Effect of various rinsing protocols after use of amine fluoride/stannous fluoride toothpaste on the bacterial composition of dental plaque.

Abstract

This clinical study evaluated the effect of different oral hygiene protocols on the bacterial composition of dental plaque. After a 2-week period of using fluoride-free toothpaste, 30 participants followed three 1-week experimental protocols, each followed by 2-week fluoride-free washout periods in a randomized crossover examiner-blind controlled trial. The 1-week experimental protocols comprised the use of AmF/SnF(2) toothpaste twice daily, after which participants either (1) rinsed with tap water, (2) did not rinse but only spat out the toothpaste, or (3) rinsed with an AmF/SnF(2) mouthwash. In the fluoride-free washout periods, the participants brushed their teeth with fluoride-free toothpaste without further instructions. Six hours after the last brushing (+/- rinsing) of each period, buccal plaque samples in the upper molar region were taken. The microbiota composition of the plaque samples was analyzed by checkerboard DNA:DNA hybridization. A statistically significant reduction was found in the total amount of DNA of the 39 major plaque species measured, and in the proportions of some acid-producing bacterial strains after the period having used the AmF/SnF(2) toothpaste + AmF/SnF(2) mouthrinsing. The results indicate that using the AmF/SnF(2) toothpaste and rinse combination could result in plaque of lower cariogenicity.

Full text

Page 1

Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Original Paper
Caries Res 2009;43:462–467
DOI: 10.1159/000264683
Effect of Various Rinsing Protocols after Use of
Amine Fluoride/Stannous Fluoride Toothpaste on
the Bacterial Composition of Dental Plaque
C. van Loveren a V.A.M. Gerardu a C.H. Sissons b M. van Bekkum
J.M. ten Cate a
b
a Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam,
Amsterdam , The Netherlands; b Department of Pathology and Molecular Medicine, Wellington School of Medicine
and Health Sciences, University of Otago, Wellington , New Zealand

AmF/SnF 2 toothpaste + AmF/SnF 2 mouthrinsing. The results
indicate that using the AmF/SnF 2 toothpaste and rinse com-
bination could result in plaque of lower cariogenicity.
Copyright © 2009 S. Karger AG, Basel
The effect of active ingredients of oral care products
depends in part on the oral substantivity of these ingre-
dients. Important factors in determining oral substantiv-
ity are the release characteristics of the ingredient from
the delivery vehicle, the physicochemical properties of
the ingredient, and factors related to the individual user.
Recently, attention has focused on post-use behavior fol-
lowing the toothbrushing of the individual user. This is
potentially a major determinant of efficacy. It has been
demonstrated that more fluoride is retained in the oral
cavity after toothbrushing when individuals do not rinse
and spit out the toothpaste, but rather use retained tooth-
paste to rinse the mouth or use a fluoride rinse after
toothbrushing [Sjögren and Birkhed, 1994]. Heijnsbroek
et al. [2006] reported increased salivary fluoride concen-
trations after brushing with toothpaste containing amine
and stannous fluoride (AmF/SnF 2 ) when the individuals
used an AmF/SnF 2 rinse afterwards. Gerardu et al. [2006]
showed that this combination of toothpaste and rinse was
effective in reducing the bacterial acid metabolism in
Key Words
AmF/SnF 2 ? Checkerboard analysis ? Clinical trial ? Plaque
composition
Abstract
This clinical study evaluated the effect of different oral hy-
giene protocols on the bacterial composition of dental
plaque. After a 2-week period of using fluoride-free tooth-
paste, 30 participants followed three 1-week experimental
protocols, each followed by 2-week fluoride-free washout
periods in a randomized crossover examiner-blind con-
trolled trial. The 1-week experimental protocols comprised
the use of AmF/SnF 2 toothpaste twice daily, after which par-
ticipants either (1) rinsed with tap water, (2) did not rinse but
only spat out the toothpaste, or (3) rinsed with an AmF/SnF 2
mouthwash. In the fluoride-free washout periods, the par-
ticipants brushed their teeth with fluoride-free toothpaste
without further instructions. Six hours after the last brushing
( 8 rinsing) of each period, buccal plaque samples in the up-
per molar region were taken. The microbiota composition of
the plaque samples was analyzed by checkerboard DNA:
DNA hybridization. A statistically significant reduction was
found in the total amount of DNA of the 39 major plaque
species measured, and in the proportions of some acid-pro-
ducing bacterial strains after the period having used the
Received: December 5, 2008
Accepted after revision: September 24, 2009
Published online: December 10, 2009
C. van Loveren
Academic Centre for Dentistry Amsterdam (ACTA)
Department of Cariology Endodontology Pedodontology , Louwesweg 1
NL–1066 EA Amsterdam (The Netherlands)
Tel. +31 20 518 8662, Fax +31 20 669 2881, E-Mail c.van.loveren @ acta.nl
© 2009 S. Karger AG, Basel
0008–6568/09/0436–0462$26.00/0
Accessible online at:
www.karger.com/cre

Page 2

Oral Hygiene Procedures and Plaque
Composition
Caries Res 2009;43:462–467
463
dental plaque and the tongue flora at least up to 6 h after
brushing. Previously, also shifts in plaque composition
were reported after having used AmF/SnF 2 -containing
toothpaste and rinse. Mengel et al. [1996] and Zimmer-
mann et al. [1993] showed that the proportion of cocci
increased, and the proportions of rods, spirochetes, fila-
ments and fusiforms decreased.
The present study was part of the study reported by
Gerardu et al. [2006], undertaken to explore the effect of
various rinsing protocols after brushing with AmF/SnF 2
toothpaste on plaque acidogenicity. The three rinsing
protocols studied were a water rinse, no rinse and a rinse
with an AmF/SnF 2 solution. This study reports on the
plaque microbiota.
Materials and Methods
A full description of the subjects, experimental design, oral
hygiene protocols, toothpastes, rinses and sampling can be found
in Gerardu et al. [2006]. In brief, after a 2-week period of using a
NaF toothpaste (Prodent Cool Mint 1,450 ppm F; Sara Lee,
Veenendaal, The Netherlands) and a 2-week fluoride-free tooth-
paste (Ultradent; Kruidvat, Renswoude, The Netherlands) peri-
od, 30 volunteers (22 women, 8 men; mean age: 26.8 8 7.3 years)
were enrolled in a randomized crossover examiner-blind con-
trolled clinical trial with 1-week experimental periods each fol-
lowed by a 2-week fluoride-free washout period. During the ex-
perimental weeks, the participants used AmF/SnF 2 toothpaste
(meridol; Gaba International AG, Therwil, Switzerland) contain-
ing 350 ppm F as Olaflur and 1,050 ppm F as stannous fluoride
twice daily. After brushing, they either (1) rinsed once for 30 s
with tap water, (2) spat out only and did not rinse their mouth, or
(3) rinsed for 30 s with 10 ml AmF/SnF 2 mouthwash (meridol,
Gaba International AG; 125 ppm F from Olaflur, 125 ppm F from
SnF 2 ). The individuals were only included in the experiment after
informed consent letters had been signed. The study protocol was
approved by the ethical board of the dental faculty of the Aca-
demic Centre for Dentistry Amsterdam.
Sampling Procedures
All samples were collected treatment-blind by 1 examiner. Six
hours after the last toothbrushing, all buccal plaque from 2 mo-
lars in the second quadrant was collected with a microbrush (Mi-
crobrush International, Grafton, Wisc., USA) for checkerboard
analysis after a sucrose rinse as described by Gerardu et al. [2006].
The microbrushes were collected in a 1:
and Tris-EDTA buffer (20 m M Tris-Cl, 1 m M EDTA; pH 8.0) and
stored at –80 ° C. At the end of the experiment the samples were
shipped by courier to the collaborating laboratory for checker-
board analysis.
1 mixture of 0.5 M NaOH
DNA:DNA Checkerboard Analysis of Plaque Samples
The method was used as previously described by Wall-Man-
ning et al. [2002] and Gellen et al. [2007]. In brief, the plaque
samples were vortex-mixed to disperse the bacteria from the mi-
crobrushes. The samples were denatured and lysed for 5 min at
95 ° C and neutralized (NH 4 acetate). Whole-genome DNA probes
labeled with digoxigenin were prepared from the 39 key species
shown in table 1 (plus Micromonas micros, deleted because of lost
standard). Samples (100 ? l) and flanking sets of DNA standards
were laid in thin (approximately 0.8 mm) lines on a 15 ! 15 cm
square of positively charged nylon membrane (Roche Diagnos-
tics) using a minislot device (Immunetics Inc., Boston, Mass.,
USA) and cross-linked to the membrane by UV light. The mem-
branes were hybridized with probes from the 40 species in a 45-
channel miniblotter (Immunetics) overnight at 42 ° C. Two strin-
gency washes removed excess probe. The membrane, equilibrated
with blocking buffer, was incubated with anti-digoxigenin-AP
antibody and washed to remove unbound antibody. Probe-target
hybrids were detected by hydrolysis of CDP-Star in detection
buffer. Chemiluminescence was captured by a ChemiGenius 2
digital camera and Genesnap software (Syngene, Cambridge,
UK).
The density of each hybridization signal was converted to an
equivalent cell number by comparison with the DNA standards,
using proprietary software (Fluor version 1). The cell numbers for
each species probed were summed to give the total probe count.
Each species was then converted to a percentage of the total probe
count for each sample (percent DNA probe).
Statistics
Differences in the proportion of each species in the total sam-
ple between the experimental groups were tested by discriminant
analysis entering all independent variables together. Discrimi-
nant analysis determines linear combinations of the independent
variables (the individual bacterial species) which best discrimi-
nate between the groups. The linear combinations of the indepen-
dent variables are so-called discriminant functions. For each
function it is calculated whether it significantly contributes to the
differences between the groups. Within a function, the relative
contribution of each independent variable and the correlation
with the function are calculated. The total amount of DNA and
the proportions of the individual strains were compared using
general linear model repeated measurements with Bonferroni ad-
justment. The level of significance was set at p ! 0.05. SPSS 14.0
software was used for statistical analysis.
Results
After all experimental periods, the average total DNA
probe count was statistically significantly lower than in
the pretreatment sample. After the period when the AmF/
SnF 2 toothpaste + AmF/SnF 2 rinse had been used, the
average total DNA probe count was statistically signifi-
cantly lower than after all other periods, except when the
brushing was followed by water rinse ( fig. 1 ).
Discriminant analysis revealed significant differences
in the composition of the dental plaque between the ex-
perimental periods. Discriminant function 1 explained
62% of the variance, function 2 explained 19% and func-
tion 3 explained 8%, with only function 1 being statisti-

Page 3

van Loveren /Gerardu /Sissons /
van Bekkum /ten Cate
Caries Res 2009;43:462–467
464
Table 1. Within-group correlations between the proportions of the various bacterial species and discriminant function 1 (column 2),
and the proportions of each species in the various treatment groups (columns 3–6)
Proportion of each species in plaque after the various treatment periods, %
correlation
with function 1out period of the
use of NaF paste
during experiment
after first wash-
after 3 wash-
out periods
after water
rinse and
no rinse
after AmF/
SnF2 rinse
Streptococcus oralis
Streptococcus mitis 1
Streptococcus gordonii
Streptococcus mitis 2
Streptococcus sanguinis
Actinomyces israelii
Neisseria mucosa
Lactobacillus rhamnosus
Veillonella parvula
Actinomyces naeslundii
Streptococcus sobrinus
Capnocytophaga gingivalis
Streptococcus anginosus
Aggregatibacter actinomycetemcomitans
Streptococcus vestibularis
Streptococcus parasanguinis
Corynebacterium matruchotii
Campylobacter rectus
Lactobacillus acidophilus
Rothia dentocariosa
Fusobacterium nucleatum
Prevotella melaninogenica
Lactobacillus fermentum
Streptococcus intermedius
Haemophilus parainfluenzae
Prevotella nigrescens/intermedia
Eikenella corrodens
Porphyromonas gingivalis
Bifidobacterium dentium
Actinomyces odontolyticus
Peptostreptococcus asaccharolyticus
Selenomonas noxia
Gemella morbillorum
Streptococcus mutans
Leptotrichia buccalis
Candida albicans
Lactobacillus plantarum
Actinomyces gerencseriae
Eubacterium saburreum
0.405
0.332
0.264
0.245
0.213
0.208
0.181
0.152
0.133
0.131
0.100
0.092
0.091
0.085
0.082
0.061
0.054
0.026
0.017
–0.012
–0.018
–0.034
–0.037
–0.042
–0.051
–0.067
–0.074
–0.087
–0.088
–0.099
–0.108
–0.115
–0.117
–0.119
–0.146
–0.148
–0.161
–0.161
–0.197
1.8 (0.6)a
3.7 (1.9)a
1.2 (0.7)a
1.4 (0.5)a
3.0 (0.5)a
2.8 (0.7)a
2.5 (1.0)a
0.4 (0.2)a
2.2 (0.6)
2.9 (0.7)
1.0 (0.5)
3.0 (0.7)
2.9 (0.9)
0.6 (0.4)
2.2 (0.6)
3.2 (0.6)
1.7 (0.8)
1.1 (1.2)
2.1 (1.6)
3.6 (0.7)
4.3 (3.6)
1.5 (0.8)
4.1 (1.1)
2.2 (0.5)
7.9 (3.5)
1.3 (0.4)
4.5 (1.4)
0.4 (0.3)
2.3 (0.5)
1.9 (0.4)
3.9 (1.4)a
1.1 (1.3)
3.8 (1.9)a
2.4 (0.7)
3.2 (1.2)a
1.7 (0.6)a
1.6 (0.7)a, b
5.3 (1.4)a
3.3 (0.9)
1.3 (0.7)b
2.1 (1.5)b
0.8 (0.5)b, c
1.1 (0.6)b
2.8 (0.7)a, b
2.8 (0.7)a
2.7 (1.1)a
0.3 (0.2)a
2.1 (0.8)
2.9 (0.7)
0.8 (0.5)
2.6 (1.2)
2.5 (1.0)
0.6 (0.5)
2.1 (0.5)
2.8 (0.8)
1.8 (0.9)
1.0 (1.3)
1.9 (2.1)
3.6 (1.1)
5.1 (4.1)
1.6 (0.9)
4.3 (1.5)
2.3 (0.6)
8.5 (3.5)
1.3 (0.5)
5.0 (1.5)
0.5 (0.3)
2.5 (0.6)
2.0 (0.4)
4.2 (1.4)b
1.4 (1.3)
4.1 (2.2)a, b
2.6 (0.7)
3.4 (1.1)a, b
1.8 (0.6)a, b
1.7 (0.6)a
5.4 (1.4)a
3.6 (1.2)
1.1 (0.7)b
1.7 (1.3)b, c
0.8 (0.7)b
1.2 (0.7)a, b
2.8 (0.7)a, b
2.6 (0.7)a
2.6 (1.0)a
0.4 (0.4)a
1.9 (0.9)
2.9 (0.7)
0.8 (0.5)
2.2 (1.1)
2.5 (1.0)
0.7 (0.6)
2.0 (0.6)
2.6 (0.8)
1.8 (0.8)
0.5 (1.0)
2.4 (2.6)
3.4 (1.3)
5.2 (4.2)
1.7 (0.9)
4.4 (1.5)
2.3 (0.7)
8.3 (3.0)
1.5 (0.6)
5.1 (1.5)
0.4 (0.3)
2.5 (0.6)
2.0 (0.5)
4.4 (1.3)b
0.5 (1.4)
4.2 (2.5)a, b
2.7 (0.8)
3.5 (1.1)a, b
2.0 (0.6)a, b
1.7 (0.7)a, b
5.4 (1.5)a
3.6 (1.1)
0.5 (0.4)c
1.3 (0.8)c
0.4 (0.4)c
0.7 (0.5)c
2.3 (0.7)b
2.2 (0.7)b
1.7 (0.7)b
0.2 (0.2)b
1.7 (0.9)
2.5 (0.7)
0.7 (0.3)
2.5 (1.2)
2.4 (1.2)
0.4 (0.3)
1.9 (0.7)
2.9 (1.0)
1.5 (0.8)
0.5 (0.5)
1.8 (1.4)
3.7 (1.4)
4.8 (4.2)
1.6 (1.1)
4.4 (1.8)
2.4 (0.7)
8.8 (3.2)
1.5 (0.6)
5.1 (1.4)
0.6 (0.4)
2.6 (0.9)
2.1 (0.5)
4.6 (1.5)b
0.6 (0.5)
5.1 (3.0)b
2.8 (0.9)
4.0 (1.6)b
2.1 (0.8)b
2.1 (0.8)b
6.5 (2.0)b
5.2 (4.6)
Values denote means (SD), unless specified otherwise. Means sharing the same superscript letter are not statistically significantly
different. Data for the 3 fluoride-free periods after AmF/SnF2 toothpaste use (column 4) are averaged, as are data for the AmF/SnF2
toothpaste + water rinse and no rinse groups (column 5).

Page 4

Oral Hygiene Procedures and Plaque
Composition
Caries Res 2009;43:462–467
465
cally significant (p ! 0.001). The average plaque composi-
tion in relation to discriminant functions 1 and 2 is rep-
resented in figure 2 . AmF/SnF 2 toothpaste (markers 5–7)
yielded a modified plaque composition compared to that
after the first washout period after the NaF toothpaste
use (marker 1). After brushing, mouthrinse with AmF/
SnF 2 (marker 7), but not the water rinse or no rinse pro-
cedures (markers 5–6), induced a further shift in micro-
biota composition that disappeared after the 2-week fluo-
ride-free washout period (markers 2–4).
Table 1 presents the within-group correlations be-
tween the proportions of the various bacterial species and
function 1. Additionally, the proportions of the species
are given after the various experimental periods. The
proportions for the 3 fluoride-free periods after the peri-
ods during which AmF/SnF 2 toothpaste had been used
were not significantly different from each other (markers
2–4 in fig. 2 ) and are presented averaged (column 4 of
table 1 ). This also applies to the proportions in the water
rinse and no rinse groups (markers 5 and 6 in fig. 2 ),
which are averaged in column 5 of table 1 . The modified
plaque composition after AmF/SnF 2 toothpaste use (col-
umn 5 of table 1 ) compared to that after the first washout
period after the NaF toothpaste use (column 3 of table 1 )
comprised a decrease in S treptococcus oralis, S. mitis bio-
200
DNA probe count (ng)
250
300
350
400
450
a
1st F-free
b
b
b
b, c
b
c
F-free after
AmF/SnF
paste +
H O
2
2
F-free after
AmF/SNF
paste + spit
2
F-free after
AmF/SnF
paste +
AMF/SnF
rinse
2
2
AmF/SnF
paste +
H O
2
2
AmF/SnF
paste + spit
2
AMF/SnF
paste +
AmF/SnF
rinse
2
2
Fig. 1. Mean DNA probe count in the
plaque samples collected after the various
experimental periods. Error bars indicate
standard errors. Means sharing the same
letter are not significantly different (p 1
0.05).
–3
Function 2
–2
–1
0
1
2
3
–3
–2
–10 123
Function 1
7
3
4
2
5
6
1
Fig. 2. Discriminant analysis of the checkerboard percent contri-
bution counts. Marker 1: plaque composition after the fluoride
(F)-free period at the start of the experiment. Marker 2: after the
F-free period after the AmF/SnF 2 paste + water rinse period.
Marker 3: after the F-free period following the AmF/SnF 2 paste +
no rinse period. Marker 4: after the F-free period following the
AmF/SnF 2 paste + AmF/SnF 2 rinse period. Marker 5: after the
AmF/SnF 2 paste + water rinse period. Marker 6: after the AmF/
SnF 2 paste + no rinse period. Marker 7: after the AmF/SnF 2 paste
+ AmF/SnF 2 rinse period.
Color version available online

Page 5

van Loveren /Gerardu /Sissons /
van Bekkum /ten Cate
Caries Res 2009;43:462–467
466
types 1 and 2, and S. gordonii, and an increase in Pepto-
streptococcus asaccharolyticus. After the use of AmF/SnF 2
rinse (column 6 of table 1 ), but not after the water rinse
and no rinse procedures (column 5 of table 1 ), a further
decrease was observed in S. oralis, S. mitis biotypes 1 and
2, S. gordonii, S. sanguinis, Actinomyces israelii, Neisseria
mucosa and Lactobacillus rhamnosus, while the propor-
tions of A. gerensceriae, L. plantarum, Candida albicans,
Leptotrichia buccalis, Gemella morbillorum and P. asac-
charolyticus had further increased.
Discussion
The main effects of the AmF/SnF 2 protocols were a
reduction of the amount of plaque, as estimated from the
total probe count of the plaque samples, and a relative
decrease in some putative caries pathogens and other lac-
tic-acid-producing species. The greatest effect was ob-
served after the period that AmF/SnF 2 toothpaste + AmF/
SnF 2 rinse had been used. Even though not all acidogen-
ic organisms decreased in proportion (in particular, S.
mutans and Lactobacilli did not), the data suggest that the
plaque had turned less cariogenic. Gerardu et al. [2006]
have shown that the protocol of AmF/SnF 2 toothpaste +
AmF/SnF 2 rinse reduced plaque acidogenicity, which
may now be related to a reduced proportion of some acid-
producing bacteria.
The decrease in the total probe count in the AmF/SnF 2
toothpaste + AmF/SnF 2 rinse group correlates with clin-
ical findings showing a decrease in plaque index after the
use of these products [Zimmermann et al., 1993; Mengel
et al., 1996]. The total probe count of the plaque samples
after the AmF/SnF 2 toothpaste + water rinse period was
between those after the toothpaste + AmF/SnF 2 rinse and
after the toothpaste + no rinse periods. This was not an-
ticipated because a greater effect was expected when
toothpaste would be less effectively cleared from the
mouth, e.g. in the no rinse period. Our finding might
therefore indicate that rinsing itself had a small addition-
al cleansing effect on the surfaces by retarding subse-
quent plaque regrowth.
The experiment was designed as a randomized exam-
iner-blind crossover controlled trial. However, the ran-
domization started only after the first fluoride-free pe-
riod after the period of having used NaF toothpaste.
Therefore, it cannot be excluded that the differences be-
tween this and the other periods are partly due to the so-
called Hawthorne effect: people change their behavior as
soon as they realize they are participating in an experi-
ment [Mayo, 1933; Feil et al., 2002]. The difference in
composition between the first fluoride-free period and
the other fluoride-free periods may, in addition to the
Hawthorne effect, be related to the fact that the first pe-
riod was preceded by a 2-week period in which NaF
toothpaste had been used, while the others were preceded
by a period when AmF/SnF 2 toothpaste had been used.
Clinical studies have shown that AmF/SnF 2 exerts a dif-
ferent pressure on plaque composition than NaF [Zim-
mermann et al., 1993; Mengel et al., 1996]. The differ-
ences between the other periods, which were random-
ized, should be fully related to the various experimental
conditions.
Marsh and Bradshaw [1990] demonstrated in continu-
ous culture experiments with 9 oral species that an addi-
tion of fluoride modified the pH-mediated disruption of
the bacterial community permitting S. mitior (S. oralis)
to compete with S. mutans within the ecosystem. Kilian
et al. [1979b] suggested that changes in plaque under flu-
oride pressure have to be explained by direct effects of
fluoride on bacterial processes favoring fluoride-resis-
tant strains over fluoride-sensitive ones. Li and Bowden
[1994] showed that biofilm formation by S. mutans and
by A. naeslundii on fluoridated hydroxyapatite could be
significantly less than on nonfluoridated hydroxyapatite.
Van der Mei et al. [2008] demonstrated that adhesion to
salivary pellicles and subsequent biofilm growth de-
creased after treatment with amine fluoride for S. mutans
but not for A. naeslundii or S. oralis. However, all this in-
formation does not contribute to explaining the outcome
of our experiment. Discrepancies between model systems
and in vivo natural plaque, as in our experiment, may be
related to the complexity of the community interrelation-
ships between bacteria [Gilbert et al., 1997; Filoche et al.,
2004].
The experimental toothpaste contained AmF and
SnF 2 . The cations of these compounds have antimicrobial
activity [Kilian et al., 1979a; Meurman et al., 1989]. Oral
hygiene products containing SnF 2 have been shown to re-
tard plaque regrowth [Gross and Tinanoff, 1977; Kilian et
al., 1979a; Benjasupattananan et al., 2005; Paraskevas and
van der Weijden, 2006; White et al., 2008]. Li et al. [2004]
followed initial plaque formation on pumiced buccal and
labial surfaces with the checkerboard DNA:DNA hybrid-
ization technique, with 17 species in common to our pan-
el. In the 6 h of plaque growth, they observed an increase
in the proportion of streptococci, in particular S. mitis
and S. oralis, at the expense of Actinomyces species. Por-
phyromonas gingivalis and Aggregatibacter actinomy-
cetemcomitans were present in low levels at all examined