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Copyright C Blackwell Munksgaard 2002

J Clin Periodontol 2002: 29: 10231028

Printed in Denmark . All rights reserved


Porphyromonas gingivalis,
Bacteroides forsythus and other
putative periodontal pathogens in
subjects with and without
periodontal destruction

A. J. van Winkelhoff1, B. G. Loos2,

W. A. van der Reijden1 and
U. van der Velden2

Department of Dental Basic Sciences,

section Oral Microbiology and 2Department of
Periodontology, Academic Centre for
Dentistry Amsterdam, the Netherlands

van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U.
Porphyromonas gingivalis, Bacteroides forsythus and other putative periodontal
pathogens in subjects with and without periodontal destruction. J Clin Periodontol
2002; 29: 102301028. C Blackwell Munksgaard, 2002.
Background and aims: Bacteria play an essential role in the pathogenesis of destructive periodontal disease. It has been suggested that not all bacteria associated with periodontitis may be normal inhabitants of a periodontally healthy
dentition. In particular, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans have been isolated infrequently from subjects without periodontitis.
The aim of the present study was to compare prevalence and proportions of a
number of periodontal bacteria in periodontitis patients and control subjects.
Material and methods: In all, 116 consecutive subjects diagnosed with moderate
to severe periodontitis (mean age 42.4) and 94 subjects without radiographic
evidence of alveolar bone loss (mean age 40.4) were recruited for the study. The
gingival condition in the control group varied between gingival health and various
degrees of gingivitis. In patients, the deepest pocket in each quadrant was selected
for microbiological sampling. In control subjects all mesial and distal sites of all
first molars were selected for sampling. All paper points from a patient were
pooled and processed for anaerobic cultivation within 6 h after sampling. Clinical variables of sampled sites included bleeding index, probing pocket depth and
clinical attachment level.
Results: A. actinomycetemcomitans, P. gingivalis, Prevotella intermedia, Bacteroides forsythus, Fusobacterium nucleatum and Peptostreptococcus micros were
significantly more often prevalent in patients than in controls. The highest odds
ratios were found for P. gingivalis and B. forsythus (12.3 and 10.4 resp.). Other
odds ratios varied from 3.1 to 7.7 for A. actinomycetemcomitans and P. micros,
respectively. Absolute numbers of target bacteria were all higher in patients, but
only the mean percentage of B. forsythus was significantly higher in patients in
comparison to controls (P 0.001).
Conclusions: A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus,
F. nucleatum and P. micros are all significant markers for destructive periodontal disease in adult subjects. Based on calculated odds ratios, B. forsythus
and P. gingivalis are the strongest bacterial markers for this disease and are
infrequently cultured from subjects without periodontal bone loss.

Key words: bateria; periodontics; P.

gingivalis; periodontal health
Accepted for publication 12 November 2001


van Winkelhoff et al.

Periodontitis is a disease that affects the

tooth supporting tissues and is characterised by loss of periodontal attachment including alveolar bone. The aetiology of the disease is multifactorial
and bacterial deposits play an essential
role in the pathogenesis (Page & Kornman 1997). The bacteria that are involved in periodontitis accumulate in
the subgingival plaque that comprises
predominantly of Gram negative strict
anaerobic rods. The group of dark-pigmented anaerobic rods is strongly associated with destructive periodontal
infections and the major pathogen in
this group is Porphyromonas gingivalis
(van Winkelhoff et al. 1989, Haffajee &
Socransky 1994). While most bacterial
species in the subgingival plaque of
periodontitis patients can also be found
in subjects without periodontitis. P. gingivalis is detected infrequently from individuals without periodontal breakdown (Slots & Ting 1999). This has led
to the hypothesis that this microorganism may not be a normal inhabitant of the oral cavity (Van Winkelhoff
et al. 1996). Detection of this putative
pathogen in periodontal health and disease strongly depends on the techniques
used and most frequently anaerobic culture techniques have been applied. This
has the disadvantage of a relatively low
sensitivity in comparison to DNAbased testing. Recently, Griffen et al.
(1998) have used a sensitive PCR assay
to test for the presence of P. gingivalis
in age-matched groups of periodontally
healthy (n 181) and periodontitis
affected individuals (n 130). To maximise the possibility to detect P. gingivalis, mesial sites of all teeth of the participants were sampled. The difference
in prevalence of this microorganism between healthy individuals and periodontitis patients appeared highly significant (P 0.0001), the odds ratio for
being infected with P. gingivalis was
11.2 times greater in the periodontitis
group than in the healthy group (95%
confidence interval, 6.519.2). The
authors concluded that P. gingivalis
should be considered a pathogen in destructive periodontal disease and they
suggested that this organism might not
be a normal inhabitant of a periodontally healthy dentition. Such an experiment, comparing the prevalence of
P. gingivalis in periodontal health and
disease in a reasonable group of individuals, has not been performed using
anaerobic culture techniques (Slots &
Ting 1999). Therefore, the aim of this

investigation was to study, by anaerobic

culturing, the prevalence of P. gingivalis
and other putative periodontal pathogens in a group of subjects with various
degrees of gingivitis but without periodontal destruction and to compare
this with a group of adult patients with
untreated periodontitis.

Material and methods

Study population

The study population included 116 consecutive adult patients who were referred to the Department of Periodontology for diagnosis and treatment of
periodontitis. A further 94 adult control subjects volunteered who were
registered for restorative dental procedures or who visited the dental school
for regular dental check-ups.
All subjects were both verbally and
written informed about the purpose of
the investigation. The study was approved by the Medical Ethical Committee of our institution.
For each periodontitis patient a set of
full mouth dental radiographs was
available and the individual number of
teeth present was determined. Subsequently, for each patient all teeth were
radiographically examined on the mesial and distal aspects. Patients suffered
from moderate to severe periodontitis
according to criteria by Kornman et al.
(1997). Control subjects were included
if they were not missing more than one
tooth per quadrant (the 3rd molar excluded), and if they had at all interproximal sites a distance between the
cemento-enamel junction and the alveolar bone crest of 2 mm on bitewing radiographs 1 year old. The gingival condition of the control subjects
varied between gingival health and various degrees of gingivitis.
Clinical and sampling procedures

The standardised clinical and microbiological examination included for the

periodontitis patients the selection of
the deepest pocket per quadrant based
on the probing pocket depth measurements as carried out during the first referral visit at ACTA (Mombelli et al.
1991). At these four sites the presence
or absence of plaque was evaluated at
the gingival margin by means of a periodontal probe. In the subjects of the
healthy/gingivitis group, all mesial and
distal sites of all first molars from the

buccal aspect were selected for microbiological sampling.

Sample sites were isolated with cotton rolls and supragingival plaque was
removed with curettes and cotton pallets. Subsequently, two paper-points
were inserted and left in place for 10 s.
All paper points were transferred to the
same vial containing 2 mL of reduced
transport medium (Syed & Loesche
1972). The samples were processed
within 6 h. After microbiological sampling, the following clinical parameters
for sampled sites were evaluated:
O Bleeding index (BI). Bleeding on
probing: 0: no bleeding; 1: pin prick
bleeding; 2: immediate and overt
bleeding; (Silness & Le 1964)
O Probing pocket depth (PPD);
O Clinical attachment loss (CAL).
Microbiological procedures

Tenfold serial dilutions were prepared

in sterile phosphate buffered saline. Appropriate dilutions were plated onto
non-selective 5% horse blood agar
plates (Oxoid no. 2; Oxoid Ltd, Basingstoke, England) supplemented with
haemin (5 Fg/L) and menadione (1 Fg/
L) (BA) and on trypticase soy-serumbacitracin-vancomycin (TSBV) plates
(Slots 1982) for selective isolation and
growth of Actinobacillus actinomycetemcomitans.
TSBV plates were incubated at 37 EC
in air 5% CO2 for 5 days, blood agar
plates were incubated at 37 EC in 80%
N2, 10% H2 and 10% CO2 for up to 3
The total number of colony forming
units on the blood agar plates was determined and converted to CFU/mL.
Identification of anaerobic isolates
The presence and proportions of P. gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus
micros, Bacteroides forsythus and
Campylobacter rectus were determined
on the blood agar plates. These isolates
were all purified and identified to species level. Identification of strictly anaerobic species was based on microscopic morphology, gram-reaction and
the profile in the API 32 A system (Biomerieux, La Balme Les Grottes,
France). Additional tests for identification included detection of a trypsinlike activity based on the degradation
of benzoyl-DL-arginine-2-naphthylamide (Sigma, St Louis, MO, USA) for the

Periodontal pathogens
Table 1. Age, gender and clinical characteristics of sampled sites of a control group and an
adult group of periodontitis patients. Standard deviations are expressed in brackets

Gender (female/male)
No. teeth
Mean Plaque Index (%)
Mean Bleeding Index
Mean Pocket Depth (mm)
Mean Attachment Loss (mm)




40.4 (11.9)
27.6 (1.7)
70.7 (27.0)
0.87 (0.57)
3.1 (0.43)

42.4 (9.8)
26.5 (3.2)
66.9 (34.7)
1.11 (0.40)
6.3 (1.26)
6.9 (1.50)


cies the mean percentage in controls

and patients were not different. When
individuals with detectable P. gingivalis
and/or A. actinomycetemcomitans were
excluded from the statistical analysis,
the mean percentages of target species
were also not significantly different between patients and controls with the exception of B. forsythus for which the
mean percentage was statistically higher
in patients (P 0.001).

*This measurement has not been taken in controls (see Materials and methods).
NS, not statistically significant.

identification of P. gingivalis (van Winkelhoff et al. 1988) and B. forsythus

(Braham & Moncla 1992, Tanner et al.
1986). Identification of A. actinomycetemcomitans was based on the characteristic colony morphology (star-like inner structure) on the TSBV plate, and a
positive catalase reaction upon exposure to 3% H2O2. The percentage of
A. actinomycetemcomitans was calculated on the basis of the number of
CFU on TSBV plates.
Statistical analysis

Difference in mean age of both test

groups was tested with an unpaired ttest. A chi-square-test was used to test
for difference in gender distribution.
Clinical data are expressed as means
and standard deviations (SD) and
tested for significant differences between control subjects and periodontitis
patients using the MannWhitney Utest. Differences in prevalence of the
various periodontal pathogens between
control subjects and periodontitis patients were determined using the
Fishers Exact test. The mean proportions of the various bacterial species
between the two groups were compared
with the MannWhitney U-test.

The mean age of the controls was 40.4

and for the patients the mean age was
42.4 (Table 1). This difference was not
significant. The gender distribution in
both groups was also similar. The mean
number of teeth was slightly, but significantly, lower in the periodontitis
group. The mean bleeding index and
mean pocket probing depth were significantly higher in the periodontitis
group. The mean plaque index at the



If a bacterial species is considered a

periodontal pathogen one would expect
this bacterium to be present in most patients with periodontitis and significantly less frequently detected in subjects without periodontal destruction.
To investigate this hypothesis a group
of individuals without periodontal destruction and a group of individuals
with overt periodontitis were identified.
Both groups were age matched. Microbiological testing was performed with
anaerobic culture techniques. It was
found that all target bacteria were
strongly associated with periodontitis,
with the exception of C. rectus. The species that were most strongly associated
periodontal destruction were P. gingivalis and B. forsythus, the odds of detecting these microorganism being 12.3
and 10.4 greater in the periodontitis
group than in the control group. This
observation strongly suggests that P.
gingivalis and B. forsythus are true
pathogens in adult patients with periodontitis. Our observations confirm the
findings of Griffen et al. (1998), who
found an odds ration of 11.2 for P. gingivalis in an adult periodontitis group
of 130 subjects. Griffen et al. (1998)
found a healthy carrier rate for P. gingivalis of 25%, whereas this amounted

sampled sites in both groups was similar (Table 1).

In Table 2 the number and proportions of individuals with detectable
target bacteria is shown. All species,
with the exception of C. rectus, were
significantly more frequently isolated
from subjects with periodontitis in comparison to control subjects. The highest
odds ratios were found for P. gingivalis
and B. forsythus, 12.3 and 10.4, respectively. Other odds ratios varied from 3.1
to 7.7 for A. actinomycetemcomitans
and P. micros, respectively (Fig. 1). The
total colony forming units (CFU) and
the number of the target bacteria are
summarised in Table 3. The total CFU
and the total number of target species
were all significantly higher in patients
than in controls.
Table 4 shows the mean percentages
of target species in culture positive subjects. The percentage of B. forsythus was
significantly higher in patients than in
controls (P 0.001). Periodontitis patients had on average a higher mean
percentage of P. gingivalis but the difference did not reach the level of significance (P 0.052). For all other spe-

Table 2. Number (%) of subjects culture positive for distinct bacterial species in the subgingival
plaque of a control group and a group of adult patients with periodontitis
Porphyromonas gingivalis
Prevotella intermedia
Bacteroides forsythus
Peptostreptococcus micros
Fusobacterium nucleatum
Campylobacter rectus

12 (12.8)


36 (31.0)

NS, not statistically significant. *P 6.44E-14.




Odds ratio
(95% CI)
3.1 (1.56.3)



van Winkelhoff et al.

Fig. 1. Odds ratios and 95% confidence intervals of different periodontal bacterial species in
adult periodontitis.

10.6% in our group of healthy subjects.

This may be explained in part by the
more sensitive PCR technique used in
their study and the greater number of
samples analysed per individual. Also,
the criteria they applied to select periodontally healthy individuals were different from the present study. They accepted a maximum probing pocket
depth and clinical attachment loss of
5.5 mm, whereas in the present study
the subjects of the healthy/gingivitis

group were selected on the basis of absence of radiographic alveolar bone

loss. Griffen et al. (1998) found a
prevalence of P. gingivalis in periodontitis patients of 79%, which is
somewhat higher than the 60% in the
present study. The more sensitive PCR
technique and the clinical periodontal
status as stated before may account for
this difference. Thus, using anaerobic
culture techniques with a limited detection level for periodontal pathogens,

Table 3. Total CFU and mean CFU (SD) of bacterial species in the subgingival plaque of a
control group and of a group of adult patients with periodontitis in culture positive subjects
CFU 104




Total CFU
A. actinomycetemcomitans
P. gingivalis
P. intermedia
B. forsythus
P. micros
F. nucleatum
C. rectus

3377.6 (12873.4)
5.6. (9.0)
152.6 (267.3)
71.5 (148.2)
44.2 (107.1)
177.2 (739.2)
80.2 (261.8)
3.6 (3.9)




Table 4. Mean percentages (SD) of bacterial species in the subgingival plaque of culture positive subjects of a control group and of a group of adult patients with periodontitis

A. actinomycetemcomitans
P. gingivalis
P. intermedia
B. forsythus
P. micros
F. nucleatum
C. rectus









the results of the study of Griffen et al.

(1998) and the present study show a
striking similarity in the odds ratio to
detect P. gingivalis in periodontitis patients.
The mean number of total CFU and
the mean numbers of target bacteria
were all higher in patients than in controls. However, the mean proportion of
P. gingivalis was not significantly different in both groups (17.4% 21.8 and
28.7% 21.3, respectively). This shows
that the mere presence of P. gingivalis in
the subgingival area does not necessarily lead to destructive periodontal infection and confirms the hypothesis
that periodontal pathogens are essential
in the pathogenesis of periodontitis but
not enough to initiate destruction of the
periodontium. Apparently, a susceptible
host and/or the presence of other
pathogenic microorganisms are needed
to initiate the disease. The marked difference in prevalence of P. gingivalis in
controls and patients in the present as
well as in the Griffen et al. (1998) study
may be a geographical phenomenon
and may not be observed in other populations on the world. For instance, if the
inclusions criteria for periodontal
health, i.e. 5.5 mm of clinical attachment loss, used by Griffen et al. (1998)
are applied on a population in Indonesia (Timmerman et al. 1998) up to 68%
of the healthy subjects would be positive for P. gingivalis. Moreover, in Indonesian individuals with 02 mm of
clinical attachment loss the prevalence
of P. gingivalis amounted 65%. The observation of this high prevalence of P.
gingivalis in this particular population
suggests that the carrier rate of this
microorganism in subjects with minimal periodontal breakdown may be related to the oral hygiene level and the
amount of dental care provided.
Since culture techniques were used in
this study, putative periodontal pathogens other than P. gingivalis could be
detected and their association with periodontitis established as well. With the
exception of C. rectus, all target bacteria were positively associated with
periodontitis with odds ratios ranging
from 3.1 for A. actinomycetemcomitans
to 10.4 for B. forsythus.
B. forsythus has been implicated in
progressive periodontitis (Dzink et al.
1988, Haffajee et al. 1988, Socransky
et al. 1988) and has been associated
with refractory periodontitis (Winkel
et al. 1997). Not only was there a strong
relationship between B. forsythus and

Periodontal pathogens
periodontitis, also the mean number of
CFU and the mean proportion of this
microorganism were significantly lower
in controls compared to periodontitis
patients (Tables 3 and 4). This implies
that this microorganism is a strong
marker for destructive periodontal disease (AAP position paper, American
Academy of Periodontology 1996). Recently, Tran et al. (2001) found a prevalence of 81.8% of B. forsythus in subjects with a low prevalence and severity
of adult periodontitis. They showed
that in these subjects, repeated detection of B. forsythus was correlated with
attachment loss at the subject but not
on a site level (odds ratio 5.3, P 0.038,
CI 1.322.5).
Mean proportions for all other bacterial markers were not different in control subjects and periodontitis patients.
This may indicate that, with the exception of B. forsythus and possibly P. gingivalis, there is little proportional
change in the composition of the subgingival microflora between periodontal health and periodontitis.
In summary, based on the present
culture data, the odds of detecting P.
gingivalis were 12.3-fold higher in subjects with periodontitis than in agematched controls. Also, B. forsythus
showed a strong relationship with periodontitis, with an odds ratio of 10.4.
Both microorganisms seem the strongest markers of destructive periodontal
disease. Lower, but significant, odds ratios were also found for A. actinomycetemcomitans, P. intermedia, F. nucleatum and P. micros.

(Durchschnittsalter 40,4 Jahre) ohne rntgenologisch sichtbaren Alveolarknochenabbau

fr die Studie rekrutiert. In der Kontrollgruppe variierte der Gingivazustand zuwischen gesunder Gingiva und verschiedenen
Graden der Gingivitis. Bei den Patienten
wurde in jedem Quadranten von der tiefsten
Tasche eine mikrobiologische Probe entnommen. Bei den Kontrollpersonen wurde von
der mesialen und distalen Flche jedes ersten
Molaren eine Probe entnommen. Alle
Papierspitzen der Patienten wurden gepoolt
und innerhalb von 6 Std. nach der Probenentnahme fr die anaerobe Kultivierung
weiterverarbeitet. Die klinischen Parameter
der Entnahmestellen waren Blutungsindex,
klinische Sondierungstiefe und klinisches Attachmentniveau.
Ergebnisse: A. actinomycetemcomitans, P.
gingivalis, Prevotella intermedia, Bacteroides
forsythus, Fusobacterium nucleatum und
Peptostreptococcus micros zeigten bei den
Patienten eine signifikant hhere Prvalenz
als bei den Kontrollen. Die hchsten OddsRatios wurden fr P. gingivalis und B. forsythus (12,3 bzw. 10,4) vorgefunden. Andere
Odds-Ratios variierten von 3,2 bis 7,7 fr A.
actinomycetemcomitans bzw. P. micros. Alle
absoluten Zahlen der Zielbakterien waren bei
den Patienten hher. Nur fr B. forsythus
war der durchschnittliche Prozentsatz bei den
Patienten im Vergleich zu den Kontrollen hher (p0,001).
Schlussfolgerungen: Bei Erwachsenen sind A.
actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus, F. nucleatum und P.
micros alle signifikante Marker einer destruktiven Parodontalerkrankung. Auf der
Grundlage der berechneten Odds-Ratios sind
B. forsythus und P. gingivalis die strksten
bakteriellen Marker dieser Erkrankung und
knnen selten bei Personen ohne parodontalen Knochenabbau kultiviert werden.

Porphyromonas gingivalis, Bacteroides forsythus und andere mutmaliche Parodontalpathogene bei Patienten mit oder ohne parodontalen Abbau
Grundlagen und Ziele: Bakterien spielen in
der Pathogenese der destruktiven Parodontalerkrankung eine essentielle Rolle. Es wurde
behauptet, dass nicht alle mit Parodontitis
assoziierten Bakterien normale Besiedler eines parodontal gesunden Gebisses seien. Speziell Porphyromonas gingivalis und Actinobacillus actinomycetemcomitans wurden selten bei Personen ohne Parodontitis gefunden.
Das Ziel der vorliegenden Studie war es, bei
Parodontitispatienten und Kontrollpersonen,
fr eine Anzahl von parodontalen Bakterien,
die Prvalenz und den relativen Anteil an der
Gesamtflora zu vergleichen.
Material und Methoden: Insgesamt wurden
116 aufeinanderfolgende Patienten (Durchschnittsalter 42,4 Jahre) mit moderater bis
schwerer Parodontitis und 94 Personen

Le Porphyromonas gingivalis, le Bacterodes

forsythus et dautres pathoge`nes parodontaux
putatifs chez des sujets avec ou sans destruction parodontale
Les bacteries jouent un role essentiel dans la
pathogene`se de la destruction de la maladie
parodontale. Seul une partie des bacteries associees a` la parodontite seraient des hotes
normaux dune dentition saine parodontalement. Specialement le Porphyromonas gingivalis (P.g.) et lActinobacillus actinomycetemcomitans (A.a.) ont ete peu souvent isoles de
sujets sans parodontite. Le but de cette etude
a ete de comparer la frequence globale et les
proportions dun nombre de bacteries parodontales chez des patients avec parodontite
et des sujets controles. Cent-seize patients
diagnostiques avec une parodontite moderee
a` seve`re ages en moyenne de 42,4 ans et 94
sujets sans evidence radiographique de perte
osseuse (40,4 ans) ont ete inclus dans cette
etude. La condition gingivale dans le groupe
controle variait entre des gencives saines et
differents degres de gingivite. Chez les pa-


tients, la poche la plus profonde de chaque

quadrant etait selectionnee pour lechantillonnage microbien. Chez tous les sujets tous
les sites mesiaux et distaux des premie`res molaires ont ete selectionnes pour lechantillonnage. Toutes les pointes de papier dun patient etaient rassemblees et mises en culture
anaerobie dans les six heures apre`s lechantillonnage. Les variables cliniques des sites
echantillonnes incluaient le saignement au
sondage la profondeur de poche au sondage
et le niveau dattache clinique. A. actinomycetemcomitans , P.gingivalis, Prevotella intermedia, Bacterodes forsythus, Fusobacterium
nucleatum et Peptostreptococcus micros
etaient significativement plus souvent retrouves chez les patients que chez les controles.
Les facteurs de risque variaient de 3,1 a` 7,7
pour A. actinomycetemcomitans et P. micros.
Les nombres absolus des bactericides etaient
tous plus importants chez les patients mais
seulement le pourcentage moyen de B.forsythus, etait significativement plus important
chez les patients en comparaison des controles (p0,001). A. actinomycetemcomitans, P.
gingivalis, P. intermedia, B. Forsythus, F. nucleatum et P. micros sont tous des marqueurs
significatifs de la maladie parodontale destructrice chez les sujets adultes . Bases sur les
facteurs de risque, B. forsythus et P. gingivalis sont les marqueurs bacteriens les plus
forts pour cette maladie et sont trop peu souvent mis en culture chez les sujets sans perte
osseuse parodontale.

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A.J. van Winkelhoff
Department of Dental Basic Sciences
Section Oral Microbiology
Academic Centre for Dentistry Amsterdam
Van der Boechorststraat 7
1081 BT Amsterdam
The Netherlands
Tel: 31 20 4448678
Fax: 31 20 4448318