Citation for published version (APA): Otten, M. P. T. (2011). Oral Biofilm as a Reservoir for Antimicrobials Groningen: University of Groningen

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1 University of Groningen Oral Biofilm as a Reservoir for Antimicrobials Otten, Marieke Petronella Theodora IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2011 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Otten, M. P. T. (2011). Oral Biofilm as a Reservoir for Antimicrobials Groningen: University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 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

3 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 PCRdenaturing 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. 76

4 Microbial composition of oral biofilm Introduction The oral cavity, and oral biofilm in particular, hosts at an estimate, between 400 and 1000 bacterial species 1. 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 biofilms 2 and between persons with or without oral diseases like periodontitis 2;3 and caries 4. But also external influences like dental flossing 5, smoking 6, drinking habits 7, wearing fixed orthodontic appliances 8 and tongue piercings 9 can influence the oral microbiome. Literature indicates that both mechanical 5;10 and chemical 11;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 volunteers 13. Unfortunately, it is believed that half of the oral bacterial species cannot be cultured 14. 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 molecules 15, on a polyacrylamide gel where each band corresponds to a predominant member of the oral microbiome 3. 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 77

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 biofilms 16;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 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. 78

6 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 (List) Meridol (Mer) Crest Pro Health mouthrinse (CPHm) Alcohol Phenols and essential oils Amine fluoride Stannous fluoride Cetylpyridinium chloride Pfizer Consumer Healthcare, Morris Plains, NJ, USA GABA Group, Basel, Switzerland Procter & Gamble, Cincinnati, USA Toothpaste Prodent Coolmint (control) Colgate Total (CT) Zendium Classic (ZC) Crest Pro Health toothpaste (CPHt) Sodium fluoride Sodium Lauryl Sulphate (SLS) Triclosan Polyvinyl methylether maleic acid Sodium fluoride SLS Sodium fluoride Colostrum Lactoperoxidase Lysozyme Glucose oxidase Amyloglucosidase Stannous fluoride Sodium hexametaphosphate SLS Sara Lee Household & Bodycare, Exton, USA. Colgate-Palmolive Company, Piscataway, USA Sara Lee Household & Bodycare, Exton, USA Procter & Gamble, Cincinnati, USA 79

7 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, 80

8 Microbial composition of oral biofilm 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, GENOtronics 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 ) μl of each PCR mixture contained 12.5 μl PCR Master Mix (0.05 units/μl Taq DNA polymerase in reaction buffer, 4 mm MgCl 2, 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. 81

9 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 nitrate 21. 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 The reference strains consisted of common bacterial species associated with oral health and disease 22;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 averages 7. Next, the occurrence of strains in the oral biofilm corresponding with the reference strains, collected after 82

10 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 83

11 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 Crest Pro Health toothpaste Similarity (%) Zendium Classic toothpaste Colgate Total toothpaste 6h 60 ± ± 7 77 ± 7 12h 67 ± ± 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 Crest Pro Health mouthrinse Similarity (%) Listerine Mouthrinse Meridol mouthrinse 6h 63 ± ± ± 14 84

12 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. 85

13 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 ATCC /5 1/5 1/5 1/5 5/5 5/5 S. sanguis ATCC /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 /5 1/5 0/5 1/5 1/5 2/5 S. mutans ATCC /5 0/5 0/5 0/5 0/5 0/5 A. naeslundii ATCC /5 0/5 0/5 0/5 0/5 0/5 86

14 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 /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 /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 /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 /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 /6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

15 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 microbiome 3;15 and compositional changes as a result of external factors, like the use of chlorhexidine mouthrinses 11;12. Besides advantages, there are also some limitations of DGGE. The presence of multiple bands from one single bacterial species on the gel 21 or the migration of bands from different species to the same location on the gel 15;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 biofilm 12. 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. 88

16 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 toothpastes 18;25 and mouthrinses Listerine, Meridol and Crest Pro Health 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 89

17 Acknowledgements The authors like to thank J. Atema-Smit and G.I. Geertsema-Doornbusch for their assistance with the PCR-DGGE. 90

18 Microbial composition of oral biofilm Reference List 1. Van der Weijden GA, Echeverria JJ, Sanz M et al. Mechanical Supragingival Plaque Control. In: Lindhe J, Lang NP, Karring T, eds. Clinical Periodontology and Implant Dentistry. Copenhagen: Blackwell Munskgaard; 2008: Haffajee AD, Socransky SS, Patel MR et al. Microbial complexes in supragingival plaque. Oral Microbiol. Immunol. 2008;23: Zijnge V, Harmsen HJ, Kleinfelder JW et al. Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients. Oral Microbiol. Immunol. 2003;18: Li Y, Ge Y, Saxena D et al. Genetic profiling of the oral microbiota associated with severe early-childhood caries. J. Clin. Microbiol. 2007;45: Corby PM, Biesbrock A, Bartizek R et al. Treatment outcomes of dental flossing in twins: molecular analysis of the interproximal microflora. J. Periodontol. 2008;79: Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. J. Clin. Periodontol. 2001;28: Signoretto C, Bianchi F, Burlacchini G et al. Drinking habits are associated with changes in the dental plaque microbial community. J. Clin. Microbiol. 2010;48: Van Gastel J, Quirynen M, Teughels W et al. Longitudinal changes in microbiology and clinical periodontal parameters after removal of fixed orthodontic appliances. Eur. J. Orthod. 2011;33: Ziebolz D, Hornecker E, Mausberg RF. Microbiological findings at tongue piercing sites: implications to oral health. Int. J. Dent. Hyg. 2009;7:

19 10. Haffajee AD, Teles RP, Socransky SS. The effect of periodontal therapy on the composition of the subgingival microbiota. Periodontol ;42: Sekino S, Ramberg P, Uzel NG et al. The effect of a chlorhexidine regimen on de novo plaque formation. J. Clin. Periodontol. 2004;31: McBain AJ, Bartolo RG, Catrenich CE et al. Effects of a chlorhexidine gluconate-containing mouthwash on the vitality and antimicrobial susceptibility of in vitro oral bacterial ecosystems. Appl. Environ. Microbiol. 2003;69: Lorenz K, Bruhn G, Netuschil L et al. How to select study designs and parameters to investigate the effect of mouthrinses? Part I: rationale and background. J. Physiol Pharmacol. 2009;60 Suppl 8: Aas JA, Paster BJ, Stokes LN et al. Defining the normal bacterial flora of the oral cavity. J. Clin. Microbiol. 2005;43: Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rrna. Appl. Environ. Microbiol. 1993;59: Addy M, Moran J. Chemical Supragingival Plaque Control. In: Lang NP, Lindhe J, eds. Clinical periodontology and implant dentistry. Vol 2. Oxford: Blackwell Munskgaard; 2008: White DJ, Barker ML, Klukowska M. In vivo antiplaque efficacy of combined antimicrobial dentifrice and rinse hygiene regimens. Am. J. Dent. 2008;21: Van der Mei HC, White DJ, Atema-Smit J et al. A method to study sustained antimicrobial activity of rinse and dentifrice components on biofilm viability in vivo. J. Clin. Periodontol. 2006;33: Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl. Microbiol. 1972;24:

20 Microbial composition of oral biofilm 20. Di Cagno R, Rizzello CG, Gagliardi F et al. Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease. Appl. Environ. Microbiol. 2009;75: Zijnge V, Welling GW, Degener JE et al. Denaturing gradient gel electrophoresis as a diagnostic tool in periodontal microbiology. J. Clin. Microbiol. 2006;44: Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv. Dent. Res. 1994;8: Marsh PD. Dental plaque as a biofilm and a microbial community - implications for health and disease. BMC. Oral Health 2006;6 Suppl 1:S Zaura E, Keijser BJ, Huse SM et al. Defining the healthy "core microbiome" of oral microbial communities. BMC. Microbiol. 2009;9: Scheie AA, Petersen FC. Antimicrobials in caries control. In: Fejerskov O, Kidd E, eds. Dental caries: The Disease and its Clinical Management. Oxford: Blackwell Munskgaard; 2008: Gunsolley JC. Clinical efficacy of antimicrobial mouthrinses. J. Dent. 2010;38 Suppl 1:S Pan PC, Harper S, Ricci-Nittel D et al. In-vitro evidence for efficacy of antimicrobial mouthrinses. J. Dent. 2010;38 Suppl 1:S16-S Otten MP, Busscher HJ, Van der Mei HC et al. Retention of antimicrobial activity in plaque and saliva following mouthrinse use in vivo. Caries Res. 2010;44:

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