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JOURNAL OF ENDODONTICS
Copyright © 1992 by The American Association of Endodontists
Ecology of the Root Canal Flora
G. Sundqvist, DDS, PhD
The root canal represents a special environment in
which selective pressures result in the establish-
ment of a restricted group of the oral flora. Popula-
tion shifts occur over time with obligate anaerobes
ultimately dominating the bacterial mix. Bacterial
interrelationships and the nutritional supply are key
factors in determining the outcome of the infection.
Endodontic treatment, apart from directly eliminat-
ing bacteria, can completely disrupt the delicate
ecology and deprive persisting bacteria of their nu-
tritional source.
BACTERIA AND T H E PERIAPICAL L E S I O N
More than 300 bacterial groups or species of bacteria are
today recognized as normal inhabitants of the oral cavity (1,
2). In recent years many new species have been described, but
many bacteria isolated from the human oral cavity remain
yet to be classified. The bacteria present in infected root canals
include a restricted group of species compared with the total
flora of the oral cavity. Most of the species found in infected
root canals have also been isolated from periodontal pockets,
but the root canal flora is not as complex as the gingival flora
(2). The number of bacterial species in root canals may vary
from 1 to more than 12, and the number of bacterial cells
recovered between < 102 to > 108. A correlation seems to exist
between the size of the periapical lesion and the number of
bacterial species and cells present in the root canal. Thus,
teeth with large lesions usually harbor more bacterial species
and have a higher density of bacteria in their root canals than
teeth with small lesions.
T H E SELECTIVE E N V I R O N M E N T IN T H E R O O T
CANAL
The dynamics of root canal infections have been studied
in a series of experiments on monkeys (3). Indigenous oral
bacteria of the monkey were introduced into root canals with
devitalized pulp tissue in two different ways, i.e. sponta-
neously from the saliva or as pure cultured strains. The access
cavities were sealed. After various periods, up to 3 yr, bacte-
riological samples were taken from the canals and bone blocks
were examined radiographically and histologically at the end
of the experimental period. In the canals infected by saliva,
facultatively anaerobic bacteria were present in greater pro-
427
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VOL. 18, NO. 9, SEPTEMBER1992
portion than anaerobic bacteria. Even at this early stage of
the experiment, selective mechanisms operated in these spon-
taneously infected root canals. Thus, staphylococ~.i, Neisseria,
lactobacilli, and fungi were very seldom found in spite of the
fact that they were normally present in saliva. Subsequently,
the relative proportion of anaerobic bacterial strains and cells
increased with time. Over a 6-month period, the facultative
bacteria were progressively outnumbered and, as the obser-
vation time increased further, the dominance of the strictly
anaerobic bacteria became even more pronounced.
When combinations of bacterial strains, originally isolated
from an endogenously infected root canal, were inoculated
into further canals with devitalized tissue, a dominance of
anaerobic bacteria was again established. Interestingly, the
original proportion of the bacterial strains was reproduced in
the canals despite the fact that they were inoculated in equal
quantities into the canals. The results indicate that selective
mechanisms occur in which certain bacteria are more capable
of surviving and multiplying in root canals than others. These
experiments also show that the endodontic milieu is selective
for the development of specific proportions of the anaerobic
microflora. Further studies of this type are, however, necessary
to elucidate the mechanisms behind the observed selection.
BACTERIAL I N T E R R E L A T I O N S H I P S
It is striking, when samples from infected root canals are
cultivated, that certain bacterial species occur so often to-
gether. This has also been found in samples from periodontal
pockets (4). This indicates that there are interrelationships
between certain bacteria that can be commensal or antago-
nistic. Microbial associations between species have been de-
termined by computing the odds ratio of detecting one species
in the presence or absence of another species (4). Positive
associations were considered to be relationships in which the
odds ratio of detecting one species in the presence of another
species was above 2, while two species were considered to be
negatively associated if the odds ratio of detecting one species
in the presence of the other was <0.5.
When the root canal samples taken at this department (5-
7) were subjected to such analysis, very strong positive asso-
ciations were found between certain bacterial species (Fig. 1).
Thus, Fusobacterium nucleatum, which was the most preva-
lent species, was positively associated with Peptostreptococcus
micros, Wolinella recta, Porphyromonas (formerly Bacteroi-
des) endodontalis, and Selenomonas sputigena. Bacteroides
intermedius, which was found in 34% of the canals, was
428 Sundqvist Journal of Endodontics

positively associated with Peptostreptococcus anaerobius, P.
micros, and Eubacterium sp. There was also a strong positive
association between Eubacterium alactolyticum and P. ana-
erobius. In general species of streptococci, Propionibacterium
propionicus (formerly Arachnia propionica), Capnocytophaga
ochracea, and Veillonella parvula showed no or negative
associations with the other bacteria. The odds ratios for Act#
nomyces israelii was less than 2 against all other species except
P. propionicus, for which the latter was also positively asso-
ciated with other Actinomyces sp.
tions may be dependent upon a food chain type of metabolism
where the metabolism of one species supplies essential nu-
trients for growth of other members of the populations
(Fig. 3).
Many of the microorganisms in infected root canals utilize
amino acids and simple peptides as energy sources and thereby
carboxylic acids, ammonia, and hydrogen sulfide are pro-

NUTRITIONAL SUPPLY

There are many factors which can influence the growth

and colonization of bacteria in root canals. The availability
of nutrients, the low oxygen tension in root canals with
necrotic pulps, and bacterial interactions must be important
ecological determinants. Conditions exist in the root canal
which permit the growth of anaerobic bacteria capable of
fermenting amino acids and peptides, whereas bacteria that
primarily obtain energy by fermenting carbohydrates may be
more restricted by lack of available nutrients. Disintegrated
pulp tissue and the tissue fluids constitute essential sources of
nutrients in the root canal. It is likely that the availability of FIG 1. Associations between prevalent bacterial species in root canal
serum-like substrates is the most important ecological factor infections. An odds ratio above 2 indicates a positive association
in the root canal. between two species.
Important information with relevance to the dynamics of
root canal infections can be obtained from the recent studies 50
of ter Steeg and van der Hoeven (8). They studied the succes-
sion of species during enrichment growth of subgingival %
of
plaque organisms in serum. Three phases could be distin- Flor
guished during growth (Fig. 2). First, the low content of
carbohydrates in serum was consumed by rapidly growing
saccharolytic bacteria, leading to lactic and formic acid pro-
duction. In a second phase, proteins were hydrolyzed, some
amino acid fermentation took place, and the remaining car-
bohydrates were used. The carbohydrates were split off from o i i
0 Time (hours) loo
the serum glycoproteins. Growth during this phase was dom-
inated by B. intermedius, V. parvula, Eubacterium sp., and
F. nucleatum. In a final phase, progressive protein degradation
and extensive amino acid fermentation took place. The pre- Fermentat on Phase I
dominant species during this phase were P. micros, F. nuclea-
tum, and Eubacterium sp. In another study P. micros has also FiG 2. Diagrammatic representation of growth phases of plaque
been found to dominate serum cultures originating from bacteria in serum, after ter Steeg and van der Hoeven (8).
subgingival microbiotas (9). The ecological niche of P. micros
may be related to its wide range ofpeptidase activities making
amino acids and peptides available from serum glycoproteins
(8). These amino acids and peptides can be used in the
metabolism of P. micros, but may also be utilized in the
metabolism of other bacteria, which have little or no proteo-
lytic activity in serum. The black-pigmented anaerobic rods
B. intermedius, P. (Bacteroides) endodontalis, and Porphyro-
monas (Bacteroides) gingivalis have a high ability to degrade
serum proteins and make peptides and amino acids available
for fermentation (10, 11).
The strong positive association among Peptostreptococcus
sp., Eubacterium sp., Bacteroides sp., and F. nucleatum is
mainly related to their nutritional demands. However, the
wide range of nutritional interrelationships that exist among
oral bacteria may also influence the associations between FIG 3. Possible nutritional relationships between bacteria in the root
certain species (12-14). Growth of mixed bacterial popula- canal.
VoL 18, No. 9, September 1992
duced. The composition of the root canal microbiota will
significantly influence the toxicity of the metabolic products,
as some products can be metabolized by some species, while
others are left to accumulate and reach toxic levels for other
bacteria. Ammonia is toxic in high concentrations, but is also
a very important source of nitrogen for many bacteria. Carbon
dioxide is essential for many bacteria and the capnophilic
bacteria (Capnocytophaga sp., Eikenella corrodens) will not
grow unless carbon dioxide is available. Lactate, formate, and
hydrogen are excreted by many bacteria as a result of their
energy metabolism. Such organisms as Wolinella recta and
Bacteroides gracilis use hydrogen and formate. W. recta is
strictly dependent on a respiratory metabolism in which only
formate and hydrogen can serve as electron donors and
fumarate, nitrate, or oxygen as electron acceptors. This makes
the organism dependent on formate- or hydrogen-producing
bacteria. The positive associations observed in the root canals
between Wolinella sp. and the peptostreptococci, Eubacte-
rium sp., and F. nucleatum (Fig. 1) may partly be dependent
on the production of formate by these bacteria. With respect
to electron acceptors, fumarate or nitrate may be expected
not to occur in the oral ecosystem (15). Recently, it was found
that W. recta can utilize the amino acids aspartate and aspar-
gine as electron acceptors by converting them to fumarate
intracellularly (15). This may indicate that the growth of W.
recta is dependent not only on formate-producing organisms
but also on proteolytic organisms which can degrade tissue
and serum proteins to give amino acids which can serve as
electron acceptors. In root canals W. recta is positively asso-
ciated with P. micros (Fig. 1) and with P. endodontalis, which
both have a strong ability to degrade serum proteins into
amino acids (8, 10, 11).
BACTERIOCINS
Another factor which may be important to the bacterial
ecology is the production of bacteriocins. A bacteriocin is a
protein, produced by a microorganism, which has the capacity
to inhibit the growth of a limited number of other species.
Although bacteriocins produced by oral bacteria have not yet
been fully characterized, it is possible that they may influence
the microbial ecology in root canals by suppressing the growth
of some species that compete for the same ecological niche.
It has been noted that the black-pigmented Bacteroides pro-
duce bacteriocins, which are able to suppress not only Gram-
positive bacteria, but have an inhibitory influence on other
Bacteroides strains as well (16). Van Winkelhoff et al. (17)
have actually shown that P. endodontalis inhibits the growth
of B. intermedius in vitro. This may explain why these species
are negatively associated in the root canal. Interestingly, strep-
tococci also have the capacity to inhibit the growth in vitro
of many anaerobic bacteria. The inhibition is mediated
through the production of hydrogen peroxide by the strepto-
cocci. It is possible that such an inhibition can take place in
the coronal parts of the root canal in teeth with pulps exposed
to the oral cavity where there is sufficient availability of
oxygen.
COAGGREGATION
It has been observed that certain bacteria often occur
together by their ability to coaggregate. Coaggregation is de-
Root Canal Flora 429
fined as the recognition between surface molecules on two
bacterial cell types so that a mixed cell aggregate is formed,
which may serve a nutritive or protective function. It may be
an important prerequisite for a metabolic interaction between
bacteria. Many oral coaggregation pairs have been identified
(18). Of the prevalent species in root canals, F. nucleatum has
been shown to have a high ability to coaggregate in vitro with
many of the oral bacteria. Such coaggregation may play some
role in the association ofF. nucleatum with P. micros and W.
recta (Fig. 1), but coaggregation is probably more important
for the colonization of the gingival pocket than in the root
canal ecology.
ENDODONTIC THERAPY
In the root canal, substrate limitation and a build up of
metabolic and growth inhibitory products give rise to the
diverse community of primary and secondary nutrient utiliz-
ers with a variety of bacterial interactions. Endodontic treat-
ment interferes dramatically with this system. The anaero-
biosis is broken when the canal is opened and biomechanical
treatment eliminates bacteria as well as deprives the canal of
nutrients and interferes with bacterial interactions. However,
after the canal is sealed the anaerobiosis is restored and an
influx of tissue fluid into the canal can support the regrowth
of bacteria. It has been shown that anaerobic bacteria which
have survived the biomechanical treatment may multiply to
high numbers in the canal between appointments if no intra-
canal dressing is used (6). Thus, the fully developed root canal
infection with its dominance of anaerobic bacteria and estab-
lished interactions is susceptible to treatment. The significance
of this infection for clinical treatment is that the root canal
should ideally be completely cleaned at the initial treatment
visit when the bacteria are particularly vulnerable to eradica-
tion by a disturbance of their sensitive ecology. After the canal
has been completely debrided, the application of an intracanal
dressing is essential to eliminate any bacterial cells which have
survived the biomechanical treatment (19). Otherwise some
bacteria such as Enterococcusfaecalis, which are more resist-
ant to treatment, but which make up a small percentage of
the flora in the original infection, may be favored by the
changed ecology in the root canal and establish infections
which are difficult to treat (6, 20).
I thank D. Figdor, Melbourne, for valuable criticism of the manuscript and
for making the illustrations.
Dr. Sundqvist is professor, Department of Endodontics, University of Umea,
Umea, Sweden. Address requests for reprints to Dr. G. Sundqvist, Department
of Endodontics, Faculty of Dentistry, University of Umea, Umea S-90187,
Sweden.
References
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odontal disease: cultural studies. In: Genco RJ, Mergenhagen SE, eds. Host-
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2. Moore WEC, Holdeman LV, Cato EP, et al. Comparative bacteriology of
juvenile periodontitis. Infect Immun 1985;48:507-19.
3. Fabricius L. Oral bacteria and apical periodontitis. An experimental study
in monkeys [Dissertation]. Gothenburg, Sweden: University of Gothenburg,
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