Advances in Dental Research
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Physiological Approaches to the Control of Oral Biofilms
Philip D. Marsh and David J. Bradshaw
Adv. Dent. Res. 1997; 11; 176
DOI: 10.1177/08959374970110010901
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http://adr.sagepub.com/cgi/content/abstract/11/1/176
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PHYSIOLOGICAL APPROACHES
TO THE CONTROL OF ORAL BIOFILMS
PHILIP D. MARSH 1 2
DAVID J. BRADSHAW1
Centre for Applied Microbiology & Research
Research Division
Salisbury, SP4 OJG, and
2
Leeds Dental Institute
Leeds LS2 9LU, UK
Adv Dent Res 11(1): 176-185, April, 1997
Abstract—Evidence that physiological strategies may be
potential routes for oral biofilm control has come from (i)
observations of the variations in the intra-oral distribution of
members of the resident oral microflora, (ii) changes in
plaque composition in health and disease, and (iii) data from
laboratory model systems. Key physiological factors that
were identified as significant in modulating the microflora
included the local pH, redox potential (E h ), and nutrient
availability. Increases in mutans streptococci and lactobacilli
occur at sites with caries; growth of these species is
selectively enhanced at low pH. In contrast, periodontal
diseases are associated with plaque accumulation, followed
by an inflammatory host response. The increases in Gramnegative, proteolytic, and obligately anaerobic bacteria
reflect a low redox potential and a change in nutrient status
due to the increased flow of gingival crevicular fluid (GCF).
Consequently, physiological strategies for oral biofilm
control should focus on reducing the frequency of low pH in
plaque by (i) inhibiting acid production, (ii) using sugar
substitutes, and (iii) promoting alkali generation from
arginine or urea supplements. Similarly, strategies to make
the pocket environment less favorable to periodontopathogens include (i) anti-inflammatory agents to reduce the
flow of (and hence nutrient supply by) GCF, (ii) bacterial
protease inhibitors, and (iii) redox agents to raise the E h
locally. Most laboratory and clinical findings support the
concept of physiological control. However, some data
suggest that the ordered structure and metabolically
interactive organization of mature dental plaque could
generate a community with a high level of homeostasis that
is relatively resistant to deliberate external manipulation.
Key words: biofilm, ecology, pH, antimicrobial agents,
fluoride, urea, arginine, redox potential, homeostasis.
Presented at the 14th International Conference on Oral
Biology, "Biofilms on Oral Surfaces: Implications for Health
and Disease", held March 18-20, 1996, in Monterey,
California, organized by the International Association for
Dental Research and supported by Unilever Dental Research
176
D
ental plaque is the biofilm that forms on the
surfaces of teeth, and is comprised of a diverse
community of bacteria, embedded in a matrix of
polymers of microbial and host origin. Plaque
develops naturally, and is generally considered of benefit to
the host because of its ability to prevent colonization by
exogenous (and often pathogenic) micro-organisms (Marsh,
1989). The formation of plaque involves a number of welldefined phases. Early colonizers, such as members of the
genera Streptococcus, Actinomyces, Hemophilus, Neisseria,
and Veillonella, adhere to a conditioning film (the acquired
enamel pellicle) by specific and non-specific molecular
interactions between adhesins on the bacterial cell and
receptors on the substratum (Gibbons, 1989; Busscher et ai,
1992). Subsequently, other bacteria adhere specifically to
already-attached cells (co-aggregation; Kolenbrander and
London, 1993), thereby increasing the diversity of the
microbial community, and providing a mechanism to
regulate bacterial succession during biofilm formation. In
this way, late colonizers (many of which are obligately
anaerobic and nutritionally fastidious species) would attach
when the physiological conditions in the biofilm are more
likely to support their growth.
ECOLOGY OF DENTAL PLAQUE
IN HEALTH AND DISEASE
The plaque microflora at anatomically distinct surfaces on
teeth varies quite markedly in its composition. The reasons
for this can include the degree of protection from mastication
and saliva flow, etc., to differences in key local environmental conditions. For example, the gingival crevice has a
low redox potential and, during inflammation, is provided
with gingival crevicular fluid, GCF, which is rich in potential
nutrients for proteolytic bacteria. Not surprisingly, this site
has higher numbers of asaccharolytic, Gram-negative
anaerobes than plaque from fissures, the composition of
which is influenced more by saliva.
Once established at a site, the microflora remains
relatively stable over time, despite regular minor
perturbations to the oral environment (Marsh, 1989). This
stability (termed "microbial homeostasis") stems not from
any metabolic indifference among the components of the
microflora, but rather results from a dynamic balance arising
out of numerous, coupled microbial interactions (Sanders and
Sanders, 1984). The precise mechanisms responsible for
maintaining this homeostasis in plaque are not fully
understood, but include both synergistic and antagonistic
interactions. Microbial stability is enhanced through the
development of nutritional inter-relationships, such as food
chains, and the need for microbial collaboration in the
catabolism of complex endogenous nutrients (ter Steeg et al.,
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VOLII(I)
PHYSIOLOGICAL
A
177
CONTROL OF BIOFILMS
ECOLOGICAL PLAQUE HYPOTHESIS
AND PREVENTION OF CARIES
FERMENTABLE
SUGAR
r *\
NEUTRAL
\
\ 1 [ENVIRONMENTAL )
I
[ [
\ ^
ACID
B
^
CHANGE
LOW
pH
J
^ /
f
|
(
I \
\ ^
S. oralis
S. sanguis
^
REMINERALIZATION
^
DEMINERALIZATION
ECOLOGICAL
SHIFT
MS
Lactobacilli
ECOLOGICAL PLAQUE HYPOTHESIS AND
PREVENTION OF PERIODONTAL DISEASE
u
CD < j
Plaque
Reduction
Plaque
i r Accumulatio
Reduced
Inflammatio
Low GCF flow
Higher E h
INFLAMMATORY
RESPONSE
ENVIRONMENTAL
CHANGE
Increased
Inflammatio
High GCF flow
Lower Eu
CO
Predominantly
G+ve microflora
Facultative
Anaerobes
ECOLOGICAL
SHIFT
^
Predominantly
G-ve microflora
Obligate
Anaerobes
o
o
Fig. 1—The ((ecological plaque hypothesis" provides a physiological approach to the control of biofilms associated with (a)
caries and (b) periodontal disease. The postulated dynamic relationship between environmental change and ecological shifts
within the biofilm implies that disease could be prevented not only by direct inhibition of the putative pathogens, but also by
interfering with the key environmental!physiological factors driving the ecological shift (Marsh, 1994).
1988; Bradshaw etal., 1994).
On occasions, homeostasis can break down at a site, and
disease can occur. Disease is associated with major shifts in
the balance of the resident plaque microflora and alterations
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178
MARSH
&
BRADSHAW
ADV DENT RES APRIL 1997
H Pre-pulse
• Planktonic
• Biofilm
a
Z3
o
O
I
L.rhamnosus
S.mutans
S.oralis
Species
Fig. 2—Comparison of selected biofilm and planktonic bacteria growing in a 10-species community, before and after 10 daily
pulses of glucose, in the absence ofpH control.
in the metabolism at a site following changes to the habitat.
Dental caries is associated with an increased frequency of
consumption of fermentable sugars in the diet, which results
in an increase in the isolation and proportions of acidogenic
and aciduric species such as mutans streptococci and
lactobacilli (Loesche, 1986). In contrast, periodontal diseases
involve an inflammatory host response to the accumulation of
plaque around the gingival margin. This response leads to an
increased flow of GCF which, in addition to introducing
components of the host response, also provides a novel
source of potential nutrients for the microflora. In more
advanced forms of periodontal disease, there are significant
increases in the prevalence of obligately anaerobic Gramnegative bacilli, especially proteolytic species. Many of these
bacteria are not detected in health, or are present at low (and
clinically insignificant) levels.
The concept that (i) a direct relationship exists between
the environment and the balance and behavior of the resident
plaque microflora, (ii) changes to that environment can lead
to the selection or enrichment of previously minor
components of this oral biofilm, and (iii) such an enrichment
can result in clinical changes to host tissues led to a modified
hypothesis (the "Ecological Plaque Hypothesis"; Marsh,
1991, 1994) being proposed to explain the transition of the
plaque microflora from having a commensal to a pathogenic
relationship with the host (Fig. 1). The basic tenet of this
hypothesis is that disease prevention can be achieved not only
by inhibiting the causative bacteria directly, but also by
identifying and controlling the ecological/physiological
factors driving these transitions. Manipulation of these key
physiological factors could lead to some degree of control
over the composition of the plaque community, and lead to
the identification of new physiological strategies to maintain
the beneficial properties of the biofilm.
PHYSIOLOGICAL FACTORS
AFFECTING DENTAL PLAQUE
Fermentable sugars and low pH
As stated previously, individuals who consume a diet rich in
fermentable carbohydrates generally have higher levels of
cariogenic bacteria and an increased incidence of dental
caries. Whether such a response by these bacteria is due to
their greater ability to (i) catabolize dietary sugars or (ii)
tolerate the low pH generated from glycolysis has been
debated for some time. Evidence suggesting that low pH was
the major physiological force was provided when human
volunteers rinsed with buffers of different pH (Svanberg,
1980). Buffer of neutral pH had no impact on the indigenous
levels of mutans streptococci, whereas rinsing with a low pH
buffer led to the enrichment of these bacteria. Definitive
proof of the selective influence of low pH came from studies
of microbial communities grown under defined conditions in
chemostat models (Bowden and Hamilton, 1987, 1989;
Bradshaw et ah, 1989b). For example, pulses of glucose had
no effect on the proportions of S. mutans and L. casei when
the pH was maintained automatically at neutral values,
whereas there was a gradual increase in the levels of these
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PHYSIOLOGICAL CONTROL OF BIOFILMS
two species, at the expense of organisms associated more
with dental health, when the pH was allowed to fall after
successive pulses (Bradshaw et al, 1989b). Indeed, when the
degree of pH change was controlled, an inverse relationship
was seen between the terminal pH and the viable counts of S.
mutans and L. casei (Bradshaw et al, 1989a). A recent study
has confirmed this observation in model biofilms, although
the selection of potentially cariogenic bacteria and the
inhibition of most acid-sensitive species were less marked
than in planktonic cultures (Fig. 2) (Bradshaw et al, 1996b),
demonstrating that susceptible bacteria could gain some
protection within the biofilm.
Redox potential
Despite the mouth's ready access to air, with an oxygen
concentration of approximately 20%, it is perhaps surprising
that the plaque microflora is comprised of few, if any, truly
aerobic species. Indeed, obligately anaerobic bacteria are
isolated more commonly, although their distribution is not
uniform, and proportions are highest at stagnant sites where
plaque can accumulate. In periodontal disease, the diversity
and number of such anaerobes increase still further, and
many nutritionally fastidious bacteria can be recovered (see
next section). Gradients generated within such biofilms can
produce an environment sufficiently reduced to permit the
growth of even strict anaerobes. Animal studies in diabetic
rats indicated that progression of periodontal disease was
associated with a reduced oxygen tension (McNamara et al,
1982), and studies of humans found a link between reduced
oxygen tension, increased periodontal pocket depth, and
bleeding, with the presence of spirochetes and other putative
periodontopathogens (Loesche et al., 1983).
Nutrients
Populations within a microbial community are dependent
solely on the habitat for the nutrients essential for their
growth. Therefore, the association of an organism with a
particular habitat is direct evidence that all of the necessary
nutrients required for growth are being provided. The
collective evidence from numerous in vitro and in vivo
studies has demonstrated that the persistence and diversity of
the resident oral microflora are due to the endogenous
nutrients provided by the host (Littleton et al., 1967; Beckers
and van der Hoeven, 1982a,b, 1984; Beighton et al, 1986;
Beighton and Hay day, 1986). In particular, salivary mucins
(glycoproteins) act as the main source of carbohydrates for
plaque bacteria, and the degradation of the oligosaccharide
side-chains depends on the concerted action of consortia of
different species, each with complementary profiles of
glycosidase activity (Beighton et al, 1986; ter Steeg et al,
1988; van der Hoeven and Camp, 1991; Bradshaw et al,
1994). Furthermore, the growth of some species can be
dependent on the provision of nutrients by other oral bacteria.
Thus, secondary feeders use the products of metabolism of
other species (e.g., lactate or succinate), while others
scavenge peptides or free amino acids generated during the
breakdown of complex host molecules by other organisms. In
this way, certain species occupy key niches (or metabolic
TABLE 1
PHYSIOLOGICAL STRATEGIES
FOR THE CONTROL OF ORAL BIOFILMS
Control of plaque pH
• inhibition of acid production
— fluoride
— sugar substitutes
— antimicrobial agents
• stimulation of base production
— arginine
— urea
— peptides
Control of redox potential
• redox agents
• oxygenating agents
Control of nutrients
• addition of base-generating nutrients
— arginine
• reduction of GCF flow
• anti-inflammatory agents
• inhibition of key microbial enzymes
roles; Alexander, 1971) within the consortium, and their
levels or activity may dictate, in part, the final balance of the
microbial community. Such species may provide valuable
targets for physiological control strategies.
The isolation and the elevated levels of some of the major
groups of periodontal pathogens may be related to the
provision of increased levels of essential nutrients in the
gingival crevice. For example, black-pigmented anaerobes
require hemin for growth, and they probably obtain this key
cofactor by degrading heme-containing host molecules
(hemoglobin, hemopexin, haptoglobin) present in GCF, the
flow of which is increased during inflammation. The
detection and levels of periodontal pathogens were enhanced
when enrichment cultures of subgingival plaque were carried
out on human serum, which was used to simulate growth on
GCF (ter Steeg et al, 1987, 1988). There are also
(contradictory) data implying that the rise in subgingival
levels of Prevotella intermedia seen sometimes during
pregnancy is due to the metabolism of the hormones estradiol
and progesterone (Kornman, 1982), which can be detected in
GCF, while increases in Prevotella spp. have also been
reported to occur in the plaque of women taking oral
contraceptives (Jensen et al, 1981).
PHYSIOLOGICAL APPROACHES
TO THE CONTROL OF DENTAL PLAQUE
The main impetus behind the desire to control the bacterial
composition of dental plaque is to prevent or reduce the
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180
MARSH &
TABLE 2
EFFECT OF DIET ON CFU IN DENTAL PLAQUE
OF DI-ASSOCIATED RATS
(A = S. mutans and "S. milleri"; B = S. mutans and S. sanguis)
Diet
(CFU.105 [mean ± SE] on 3 molars)
A
Basal
Sucrose
Arginine
S. mutans
53 ± 7
294 ± 46**
17 ± 8**
"S. milleri"
259 ±31
81 ± 10**
498 ± 79**
Total CFU
B
Basal
Sucrose
Arginine
S. mutans
76 ±10
274 ± 38**
70 ±15
S. sanguis
130 ±14
90 ±16
Total CFU
206 ±15
365 ± 49*
258 ± 20*
311 ±30
375 ± 50
516 ±82*
188 ±15**
Significant differences in 0.01 < p < 0.05,
compared with basal diet.
From van der Hoeven et al (1985).
0.01 > p,
incidence of caries or periodontal diseases. As discussed in
the previous sections, the main physiological targets for these
diseases would include a reduction in conditions of low pH in
supragingival plaque and an alteration to the environment
(redox potential, nutrient availability) in a periodontal pocket
(Table 1). Some potential strategies to achieve these aims
will now be discussed.
Prevention of low pH
Bacterial homeostasis in plaque biofilms would be less likely
to be disrupted if the frequency and depth of acidic
conditions following sugar intake could be reduced. This
could be achieved physiologically by (a) inhibitors of acid
production, (b) consumption of food or drinks containing
non-fermentable sweeteners, and (c) the local generation of
base (alkali) in plaque. This strategy would not only remove
the major environmental pressure selecting for cariogenic
(aciduric) species, but also prevent the inhibition of the
species predominating in health, which are generally acidsensitive. Strategies to stimulate saliva flow would also help
control plaque pH at favorable values, and increase clearance
of fermentable substrates.
Inhibitors of acid production. The principal mode of
action of fluoride is to increase the resistance of enamel to
demineralization and to promote remineralization. However,
low (sub-MIC) levels of fluoride can also reduce glycolysis,
and this inhibitory activity is enhanced as the environmental
pH falls. Studies of microbial communities grown in a
chemostat have shown that sub-MIC levels of fluoride have
no effect on the proportions of individual species during
growth at neutral pH, but could prevent the selection of S.
mutans during pH-fall conditions (Bradshaw et al, 1990).
The rate of acid production by the microbial community was
BRADSHAW
ADV DENT RES APRIL 1997
reduced, enabling acid-sensitive species to persist at higher
levels than in the absence of fluoride. Some researchers have
questioned whether fluoride bound to enamel could be
released during glycolysis in vivo in sufficient concentrations
to modulate the rate of acid production. However, recent data
have shown that biofilms of mono-cultures of several oral
bacteria, when grown on fluoride-bound hydroxyapatite,
could release sufficient fluoride from the substratum to
reduce the growth and accumulation of cells (Li and Bowden,
1994), while sufficient fluoride was released from
fluorhydroxyapatite by bacterial fermentation to inhibit lactic
acid production by S. mutans (Guha-Chowdhury et al., 1995).
Thus, the anti-caries properties of fluoride may also include
the stabilization of the plaque microflora by slowing
glycolysis and thereby removing the transient advantage
given to mutans streptococci during periods of low pH within
the biofilm (Hamilton and Bowden, 1982; Bradshaw et al.,
1990).
Antimicrobial agents are being used as an adjunct to
mechanical cleaning for plaque control (Addy, 1990). Such
agents delivered from dental products are present in the
mouth for considerable periods at sub-MIC levels. At such
concentrations, agents such as chlorhexidine and Triclosan
can interfere with sugar transport and glycolysis (Marsh et
al, 1983; Scheie, 1989), and may still be of therapeutic
value, therefore, by helping to stabilize the biofilm
community.
Sugar substitutes. Sugar substitutes could play a role in
reducing the impact of low pH on microbial homeostasis in
plaque in a number of ways. Their consumption in snack
foods would reduce the frequency of acidic conditions in the
biofilm, while their ability to stimulate saliva flow will
provide increased buffering capacity, substrate clearance, and
remineralization potential to enamel. Saliva also contains
antimicrobial agents, as well as urea and peptides from which
base (alkali) can be generated. Again, this combination of
physiological factors would tend to stabilize the resident
plaque community and reduce the competitive advantages
normally given to cariogenic species during regular sugar
consumption.
Some sugar substitutes, such as aspartame, saccharin, and
xylitol, also have the potential to inhibit bacterial growth
(Grenby and Saldanha, 1986), with saccharin and xylitol
being particularly effective against mutans streptococci (Best
and Brown, 1987; Makinen, 1989; Scheie, 1989). Delivery of
xylitol with glucose to a consortium of oral bacteria resulted
in a reduced rate of acid production and a selective
suppression of S. mutans under conditions where this species
would otherwise flourish (Bradshaw and Marsh, 1994).
Clinical studies using xylitol-containing products, ranging
from chewing gum to dentifrices, have also reported a
selective inhibition of mutans streptococci (Isokangas et al.,
1991; Petersson et al, 1991; Svanberg and Birkhed, 1991).
Alkali generation in dental plaque. Several endogenous
and exogenous compounds can be metabolized by plaque
bacteria to yield base (principally ammonia). Arginine-
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PHYSIOLOGICAL CONTROL OF BIOFILMS
MICROHABITATS & PHYSIOLOGICAL
CONTROL OF BIOFILMS
REDOX
AGENTS
SUQAR
SUBSTITUTES
ANAEROBIC
ACTDURIC
DOMAIN
pH
CONTROL
AGENTS
I
DOMAIN
Fig. 3—Schematic representation of the development in biofilms of gradients in substrates and products over relatively short
distances due to bacterial metabolism. This would result in a spatial organization within the biofilm of consortia of bacteria
with specific metabolic preferences. This could result in metabolic "activity domains" which, depending on the architecture of
the biofilm, may restrict penetration of substrates for physiological control strategies. Similar "domains" may exist in
neighboring areas of the biofilms. Such "domains" may give rise to a mosaic of micro-environments.
artificial mouth model inoculated with human plaque.
containing peptides and urea in saliva can be hydrolyzed to
Supplementation of a basal medium (simulating the
ammonia, and this activity may account, in part, for the
composition of saliva) with low concentrations of urea or
relatively high pH in "resting" or "sugar-starved" plaque
arginine led to a rise in the resting pH of the biofilms, and an
(Theilade, 1990). In addition, ornithine is produced which
increase in urease activity (Sissons et al., 1991). This change
can be decarboxylated, particularly at low pH, leading to a
in the biofilm metabolism was associated with only minor
rise in pH. Supplementation of carbohydrates with arginine
shifts in the microbial composition of the community,
when pulsed to arginolytic bacteria (e.g., S. sanguis) resulted
although the flora was not analyzed at the species level, so
in a higher terminal pH (Ferro et al., 1983), while arginine as
that any shifts in the principal ureolytic organism in plaque
either the free amino acid or as part of a short peptide can
(S. salivarius) would not have been detected in this study.
stimulate the gcowth of streptococci associated with sound
The
pH response of these biofilms to urea was doseenamel, such as S. sanguis and members of the "S. milleri"
dependent; the metabolism of arginine, however, produced
group (Rogers etal, 1987a; Rogers, 1990).
only half the pH-rise response as the same concentration of
These findings have prompted attempts to manipulate the
urea. Subsequent studies on deep biofilms (maximum
composition of the plaque microflora by augmentation of
thickness, 5-8 mm) showed that urea-induced pH gradients
endogenous levels of base-generating compounds with pulses
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by ondevelop
September 28, 2009
can
and last for several hours (Sissons et al., 1994b).
of arginine or urea. Several studies have been Downloaded
conducted
in an
182
MARSH
TABLE 3
EFFECTS OF A REDOX AGENT (METHYLENE BLUE)
ON THE MICROFLORA OF PERIODONTAL POCKETS
Decrease
Total anaerobes
Gram-negative anaerobes
Spirochetes
Motile bacteria
GCF flow
Increase
Facultative anaerobes
Coccal cells
Methylene blue (0.1% w/v) was applied subgingivally, daily
for 7 days, to 25 sites in seven patients (ages, 31 to 57 yrs)
(Wilsons a/., 1992).
The initial pH gradients were alkaline at the biofilm surface,
and only eventually did the inner layers became alkaline.
Such slow changes in pH gradient were probably due to the
slow penetration of urea to the inner layers of the biofilm, its
rapid metabolism by the outer layers of the biofilm, and a
rate-limiting clearance of ammonia (Sissons et al., 1994a).
The microbial organization of a biofilm will be of
significance in attempts to alter the physiology of the
community by the addition of nutrients. Recent studies have
shown that model oral biofilms have an ordered structure—
for example, with oxygen-consuming species located on the
surfaces of the biofilm—thereby facilitating and protecting
the growth of obligately anaerobic bacteria in the depths of
the biofilm (Kinniment et al., 1996). The success of a
physiological strategy may depend on overcoming highly
coupled microbial interactions within highly structured
biofilms, especially in mature biofilms.
Studies of simple mixed cultures of plaque bacteria in
chemostats and gnotobiotic rats have confirmed that the
potential does exist for shifting the composition of a
simplified microbial community by manipulating the
concentrations of key substrates. Steady-state levels of S.
mutans, S. sanguis or "S. milleri" in mixed continuous
culture could be regulated by the controlled addition of
critical concentrations of glucose or arginine (Rogers et al,
1987a). Likewise, viable counts of S. sanguis or "S. milleri"
could be increased, and those of S. mutans decreased, on the
dentition of rats fed an arginine-supplemented diet, whereas
the converse occurred when the diet was switched to one
containing sucrose (Table 2; van der Hoeven et al., 1985).
Alteration of subgingival environment
Redox potential. The Gram-negative anaerobes associated
with periodontal disease require a low redox potential for
growth. Therefore, a physiological route to the reduction of
disease could involve the modification of the pocket Eh by
the delivery of either oxygen or oxygen radicals to a site, or
of compounds which increase the local redox potential.
Addition of a redox agent, methylene blue, to reduced
&
BRADSHAW
ADV DENT RES APRIL 1997
bacterial cultures raised the Eh of the medium to -125 mV
and prevented the growth of the periodontopathogen
Porphyromonas gingivalis (Fletcher and Wilson, 1993). In a
clinical trial, a seven-day treatment regime involving
methylene blue led to a decrease in the proportions of Gramnegative anaerobes (including spirochetes) and motile
bacteria and a reduction in the flow of GCF, while bacteria
associated with gingival health increased (Table 3; Wilson et
al., 1992), suggesting that this approach has genuine
potential.
Similar approaches to manipulation of the community
composition of a laboratory microbial community via Eh
were made with the use of aeration, with unexpected results
(Bradshaw et al., 1996a). A two-stage chemostat model
system was used to grow (i) a 10-species consortium
anaerobically in the first stage, which was fed into (ii) an
aerated second stage, which contained hydroxyapatite discs
for biofilm formation. It was anticipated that anaerobes
would be unable to grow in the second stage, except in the
biofilms, and only when gradients developed and conditions
within the biofilm became reduced. However, all obligate
anaerobes persisted, both in the biofilms and in the
planktonic phase, albeit initially at lower levels, but the
predominant organism was an aerobe, Neisseria subflava.
When the experiment was repeated without the aerobe, the
anaerobes still grew, but there was an initial increase in
facultative species, especially streptococci. The consortium
was able to protect anaerobes, even in planktonic culture, for
example, by consuming oxygen, emphasizing the
significance of interactions among members of microbial
communities in the maintenance of homeostasis.
pH and nutrients. Other physiological targets within the
pocket environment could include altering the local pH or
reducing the availability of endogenous nutrients from GCF.
A small rise in pH (from pH 7.0 to 7.5) can occur within the
gingival crevice during inflammation (Eggert et al., 1991),
presumably due to proteolysis by the asaccharolytic
microflora. A similar shift in pH dramatically altered the
balance of a community of three black-pigmented anaerobes,
with the proportions of P. gingivalis increasing from < 1% at
pH 7.0 to > 99% of the cultivable microflora at pH 7.5
(McDermid et al., 1990). Similarly, growth of subgingival
plaque on human serum (used as a substrate to mimic GCF)
resulted in the enrichment of periodontal pathogens (ter Steeg
et al., 1987, 1988). GCF and serum would provide peptides
and heme-containing molecules which are essential for the
growth of many periodontopathogens. These data suggest
that strategies to reduce inflammation could also indirectly
modify the biofilm community by restricting the availability
of key nutrients.
The use of antimicrobial agents could also indirectly affect
the physiology of the subgingival community. Chlorhexidine
and Triclosan, which are widely used in anti-plaque products,
can inhibit bacterial proteases when present at sub-MIC
levels (Rogers et al, 1987b; Scheie, 1989; Cummins, 1991;
Grenier, 1993). Such an inhibition may affect the ability of
proteolytic anaerobes to acquire nutrients for growth, and
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VoLll(l)
PHYSIOLOGICAL CONTROL OF BIOFILMS
prevent their deregulation of the host-inflammatory response.
CONCLUDING REMARKS
It is clear from data on the site distribution of the oral
microflora, and on the shifts in the balance of the
predominant bacteria in plaque that occur in disease, that
physiological processes can modify the composition and
metabolism of oral biofilms. Therefore, it may be possible to
design strategies to modify the composition of plaque
deliberately more toward that found in health than in disease,
and there is a limited amount of laboratory and clinical data
to support this contention.
However, some laboratory data also suggest that some of
the biological features of plaque may make it less easy to
predict the outcome of some strategies for control. Plaque is a
biofilm with a high species diversity and an ordered structure.
This diversity is maintained, in part, through highly coupled
metabolic interactions, one consequence of which may be to
increase the strength of homeostasis within the biofilm,
making it less susceptible to manipulation by some
physiological approaches.
The use of substrates that can be metabolized appears to
generate gradients over relatively short distances (several
microns) within a biofilm (Costerton et al., 1994). Therefore,
the influence of such molecules on relatively deep biofilms
(several 100 microns) requires further study, especially on
their distribution within and penetration of the biofilm.
Direct observations of the structures of living biofilms
from aquatic environments have demonstrated a loose, open
structure, with water channels which may enable small
molecules to penetrate deep into the biofilm (Costerton et al,
1995). Whether biofilms from relatively nutrient-rich
habitats, such as the mouth, have a similar architecture
remains to be determined, but this aspect may be of critical
importance in determining the outcome of some of the
physiological approaches to control described in this review.
Such studies should also be complemented by research into
the fundamental properties of oral bacterial consortia, since it
is clear that the metabolic potential of communities is greater
than the sum of the individual populations. Further work on
the influence of surface growth on the behavior of plaque
communities will also be needed before the full potential of
physiological approaches to biofilm control will be realized.
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