Chapter 52

Dental Caries
Richard J. Lamont1 and Paul G. Egland2
1
University of Louisville, Louisville, KY, USA, 2Augustana College, Sioux Falls, SD, USA

Dental caries, or tooth decay, is an infectious disease of

Sa

uc gth
the dentition characterized by localized destruction of the liv

n
od n
tio
ar

pr stre
tooth. Organic acids produced by tooth-dwelling bacteria y
flo
dissolve the mineralized tissues of the tooth, and the

ac nd
w
ra

of vel a
resulting carious lesion progresses inward from the tooth te

surface. Left untreated, caries will progress to involve the
dentin and eventually the pulp chamber which becomes
necrotic. Infection can then spread out to the periodontal
tissue at the root apex, leading to periapical abscesses,
and in rare cases a serious systemic infection can occur.
Dental caries is a multifactorial disease involving
interactions among three principal factors: the host, pri-
marily the teeth and saliva; the microbiota, primarily
acid-producing bacteria on the tooth surface; and the diet,
primarily the availability of fermentable carbohydrate.
Traditionally these factors are represented diagrammati-
cally as three overlapping circles (Fig. 52.1), and for
caries to occur all three factors must be conducive to the
onset of disease. In addition, caries is usually slowly pro-
gressing and reversible in the early stages, and so an
observable carious lesion requires that the three factors be
operational for a sufficient period of time.
STRUCTURE OF TEETH
Teeth consist of the visible coronal structure (crown)
above the cementoenamel junction (CEJ) at the gumline
(supragingival), and a root structure below the CEJ (sub-
gingival). The surface layer of the crown is composed of
enamel, mainly hydroxyapatite (crystalline calcium phos-
phate). Enamel is highly mineralized, about 96% by
weight, and is the hardest biological substance in the
human body. Enamel surfaces can be smooth, or, on the
occlusal surfaces of molars and pre-molars, in depressions
and clefts called pits and fissures. Supporting and directly
underneath the coronal enamel is dentin, a less mineralized
organic matrix containing about 70% hydroxyapatite with
collagenous proteins, phosphoproteins and proteoglycans.
Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00052-4
© 2015 Elsevier Ltd. All rights reserved.
id
Le
Microbiota Host
Caries
Diet
Fermentable carbohydrate intake
FIGURE 52.1 Three overlapping circles diagram showing requirement
for cariogenic bacteria, a fermentable substrate in the diet, and a suscep-
tible host to result in caries. Primary contributors to each criterion are
indicated.
The pulp chamber is directly beneath the dentin and
houses blood vessels and nerves which extend into the root
as root canals. Roots are made of dentin with a surface of
cementum which is a hydroxyapatite (about 50%) and col-
lagen matrix. Dentin contains microscopic fluid-filled
channels, the dentinal tubules, which radiate out from the
pulp chamber to the exterior enamel or cementum border.
Caries can initiate on enamel or cementum surfaces.
The initial carious lesion is subsurface due to diffusion
of bacterial organic acids. Indeed, in the early stages of
enamel caries there is little visible damage to the outer
surface despite considerable demineralization below.
The first sign of enamel caries is what is known as a
‘white spot’, an area where subsurface demineralization
causes the enamel to manifest an opaque or white col-
our. As the crystal structure is still intact, a white spot
lesion can be remineralized; however, further deminer-
alization will eventually cause loss of crystal structure
and cavitation.
945
946 PART | 8 Anaerobic Infections
TYPES OF DENTAL CARIES
Demineralization is a physicochemical process of dissolu-
tion of hydroxyapatite crystals by bacterial acids [1]. As
the relationship between hydroxyapatite solubility and pH
is exponential, small reductions in pH can have major
effects. The degree of mineralization and the topology of
the dental tissues influence both bacterial colonization
and the degree of sustained low pH challenge necessary
for pathology. Examples of different types of caries are
shown in Figure 52.2.
1. Enamel smooth surface caries. Smooth surfaces are
cleansed by salivary flow and the mechanical action
of the tongue and lips. Oral hygiene procedures such
as tooth brushing also effectively clean these surfaces.
Hence, bacterial colonization is challenging, and
smooth surface caries reflects a breakdown in oral
hygiene or host defence, along with a high carbohy-
drate (total amount or frequency) intake.
2. Pit and fissure and interproximal caries. These areas
are physically retentive allowing both acid accumula-
tion and entrapment of bacteria in the absence of spe-
cialized adherence mechanisms.
3. Root caries. Roots normally have a cementum surface.
However, this is often lost after periodontal proce-
dures such as scaling and root planing, and dentin is
exposed. Both cementum and dentin are less mineral-
ized then enamel and thus more easily dissolved.
Fortunately, the bacteria that colonize the subgingival
area are generally less cariogenic than those on supra-
gingival enamel surfaces. However, root surfaces can
become in essence supragingival, and exposed to more
(a) (b)
(c)
cariogenic bacteria, as the gingiva recedes with age or
after periodontal surgery.
4. Recurrent (secondary) caries. A large proportion, as
much as 50%, of restored carious lesions can develop
recurrent caries around the restorations.
5. Rampant caries. In cases where salivary flow is reduced,
a condition known as xerostomia, widespread and severe
carious lesions develop. The lack of saliva compromises
the buffering and flushing of acids which are major pro-
tective functions of saliva. Xerostomia can be geneti-
cally determined, as in the case of the autoimmune
disease Sjögren’s syndrome, occur as a side effect of
some medication, or result from radiation therapy for
head and neck cancer which destroys the salivary glands.
Methamphetamine also reduces salivary flow which
contributes to the rampant caries seen in ‘meth mouth’.
6. Early childhood caries (ECC; nursing or baby bottle car-
ies). This is rampant caries of the primary dentition of
infants and toddlers caused by sweetened liquids remain-
ing on the teeth for prolonged periods. Unrestricted
access to night-time bottles with fruit juice or sweetened
formula is a major risk factor as the fermentable substrate
is provided when salivary flow is lowest.
CARIES IN POPULATIONS
Dental caries is one of the oldest and most common infec-
tions of humans. Caries incidence increased dramatically
in post-industrial societies with increasing affluence, and
in particular with the availability of processed sugar. In
more recent times, improved oral hygiene practices cou-
pled with fluoridation of the public water supply along
FIGURE 52.2 (a) Extensive occlusal surface car-
ies. (b) Early (discoloration) and late stages of root
caries. (c) Severe early childhood caries (ECC),
caries has progressed to the point where the teeth
have decayed and lost at the gumline. Images
courtesy of Dr. Masae Kuboniwa, Osaka
University and Dr. Ed Zapert, Florida State
Department of Health.
 Chapter | 52 Dental Caries 947

with the presence of fluoride in dentrifices, mouth rinses
and processed foods, have greatly reduced the prevalence
of dental caries in the population. Nevertheless, caries,
and in particular ECC, remains a significant problem in
some sections of society, especially those with low socio-
economic status and with limited access to healthcare. In
the USA, caries remains a significant childhood disease
and around 20% of the population experiences 80% of the
carious lesions. The prevalence of root caries is different
from that of coronal caries. Although root caries can occur
in young individuals, prevalence is correlated with increas-
ing age. With the growing geriatric population in devel-
oped countries and as more of the elderly remain dentate,
root caries is increasing in incidence and significance.
IMPORTANT BACTERIA IN CARIES
Streptococcus mutans
The primary pathogen in all forms of caries has been rec-
ognized for many years as S. mutans, a Gram-positive,
alpha-haemolytic, facultative anaerobe [2]. What was
originally one species is now considered a group of
organisms (the mutans streptococci), with differing host
range and cariogenic potential, which are not closely
related at the 16S rRNA level (Table 52.1). S. mutans and
S. sobrinus are the predominant pathogenic species in
humans, with S. mutans serotype c being the most
common isolate (70 80%) from carious lesions. Serotype
is based on the chemical compositions of surface
TABLE 52.1 Classification of Mutans Streptococci
Species Serotypes Host
S. mutans c, e, f, k Human
S. sobrinus d, g Human
S. ratti b Rats
S. ferus c Rats
S. criceti a Hamsters
S. doweni h Monkeys
polysaccharides, which are composed of a rhamnose
backbone and glucose side chains. S. mutans possesses
specialized mechanisms for attachment to, and retention
on, tooth surfaces. In addition to adherence, the key viru-
lence attributes of the organism are efficient production
of lactic acid from sugar fermentation and resistance to
the adverse effects of low pH (acidurance) as will be dis-
cussed in more detail later.
Acquisition of S. mutans
S. mutans preferentially colonizes the hard surfaces of the
teeth and hence oral colonization occurs shortly after the
eruption of the primary teeth, the so-called ‘window of
infectivity’ around the age of two [3]. Saliva from the
mother or other primary caregiver is the predominant
source of S. mutans transmission as established by
restriction-fragment length polymorphisms (RFLP) of
S. mutans isolated from mother infant pairs [4].
Interestingly, delaying the age at which a child becomes
infected can reduce subsequent caries risk. An infant may
initially carry only one clonal line of S. mutans but by
adulthood they can have up to seven different clonotypes.
The S. mutans population is remarkably stable once estab-
lished, and resistant to superinfection with other S. mutans
clonal types even after exchange of saliva with other peo-
ple [5]. In developed countries it is likely that almost the
entire population is colonized to some degree with mutans
streptococci.
Lactobacilli
A number of Lactobacillus species inhabit the oral cavity
and these organisms both efficiently produce lactic acid
and are tolerant to its adverse effects [6,7]. However,
lactobacilli lack adherence mechanisms for enamel sur-
faces and thus are not major contributors to the initiation
of smooth surface caries. Lactobacilli play a bigger role
in pit and fissure caries where they can become physically
entrapped, and as secondary invaders of established
lesions where they can outcompete less acid-tolerant
organisms.

S. macacae c Monkeys Polymicrobial Communities

S. orisuis c Pigs Molecular approaches such as 16S sequencing and high-
S. dentirousetti d Bats, chimpanzees throughput genome sequencing have demonstrated that
carious lesions contain complex microbial communities
S. devriesei Unknown Horses and that S. mutans is not always present [8 10]. Other
S. dentapri c Boars potential acid producers that can be found at high levels
in these subjects include strains of the following species:
S. ursoris Unknown Bears
Selenomonas, Neisseria, Propionibacterium, Actinomyces,
S. troglodytae c Chimpanzees Bifidobacterium, Atopobium and low-pH, non-mutans
streptococci. The view of the aetiology of dental caries is
948 PART | 8 Anaerobic Infections
thus changing to one of an acidogenic, aciduric bacterial
community, the constituents of which may vary among
individuals, sites and stage of lesion.
VIRULENCE MECHANISMS OF S. MUTANS
Although communities may be more important than single
species in the aetiology of dental caries, S. mutans is the
only organism for which the virulence factors are well-
defined. However, it is likely that other cariogenic organ-
isms will possess similar attributes.
1. Adhesion. Bacterial colonization of sites subject to
mechanical clearing forces requires specific, high-
affinity adherence for retention. Newly erupted or pro-
fessionally cleaned tooth surfaces rapidly become
coated with a layer or pellicle of salivary molecules,
and it is to these salivary molecules that initial coloni-
zers adhere. The major adhesin of S. mutans is an
B185-kDa surface protein known variously as
antigen (Ag) I/II, antigen B, PAc, P1, and SpaP
[11 13]. Antigen I/II contains several distinct
domains: the signal (leader) adjacent to an N-terminal
region comprising alanine-rich repeats; a central vari-
able (V) region; and a C-terminal proline-rich region.
The N-terminal region is predicted to form α-helical
coiled coil structures resembling group A streptococ-
cal M protein. The V region is predicted to carry a
lectin-like domain for carbohydrate binding. The C-
terminal proline-rich repeats of 39 amino acid residues
are required for secretion and stability. Antigen I/II is
linked to the cell wall through a C-terminal sortase-
processed LPXTG motif. Crystal structure shows that
the protein forms an elongated structure comprised
of a globular base region from which extend structures
resembling a stalk and a head (Fig. 52.3). The salivary
pellicle receptor with the highest affinity for antigen
I/II is gp-340, a highly glycosylated (B25% carbohy-
drate) glycoprotein (molecular mass 340 kDa) pro-
duced at several mucosal surfaces, and involved in the
regulation of cellular immune responses and epithelial
differentiation. Antigen I/II has multiple binding sites
for both the oligosaccharide and the peptide backbone
sequences of gp-340, and this multivalency allows gp-
340 to also aggregate bacterial cells after initial
attachment to pellicle. As gp-340 can interact with
other oral bacteria, this bridging process can facilitate
the development of a heterotypic microcolony. Indeed
another property of gp-340 in the fluid phase of saliva
is the aggregation of bacteria promoting clearance
through expectoration or swallowing. Antigen I/II is
functionally versatile and can also bind to collagen
and fibronectin which may allow S. mutans to colo-
nize soft tissues in the oral cavity or other sites in the
FIGURE 52.3 Model of AgI/II structure, showing the interaction of
alanine (A1 2) and proline (P1 3) regions which fold back upon them-
selves to generate a stalk-like structure presenting a variable (V) region
head. From Larson et al. Proc. Natl. Acad. Sci. USA 2010, 107:
5983 5988, with permission.
body if the organism gains access systemically. Direct
binding to other bacteria and stabilization of multispe-
cies community structure is an additional feature of
antigen I/II.
2. Polysaccharide production. Subsequent to antigen
I/II gp-340-mediated attachment, accumulation and
retention of S. mutans-containing microcolonies is
enhanced by the production of large amounts of
extracellular polysaccharide (Fig. 52.4a) [2,14 16].
Reflecting the close relationship between the
organism and sucrose consumption, the β-2,1-linked
fructose glucose disaccharide sucrose is the predomi-
nant substrate for polymer production. S. mutans
produces glucosyltransferases (Gtf) and a fructosyl-
transferase (Ftf) on the cell wall. These enzymes con-
tain two functional domains. An N-terminal catalytic
domain (800 amino acid residues) hydrolyses sucrose
into the monosaccharide constituents glucose and fruc-
tose while conserving the relatively high energy from
the bond between the C1 of glucose and the C2 of
fructose. The catalytic domain of the Gtf is related to
the α-amylase superfamily, i.e. glycoside hydrolase
family 13, and is predicted to contain alternating
β-strands and α-helices in a (β/α)8 barrel motif. The
free energy of hydrolysis is then used by the
C-terminal binding domain (500 amino acid residues)
to transfer the glucosyl or fructosyl units to the grow-
ing polymer chains of glucose (glucans) or fructose
(fructans), respectively. The binding domain possesses
 Chapter | 52 Dental Caries 949

(a) (b) AgI/II Gtf Glucan

Salivary
pellicle gp-340
Gbp

T
O
O
T
H

FIGURE 52.4 (a) Exopolysaccharide production by S. mutans grown in sucrose. Fluorescent cross-sectional image of S. mutans (green) enmeshed
and surrounded by an intricate exopolysaccharides (EPS) matrix (red). From Xiao et al., PLoS Pathogens 2012;8(4):e1002623. (b) Schematic represen-
tation of colonization of the tooth surface by S. mutans. The Ag I/II adhesin of S. mutans (blue shading) binds to gp-340 in the enamel salivary pellicle
and adsorbed to other bacteria (red shading). Extracellular insoluble glucans are produced by glucosyltransferases (Gtf) and mediate intercellular bind-
ing with both the Gtf themselves and glucan-binding proteins (Gbp). Gtf can be released extracellularly and adsorb to the tooth surface where they
produce additional glucan which can mediate attachment.

a series of YG repeats, each of which comprises a
21-amino-acid-residue sequence with one or more
aromatic residues, that have arisen from multiple
duplication events. Glucan-binding domains exhibit a
flexible structure with a high content of β-sheet and
random coil structure. An interesting corollary of the
synthesis mechanism is that glucose or fructose them-
selves cannot be used for polymer production as the
necessary energy for polymerization is not provided.
Additionally, other disaccharides such as maltose and
lactose have a lower free energy of hydrolysis com-
pared to sucrose and cannot be used as glucosyl
donors. There are three S. mutans glucosyltransferases
designated GtfB, GtfC and GtfD. GtfD produces
mainly soluble linear glucan with α-1,6 glycosidic lin-
kages. GtfB produces insoluble glucan rich in α-1,3
branch linkages. GtfC produces a mixture of low-
molecular-mass insoluble and soluble glucans.
Sucrose induces the expression of gtfB, while glucose
and fructose repress expression at acidic pHs.
Repression is thought to be mediated by the RegM
transcriptional regulator which shows homology to
catabolite control protein A (CcpA). A two-
component system (TCS), VicRK, positively regulates
the expression of GtfBCD, Ftf, and the glucan-binding
protein GbpD (see below). Glucans with α-1,4 lin-
kages are not produced, presumably these have been
selected against over time as they would be a substrate
for salivary amylase. The fructan synthesized by Ftf is
a soluble polymer with linear β-2,1 and β-2,6 linkages.
Fructan along with the soluble α-1,6-linked glucans,
are degraded rapidly by a fructanase (FruA) and dex-
tranase, respectively, and act as an extracellular, read-
ily metabolizable, reserve energy source. FruA is an
exo-β-D-fructosidase controlled by the four-component
regulatory system LevSRQT. Dextranase is a member of
the glycoside hydrolase family 66, and produces
isomalto-oligosaccharides of various sizes by intramo-
lecular transglycosylation. The insoluble glucans can
also serve as an extracellular longer-term energy source.
However, their primary purpose is to enclose the bacte-
rial microcolonies in a retentive matrix. Gtf enzymes are
also secreted extracellularly and can adsorb to salivary
pellicle and other bacteria where they function to pro-
vide additional cohesive microenvironments (summa-
rized in Fig. 52.4b). Glucose inside the bacterial cell can
be polymerized into a glycogen-like intracellular poly-
saccharide (IPS) that also functions as a reserve energy
source. Mobilization of IPS for glycolysis will extend
the duration of acid production during periods when die-
tary carbohydrates are lacking [2,15,17 23].
3. Glucan-binding proteins. In order to attach to the glu-
can produced by Gtf, S. mutans expresses lectin-like
molecules, the glucan-binding proteins (Gbp) [16].
Four Gbp, A D, have been identified with GbpC
being the principal glucan receptor. Gbp are necessary
950 PART | 8 Anaerobic Infections
for S. mutans to accumulate into complex communi-
ties and they also determine the architecture of the
community. The transcriptional regulator CovR
represses gbpC, as well as the glucosyl transferase
genes [24]. Apart from their affinity for glucan, the
Gbp have little in common and differ in structure,
function and immunological properties, although
GbpA and GbpD contain the YG repeat domains
found in the glucan-binding domain of Gtf, evidence
for a functional selection of conserved residues for
carbohydrate binding. GbpB may also function as a
peptidoglycan hydrolase and contribute to cell wall
cycling and synthesis.
4. Acid production. While adhesion to the tooth surface is
essential for allowing contact of bacteria with the
enamel surface that is damaged during caries, the pro-
duction of organic acids by oral bacteria is the ultimate
virulence factor in caries formation. When the pH
drops below approximately 5.5 the balance between
enamel mineralization and solubility shifts, and
hydroxyapatite crystal structure is lost. Ultimately, the
destruction of enamel allows bacteria access to the
interior tissue of the teeth. These acids are the products
of fermentation. Fermentable sugars include not only
those present in the human diet, but also sugars derived
from host tissues. The latter can be liberated and made
available by the action of bacterial exoglycosidases
that cleave the sugars from host salivary glycoproteins.
Sucrose is considered the most cariogenic sugar
because it can be converted into polysaccharides
(described above) and fermented into organic acids,
among the strongest of which is lactic acid with an
ionization constant (pKa) of 3.5. Lactic acid is thus
considered the primary organic acid associated with
dental caries. Sucrose and other sugars can be trans-
ported into the S. mutans cell by several distinct
mechanisms [17,19,20]. The sugar:phosphotransferase
system (PTS) utilizes a high-affinity permease, EIIScr
(ScrA) located in the cell membrane which interna-
lizes sucrose as sucrose-6-phosphate. Intracellular
sucrose-6-phosphate is then hydrolysed to glucose-
6-phosphate and fructose by ScrB, a sucrose-
6-phosphate hydrolase. The scrA and scrB genes are
tandemly arranged on the S. mutans chromosome but
transcribed in opposite directions from individual pro-
moters. A fructokinase, encoded by scrK which is
adjacent to, and downstream of, scrA, converts fruc-
tose to fructose-6-phosphate which can be mobilized
through the glycolytic pathway. The GalR-LacI-type
transcription factor ScrR (the gene for which is imme-
diately downstream of scrB) negatively regulates
expression of scrA and scrB, and is repressed in the
presence of sucrose. A second PTS permease, EIITre,
while having a higher affinity for trehalose, can also
transport sucrose. The related binding-protein-
dependent ABC-type transport systems MSM (multi-
ple-sugar metabolism system) and Mal (maltose sys-
tem) internalize several sugars including sucrose and
maltose, which is a derivative of starch. Cytoplasmic
sucrose is converted to glucose-1-phosphate and fruc-
tose by the sucrose phosphorylase GtfA.
Prioritization of sugar utilization by S. mutans is
likely accomplished by several mechanisms. Carbon
catabolite repression (CCR) in S. mutans occurs
through CcpA [25], which regulates gene expression
by binding to conserved catabolite response elements
(CRE) in the promoter regions of CCR-sensitive
genes. In addition, the affinities and expression levels
of the PTS phosphocarrier protein HPr and the EII
permeases regulate CCR.
When grown with excess glucose, S. mutans is
homofermentative, generating lactic acid as the pri-
mary end product. This is achieved by processing glu-
cose using the Embden Meyerhof Parnas pathway
of glycolysis, to produce pyruvate [18,26]. This is fol-
lowed by fermentation of pyruvate to lactic acid, using
the enzyme lactate dehydrogenase, to regenerate
electron-accepting NAD1 needed for further ATP for-
mation (Fig. 52.5). In addition to lactic acid, S. mutans
can produce mixed acids by carrying out heterofer-
mentative growth, yielding formate, ethanol and ace-
tate. This occurs when sugars other than glucose are
used for growth. The amount of oxygen present also
Glucose
2 NAD+ 2 ADP
2 ATP
2 NADH
2 Pyruvate
2 NAD+
2 Lactic acid
Acetyl-CoA + Formic acid
Acetyl-phosphate
ADP
ATP Ethanol
Acetic acid
FIGURE 52.5 Acid production by S. mutans. Summary pathways of
glycolysis via the Embden Meyerhof Parnas pathway (black), lactate
fermentation (red) and mixed-acid fermentation (blue) are shown. Acid
products are shown in boxes. The fate of pyruvate depends on substrate
sugars and environmental oxygen levels.

influences whether homofermentative or heterofer-
mentative growth occurs. S. mutans must use the
homofermentation pathway in the presence of oxygen
because enzymes in the heterofermentative pathway
are oxygen-sensitive. It is likely that recently identi-
fied bacteria from carious lesions (see above) can also
produce lactate or other organic acids. There is also
evidence that organisms in plaque such as Veillonella
spp. can ferment lactic acid produced by streptococci
and thus potentially raise the pH. It is the outcome of
bacterial community metabolism that will ultimately
determine plaque pH.
5. Acid tolerance. Low pH levels are toxic to many bac-
terial cells, and hence, as a carious lesion develops,
bacteria that are resistant to the adverse effects of low
pH will be selected. S. mutans has developed a num-
ber of mechanisms to tolerate acidic conditions and
prevent acid damage. Growth of S. mutans can then
recommence when environmental pH levels return to
values above about pH 5.0. [27 29].
i. S. mutans maintains the internal pH (pHi) at about
1 unit above the external environment by pumping
hydrogen ions out of the cell. The membrane-
bound F-ATPase is the primary mechanism by
which protons are extruded to prevent acidifica-
tion of the cytoplasm and maintain pH homeosta-
sis. The elevated pHi allows the acid-sensitive
glycolytic enzymes to continue to function under
conditions where the external pH is below levels
compatible with growth. The S. mutans F-ATPase
has a pH optimum of 6.0 which correlates with
acid tolerance levels for growth of the organism.
However, S. mutans can continue to perform gly-
colysis at pH values close to 4, although cell divi-
sion stalls and the ATP produced is used to fuel
the F-ATPase pump. Structurally, the enzyme
contains an Fo component embedded in the mem-
brane and an F1 component that protrudes from
the membrane into the cytoplasm. The Fo compo-
nent consists of one a, two b, and usually about
12 c subunits. The F1 component, which can cata-
lyse either hydrolysis or synthesis of ATP, has
three α subunits, three β subunits, and single cop-
ies of the γ, δ and ε subunits. Proton movement is
a mechanical process in which the c subunits
rotate while the ab2 subunits form a stator. The γ
and ε subunits also spin and translate the rotation
to the complex of alternating α and β subunits.
ii. Acid tolerance also involves increased levels of
long chain and unsaturated fatty acids in the cell
membrane. This process requires the activity of
the FabM protein which is responsible for
biosynthesis of mono-unsaturated fatty acids in
S. mutans.
Chapter | 52 Dental Caries 951
iii. S. mutans possesses an agmatine deiminase sys-
tem (AgDS) which detoxifies and converts agma-
tine to putrescine, yielding ammonia, CO2 and
ATP. The AgDS system is active at low pH
values and the ammonia produced will raise cyto-
plasmic pH while ATP can be used for the
F-ATPase pump. Transcription of AgDS is
affected by several TCS, including the CiaRH sys-
tem. The CiaRH TCS involves a third protein,
CiaX, a double glycine-containing calcium-sens-
ing peptide. In the calcium-limiting conditions
found in saliva, CiaX may serve as a calcium
scavenger to help maintain the optimal physiology
of S. mutans.
iv. A second system for alkali production by
S. mutans involves fermentation of malolactic
acid, a major acid in fruits such as apples. In this
process, dicarboxylic L-malic acid is decarboxy-
lated and catabolized to L-lactate and CO2. This
results in cytoplasmic alkalinization and also ATP
synthesis by means of the F-ATPase acting in the
synthase mode. Moreover, as CO2 can be rapidly
converted to bicarbonate via carbonic anhydrase,
MLF enhances the buffering capacity of the bacte-
rial cell.
6. Biofilm adaptation strategies. As mentioned, the pre-
dominant mode of existence of S. mutans on tooth sur-
faces is as a component of a complex biofilm
community known as dental plaque. S. mutans experi-
ences a number of stressors in the biofilm situation,
including nutrient fluctuation, oxygen tension and inter-
bacterial competition, in addition to acidic conditions. In
response, biofilm cells of S. mutans show a general coor-
dinated series of regulatory events that control molecular
chaperones and DNA repair systems along with more
specific systems discussed below [28,30].
Within a biofilm S. mutans cells show an increase
in competence, promoting increased uptake of DNA
either as a nutrient source or as a means of increasing
genetic diversity. Competence is controlled by compe-
tence stimulating peptide (CSP), a secreted double-
glycine quorum sensing signal. CSP is the product of
the comC gene and binds to a sensor histidine kinase,
ComD, which, with the response regulator ComE,
form a TCS that initiates temporally distinct waves of
transcriptional activity. In addition to competence,
these coordinated transcriptional activities serve to
control bacteriocin production and stress responses,
along with community development and architecture.
Autolysis is also linked to the competence control
pathway, ensuring a supply of extracellular
DNA which also plays an important role in stabilizing
biofilm structure. Other TCS interface with the
952 PART | 8 Anaerobic Infections
competence control network. The HK sensor kinase of
the HK/RR11 TCS may act as a second receptor for
CSP, and influence S. mutans community architecture.
VicRK has also been implicated in competence devel-
opment as well as responses to oxidative and acid
stress. The VicRK system includes the VicX protein
which shares significant homology with metal-
dependent hydrolases belonging to the β-lactamase
superfamily and may integrate cell wall remodelling
and cell division with stress responses [18,31].
A second quorum sensing system in S. mutans
involves the species-non-specific autoinducer (AI)-2
signal. AI-2 is more accurately a family of structurally
closely related furnanones produced by the enzyme
LuxS. Mutations in luxS affect community architec-
ture, and LuxS also impacts S. mutans genes that con-
tribute to a general stress response and production of
the mutacin I bacteriocin. Convergence of these path-
ways may depend on the transcriptional regulator IrvA
which is also involved in the control of GbpC
[18,28,32].
A general bacterial strategy for adaptation to envi-
ronmental changes, shared by S. mutans, is the strin-
gent response, which is initiated by accumulation of
(p)ppGpp GDP- and GTP-derived molecular alar-
mones. (p)ppGpp accumulates when aminoacyl tRNA
pools fail to keep pace with the demands of protein
synthesis, signalling nutritional stress that modulates
gene expression and the inhibition of stable rRNA and
tRNA synthesis. The RelA, RelP and RelQ enzymes
are responsible for the production of the (p)ppGpp in
S. mutans, and the reduction in growth rate and
biofilm accumulation triggered by the stringent
response may minimize damage from oxygen radicals
produced by metabolism. In S. mutans the stringent
response also impacts tolerance of acid and oxidative
stress [27,28].
Within a mixed-species community S. mutans
competes with related species of oral streptococci for
space and resources. For example, S. sanguinis and
S. gordonii produce hydrogen peroxide more effi-
ciently than S. mutans, and consequently S. mutans is
inhibited in communities of these three species. On
the other hand, biofilm cells of S. mutans increase the
production of bacteriocins that target S. sanguinis and
S. gordonii. While this would tend to restore a compet-
itive advantage to S. mutans, it is counterbalanced by
the production of a protease (Sgc) from S. sanguinis
and S. gordonii which can diminish bacteriocin produc-
tion by S. mutans. These multilevel antagonistic inter-
actions lead to competitive exclusion whereby
establishment of S. gordonii/S. sanguinis or of
S. mutans in a niche inhibits colonization by the antag-
onistic species [18,32 34].
Community adaptation in S. mutans thus involves
complex interactions among transcription factors and
regulators which constitute global regulatory networks
that control responses to environmental stress, the
transport and metabolism of carbohydrates, and inter-
actions with other bacteria. In this manner, growth and
survival decisions are integrated with stress tolerance
in this cariogenic pathogen.
HOST FACTORS IN CARIES
Many factors in the oral environment modulate the sus-
ceptibility of the host to caries. The tooth itself becomes
more resistant to demineralization over time, which is
part of the reason caries is primarily a disease of child-
hood. This post-eruptive resistance involves incorporation
of fluoride into the enamel. When hydroxyapatite remi-
neralizes in the presence of fluoride, hydroxyl ions can be
substituted by fluoride ions and the resulting fluorapatite
is less soluble and more resistant to acid attack [1].
The overwhelmingly most important host factor in car-
ies is saliva which affects the caries process in a number
of ways [35]:
1. Salivary flow rate. As saliva flows around the teeth it
dilutes and removes acid and washes away weakly
bound bacteria.
2. Salivary buffering capacity. There are two major buff-
ering systems in saliva based on phosphate and
bicarbonate-carbonic acid, of which the latter is the
more significant. In aqueous solution, carbonic acid
dissociates into a bicarbonate ion and a proton, or into
carbon dioxide and water, as illustrated by the
equation:
CO2 1 H2 O2H2 CO3 2HCO32 1 H1
When acid and thus hydrogen ions increase, the
reaction runs toward carbon dioxide and water,
thus removing the protons and increasing pH. The
bicarbonate-carbonic acid system buffers at pHs
that are found in the mouth and the amount of
bicarbonate increases as salivary flow increases.
Ammonia and urea in saliva also contribute to neu-
tralization of acids.
3. Supersaturation. Calcium and phosphate are supersatu-
rated in saliva due to binding to proline-rich proteins
and statherin. Supersaturation inhibits dissolution of
the hydroxyapatite mineral.
4. Direct antibacterial effectors. Saliva contains several
molecules with antimicrobial activity (see Table 52.2)
which help prevent bacterial and fungal overgrowth.
These mechanisms are only partially effective against
the resident microbiota of the oral cavity including
S. mutans.
 Chapter | 52 Dental Caries 953

TABLE 52.2 Antibacterial Properties of Saliva

Molecule Major Function(s)

Lysozyme Digests the peptidoglycan in Gram-positive bacterial cell walls by breaking the β (1 4) bond
between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan. Lysozyme can also
activate cell wall autolysins and aggregate bacteria

Salivary peroxidase Catalyses the oxidation of thiocyanate (SCN-) to hypothiocyanite (OSCN-) by bacterial hydrogen
peroxide. At acid pH, OSCN- is protonated and traverses bacterial membranes. Hypothiocyanite
oxidizes SH groups in bacterial enzymes and inhibits metabolism

Lactoferrin An iron-binding glycoprotein which inhibits bacterial growth by binding and sequestering Fe21 ions.
In the apo (iron-free) state lactoferrin is also toxic to bacteria

Calgranulin A and B Metal ion-binding proteins that inhibit bacterial growth by scavenging divalent cations

Cystatins Cysteine-rich peptides which inhibit bacterial cysteine proteinases

Histatins Cationic histidine-rich proteins that are primarily antifungal but also inhibit bacterial growth

Mucins High-molecular-weight glycoproteins (30 80% carbohydrate) that aggregate bacteria and promote
clearance

gp-340 A large glycoprotein (340 kDa) that aggregates bacteria

Surfactant protein A A bacterial agglutinin and member of the collectin family

β2 microglobulin Aggregates S. mutans

Peptidoglycan recognition A family of large (90 115 kDa) antimicrobial proteins that bind to cell wall peptidoglycan
proteins 3 and 4

Secretory (S)-IgA Composed of an IgA dimer (300 kDa), J chain (15 kDa), and secretory component (70 kDa).
Two isoforms: IgA1 (mostly recognizes proteins) and IgA2 (mostly recognizes carbohydrates).
Proteases produced by some bacteria (but not S. mutans) cleave S-IgA1. S-IgA2 has a deletion
in the hinge region that renders it resistant to these proteases

Antimicrobial Strategies in Caries bacterial acid production by inhibiting metabolic enzymes

Dental caries remains a significant public health problem.
Effective oral hygiene procedures have controlled the
incidence in the adult populations of developed countries.
However, prevention of caries in children and at risk
adults can require further intervention. In addition to con-
trol of carbohydrate intake and mechanical procedures
such as professional cleaning and sealants, there are a
number of strategies that target S. mutans and other acid-
producing bacteria.
Water Fluoridation
Community-based water fluoridation is one of the most
successful public health interventions both in terms of dis-
ease reduction and cost-effectiveness. There are a variety
of mechanisms by which fluoride exerts anticaries proper-
ties. Fluoride present in the aqueous phase of enamel
interacts with calcium in the calcium phosphate matrix
of enamel and both inhibits demineralization and pro-
motes remineralization of enamel. Topical fluoride also
has antimicrobial properties. Fluoride interferes with
and by disrupting the proton gradient across bacterial
membranes, thus rendering the organisms more suscepti-
ble to acidic conditions [36,37].
Topical Antimicrobials
Fluoride in dentifrices, varnishes and mouth rinses pro-
vides high levels of fluoride more directly to tooth
enamel. The broad-spectrum antimicrobial triclosan is
included in some dentifrices. Triclosan’s primary action is
through inhibition of fatty acid synthesis by targeting the
active site of the enoyl-acyl carrier protein reductase
enzyme. Chlorhexidine-containing mouth rinses are effec-
tive antimicrobials, and chlorhexidine has the property of
substantivity whereby it binds to oral surfaces and
remains active over extended times. Chlorhexidine works
mainly by membrane disruption [36,38].
Sugar Alcohols
S. mutans cannot ferment sugar alcohols and so when
used as sugar substitutes these compounds are not
954 PART | 8 Anaerobic Infections

TABLE 52.3 S. mutans Immunization Strategies

Immunogen Strategies and Outcomes

Killed whole cells Parenteral immunization in monkeys protected against caries. Potential for induction of heart
cross-reactive antibodies

Antigen I/II protein or fragments
Parenteral immunization in monkeys and rodents protected against caries. Cross-reactivity with
heart muscle is controversial. N-terminal fragment fused to CtxB as an adjuvant or delivered
by an attenuated Salmonella vector protected against caries in rodents when administered
intranasally. Oral passive immunization with antigen I/II antibodies (monoclonal or produced
in plants) prevented S. mutans colonization in non-human primates

Gtf enzyme or fragments Parenteral, subcutaneous and oral immunization in rodents protected against caries. Passive
immunization with antibodies produced in hens’ eggs or cows’ milk prevent S. mutans colonization

GbpB Parenteral immunization in rodents protected against caries

Chimeric Gtf-Antigen I/II Small segments of the glucan-binding domain of Gtf and antigen I/II fused and delivered
fragments intranasally in liposomes protected rodents against caries

DNA DNA encoding both the glucan-binding region of Gtf and AgI/II segments protected against caries
in rodents

cariogenic. Xylitol has additional anti-S, mutans proper-
ties, as the bacteria can incorporate xylitol through the
phosphoenolpyruvate phosphotransferase system and con-
vert it to xylitol-5-phosphate. Xylitol-5-phosphate inhibits
glycolytic enzymes, impeding both growth and acid pro-
duction. Moreover, this energy-consuming futile xylitol
metabolic cycle retards S. mutans growth [39].
Immunization
The search for a caries vaccine has been ongoing for sev-
eral years, utilizing a variety of immunogens and delivery
routes (Table 52.3) [40 42]. Although there has been
success in animal models, this has yet to be translated
into a human vaccine. The primary objective for caries
vaccine development has been to induce production of
protective salivary S-IgA antibodies. In the absence of
significant opportunity, or of functionality, to fix comple-
ment or opsonize, the action of S-IgA antibodies in saliva
is restricted to promoting aggregation, blocking adhesion
and inhibiting bacterial extracellular enzyme activity.
Hence immunogens are selected to elicit antibodies that
can block the sucrose-independent or sucrose-dependent
colonization mechanisms of mutans streptococci. On a
societal level, it remains to be determined if a caries vac-
cine will be considered sufficiently safe for use against
what is normally a non-life-threatening disease.
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