prevent dental caries, the studies have contributed substantially to
our understanding of the disease and S. mutans and are foundational in guiding current and
future research. Dental caries remains a serious public health concern and one that could
strongly benefit from a precision therapy, such as STAMPs, for targeting a specific pathogen,
or pH-responsive, acid-activated antimicrobials for controlling a group of acid-producing,
cariogenic, biofilm-forming bacteria, to supplement current recommended fluoride and
hygienic regimens.
7.4. Challenges to Antimicrobial Approaches for Caries Management

The etiology of dental caries includes complex multidimensional interactions over
time between acid-producing bacteria, fermentable carbohydrates, and host factors includ-
ing genetics, tooth mineralization status, saliva composition, nutrition, immune response,
and oral hygiene [321]. Caries risk in individuals can change to high risk at any time [322]
depending on shifts in the symbiotic–dysbiotic supragingival microbiome and the nat-
ural demineralization–remineralization balance at the enamel–biofilm interface, which
is influenced by sugar intake. The “cariogenic microbiota” has evolved not only to in-
clude increased numbers of acidogenic and acid-tolerant microorganisms identified in the
biofilm [323] but also an extended list of functional traits and molecules that are involved
in the oral microbial community and ecological system of disease [324,325].
Lastly, dental caries is a biofilm-mediated, sugar-driven, and behavior-modifiable

disease. Host–microbiota–diet interactions in the disease pathophysiology bring together
additional aspects of the complexity of studying the caries microbiome. Current research
focuses on the human oral microbiome, metaproteome, and metatranscriptome of dental
caries. A group of discriminant bacterial species and a list of bacterial and human proteins
(involved in pH buffering, exopolysaccharide synthesis, protein synthesis, iron metabolism,
and immune response) were used to create models to effectively separate clusters of healthy
and diseased individuals [326] and to predict risk for caries onset [327]. These exploratory
multi-omic studies will provide new insights into the possibility of developing cariogenic
biomarkers for caries diagnosis, treatment, and prevention. Nevertheless, further large-
scale clinical studies are needed to test and validate the findings and to achieve a better
understanding of the mechanisms determining the interrelationship of those biomarkers.
8. Conclusions
We conclude that while dental caries remains an important clinical problem world-
wide, our understanding of the associated microbial biofilms has greatly advanced. The
microbiology of dental caries was built on the concepts of the golden age of microbiol-
ogy, using the one-bacterium-for-one disease hypothesis. In caries research, studies of
cariogenic pathogens have focused mainly on S. mutans and lactobacilli. Cultural, and
especially molecular, approaches in microbiological analyses have evolved with associated
conceptual changes to focus on the biofilm community rather than individual species.
Health is now recognized as being associated with a balanced symbiotic community, includ-
ing acid-balancing microbial strategies, which is lost in the unbalanced dysbiotic change
associated with disease. The caries-associated microbiome is not characterized by a single
microbiota but varies between individuals, tooth locations, and rates of disease progres-
sion, with some evidence that the most aggressive caries is associated with the highly
acidogenic, acid-tolerant S. mutans and Scardovia species. Functional characterization of the
caries-associated microbiome can lead to a better understanding of microbial activities at a
community level. The study of the molecular function of the oral microbiome is leading
to an expanding characterization of caries as it relates to community activity. Prevention
and treatment approaches for caries have moved away from suppressing the whole com-
munity with antimicrobials, fluoride, and silver-containing agents, which had limitations
despite many treatment successes. Modern therapeutic approaches focus on targeting the
pathogenic components of the microbiome, including acidogenic species such as S. mutans,
to achieve ecological rebalancing of the dysbiotic community. We look forward to an
ever-increasing understanding of dental caries and strategies for its treatment, prevention,
and final eradication.
Author Contributions: This review was planned and written jointly by all authors. All authors have
read and agreed to the published version of the manuscript.
Funding: We acknowledge support from the following sources: Grace Spatafora: NIH-NIDCR
R01 DE014711; Yihong Li: NIH-NIDCR R01 DE019455; Xuesong He: NIH-NIDCR R01 DE023810,
NIH-NIDCR R01 DE030943, NIH-NIDCR R01 DE029479; Anne Tanner: William Bingham II Trust.
Data Availability Statement: Data are from literature sources as cited in the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
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microorganisms

1 Biology and Program in Molecular Biology and Biochemistry, Middlebury College,

Keywords: caries; microbiology; ecology; treatment; Streptococcus mutans

Microorganisms 2024, 12, 121. https://doi.org/10.3390/microorganisms12010121 https://www.mdpi.com/journal/microorganisms

assessment (CAMBRA® v.4.0) model (cda.org/CAMBRA4) [29] or the Cariogram software

2.2. Diet and Dental Caries

2.3. Plaque pH and Host Factors

3. Microbiome of Dental Caries

3.1. History of Caries Microbiology

3.2. Development of the Tooth-Associated Microbiome Leading to Caries

3.2.1. Pioneer Species

3.2.2. Early Colonizers

3.2.5. The Mature Biofilm

Ultimately, the tooth-adherent microbial community reaches maturation when the

3.3. Culture and Genetic Analysis of the Cariogenic Microbiome

4. Caries Microbiome at Different Lesion Sites

lesions progress. Studies involving sufficient participants to overcome variations based

4.1. Initial Caries

4.2. Dentin Caries

4.2.1. Root Caries

4.2.2. Deep Dentin Caries

4.3. Caries Microbiome of Aggressive Lesions

such as Actinomyces, Dialister, Fusobacterium, Neisseria, Peptostreptococcaceae, Tannerella, and

4.4. Yeasts and Dental Caries

5. Principal Caries-Associated Bacteria

5.2. Lactobacillus Species

Microorganisms 2024, 12, 121 21 of 47

Lactobacillus species were frequently detected in advanced dentin carious lesions

Vaginal (n=194) Gingival (n=194)

6. Microbiome: Beyond Microbial Composition and the Oral Microbiome:

capable of generating information on the functional activity of whole microbial commu-

7. Treatment of Caries as a Microbial Disease

7.1. Fluoride and Anticaries Activity

7.1.2. Antimicrobial Properties of Fluoride

7.1.3. Chemical Action of Fluoride to Strengthen Teeth

7.2. Silver Nanoparticles

7.3. Approaches for Caries Control Targeting the Microbiome

7.4. Challenges to Antimicrobial Approaches for Caries Management

Lastly, dental caries is a biofilm-mediated, sugar-driven, and behavior-modifiable

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