University of Groningen

Oral Biofilm as a Reservoir for AntimicrobialsOtten, Marieke Petronella Theodora

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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

76

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.

Microbial composition of oral biofilm

77

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

Chapter 5

78

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.

Microbial composition of oral biofilm

79

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

(List)

Alcohol Phenols and essential oils

Pfizer Consumer Healthcare, Morris Plains, NJ, USA

Meridol®

(Mer) Amine fluoride Stannous fluoride

GABA Group, Basel, Switzerland

Crest Pro Health® mouthrinse (CPHm)

Cetylpyridinium chloride Procter & Gamble, Cincinnati, USA

Toothpaste Prodent Coolmint®

(control)

Sodium fluoride Sodium Lauryl Sulphate (SLS)

Sara Lee Household & Bodycare, Exton, USA.

Colgate Total®

(CT) Triclosan Polyvinyl methylether maleic acid Sodium fluoride SLS

Colgate-Palmolive Company, Piscataway, USA

Zendium Classic®

(ZC) Sodium fluoride Colostrum Lactoperoxidase Lysozyme Glucose oxidase Amyloglucosidase

Sara Lee Household & Bodycare, Exton, USA

Crest Pro Health® toothpaste (CPHt)

Stannous fluoride Sodium hexametaphosphate SLS

Procter & Gamble, Cincinnati, USA

Chapter 5

80

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 10°C (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 -20°C.

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,

Microbial composition of oral biofilm

81

Hamburg, Germany) and subsequently washed and vortexed with 200 μl 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 μl 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’-

CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-3’)21 to make

it suitable for DGGE, and R-518 (reverse primer, 5’-ATTACCGCGGCTGCTGG-

3’)22. 25 μl of each PCR mixture contained 12.5 μl PCR Master Mix (0.05 units/μl

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 μl of both

forward and reverse primer (1 μM), and 100 ng DNA (in a volume of 10.5 μl, see

“DNA isolation and extraction”). The temperature profile included an additional

denaturing step of 5 min at 94°C, followed by a denaturing step at 94°C for 45 s, a

primer annealing step at 58°C for 45 s, an extension step at 72°C for 1 min and a

final extension step of 72°C for 5 min. PCR products were analyzed by

electrophoresis on a 2.0% agarose gel containing 0.5 μg/ml ethidium bromide.

Chapter 5

82

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 60°C. 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 4°C. 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

Microbial composition of oral biofilm

83

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

Chapter 5

84

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 Time

Similarity (%)

Crest Pro Health toothpaste

Zendium Classic toothpaste

Colgate Total 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 Time

Similarity (%)

Crest Pro Health mouthrinse

Listerine Mouthrinse

Meridol mouthrinse

6h

63 ± 14

59 ± 21

59 ± 14

Microbial composition of oral biofilm

85

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.

Chapter 5

86

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 Marker Species

Mouthrinses

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 ATCC 35037

2/5

1/5

1/5 1/5

5/5

5/5

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

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.

Toothpastes 6h 12h

Bacterial Marker Species

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 35037

1/6

1/6

1/6

2/6

1/6

1/6

5/6

6/6

6/6

6/6

6/6

5/6

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

88

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.

Microbial composition of oral biofilm

89

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

Chapter 5

90

Acknowledgements

The authors like to thank J. Atema-Smit and G.I. Geertsema-Doornbusch for their

assistance with the PCR-DGGE.

Microbial composition of oral biofilm

91

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