CONTROVERSY
Research on dental caries has a long history, and the biology of its development has proved to be far more complex than
anyone might have thought. Specificity and non-specificity of the causative bacteria have been at the center of the controversy of how this disease occurs and how it should be treated. From this article, it appears that the composition, metabolism, and
caries-causing capability of applicable bacterial mixtures are integrated, and that they are affected collectively by oral ecological conditions and changes. This is an area of Oral Biology that needs further exploration and could provide significant dividends in the development of our understanding of human ecological infections in general.
–Olav Alvares, Editor
A MIXED-BACTERIA ECOLOGICAL APPROACH
TO UNDERSTANDING THE ROLE OF THE ORAL
BACTERIA IN DENTAL CARIES CAUSATION:
AN ALTERNATIVE TO STREPTOCOCCUS MUTANS
AND THE SPECIFIC-PLAQUE HYPOTHESIS
I. Kleinberg
Department of Oral Biology and Pathology, State University of New York, Stony Brook, New York 11794-8702
ABSTRACT: For more than 100 years, investigators have tried to identify the bacteria responsible for dental caries formation and to
determine whether their role is one of specificity. Frequent association of Lactobacillus acidophilus and Streptococcus mutans with caries
activity gave credence to their being specific cariogens. However, dental caries occurrence in their absence, and the presence of other
bacteria able to produce substantial amounts of acid from fermentable carbohydrate, provided arguments for non-specificity. In the
1940s, Stephan found that the mixed bacteria in dental plaque produced a rapid drop in pH following a sugar rinse and a slow pH
return toward baseline. This response became a cornerstone of plaque and mixed-bacterial involvement in dental caries causation
when Stephan showed that the pH decrease was inversely and clearly related to caries activity. Detailed examination of the pH (acidbase) metabolisms of oral pure cultures, dental plaque, and salivary sediment identified the main bacteria and metabolic processes
responsible for the pH metabolism of dental plaque. It was discovered that this metabolism in different individuals, in plaque in different dentition locations within individuals, and in individuals of different levels of caries activity could be described in terms of a
relatively small number of acid-base metabolic processes. This led to an overall bacterial metabolic vector concept for dental plaque,
and helped unravel the bacterial involvement in the degradation of the carbohydrate and nitrogenous substrates that produce the
acids and alkali that affect the pH and favor and inhibit dental caries production, respectively. A central role of oral arginolytic and
non-arginolytic acidogens in the production of the Stephan pH curve was discovered. The non-arginolytics could produce only the
pH fall part of this curve, whereas the arginolytics could produce both the fall and the rise. The net result of the latter was a less acidic
Stephan pH curve. Both kinds of bacteria are numerous in dental plaque. By varying their ratios, we were easily able to produce
Stephan pH curves indicative of different levels of caries activity. This and substantial related metabolic and microbial data indicated
that it is the proportions and numbers of acid-base-producing bacteria that are at the core of dental caries activity. The elimination of
S. mutans, as with a vaccine, was considered to have little chance of success in preventing dental caries in humans, since, in most cases,
this would simply make more room for one or more of the many acidogens remaining. An understanding of mixed-bacterial metabolism, knowledge of how to manipulate and work with mixed bacteria, and the use of a bacterial metabolic vector approach as
described in this article have led to (1) a more ecological focus for dealing with dental caries, and (2) new means of developing and
evaluating anti-caries agents directed toward microbial mixtures that counter excess acid accumulation and tooth demineralization.
Key words. Streptococcus mutans specificity, dental caries causation, mixed-bacteria metabolism, dental plaque pH, alkali formation.
(I) Introduction
T
oward the end of the 19th century, the new science of bacteriology arose as a result of major advances in the formulation of growth media and the development of techniques for
isolating and studying bacteria. Micro-organisms proved to be
major causes of several diseases that were fatal to humans.
Disease occurred when a specific micro-organism reached a
target site where the tissues and conditions enabled the organism to flourish and reach the elevated numbers needed to
cause significant damage to the host and even death. W.D.
Miller (1890) learned these isolation techniques and applied
them to the examination of the many micro-organisms resident
within the oral cavity. At the same time, he found that, collec-
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13(2):108-125 (2002)
tively, the mixed bacteria contained in whole saliva were able
to produce acid from added fermentable carbohydrate when
incubated at body temperature, and that this acid was sufficient to decalcify teeth. When fermentable carbohydrate was
not added to the saliva, putrefaction replaced fermentation,
alkalinity replaced acidity, and no decalcification was seen.
These studies enabled Miller to formulate his acid decalcification theory for dental caries which, in simple terms,
involved two steps: First, the mixed bacteria resident in the
mouth produce acid from fermentable carbohydrate; and second, the acid then dissolves tooth mineral to initiate and sustain the cavity-producing process. Stimulation of acid production has been extensively studied because of its central role in
the caries process, and was confirmed many times with bacterial isolates from the oral microbiota (Stephan and Hemmens,
1947; Kleinberg et al., 1982; Denepitiya and Kleinberg, 1984;
Wijeyeweera and Kleinberg, 1989a; van Houte, 1994; van
Houte et al., 1994, 1996), with dental plaque and salivary sediment in vitro (Kleinberg et al., 1982; Singer et al., 1982; Salako
and Kleinberg, 1992), and with plaque in vivo (Stephan, 1940,
1944; Strålfors, 1950; Kleinberg, 1961; Imfeld, 1983).
Since the time of Miller, there has been continual controversy regarding the role of bacteria in dental caries causation,
particularly in humans. At issue have been (i) whether a specific bacterial species or a non-specific mixture of bacteria is
the agent responsible, and (ii) whether it is an infectious bacterial disease in the classic sense or an ecological overgrowth.
In recent years, Loesche (1986) brought focus to this controversy by proposing a specific-/non-specific-plaque hypothesis
for dental caries and concluded that specificity and classic
infection were on the correct side of the debate. Accordingly,
he argued that the way to deal with the microbial aspect of the
dental caries problem is targeted elimination from the oral
microbiota of what is generally known as Streptococcus mutans,
or the mutans streptococci (for example, with antibiotics or
vaccines; Loesche, 1986; Taubman and Smith, 1993). In framing
the argument, Loesche considers that those favoring the nonspecific side of his hypothesis believe that the oral bacteria as
a whole produce the acid from fermentable carbohydrate that
is central to dental caries formation. So the role of bacteria in
caries causation is one of non-specificity. In this case, removal
of as much as possible of the plaque that accumulates on the
teeth is a logical means of dealing with the caries problem.
To a certain degree, the author of the present article falls
into the non-specific group, since he has concluded that
Streptococcus mutans cannot be, except in a few specific cases,
the bacterial cause of dental caries. This is simply because
many non-mutans micro-organisms are sufficiently acidogenic/aciduric and numerous to produce the amounts of acid
necessary (van Houte, 1994, and others). However, and as covered below, he also considers the bacteria involved in dental
caries causation to be due to an increase in a mix or spectrum
of resident acidogenic/aciduric oral micro-organisms and/or
a decrease in a mix or spectrum of resident bacteria best able
to produce counteracting alkali. Like others, he considers such
microflora changes reactive, since they occur in response to
changes in the oral ecology (Marsh, 1989).
The present article identifies problems with S. mutans
being the causative element of the dental caries process and
has as its main thrust the thesis that dental caries results largely when the bacterial compositions of the microflora resident
in predominantly retentive dentition sites are such that they
13(2):108-125 (2002)
generate excessive amounts of acid and/or deficient amounts
of base. As I hope will become evident below, alkali deficiency
is an important element of the microbial dysfunction involved,
and bacterial alkali production is closely linked to the protection against caries afforded by saliva.
In accordance with the Loesche hypothesis, which considers certain of the mutans streptococci to be the primary cause
of dental caries, treatments and cures are directed toward
elimination of these micro-organisms. On the other hand,
where mixtures of diverse acidogens are involved, and they
arise in response to ecological change, treatments and cures
need to be directed toward correction of, or compensation for,
the oral ecological dysfunction involved. The bacteria, while
essential, are necessarily secondary. In this regard, encouraging formation of alkali by the oral bacteria is likely to be an
important aspect.
(II) Lactobacilli and Streptococci as Specific
Dental Caries Infectious Agents
The lactobacilli and streptococci are major genera of the category of bacteria generally referred to as the lactic acid bacteria
(Thompson, 1987), so it is no surprise that microbial species
within these genera have been proposed as specific agents of
the acid production that is primary to the dental caries process
(Hamada and Slade, 1980; Loesche, 1986; van Houte et al.,
1994; Liljemark and Bloomquist, 1996). In a search for a cariescausative micro-organism from among the mixed oral microbiota, Bunting et al. (1929) and Jay (1947) identified lactobacilli in general and Lactobacillus acidophilus (B. acidophilus) in particular as possible candidates. Many studies carried out by
them and thereafter by others have shown frequent association between the presence of lactobacilli and the prevalence of
dental caries, suggesting such a possibility. For example, elevation of the level of fermentable carbohydrate in the diet led
to elevated lactobacillus counts, whereas lowering of such carbohydrate resulted in lactobacillus reduction (Becks et al.,
1944; Becks, 1950). Dentition sites favoring retention of fermentable dietary carbohydrate also favored elevated numbers
of lactobacilli and the development of dental caries lesions
(Stecksén-Blicks, 1985; Crossner et al., 1989). These sites included the pits, fissures, and approximal areas of the teeth where
caries lesions are most frequently found (Klein and Palmer,
1941; Barr et al., 1957). Also observed was that the placement
of dental appliances such as orthodontic bands on dentition
sites changes the morphological conditions, which then lead to
enhanced carbohydrate retention, more lactobacilli and other
acidogens, a more acidogenic dental plaque, and, in turn, to
caries elevation (Sakamaki and Bahn, 1968; Balenseifen and
Madonia, 1970; Chatterjee and Kleinberg, 1979; Scheie et al.,
1984; Boyar et al., 1989).
Despite these relationships, other observations indicated
that lactobacilli are not essential for caries development. This
is because caries lesions can develop in the absence of lactobacilli, and other acidogens that are resident members of the
oral microbiota can provide the necessary acid. The relationship between lactobacilli and dental caries, at least in humans,
was not proven to be cause-and-effect. A better argument
could be made for its being associative (Sims, 1985).
The intensity of this difference in belief subsided until the
early 1960s. It then flared up again with S. mutans (first isolated by Clarke in 1924 from caries lesions) being identified as a
possible caries-causing candidate (Fitzgerald and Keyes, 1960;
Crit Rev Oral Biol Med
109
ble incisor teeth could produce a rapid
and substantial decrease in the pH
immediately following exposure to a
sugar challenge in the form of a rinse
with a glucose or sucrose solution. After
reaching a minimum, the pH showed a
subsequent slow rise to baseline, which
usually took about one hour. The second study was of even greater importance, since it showed that the extent of
decrease and location on the pH scale of
this sugar-challenge curve (subsequently called the Stephan curve) was
inversely related to the caries activities
of the subjects tested (Fig. 1). He measured caries activity by scoring individuals for caries lesions over a two-year
period and establishing subject groups
with caries activities ranging from
caries-free to highly caries-active (Fig.
1).
Figure 1. Relation between the Stephan pH challenge test and dental caries activity in maxillary
In a subsequent study in vivo,
and mandibular incisor plaque of subjects with different levels of caries activity. The challenge
Kleinberg
(1961) manipulated sugar
was a 10% (w/v) glucose solution administered as a rinse (25 mL) for two min, and the pH was
measured with an antimony-rod touch-type pH electrode (adapted from Stephan, 1944, and substrate availability in a wide variety
of ways and showed a clear cause-andreproduced from Kleinberg et al., 1982).
effect relationship between substrate
availability and the magnitude and
Keyes, 1960; Keyes and Jordan, 1963; Carlsson, 1968;
duration of the subsequent plaque pH fall (Fig. 2). Glucose
Guggenheim, 1968; Duchin and van Houte, 1978). To the large
was the substrate provided, and availability was defined as
majority of researchers in this field, it became the caries cause
the product of substrate concentration and the time it was
célèbre, and a massive amount of research ensued. Subsequent
available. The pH fall, the reaching of a pH minimum and the
studies led to the discovery of at least 8 serotypes of S. mutans,
slow pH rise thereafter, and the characteristics of the Stephan
which were then grouped mainly into 4 species relevant to
curve were explained in terms of the kinetics of acid produchumans and small laboratory animals (Loesche, 1986;
tion and acid removal under open system conditions and conMichalek and Childers, 1990). Serotypes c, e, and f remained S.
ditions of limited availability of sugar substrate. Delay in the
mutans; d, g, and h became S sobrinus; serotype a became S.
return of the pH to baseline was observed and explained by
cricetus, and serotype b became S. rattus. Collectively, they are
the prolongation of acid production when the availability of
now referred to as the mutans streptococci. To other investigaglucose substrate was prolonged. The extent of the plaque pH
tors, including the author, S. mutans has simply been another
decrease (i.e., how low and for how long) was attributed to the
acidogenic micro-organism of many within the human mouth
concentration of bacteria present and the carbohydrate subthat are able to produce relatively large amounts of acid. As
strate available (Kleinberg, 1961, 1970a).
indicated above, acid is the central virulence agent in dental
That the basis of the Stephan curve was essentially an
caries development, and, as seen earlier for lactobacilli, similar
enzyme-substrate type of relationship expressed in an open
associations with availability of fermentable carbohydrate in
system was supported in a follow-up study with urea
the diet and with dental caries were also observed (see
(Kleinberg, 1967b). This showed that making this nitrogenous
Kleinberg, 1977a,b, for references and discussion; Hamada and
substrate available to plaque in situ, as in the glucose study,
Slade, 1980; Loesche, 1986). In a sense and to a large degree,
gave almost exactly the same results, except that base formahistory repeated itself with the “discovery” of this new microtion and rise in the pH occurred rather than acid formation
bial acidogen.
and a fall in the pH (Fig. 2). The results with glucose and urea
were basically symmetrical and indicated that both acid and
(III) Basis and Dynamics of Acid-Base pH
base formation can contribute to the plaque pH and serve as
Change in Dental Plaque in vivo
counteracting metabolic forces in the caries process.
In the interim period between focus on species of lactobacilli,
The two Kleinberg plaque in vivo studies (1961, 1967b) led
particularly L. acidophilus, and species of streptococci, particuto the classification of plaque pH responses into two types and
larly S. mutans, significant advances were made in identifying
the recognition that they were determined by the substrate
their roles in the dental caries process of dental plaque as a
provided and its availability. They also led to the vector format
whole and a tendency to move in the direction of less specishown in Fig. 3, where the effects on the plaque pH of low and
ficity. This resulted from two landmark investigations, carried
high substrate availability can be seen. With glucose as subout by Stephan (1940, 1944), demonstrating that dental plaque
strate, the first condition produced a pH curve in which there
has the ability to produce rapid and substantial decreases in
was a rapid pH fall and a subsequent slow pH rise which
pH in vivo. The initial study showed for the first time that denoccurred once the glucose was consumed by the plaque bactetal plaque that was allowed to accumulate on readily accessiria (i.e., the Stephan pH curve response). The second (high sub-
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strate) condition also produced a curve
with a rapid pH fall, but this was followed by a “bottoming out” of the pH,
where it remained as long as there was
fermentable carbohydrate substrate still
available. In the case of urea, it was pH
rise and fall, and pH rise and plateauing, respectively.
What was striking in these
Kleinberg pH studies was that dental
plaque, a complex mixture of several
hundred species of resident bacteria
(Moore et al., 1982; Liljemark and
Bloomquist, 1996), functioned as a single entity and did so in a relatively simple and predictable manner. This led to
the recognition that pH could be used as
a master variable of this mixed-bacterial
system (Kleinberg, 1970a), and that pH
response to substrate challenges with
primary substrates such as glucose and
urea was an easy way to measure the
dynamics of overall pH (acid-base)
metabolism in real time.
(IV) Dental Plaque
as a Mixed-bacterial Entity
of Coordinated pH Metabolisms
Figure 2. The effect of the availability of glucose and urea substrate on the pH of dental plaque
in situ. Availability can be prolonged as shown here by raising the concentration of the challenge substrate or extending the time that substrate is available to the plaque bacteria (from
Kleinberg, 1970b).
In an extensive survey of the pH of dental plaque in different dentition locations throughout the mouth and its relation to meals, rates of resting salivary flow, and salivary pH,
an observation of broad significance was made (Kleinberg and
Jenkins, 1964). Plaque in different dentition sites within a
given person’s mouth, in the same dentition sites in different
individuals’ mouths, and in sites showing different caries
prevalence rates all showed the same type of pH response
after eating, except that the responses varied in magnitude of
pH change and location on the pH scale (Fig. 4a). In this study
on 85 subjects (and about 12,000 pH measurements), the pH
was highest in the morning before the subjects ate breakfast,
lower after fermentable carbohydrate became available to the
plaque bacteria throughout the mouth, and rose slowly thereafter toward baseline over a mean period of 3½ hours. The site
variation in this pH synchrony was attributed by Kleinberg
and Jenkins (1964) to two primary factors. One was variation
in the access of the dentition sites to saliva, which followed a
pattern largely determined by variation in amounts of saliva
from the three pairs of major salivary glands and the intra-oral
locations of their duct orifices (Schneyer and Levin, 1955a,b;
Sreebny, 1987, 2000). The other was attributed to variations in
tooth and dentition morphology (Kleinberg, 1978). These variations affect the amounts and types of bacteria that are able to
accumulate in the different tooth and dentition sites throughout the mouth, and substrate availability. Both are primary to
acid formation: One supplies bacterial enzymes, and the other
the substrates.
A significant observation made in the comprehensive
Kleinberg and Jenkins (1964) study with broad consequences
was that plaque in dentition sites favored by saliva, and in
individuals with a higher basal (i.e., resting) rate of salivary
flow and pH, favored higher levels of plaque pH (Fig. 4b).
13(2):108-125 (2002)
Figure 3. Vector format of the two types of acid-base pH curves that
result from short and long periods of exposure to glucose or urea as
seen in Fig. 2. A Type A response results when substrate is limited. A
Type B response results when substrate is in excess (adapted from
Kleinberg, 1977b).
Note that the resting saliva pH fell between the fasting (i.e.,
highest) pH and the minimum (i.e., lowest) pH of the plaque
pH ranges shown. Its significance is that a sustained fasting
pH so much higher than the salivary pH, as seen here, can
happen only if substantial base formation is continually taking
place within the dental plaque, particularly at this time of the
day (Kleinberg and Jenkins, 1964; Kleinberg, 1967b).
The synchrony seen in the Kleinberg and Jenkins investigation is illustrated in Fig. 5, where the pH of dental plaque of
Crit Rev Oral Biol Med
111
pH response were different, presumably because of differences in saliva
availability and amounts of plaque
that accumulate because of dentition
location and morphology.
(V) Simulation of the Stephan
pH Curve in vitro with Single
and Mixed-bacterial
Populations
As a first step toward identification of
the bacteria mainly responsible for the
Stephan curve and its simulation,
Stephan and Hemmens (1947) prepared
bacterial isolates from dental plaque and
examined each alone and in mixtures to
see whether and which could give in
vitro the rapid pH fall and subsequent
slower pH rise characteristic of the
Stephan curve in vivo. Alone, pure cultures of many of the oral bacteria tested
Figure 4. (a) Relation between time of eating and the pH of dental plaque in 18 different intercould produce an acidic pH response,
proximal and facial/lingual dentition locations throughout the human mouth. Highest pH
but few could give the curve seen in vivo
occurred in plaque in the mandibular interproximals of the lower incisors. Lowest pH occurred
for plaque as a whole. In the experiments
in the same sites of the upper incisors (see Kleinberg and Jenkins, 1964, for the specifics of the
in which they explored mixtures of the
individual sites). Mean resting saliva pH was 6.7 (from Kleinberg, 1970b). (b) Range of pH of
isolates, they opened the door to the posplaque on upper and lower approximal incisor surfaces in slower and faster salivary secreters.
The dotted lines represent the mean resting salivary pH of the two groups (from Jenkins, 1979,
sibility that bacterial mixtures as well as
based on data in Kleinberg and Jenkins, 1964).
single micro-organisms might be
Stephan pH curve simulators. It soon
became clear that mixing pure cultures
to identify the bacteria responsible for production of the Stephan
curve and its relation to dental caries activity was not an easy
task. It meant sorting through so many combinations and permutations of plaque bacterial isolates that the task of finding bacterial mixtures or combinations that were applicable was
extremely remote. Combining more than a few bacteria can be an
experimental nightmare (see, for example, Fig. 9 of Wijeyeweera
and Kleinberg, 1989b). These difficulties may have been the reason for this important work of Stephan and Hemmens (1947)
being hardly noticed and coming to an end.
Despite their inability to identify the bacteria primarily
responsible for the Stephan curve, Stephan and Hemmens (1947)
nonetheless made several important observations, one of which
was that a high concentration of oral acidogenic micro-organisms
was essential for the production of the initial rapid and oftentimes extensive pH fall seen in plaque in vivo in response to a
Figure 5. The effect on plaque pH levels of rinsing for 2 min, first with
sugar substrate challenge. Using pure culture biofilms, Strålfors
100 mL of a 1% and then with 100 mL of a 20% (w/v) glucose solu(1950) confirmed the importance of high bacterial cell concentration. Each point is the mean pH of 2 plaque sites in each of five subtions in the production of rapid acidification of the plaque pH
jects. Each rinse was divided into two 50-mL portions, and rinsing
and in the formation of the Stephan curve. The biofilms he conwith each was for 1 min (adapted from Kleinberg et al., 1981).
structed consisted of pure cultures of oral bacteria encapsulated
in agar gels formed around a glass pH electrode in a configurathree representative dentition sites was compared after two
tion that simulated dental plaque in situ and enabled the pH of
successive sugar challenges of different magnitudes. The influboth its inner and outer surfaces to be measured at the same time.
ence of saliva is relevant when maxillary and mandibular
Thereafter, Kleinberg (1967a) showed that the Stephan pH curve
interproximal plaque samples are compared (also cf. Fig. 1).
could be simulated with salivary sediment suspensions. These
The effect of plaque thickness, to a large degree, is probably
various studies were collectively extremely important, because
involved when maxillary labial and maxillary interproximal
they demonstrated that this curve could be modeled with bacteplaque samples are compared. Important to note is that the pH
ria either in suspensions or in biofilms, with pure or mixed bacchanges in these three dentition locations were in synchrony,
terial cultures, and either in vivo or in vitro.
but the locations on the pH scale and/or their magnitudes of
Subsequent studies in our laboratory proved salivary sed-
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Crit Rev Oral Biol Med
13(2):108-125 (2002)
iment to be metabolically and microbially remarkably similar
to pooled dental plaque (Kleinberg et al., 1982; Singer et al.,
1982; Salako and Kleinberg, 1992; Ryan and Kleinberg, 1995a).
Salivary sediment is easily obtained from stimulated whole
saliva by centrifugation. It consists of a sampling of the bacteria that are ready to be dislodged or shed from the surfaces of
the oral hard and soft tissues (Kleinberg and Westbay, 1992;
Liljemark and Bloomquist, 1996). The bacteria on these surfaces constitute a continuous culture of mixed bacteria, and
many are shed as part of mucosal epithelial desquamation. A
major advantage of using salivary sediment as a simulant of
dental plaque acid-base metabolism is that it is available in
abundance and provides a sampling of oral mixed bacteria
“on tap” that have grown in and are compatible with oral
environmental conditions. There is no need to grow oral isolates outside the mouth and recombine them for use in many
oral mixed-flora experiments. In the use of salivary sediment
for study, it was found important that the cell concentration be
appropriately adjusted and fixed (Singer et al., 1982), and that
donor subjects had fasted overnight before saliva collection
was made in the morning (Sandham and Kleinberg, 1969).
Overnight fasting ensured that little or no bacterial carbohydrate could be stored to affect the initial pH and the extent and
nature of the pH fall in these experiments (Denepitiya and
Kleinberg, 1984). For many experiments, a pre-incubation to
deplete such substrates was also done.
(VI) Determination of the Main Metabolic
Pathways and Acids Involved in the Lowering
of the pH when Fermentable Carbohydrate is
Catabolized by the Oral Mixed Bacteria
A series of investigations was carried out to characterize the
metabolism of the salivary sediment system under glucose
substrate conditions that produced the main types of pH
curves, like those seen in plaque in situ (Sandham and
Kleinberg, 1969, 1970a,b). Along with standardization of the
sediment concentration at the 16.7% (v/v) level, the glucose
concentration was varied between 0 and 30% (w/v), as was
done in vivo (Kleinberg, 1961), and pH, uptake of glucose, and
formation of lactic and other short-chain carboxylic acids
(SCCA) were determined along with carbon dioxide over a
four-hour period of incubation at 37°C. At the same time,
experiments were done where the carbon dioxide produced
from glucose labeled on different carbon atoms with 14C was
examined by a micro-radiorespirometric method for determination, by 14C tracing, the metabolic pathways whereby glucose is degraded (Sandham and Kleinberg, 1970b).
These experiments showed progressively greater acidic pH
responses as the glucose concentration was progressively
increased. An active Embden-Meyerhof Scheme (EMS) and an
incomplete tricarboxyllic acid (TCA) cycle were demonstrated.
Instead of an intact cycle, pyruvate arising out of the EMS pathway flows into different types of short-chain carboxylic acids,
with the types and their proportions determined by the glucose
level. At low glucose levels, when the pH rapidly fell and then
slowly rose, L(+) lactic acid correspondingly rapidly rose and
then slowly fell (Sandham and Kleinberg, 1970a; cf. dental
plaque in vivo in Geddes, 1972, 1975). Basically, it behaved like a
metabolic intermediary, since it is subsequently converted mainly to carbon dioxide and acetic acid. In contrast, at high glucose
levels, when the pH fell and reached and remained at a more
13(2):108-125 (2002)
acidic level, lactic acid rose asymptotically and behaved like an
end-product along with acetic and propionic acids, which progressively rose (Sandham and Kleinberg, 1970a). An important
factor that comes into play at high sugar concentrations and
affects the SCCA proportions is that a low pH is reached which
can inhibit or slow lactic acid formation (Iwami and Yamada,
1980), and its subsequent conversion to acetic and propionic
acids is slowed or stopped (Sandham and Kleinberg, 1970a).
An important observation was that salivary sediment,
even though comprised of many different types of bacterial
cells, evidently behaved in synchrony, and the study of its
overall metabolism was no more difficult than studying pure
cultures or simple pure culture mixtures (Traudt and
Kleinberg, 1999). Simplified for subsequent experiments was
the observation, once more, that two kinds of pH curves are
observed as in plaque in vivo, one at low sugar availability (initial fast pH fall followed by a slow pH rise) and the other at
high sugar availability (initial rapid pH fall followed by a
delayed or no pH rise). Explanations for pH curve shapes and
their relation to glucose utilization and amounts and types of
short-chain carboxylic acids generated were made evident by
these experiments (Sandham and Kleinberg, 1970a). Also evident from these studies was that the metabolic configuration
of the sediment microflora entity was an aerobic glycolysis
type of metabolism (Korayem et al., 1990), which fits in with
the low oxygen environment found in the mouth (Globerman
and Kleinberg, 1979).
(VII) Effects of Saliva on the Stephan pH Curve
Saliva is the main biological fluid bathing the oral microbiota in the oral cavity and is the medium of the salivary sediment system. My colleagues and I therefore performed
experiments to examine its effects on the Stephan pH curve
in the sediment system and on the fermentation parameters
responsible for its shape (Kleinberg et al., 1973). The effects of
saliva were profound (Fig. 6), and its manifestations at low
and high substrate levels were different. At low substrate
concentrations, there was a less-than-optimal pH fall with
saliva present; the pH reached a minimum, and then subsequently slowly rose toward baseline. When saliva was
absent, the initial pH fall was rapid as before but instead fell
to a lower level, reached a pH where it leveled off, and
remained there for much of the experimental period. In other
words, the pH fall without saliva was enhanced, and the rise
part of the Stephan pH curve was lost. Part of the pH rise loss
may be due to the absence of salivary bicarbonate
(Wijeyeweera and Kleinberg, 1989b).
At high substrate concentrations with substrate in excess,
the pH fell as before, and no pH rise was seen with or without
saliva (Fig. 6). However, examination for glucose uptake and
other metabolic parameters showed that glucose utilization
and acid formation were much greater with saliva present
(Fig. 6). Evidently, base formation and saliva buffering when
saliva is present prevent the pH from falling to a lower pH
than would otherwise have occurred. The net result is that the
glucose substrate is cleared more rapidly because of the saliva.
In vivo, clearance is enhanced in those oral sites where saliva is
continually being replenished (Dawes, 1989). It appears that
excessive fermentation and deficient saliva are two overall factors that can work together to make return of the pH from
acidity more difficult than with either alone.
Crit Rev Oral Biol Med
113
(B) CONTRIBUTION OF UREA
TO BASE FORMATION
Urea is also involved in base formation
by saliva. When its degradation and
effects on the pH were explored in the
salivary sediment system, it had minimal effects if no exogenous urea was
added. This is because the amounts present in saliva supernatant, when used in
this in vitro system, are limited. In contrast, in vivo, where saliva is continuous
and the supply of urea is therefore substantial, the effect of urea on the pH is
much greater (Kleinberg and Jenkins,
1964), and its effect on caries activity
can be considerable (Peterson et al.,
1985; Meyerowitz, 1993). For this reason, our studies on urea have been both
in vitro and in vivo (Biswas and
Kleinberg, 1971; Singer et al., 1982;
Singer and Kleinberg, 1983a,b). The
experiments on urea catabolism
showed mostly production of ammonia
and carbon dioxide, but some amino
group incorporation into amino acids
was also demonstrated (Singer and
Kleinberg, 1983a,b). The incorporation
was mostly into alanine, especially if
there was some glucose present.
Figure 6. Effects of various levels of salivary supernatant on the pH of salivary sediment incubation mixtures incubated with (a) 2.8, (b) 5.6, and (c) 28.0 mM glucose (0.05, 0.1, and 0.5%
w/v, respectively). Shown in (d) are the effects in salivary sediment incubation mixtures of 33.3%
(v/v) salivary supernatant from centrifuged whole saliva on the utilization of glucose at 2.8 and
28.0 mM glucose (adapted from Kleinberg et al., 1973).
(A) IDENTIFICATION OF THE PH-RISE FACTOR
IN SALIVA
To identify the constituents responsible for the pH-raising effect
of saliva seen in vitro, we next carried out saliva fractionation
experiments. This proved to be mostly arginine available from
small arginine peptides (Kleinberg et al., 1979). This common
amino acid, especially if supported by saliva buffering, proved to
be highly effective (Wijeyeweera and Kleinberg, 1989a). The
buffering capacity of saliva is a property that has often shown an
inverse correlation with caries prevalence (Afonsky, 1961) and is
largely due to salivary bicarbonate (Lilienthal, 1955).
The metabolic pathways involved in arginine degradation
were also explored. Its conversion to ornithine was shown to
occur via the arginine deiminase pathway (Kanapka and
Kleinberg, 1983), and although ornithine can lead to some
putrescine formation, subsequent examination indicated that
its main degradation occurred via glutamate and a-ketoglutarate to yield, ultimately, ammonia, carbon dioxide, and
acetate (Traudt and Kleinberg, 1999).
114
(VIII) Microbial Elements
Responsible for the Stephan
Curve and its Relation
to Dental Caries
Once arginine was identified, this
amino acid was all that was needed,
together with glucose, to produce the
Stephan pH curve in vitro. When provided as its bicarbonate salt to ensure
buffering like that normally present in
saliva (cf. Stephan and Hemmens,
1947), the stage was set to tackle the question of which bacteria of the resident oral microflora entity could produce
Stephan pH curves in vitro with only glucose and arginine as
the substrates provided (Kleinberg et al., 1982; Wijeyeweera
and Kleinberg, 1989b). In other words, the Stephan pH curves
seen with and without saliva could now be simulated without
the complications of using saliva supernatant, with its multiplicity of substrates and buffers that might affect the pH.
The results were dramatic (Table) and are illustrated for
some bacteria in Fig. 7, along with results where arginolytic
and non-arginolytic bacteria were combined. Many of the
major acidogenic bacteria in the survey produced a pH fall but
little or no pH rise (the non-arginolytics), whereas similar acidogenic bacteria also produced a pH fall but one that was less,
because these bacteria could produce base from arginine
(arginolytics). Mixing the two types of bacteria generally
resulted in curves intermediate in value.
Relevant to the dental caries process is that the bacterial
members that carry only the acid-forming function are those
Crit Rev Oral Biol Med
13(2):108-125 (2002)
TABLE
pH-raising Activity of Oral Micro-organisms Determined with Arginine or Arginine Peptide
(from Kleinberg et al., 1982)
pH-raising
Non-pH-raising
pH-raising
Non-pH-raising
S. mutans FA-1 (b)
BHT (b)
GF-71 (b)
130-P (b)
S. mutans OMZ-61 (a)
E-49(a)
AHT (a)
A. naeslundii ATCC 19039
A. viscosus ATCC 15987
S. sanguis G9B (Type A)
S. milleri
S. faecalis ATCC 4082
S. mutans GS-5 (c)
10449 (c)
Ingbritt (c)
S. mutans P-4 (e)
AT-10 (e)
LM-7 (e)
S. mutans OMZ-175 (f)
QP50-1 (f)
S. mutans ATCC 27353 (d/g)
OMZ-176 (d/g)
B-13 (d/g)
6715 (d/g)
S. sanguis ATCC 10557 (Type B)
S. mitior
A. odontolyticus ATCC 17982
A. israelii ATCC 27037
L. cellobiosus ATCC 11739
L. brevis ATCC 11577
L. acidophilus ATCC 4356
L. salivarius ATCC 11741
L. fermentum NCTC 6991
L. casei NCTC 6375
N. sicca ATCC 29256
N. subflava ATCC 10555
B. catarrhalis ATCC 23246
Organisms were tested in four-hour incubations (600 mL) at 37°C containing bacterial cells at a suspension concentration of 8.3% (v/v), glucose at 4.2 mM,
and arginine or the arginine peptide, lysylargine, at 3.3 mM. S. mutans serotypes are shown in parentheses. Although S. mutans AHT was obtained and
is shown as an “a” serotype, when tested with various fluorescent antisera, it was found to be a “c” serotype.
that most tend to be cariogenic. When fermentable carbohydrate becomes available in the mouth during eating, one can
expect these bacteria to produce a substantial amount of the
acid, cause a rapid fall in the pH, and keep the pH acidic for as
long as there is fermentable carbohydrate left to sustain acid
production. In contrast, the bacteria in plaque that also possess
the ability to raise the pH (by degrading arginine and producing base; Kanapka and Kleinberg, 1983) would be expected to
produce less net acid and as a consequence be less or non-cariogenic. Hence, it is interesting to note that all of the mutans
streptococci, except for S. rattus, are members of the first group,
whereas S. rattus belongs to the second group (Table). The same
two kinds of acid-producing bacteria are seen with the oral lactobacilli. Some are arginolytic, while others, such as L. acidophilus and L. casei, are not (Table). In other words, the mutans
streptococci and lactobacilli that have been related to increased
dental caries activity (Hamada and Slade, 1980; Loesche, 1986;
and others) are species that are non-arginolytic, whereas S. rattus, which has been associated both in animals and in humans
with the caries-free condition (Kilian et al., 1979), is arginolytic.
Evident from these experiments was that a higher ratio of
arginolytic to non-arginolytic bacteria in a mixture of the two
is conducive to a less cariogenic type of Stephan pH curve,
whereas the reverse ratio is more favorable to a more cariesactive type of curve. At long last, we were able to achieve
what Stephan and Hemmens (1947) had hoped to do more
than 50 years ago. Also evident is that acid and base formation both occur in dental plaque and counteract each other in
the determination of plaque pH. As a consequence, a deficiency in base formation (largely associated with saliva) can
be as important in dental caries development as can excessive
formation of acid from fermentable carbohydrate (largely
associated with the diet). Identification of arginine as a means
13(2):108-125 (2002)
of combating dental caries by enhancing base formation has
added another and important basis for saliva being protective
against this disease. Urea was identified earlier as a possibility for enhancing the formation of base, but it can have some
problems if inappropriately used as an anti-caries agent
(Jenkins and Wright, 1950, 1951).
Further experiments enabled flora shifts associated with
greater acidogenicity and caries development to be simulated
and thus effects on the pH curve to be studied. For example,
change from a non-cariogenic to a cariogenic type of microflora
is favored when the oral environment is enriched with fermentable carbohydrate from the diet (Becks et al., 1944), or when
a condition of saliva deficiency develops, as happens with onset
of a xerostomic condition (Sreebny, 1987, 2000). Simulation in
vitro is accomplished by the addition, to plaque or sediment, of
the non-arginolytic, acidogenic bacteria that might rise when
such a shift occurs (Fig. 8). The converse would simulate a
change to a non-cariogenic microflora by the addition of an
arginolytic micro-organism (Fig. 8). This is a simple means of
flora manipulation that opens the way for quantitative study of
the effects on the acidogenicity such changes could or would
produce. It identifies as well the influence, on the metabolism of
the microflora entity, of changes in the proportions of its microbial constituents, whether caused experimentally or ecologically.
(IX) Bacteria that are Significant Contributors
to the Biochemical Processes that Produce the
Plaque Acid-Base Metabolic Vectors
Extensive studies have been carried out in our laboratory to
identify which biochemical processes and which of the numerically more prominent oral bacteria are responsible for the metabolic vectors that form the foundation of plaque pH metabolism
Crit Rev Oral Biol Med
115
Figure 7. Examples of pH responses of related arginolytic and nonarginolytic bacteria (see Table) mixed in a 1:1 ratio and incubated
with glucose at 4.2 mM and arginine at 3.3 mM. Total bacterial cell
concentration was 8.3% (v/v). In all cases, the mixing of pure cultures
of oral bacteria produced an intermediate result (from Wijeyeweera
and Kleinberg, 1989b).
(Fig. 9a). From these extensive microbial-metabolic investigations, it was found that most Gram-positive bacteria and some
Gram-negatives contribute to vector 3 (Salako and Kleinberg,
1992; Ryan and Kleinberg, 1995a), which is of prime importance
in the causation of dental caries (Stephan, 1944). H. parainfluenzae, A. viscosus, A. naeslundii, and Staph. epidermidis and possibly
Strep. salivarius are key contributors to vector 1 (Sissons et al.,
1988; Salako and Kleinberg, 1989), which is important for calculus formation and dental caries resistance. The pH-rise bacteria
listed in the Table contribute to vector 4, which in this article is
considered a key element in counteracting dental caries development. A mix of Gram-positive and Gram-negative bacteria
contributes to vector 2 (unpublished data). Some of the shortchain carboxylic acids (SCCA) produced in vector 2 from amino
acids are active in eliciting cell and gingival crevicular fluid
changes associated with gingivitis (Niederman et al., 1997).
116
Figure 8. In vitro pH response of pooled dental plaque to which (a) S.
mutans GS-5 (serotype c), (b) S. sanguis II (i.e., oralis) isolated from
saliva, (c) L. casei, or (d) A. viscosus had been added. Incubation mixtures were prepared as in Fig. 7. All four strains are generally considered to be cariogenic. Note that none has pH-raising capability
(Table). In each case, the Stephan pH response was made more acidogenic with their addition (from Wijeyeweera and Kleinberg,
1989a).
(X) Combination Experiments with Pure
Cultures Relevant to Plaque pH Metabolism
Experiments with pure cultures have demonstrated that bacteria carrying one or more of the metabolic vectors shown in Fig.
9a will generally give an averaging effect when similar vectors
are combined (see Figs. 7 and 8). However, when the bacteria
mixed carry different metabolic vectors, then the result is one
of addition. This would be the case where there is, for exam-
Crit Rev Oral Biol Med
13(2):108-125 (2002)
ple, a tandem relationship between two micro-organisms. A
streptococcus that produces lactic acid from glucose in combination with a veillonella organism that utilizes the lactic acid
produced is a good example of oral bacteria in a tandem relationship (Hamilton and Ng, 1983). Another good example is
the production of hydrogen peroxide from glucose by any of
several streptococci and the utilization of the peroxide they
produce by Neisseria sicca, Haemophilus parainfluenzae and segnis, Actinomyces viscosus, and Staphylococcus epidermidis (Ryan
and Kleinberg, 1995b).
The benefit of using a metabolic vector approach is that it
“bundles” bacteria metabolically and thus makes it much easier to handle bacterial mixtures both conceptually and experimentally. Also, ecological conditions and their relation to disease-causing potential are more easily perceived and symbolized (Fig. 9b; Kleinberg and Westbay, 1992). One can look at
mixtures as functional units rather than as a collection of different kinds of bacterial cells.
Manipulations are also relatively easy. For example, one
can take pooled plaque or salivary sediment and, despite their
cellular complexities, introduce one or more bacteria with a
desired metabolic function and in desired amounts to determine if and how much the relevant metabolism of the original
microbial composition is altered (Fig. 10; Ryan and Kleinberg,
1995a). In this way, one can assess whether a particular microorganism can modify the microflora capabilities—for example,
whether acid generated from a fermentable carbohydrate substrate can elevate the production of acid to a level critical for
the development of caries lesions. By way of illustration, when
an acidogen is present at about 1 to 2% or less, its contribution
to total acid formation and its impact on the pH would generally be minimal (Sims, 1985; Wijeyeweera and Kleinberg,
1989b). Lower levels than this are the norm for S. mutans in
many caries-active situations (Loesche, 1986; van Houte, 1993);
this is another reason for considering S. mutans to be a minor
organism in human dental caries formation. Many other flora
manipulation experiments are possible, but of particular value
are modeling experiments for product development, which we
have successfully done for the treatment of oral malodor
(Kleinberg and Codipilly, 1999).
(XI) Specificity of Streptococcus mutans
in Dental Caries Formation
With the re-discovery of S. mutans, a tremendous surge in
research occurred during the 1970s and 1980s which appeared
to implicate this micro-organism as the caries-causative agent
and to establish its specificity. Much of the belief in S. mutans’
specificity arose from experimental animal studies which suggested that classic infection was the start of the process. For
more than a decade before this, studies had been carried out in
an attempt to model caries in laboratory animals, mainly rats
and hamsters (Fitzgerald and Keyes, 1960; Keyes, 1960; Keyes
and Jordan, 1963; Jordan et al., 1972; Jordan and Sumey, 1973;
Tanzer, 1981). At the time, a major frustration for researchers in
this area of investigation was that these animals could be cariesprone in one laboratory but not when transferred to another.
Loss of such susceptibility continued until Keyes and his colleagues observed that the susceptible animals lost their caries
activity when the cariogenic micro-organism(s) was lost from
the experimental animals’ mouths. However, when it was discovered that S. mutans, either as a pure culture or in plaque from
13(2):108-125 (2002)
Figure 9. (a) Main metabolic processes associated with the pH vectors that characterize dental plaque acid-base metabolism. Vectors 1
and 4 produce base; Vectors 2 and 3 produce acid. Dashed line represents neutrality, and SCCA is the abbreviation for short-chain carboxylic acids. (b) Diagrams 1 and 2 characterize plaque with balanced acid-base metabolisms; 1 is for thicker and 2 is for thinner
plaque (Imfeld, 1983). Diagram 3 characterizes plaque where alkali
formation is more dominant, whereas in 4, acid formation dominates
(modified from Kleinberg and Westbay, 1992).
a caries-active individual or even from feces from an infected
animal, was introduced into the mouths of such animals, the
non-cariogenic deficiency was corrected. Since then, it has
become routine practice in animal caries experiments to infect
the animal participants with the S. mutans micro-organism.
These findings led to the conclusion that animal caries is
basically a classic infectious process, because infection or inoculation by a causative micro-organism starts the process. One
needs to keep in mind, however, that sucrose in the diet is usually used to prime the experimental animals (Keyes and
Jordan, 1963), which in essence means that the right substrates
and the right bacteria are both needed to produce the high levels of acid required for caries generation.
Thereafter, many studies with S. mutans attempted to
show that this micro-organism was specific to the caries
process and tried to identify how this might be so in humans.
Aspects extensively investigated included its ability to: (i) produce acid rapidly from fermentable carbohydrate and lower
the pH; (ii) survive and continue to produce acid at acidic pH;
(iii) produce high levels of intra- and extracellular polysaccharides, largely as storage components to prolong acid formation
Crit Rev Oral Biol Med
117
some bacterial mixtures that do not
include either S. mutans or lactobacilli.
For example, pH levels below 3.5 have
been achieved in our laboratory by
combining S. oralis with either Neisseria
sicca or Veillonella parvula and activating the pH response with relatively
high levels of glucose (Traudt and
Kleinberg, 1999).
The ability of S. mutans to produce
large amounts of extracellular, sticky
glucans from sucrose was considered
an important part of plaque formation
(Gibbons and Nygaard, 1968; Gibbons,
1984, 1989). Adherence of S. mutans
was illustrated by its ability to stick to
the sides of glass vessels in which these
organisms were grown (Gibbons, 1984,
1989) or to wires suspended in a growing S. mutans culture (McCabe et al.,
1967). The formation of adherent extracellular dextrans from sucrose was vigorously pursued during this time and
was considered to be an essential element in mutans specificity and cariogenicity (see Nyvad, 1993). It was proposed that dextranase, an enzyme able
to hydrolyze dextrans, might be used
to disperse such gelatinous plaque, but
three clinical trials subsequently
showed this enzyme to be ineffective
(Caldwell et al., 1971; Keyes et al., 1971;
Lobene, 1971).
Although bacterial attachment to an
acquired pellicle derived from saliva is
commonly considered to be the first
step in the plaque formation process
on the more readily accessible surfaces
of freshly cleaned teeth (Egelberg,
1970), other experiments have indicated that bacteria on these and on the
poorly accessible dentition surfaces do
not necessarily need a pellicle to attach
to the tooth surface (Frank and
Brendel, 1966). With the development
Figure 10. Catabolism of 2.8 mM glucose when a rapid glucose fermenter, L. fermentum, is
of an intra-oral ultraviolet camera
added to salivary sediment in A and to dental plaque in C at a 1:1 ratio. Shown in B and D are
which enables plaque to be visualized
the results when a slow glucose fermenter, H. parainfluenzae, is similarly provided (from Ryan
without initial staining (Kleinberg et
and Kleinberg, 1995a).
al., 1978), it became patently clear that
such a debate might be largely irreleand an acidic pH; and (iv) in addition to extracellular polysacvant to the day-to-day reality where dental plaque spontacharides, produce polymers to facilitate adherence to tooth
neously re-forms when removed by oral hygiene or dislodged
surfaces and build-up of large bacterial deposits.
by food. This is because photography with this device indicatIn examining the ability of resident bacterial species to
ed that the main starting point in most plaque re-formation
lower the pH and continue to do so at acidic pH, van Houte
investigations (that involve no cellular strips) is not formation
(1993, 1994) and van Houte et al. (1994, 1996) found that many
of a pellicle and then attachment of bacteria at all. Rather, it
oral bacteria besides the mutans streptococci and lactobacilli
appears to be the result largely of growth of already-attached
were sufficiently acidogenic to be cariogenic. These other bacbacteria located in the harder-to-clean tooth surface recesses or
teria included non-mutans streptococci such as S. sanguis,
niches, perhaps with some pellicle material present
mitis, and milleri and bacteria that were neither streptococci
(Kleinberg, 1987).
nor lactobacilli, such as strains of actinomyces and bifidobacThis was evident from experiments where even careful
teria. In addition, very low pH levels can be reached with
removal of plaque by dental prophylaxis (by means of pumice
118
Crit Rev Oral Biol Med
13(2):108-125 (2002)
and disclosing solution) still left material visible to the ultraviolet camera but not to the eye (see also Kleinberg, 1977a,b;
Gwinnett et al., 1978; Kleinberg, 1978). These remains expanded
into larger deposits and progressively spread over the surfaces
of the teeth, even though normal toothbrushing was maintained. Scanning electronmicrographs taken in similar experiments indicated that bacterial growth (perhaps with apposition
of some bacteria and proteins from the salivary milieu) resulted
in the development of small mounds that preceded the spreading out over the tooth surface and in due course becoming a
continuous biofilm (Björn and Carlsson, 1964; Kleinberg et al.,
1971; Saxton, 1972). Subsequently, Theilade and Theilade (1985)
showed, by use of filter-covered films that prevented apposition
of bacteria from saliva but not the access of nutrients, that
plaque formation is largely due to growth of already-attached
bacteria. Growth evidently is the dominant factor, but there are
sufficient data to suggest that salivary protein deposits, as well
as bacteria from the oral milieu, may make some contribution.
In an attempt to understand how dental plaque is held
together, investigators conducted two studies (Silverman and
Kleinberg, 1967a,b) in which they harvested plaque from the
teeth of human volunteers and, based on earlier work by
Dobbs (1932), dispersed it by raising its pH with cold 0.1 M
sodium hydroxide. This resulted in the plaque cells and
plaque matrix proteins becoming sufficiently negatively
charged that they separated, to a large degree, by mutual
repulsion (i.e., their zeta-potentials were raised to where they
could no longer aggregate). The suspended cellular and solubilized macromolecular components were then separated by
centrifugation, and by a series of dialysis and gel filtration
steps, the macromolecular components were separated into 11
fractions and analyzed for carbohydrate, calcium, phosphorus, and nitrogen content.
In their experiments, Silverman and Kleinberg (1967b)
identified the primary means whereby plaque is held together.
At the pH levels seen in the mouth and on oral tooth surfaces,
most pH-aggregation titrations indicated that the bacteria have
a net negative surface charge. This was later confirmed by
Singer (1973) using continuous particle electrophoresis and by
Olsson and Glantz (1977) using a similar type of procedure, viz.,
particle micro-electrophoresis. When the pH, calcium, and the
11 molecular-weight components isolated from dental plaque
were examined for their ability to form aggregates with the
plaque bacteria, it was evident that calcium ion served as a key
cross-linking cation, and that the ability of the bacteria and
large-molecular-weight plaque components to aggregate with
or without calcium and with or without one another was heavily dependent upon pH. As pointed out by Olsson and Glantz
(1977), aggregation and adherence are favored when the zetapotential of particle surfaces, i.e., bacteria and aggregating
macromolecules, approaches zero millivolts. Then, secondary
short-range forces and molecules produced or modified by the
bacteria can ensure stability to the couplings that have occurred.
(XII) The Tooth-Plaque-Saliva
Demineralization-Remineralization
Relationship and its Relation to Plaque
Acid-Base Metabolism
When Stephan, in his 1944 study, showed different pH levels
of response to a sugar challenge by caries-free and cariesactive individuals (Fig. 1), analysis of the data immediately
suggested the existence of a critical pH or a critical pH range.
13(2):108-125 (2002)
Figure 11. Relation between the pH of the supernatant of whole saliva (after 10,000 g centrifugation) and aggregation of its constituents.
A high-pH plaque favors formation of a higher calcium phosphate
and lower carbohydrate protein containing aggregate; a low-pH
plaque favors the reverse. Indicated in the Fig. are pH ranges of different kinds of plaque. Possible variation in saliva pH is shown in Fig.
13 (from Kleinberg et al., 1977).
What this indicated was that tooth demineralization, step two
of the Miller acid decalcification process, would not occur
unless there was enough acid to decrease the pH to a point
where the solubility conditions in the plaque environment
became undersaturated with regard to the main mineral of the
tooth tissues, viz., the calcium phosphate salt, hydroxyapatite.
Chemical factors preventing teeth from dissolving at a pH
above the critical pH level involve two processes. Both are
rooted in saliva. One is based on the solubility of tooth enamel in saliva (Fosdick and Starke, 1939). This involves salivary
calcium and phosphate ions repressing tooth mineral dissolution by mass action, and, if the pH is sufficiently elevated,
replacing lost tooth mineral by remineralization. The other is
more complicated and also arises from saliva. It consists of the
deposition, into and onto plaque and teeth, of a salivary aggregate consisting of a calcium phosphate carbohydrate protein
complex (Fig. 11), which was subsequently named salivary
precipitin (Kleinberg et al., 1994).
This complex was fractionated by high-voltage electrophoresis and its constituents analyzed (Chatterjee and
Kleinberg, 1979).
Above neutrality, it was high in calcium phosphate (Fig. 11)
and is apparently present in an amorphous or poorly crystalline
form (Kaufman and Kleinberg, 1973). It is acid-soluble, much
more than the calcium phosphate of tooth substance (Kleinberg
et al., 1994). This means that when acid is produced by the
plaque bacteria from fermentable carbohydrate and the pH
drops, the calcium phosphate in the salivary precipitin in
plaque, or some modification or alteration thereof, would dissolve before the hydroxyapatite of the tooth (Fig. 12). As a consequence, its role is one of a surrogate source of calcium and
phosphate ions, which one might expect would make it possible
to maintain calcium phosphate saturation of plaque fluid
(Margolis, 1993). This should prevent cavity formation by mass
action, as well as provide ions for remineralization to occur.
Crit Rev Oral Biol Med
119
Figure 13. Role of saliva in the various calcium phosphate reactions
that occur at the tooth-plaque-saliva interface. Saliva provides calcium and phosphate (i) as ions and (ii) as part of a calcium phosphate
carbohydrate protein complex (i.e., salivary precipitin). Both help
provide a continuous supply of calcium and phosphate for plaque
and tooth mineralization. At acidic pH (high H+ concentration), movement of calcium and phosphate from tooth and plaque to saliva is
favored, whereas at alkaline pH, the reverse is favored. The calcium
and phosphate ions produced in the plaque from plaque calcium
phosphate during acid formation are in a position to suppress or
retard tooth solubilization by mass action as well as to facilitate tooth
remineralization (from Kleinberg et al., 1983).
Figure 12. Comparison of the solubilization rates of human tooth
enamel slices and salivary precipitin samples prepared from seven
different subjects (identified by initials) (from Kleinberg et al., 1994).
Solubilization of salivary precipitin calcium phosphate can be
expected to occur as the pH declines following plaque exposure
to fermentable carbohydrate and to cease when the pH rises
sometime thereafter, when there is no longer carbohydrate substrate for the plaque bacteria to ferment and generate acid. The
pH at which solubilization ceases should be a point of saturation, and the pH between the lowest pH reached during the pH
fall and the critical pH should be a zone of undersaturation.
In essence, the acid-base cycling by the plaque bacteria
stimulates the demineralization-remineralization cycling
among tooth, plaque, and saliva, with plaque as the intermediary (Fig. 13). The role of plaque is complex in that it involves
plaque fluid, plaque calcium phosphate, plaque calcium and
phosphate ions, and plaque pH in the midst of tooth calcium
phosphate, saliva ability to deposit plaque calcium phosphate
(Ashley, 1975; Ashley and Wilson, 1977), saliva calcium and
phosphate ions, saliva pH, and plaque acid-base metabolism
(Fig. 14). Shown in Fig. 14 are the potential plaque and saliva pH
levels that could affect tooth and plaque demineralization/remineralization. Generally, caries-free plaque will fluctuate more
toward the neutral and alkaline parts of the pH range; highly
caries-active plaque will fluctuate more toward the acidic end
(see Fig. 1). Saliva pH will range between resting and stimulated pH levels (Dawes and Jenkins, 1964; Jenkins, 1978).
Largely driving these processes are the frequency and
120
duration of the plaque pH cycling periods and periods of saliva replenishment of plaque and tooth mineral. To a large
degree, such cycling should eventually determine whether a
caries lesion develops (Kirkham et al., 1994).
(XIII) Design of New Anti-caries Compositions
Based on These Concepts
Miller’s two-stage theory of dental caries initiation and development lends itself to a two-pronged approach to caries prevention and the reversal of any damage caused, at least in the
early stages of caries lesion development. To counter the
excess acid/deficient base formation component of the caries
process (first step), arginine has been selected as the active
agent. To counter the excess demineralization/deficient remineralization component (second step), calcium has been
selected as the active agent (Kleinberg et al., 1998, 2000). When
the two are combined with one or more cariostatic anions, the
resulting composition has been named CaviStat (Kleinberg,
1999). Bicarbonate/carbonate are preferred anions.
Experiments are in progress to evaluate its suitability as an
anti-caries agent, alone and in combination with fluoride.
(XIV) Concluding Remarks
S. mutans is still considered by many to be a specific microorganism that causes dental caries. A large body of research
has been developed and assembled that can be taken as support for this thesis (see Loesche, 1986). However, the bulk of
the evidence that appears supportive of a relation between S.
mutans and dental caries in humans can be considered to be
associative and not causative (Sims, 1985). For example, S.
mutans often rises in number when there is increased availability of fermentable carbohydrate, especially sugars. Also,
it is often (but not invariably) found in those regions of the
Crit Rev Oral Biol Med
13(2):108-125 (2002)
dentition where caries lesions tend to develop. But unsupportive of its being essential for dental caries causation is that
caries lesions can develop in the absence of S. mutans, and S.
mutans numbers are often low even when caries is present
(Sims, 1985; van Houte, 1993, 1994; van Houte et al., 1994,
1996). As pointed out above, dental caries can develop with
other acid-producing bacteria, many of which are present in
numbers many-fold higher than those seen for S. mutans. In
this regard, the same reasons given before for lactobacilli not
being causative also apply to S. mutans. In animals, the situation can be different. There, S. mutans, if of the nonarginolytic type, would be causative if it has to be inoculated
for the caries process to begin, and if the oral environment is
primed beforehand (usually with elevated levels of sucrose
supplied in the diets of the experimental animals). S. rattus,
readily found in rats, is arginolytic; it should not be as cariesconducive as the other mutans streptococci, even if sugars
were supplied as a priming condition.
With regard to the non-specific part of the Loesche hypothesis, it focuses on the phenomenon that increasing amounts of
plaque accumulate on tooth surfaces when oral hygiene is
avoided (Ritz, 1967). The increase is spontaneous and includes
an increase in the numbers of acidogenic micro-organisms
along with other resident oral bacteria. All factors being equal,
such an increase in bacterial numbers will give a more acidic
Stephan pH response than before the build-up (Stephan and
Miller, 1943; Imfeld, 1983). This would make a caries-producing
condition possible or an existing caries condition worse.
Removal by physical and/or chemical means of the plaque
that has built up reverses the effects of the process and is therefore a logical method of treatment (Stephan and Miller, 1943).
Oral hygiene devices, such as toothbrushes with or without
toothpastes and the like, would be the order of the day. So might
germicides or biocides. Not knowing the causative microorganism or micro-organisms would not matter (as long as the
biocides utilized were not harmful), since removing most of the
bacteria in the process would reduce the levels of the cariescausative organism(s) and thus be effective. From the work of
Axelsson et al. (1991), frequent removal of plaque at a level necessary to be successful is difficult for a patient to achieve and
sustain, but when done in conjunction with professional help,
significant caries reduction can occur. The reason for the difficulty is that the pertinent cariogenic oral bacteria are in hard-toreach dentition sites, viz., pits, fissures, and approximal surfaces
of the teeth. Moreover, the oral bacteria grow continuously, and,
consequently, their removal needs to be frequent and efficient if
the bacterial numbers are to be reduced and kept at levels low
enough to ensure that the acid produced is insufficient to produce demineralization and hence caries lesions.
A third approach is indicated by the present article. It is
somewhat of a hybrid in that it does not quite fit into either
the specific or non-specific aspects of the Loesche hypothesis.
First, it involves mixed and diverse bacteria which may or
may not include S. mutans. For this reason alone, it has to be
considered non-specific. Second, it usually involves ecological
overgrowth of certain bacteria and reduction in the numbers
of others (e.g., Brown et al., 1975). This unequal effect indicates
some specificity and, hence, would exclude it from the nonspecific-plaque concept proposed by Loesche. The mixed bacteria that emerge and give this “passive specifity” are those
best able to respond effectively to the niche provided by the
ecological change.
13(2):108-125 (2002)
Figure 14. Relationship between pH and rate of solubilization of calcium phosphate at the tooth-plaque-saliva interface. Enamel calcium
phosphate is mostly hydroxyapatite. Plaque calcium phosphate is xray amorphous or poorly crystalline brushite and apatite (Kaufman
and Kleinberg, 1973). Since tooth and plaque in vivo are parts of an
open system, rates of solubilization are expressed as Ca or P solubilized per unit time. Because plaque calcium phosphate is solubilized
more easily than enamel hydroxyapatite, higher plaque fluid levels of
calcium and phosphate ions are ensured. As shown, one can expect
cariogenic plaque to range over the acidic part of the range shown,
calculogenic plaque over the alkaline part, and normal plaque in
between (see Fig. 11). Saliva pH covers a narrower range (from
Kleinberg et al., 1983).
Ecological changes known to be of prime importance in
caries causation include excessive sugar in the diet (Becks,
1950) and significant reduction in the salivary flow (Brown et
al., 1975). Both result in a substantial rise in the
acidogenic/aciduric bacterial component of the oral flora. Of
the two, the salivary change should also result in reduction in
the bacteria contributing to alkali formation, and if so, because
of the dual effect on the plaque microflora, this might account
for the severe caries response often seen in xerostomic patients.
A treatment identified for use in countering the ecological
dysfunction involved is the provision of alkali-producing substrates such as arginine and urea (Kleinberg et al., 1979; Rogers
et al., 1987; Burne and Marquis, 2000). Arginine is preferred
because of the spectrum of bacteria involved (Salako and
Kleinberg, 1989) and the practical problems associated with
the use of urea. If provided in a dentifrice, such agents would
not only favor alkali production to counter oral acidity but
should also help, through the physical removal of plaque, to
add a further anti-caries benefit by reducing plaque load,
which would mean recruitment of the non-specific-plaque
hypothesis approach.
Because of the wide scope of the subject dealt with in this
article and its focus on the presentation of an alternative to S.
mutans and the specific-plaque hypothesis, there are many
publications both old and new in an immense body of literature that is relevant but cannot, because of space limitations,
be incorporated or referred to here. Nonetheless, what has
been framed here should make it possible for the reader to
Crit Rev Oral Biol Med
121
identify where many such articles would apply. The author
believes that the concepts and overall thesis presented here
should serve as a guide to an understanding of a mixed-bacterial/ecological approach to dental caries causation. It should
also be of value in re-interpretation of some of the caries literature, as well as development of an understanding of mixedflora physiology and how to study it within the oral cavity and
in other microbial systems as well.
Acknowledgments
The data in this article come from work carried out in the author’s laboratories over a period of more than 40 years, the first three of which were in the
laboratory of Dr. Neil Jenkins, under whom the author was fortunate to have
done his PhD studies. Dr. Jenkins’ collegiality, insights, and depths of knowledge were then, and have since always been, extremely valuable. I would also
like to acknowledge the contributions of the more than 20 PhD students whom
the author has mentored but who have always been considered as colleagues,
and an even larger number of post-doctoral trainees, research assistants, and
associates who have shared in the many hurdles and joys of the many discoveries that have been made. Many of their names are listed in the references to
this article where there is shared authorship.
Support for the research has come from various sponsors, ranging
from the National and Medical Research Councils of Canada, the National
Institute of Dental and Craniofacial Research in the USA, and from numerous industrial supporters. Support from several of these sponsors and in particular from the late Mr. Ernie Sandler, President of IDE Interstate, and Mr.
Mitchell Goldberg, President of Ortek Therapeutics, made it possible to weather the massive S. mutans mania that encompassed the dental research community for a good part of this period. Ironically, the author has rather enjoyed
going against the conventional wisdom, since he has always believed that challenge and even adversity can be among the various joys of life.
The considerable assistance by Ms. Pat Calia in the preparation of this
manuscript and by Dr. Mark Wolff in the preparation of the figures and valuable comments is most gratefully appreciated.
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