Chapter 5

Influence of toothpastes and

mouthrinses on the microbial
composition of oral biofilm

Marieke P.T. Otten, Henk J. Busscher, Henny C. van der Mei, Frank Abbas, Chris
G. van Hoogmoed

Part of this chapter is, in combination with Chapter 4, submitted to Clinical Oral
Investigations
Chapter 5

Abstract

The aim of this study was to examine the microbial similarity between oral
biofilms collected from human volunteers after using different antibacterial
toothpastes and mouthrinses, as compared to the use of a control toothpaste without
antibacterial claims. Oral biofilms from the same volunteer were collected either
after 2 weeks use of a control toothpaste (Prodent Coolmint ) and after 2 weeks of
using an antibacterial toothpaste (Crest Pro Health, Colgate Total, Zendium
Classic) or additional use of an antibacterial mouthrinse (Listerine, Meridol, Crest
Pro Health). The bacterial compositions of the biofilms were compared using PCR-
denaturing gradient gel electrophoresis (DGGE), and compared to a reference
strain. All products influenced the bacterial composition as compared to the
control. Shifts in bacterial composition were most obvious for the three
mouthrinses used. The occurrence of bacterial strains corresponding with the
reference strains varies greatly within volunteers using the control paste and after
the use of the different products. Antibacterial toothpastes and mouthrinses
influence the bacterial composition of oral biofilms compared to biofilms collected
after brushing with a control toothpaste without antibacterial claims.

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 Microbial composition of oral biofilm

Introduction

The oral cavity, and oral biofilm in particular, hosts at an estimate, between 400
and 1000 bacterial species1. The bacterial composition of the oral biofilm at
different places and points in time is not always the same due to internal and
external influences. The bacterial composition varies between newly formed or 7
days old oral biofilms2 and between persons with or without oral diseases like
periodontitis2;3 and caries4. But also external influences like dental flossing5,
smoking6, drinking habits7, wearing fixed orthodontic appliances8 and tongue
piercings9 can influence the oral microbiome. Literature indicates that both
mechanical5;10 and chemical11;12 influences resulted in compositional changes in the
biofilm.
Different techniques exist to determine and compare the composition of oral
biofilms. One method is to measure the number of colony forming units in oral
biofilm or plaque samples taken from volunteers13. Unfortunately, it is believed
that half of the oral bacterial species cannot be cultured14. A powerful method to
include a larger variety of strains and species in the evaluation, is denaturing
gradient gel electrophoresis (DGGE)3;15. 16S rRNA gene fragments can naturally
be found in all bacteria. Although the 16S rRNA genes between different bacteria
differ, the base sequences in the beginning and end of all different genes are equal.
An universal primer can be used to isolate the gene fragment, after which
Polymerase Chain Reaction (PCR), a technique to amplify DNA from the original
plaque samples, is applied to generate copies of the original DNA-sequence to
perform DGGE. DGGE results in a pattern of bands, based on the electrophoretic
mobility of the DNA molecules15, on a polyacrylamide gel where each band
corresponds to a predominant member of the oral microbiome3.
Current mechanical cleaning methods can remove oral biofilm, although never
completely and are not targeted toward the removal of specific cariogenic or
periodontogenic pathogens. Therewith, also the composition of oral biofilm might

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Chapter 5

become more remote from what is generally called the oral microbiome at
health, although the exact composition of the oral microbiome at health is also not
well known. Antibacterial agents, added to toothpastes and mouthrinses, are
generally accepted to aid in the control of oral biofilms16;17, therewith preventing
the development of oral diseases like caries or periodontal diseases. At the same
time it is known, that different strains and species may be more or less susceptible
to these antibacterial agents and it is likely that their use will affect the composition
of oral biofilm.
Consequently, the aim of this study was to examine the microbial similarity
between oral biofilms collected from human volunteers after using different
antibacterial toothpastes and the additional use of antibacterial mouthrinses, as
compared to a control toothpaste without antibacterial claims using PCR-DGGE.

Material and methods

Selection of volunteers and products

Volunteers are healthy dentistry and oral hygiene students (7 males, 10 females,
age 19-25 years). For this study, three antibacterial mouthrinses, three antibacterial
toothpastes and one control toothpaste without antibacterial claims were used, as
listed in Table 1, together with their main active components. All products were
commercially purchased.

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 Microbial composition of oral biofilm

Table 1. Toothpastes and mouthrinses used in this study, together with their main active
components and manufacturer.
Mouthrinse Main active components Manufacturer

Listerine Alcohol Pfizer Consumer

(List) Phenols and essential oils Healthcare, Morris Plains,
NJ, USA

Meridol Amine fluoride GABA Group, Basel,

(Mer) Stannous fluoride Switzerland

Crest Pro Health Cetylpyridinium chloride Procter & Gamble,

mouthrinse Cincinnati, USA
(CPHm)

Toothpaste

Prodent Coolmint Sodium fluoride Sara Lee Household &

(control) Sodium Lauryl Sulphate Bodycare, Exton, USA.
(SLS)

Colgate Total Triclosan Colgate-Palmolive

(CT) Polyvinyl methylether Company,
maleic acid Piscataway, USA
Sodium fluoride
SLS

Zendium Classic Sodium fluoride Sara Lee Household &

(ZC) Colostrum Bodycare, Exton, USA
Lactoperoxidase
Lysozyme
Glucose oxidase
Amyloglucosidase

Crest Pro Health Stannous fluoride Procter & Gamble,

toothpaste Sodium Cincinnati, USA
(CPHt) hexametaphosphate
SLS

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Chapter 5

Experimental protocol
Volunteers brushed their teeth for 2 weeks with the control toothpaste. After those
2 weeks, teeth were brushed with an antibacterial toothpaste or the control
toothpaste with or without additional use of an antibacterial mouthrinse.
Mechanical cleaning, consisting of brushing and interdental cleaning, was done
twice a day according to the habitual routine of the volunteers. Rinsing was done
for 30 s with the appropriate amount of mouthrinse as recommended by the
manufacturer, immediately after brushing. After 2 weeks of using either the control
toothpaste, the antibacterial toothpaste or the control toothpaste supplemented with
an antibacterial mouthrinse, oral biofilm was collected 6 h or 12 h after the last
morning brushing or brushing and rinsing (see Collection of oral biofilm).

Collection of oral biofilm

Oral biofilm was collected from the buccal, lingual, palatal and interproximal sides
of the dentition with a sterile cotton swab stick and a dental instrument (Implant
Deplaquer, KerrHawe, Switzerland)18. The biofilm collected was suspended in 2 ml
sterile Reduced Transport Fluid (RTF)19. To suspend bacterial clumps, all
individual biofilm samples were vortexed and sonicated for 10 s at 30 W (Vibra
Cell model 375, Sonics and Materials Inc., Danbury, CT, USA). 1.5 ml fluid was
collected in an Eppendorf cup (Eppendorf, 1.5 ml, Hamburg, Germany) and
centrifuged at 20,000 g for 5 min at 10C (Eppendorf Centrifuge 5417R, Hamburg,
Germany). Next the supernatant was removed and 0.5 ml TE buffer (10 mM Tris-
HCL, pH 7.5, 1 mM EDTA) was added to the pellet in the Eppendorf cup. All
samples were stored at -20C.

DNA isolation and extraction

The 0.5 ml samples were used for the extraction of DNA. After thawing, the
samples were centrifuged for 5 min at 13,000 g (Eppendorf Centrifuge 5415D,

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 Microbial composition of oral biofilm

Hamburg, Germany) and subsequently washed and vortexed with 200 l TE, again
followed by centrifuging for 5 min at 13,000 g. Next, the supernatant was removed
and the pellet was subsequently placed in a microwave (500 W, 5 min), after which
it was suspended in 50 l TE. The samples were vortexed and placed on ice. The
quality and quantity of DNA samples were measured with a NanoDrop
spectrophotometer (ND-1000, NanoDrop Technologies, Inc, Wilmington, DE,
USA) at 230 nm. The final concentration of each DNA sample was adjusted to 100
ng DNA for all PCR amplifications.

PCR amplification
PCR was performed with a Tgradient thermocycler (Bio-rad I-cycler, GENO-
tronics BV, USA). For amplification of the 16S rRNA gene, the following bacterial
primers were used: F357-GC (forward primer, 5-GC clamp-
TACGGGAGGCAGCAG-3)20 containing a GC clamp (5-
21
CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-3) to make
it suitable for DGGE, and R-518 (reverse primer, 5-ATTACCGCGGCTGCTGG-
3)22. 25 l of each PCR mixture contained 12.5 l PCR Master Mix (0.05 units/l
Taq DNA polymerase in reaction buffer, 4 mM MgCl2, 0.4 mM dATP, 0.4 mM
dCTP, 0.4 mM dGTP, 0.6 mM dTTP (Fermentas Life Sciences)), 1 l of both
forward and reverse primer (1 M), and 100 ng DNA (in a volume of 10.5 l, see
DNA isolation and extraction). The temperature profile included an additional
denaturing step of 5 min at 94C, followed by a denaturing step at 94C for 45 s, a
primer annealing step at 58C for 45 s, an extension step at 72C for 1 min and a
final extension step of 72C for 5 min. PCR products were analyzed by
electrophoresis on a 2.0% agarose gel containing 0.5 g/ml ethidium bromide.

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Chapter 5

DGGE
DGGE of PCR products generated with the F357-GC / R-518 primer set was
performed as described by Muyzer et al.15, by using system PhorU (INGENY,
Goes, The Netherlands). The PCR products were applied on 8% (w/v)
polyacrylamide gel in 0.5 X TAE buffer (20 mM Tris acetate, 10 mM sodium
acetate, 0.5 mM EDTA, pH 8.3). The denaturing gradient consisted of 30 to 80%
denaturant (100% denaturant equals 7 M urea and 37% formamide). Gels were
poured using a gradient mixer. A 10 ml stacking gel without denaturant was added
on top. Electrophoresis was performed overnight at 120 V and 60C. Gels were
stained with silver nitrate21.

Similarity analysis of microbial profiles by DGGE

Each DGGE gel was normalized according to a marker consisting of 8 reference
species, and stored at 4C. The reference strains were Streptococcus sobrinus
American Type Culture Collection (ATCC) 33478, Streptococcus sanguinis ATCC
10556, Streptococcus oralis ATCC 35037, Streptococcus mitis ATCC 9811,
Streptococcus salivarius HB, Streptococcus mutans ATCC 10449, Lactobacillus
sp, Actinomyces naeslundii ATCC 51655. The reference strains consisted of
common bacterial species associated with oral health and disease22;23. DGGE gel
images were converted and transferred into a microbial database with GelCompar
II, version 6.1 (Applied Maths). The similarities in microbial diversity between the
control band profiles, i.e. biofilm samples collected after using a control toothpaste,
and the antimicrobial band profile, i.e. samples collected after using an
antimicrobial product for each volunteer were analyzed using a band based
similarity coefficient. The clustering algorithm to calculate the dendograms was a
non-weighted pair group method with arithmetic averages7. Next, the occurrence of
strains in the oral biofilm corresponding with the reference strains, collected after

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 Microbial composition of oral biofilm

the use of antibacterial health care products as compared with the control
toothpaste, was determined.

Results

Figure 1 shows a representative DGGE profile of oral biofilms from volunteers

after using the different toothpastes and mouthrinses included. Some profiles of
biofilm samples showed more distinct bands than others, on the basis of which a
mean similarity between the profiles was calculated.

Prodent Coolmint toothpaste (control)

Zendium Classic toothpaste

Colgate Total toothpaste

Crest Pro Health toothpaste

Listerine mouthrinse

Crest Pro Health mouthrinse

Meridol mouthrinse

Figure 1. DGGE profile of PCR-amplified bacterial 16S rRNA gene segments. The DGGE
gels were obtained from oral biofilm samples collected from different volunteers after 2
weeks of using a particular toothpaste or a control toothpaste without antibacterial claims
(Prodent Coolmint) supplemented with the use of a mouthrinse.

Tables 2 and 3 show the mean similarity between the control biofilm and the oral
biofilm collected after the use of antibacterial health care products. In general, the
use of a mouthrinse in addition to the control toothpaste gave larger dissimilarities
in biofilm composition than the use of antibacterial toothpastes alone. The
dissimilarity in bacterial composition with respect to the control toothpaste was
largest after the use of Crest Pro Health (Table 2). Interestingly, the dissimilarity

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Chapter 5

after use of Crest Pro Health decreased between 6 and 12 h after the last use, while
for Colgate Total the dissimilarity increased over time. The dissimilarities in
biofilm composition after use of a control toothpaste supplemented with the use of
an antibacterial mouthrinse (Table 3), did not differ significantly and ranged
between 59% to 63%.

Table 2.. Similarities based on DGGE analysis in microbial composition between control
biofilms, obtained during use of Prodent Coolmint and collected during the use of an
antibacterial toothpaste. Volunteers (n = 6) brushed for 2 weeks with a control toothpaste,
followed by 2 weeks of brushing with an antibacterial toothpaste. Values are presented as
averages standard deviations for plaques collected 6 and 12 h after the last brushing.

Collection Similarity (%)

Time
Crest Pro Health Zendium Classic Colgate Total
toothpaste toothpaste toothpaste

6h 60 14 75 7 77 7
12h 67 15 71 8 67 6

Table 3.. Similarities based on DGGE analysis in microbial composition between control
biofilms, obtained during use of Prodent Coolmint and collected during the additional use
of an antibacterial mouthrinse. Volunteers (n = 5) brushed for two weeks with a control
toothpaste, followed by 2 weeks of using an antibacterial mouthrinse in addition to
brushing with a control toothpaste. Values are presented as averages standard deviations
for plaques collected 6 h after the last brushing over 5 experiments.

Collection Similarity (%)

Time
Crest Pro Health Listerine Meridol
mouthrinse Mouthrinse mouthrinse

6h 63 14 59 21 59 14

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 Microbial composition of oral biofilm

Tables 4a and 4b summarizes the occurrence of strains in the oral biofilm

corresponding with the reference strains, collected after the use of antibacterial
health care products as compared with the control toothpaste. Note that due to
migration of bands from S. oralis and S. mitis to the same location, no distinction
could be made between their occurrence. The occurrence of strains corresponding
with the reference strains varies greatly within volunteers using the control paste
and after the use of the different products. However, due to the small group sizes, it
is impossible to draw solid conclusions about the occurrence of certain strains prior
to and after the use of a product. Within the limitations of the study, it can be seen
from Table 4a, that the supplementary use of Crest Pro Health mouthrinse
eliminated S. mutans in all three volunteers carrying this strain prior to the use of
the mouthrinse. As another interesting trend, lactobacilli appeared in three
volunteers after the use of the Meridol mouthrinse, whereas before its use these
lactobacilli were not detectable.

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Table 4a.. Occurrence of bacterial reference strains in oral biofilm obtained after the use of
Prodent Coolmint (control) and collected during the additional use of an antibacterial
mouthrinse. Volunteers (n = 5) brushed for two weeks with a control toothpaste, followed
by 2 weeks of using an antibacterial mouthrinse in addition to brushing with a control
toothpaste.
Bacterial Mouthrinses
Marker
Species
Control CPHm Control List Control Mer
Lactobacillus
Sp 1/5 1/5 0/5 1/5 0/5 3/5

S. mitis
ATCC 9811 /
S. oralis 2/5 1/5 1/5 1/5 5/5 5/5
ATCC 35037

S. sanguis
ATCC 10556 1/5 0/5 4/5 4/5 4/5 3/5

S.salivarius
HB 3/5 3/5 0/5 0/5 5/5 4/5

S. sobrinus
ATCC 33478 1/5 1/5 0/5 1/5 1/5 2/5

S. mutans
ATCC 10449 3/5 0/5 0/5 0/5 0/5 0/5

A. naeslundii
ATCC 51655 0/5 0/5 0/5 0/5 0/5 0/5

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 Table 4b.. Occurrence of bacterial reference strains in oral biofilm obtained after the use of Prodent Coolmint and collected during the use of an
antibacterial toothpaste. Volunteers (n = 6) brushed for two weeks with a control toothpaste, followed by 2 weeks of using an antibacterial
toothpaste.
Bacterial Toothpastes
Marker Species 6h 12h
Control CPHt Control ZC Control CT Control CPHt Control ZC Control CT
Lactobacillus
Sp 0/6 0/6 1/6 2/6 3/6 3/6 1/6 1/6 0/6 0/6 0/6 1/6

S. mitis ATCC
9811/
S. oralis ATCC 1/6 1/6 1/6 2/6 1/6 1/6 5/6 6/6 6/6 6/6 6/6 5/6
35037

S. sanguis
ATCC 10556 1/6 0/6 4/6 4/6 1/6 1/6 5/6 5/6 6/6 6/6 6/6 5/6

S.salivarius
HB 0/6 0/6 2/6 2/6 3/6 3/6 4/6 4/6 2/6 4/6 2/6 2/6

S. sobrinus
ATCC 33478 0/6 0/6 1/6 2/6 1/6 0/6 0/6 2/6 3/6 2/6 2/6 3/6

S. mutans
ATCC 10449 1/6 1/6 1/6 1/6 0/6 0/6 5/6 6/6 6/6 6/6 1/6 0/6

A. naeslundii
ATCC 51655 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6
Chapter 5

Discussion

DGGE profiling of the bacterial composition of oral biofilms of volunteers using

different antibacterial oral health care products resulted in different compositions
of the oral microbiome, although it is difficult to establish whether the changes
observed indicate a shift toward a more healthy microbiome or not. Recently, in a
study performed by Zaura et al.24 using next generation sequencing, it was found
that healthy individuals have around 1660 bacterial sequences in common,
suggesting that there is a core microbiome at health. The oral microbiome at
health has been studied and mapped by Aas et al.14, concluding that in general the
flora is highly diverse, site and subject specific. Therefore, our design where the
biofilm was collected from the same volunteers and identical sites in the oral cavity
is valuable for studying the effect of different antibacterial health care products.

DGGE
Several studies have shown that DGGE is a powerful tool to study the oral
microbiome3;15 and compositional changes as a result of external factors, like the
use of chlorhexidine mouthrinses11;12. Besides advantages, there are also some
limitations of DGGE. The presence of multiple bands from one single bacterial
species on the gel21 or the migration of bands from different species to the same
location on the gel15;21, as also occurred in our study for S. mitis and S. oralis,
complicate the interpretation of the DGGE profile of biofilm samples. Since also
non-viable microorganisms are part of the oral biofilm and accordingly can be
detected by DGGE, it may furthermore complicate monitoring dynamic changes in
the oral biofilm12. Therefore, the data represented in this study should be
interpreted as being a supplement to other parameters like colony forming units,
clinical plaque and gingivitis parameters and Minimal Inhibitory Concentration and
Minimal Bactericidal Concentration values against the antibacterial components in
a product.

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 Microbial composition of oral biofilm

Effects of toothpastes and mouthrinses

All antibacterial toothpastes and mouthrinses used in this study influenced the
bacterial composition compared to the control toothpaste, leading to similarity
values of at the most 77% (see Table 2 and 3). The differences in compositional
similarity of the different experimental biofilms with respect to the control biofilm
suggest, that the antibacterial components in the different pastes influenced the
bacterial composition of the biofilm in different ways. From both in vitro and in
vivo studies is known that both Crest Pro Health and Colgate Total toothpastes18;25
and mouthrinses Listerine, Meridol and Crest Pro Health26-28 have antibacterial
efficacies. Interestingly, the dissimilarity between control plaques and plaques
collected after either 6 h or 12 h after the use of the toothpastes Crest Pro Health
and Colgate Total is largest, when the viability of the biofilm is lowest (compare
with Table 2, Chapter 4).

Conclusion

In summary, we observed a significant variation in DGGE profiles of oral biofilms

in volunteers after the use of different antibacterial oral health care products. The
diversity in the bacterial composition of experimental biofilms compared to the
control was most obvious in volunteers using one of the antibacterial mouthrinses
in addition to a control paste. Remarkably, the time after which the samples were
collected influenced the compositional similarity for antibacterial toothpastes Crest
Pro Health and Colgate Total.
The results of the present study justify further investigation in larger groups of
volunteers

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Chapter 5

Acknowledgements

The authors like to thank J. Atema-Smit and G.I. Geertsema-Doornbusch for their
assistance with the PCR-DGGE.

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 Microbial composition of oral biofilm

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