**6. The role of antimicrobials in the future**

A better understanding of bacterial communities found in biofilms, such as its diversity and interactions among cells, provides opportunities for new methods to control biofilm formation (Wade, 2010). It has been shown that blocking communication mechanisms between cells in biofilms (quorum-sensing) can partially restore their susceptibility to antimicrobial agents (Bjarnsholt et al., 2005). Other benefits may include reduction of pathogenic microorganisms due to reduction in the virulence mechanism in the microorganism of interest. In the particular case of dental caries, blocking or reducing the activity of glycosyltransferase in *S. mutans* would be interesting, since these enzymes are implicated in the ability of this cariogenic bacterium.

In addition, probiotic approaches for oral use are being developed: such as the development of s *Lactobacillus paracasei* trains which maintain its co-aggregation activity with *S. mutans*  even when dead (Lang et al., 2010), or *Lactobacillus reuteri* strains that are able to reduce the number of *S. mutans* in the mouth (Caglar et al., 2008) in order to decrease the incidence of tooth decay. Other bacterium normally found in the mouth, and important in this sense, is *Streptococcus salivarius*, which produces a bacteriocin that inhibits anaerobic Gram-negative bacteria and that in vivo was shown also to reduce the level of halitosis (Burton et al., 2006).

In conclusion, microbiota analysis methods independent of culture have allowed to understanding the diversity of the oral microbiota. So far, most studies have focused on the microbiota composition in the disease, but a better understanding of this microflora in health is required and also as probiotic organisms are capable of restoring and maintaining health in such environment. Thus, future studies are still needed, especially for analyzing the interactions between species and how to use this knowledge to develop new products for prevention and treatment of oral diseases.

#### **7. References**

214 Contemporary Approach to Dental Caries

Since tooth decay is an infection, it would be logical to treat the disease with antibiotics or antimicrobials, such as antimicrobial peptides. However, most of these agents are not selective, have broad spectrum of action on the microorganisms such as chlorhexidine, iodopovidine, fluoride, penicillin or other antimicrobial/antibiotics. Importantly, the agents described above does not sterilize the oral cavity, since it is exposed to the external environment where there are many microbes, it is not a sterile space. Thus, the use of broadspectrum agents for treating dental caries can suppress the infection, but will never eliminate it entirely (Luoma et al., 1978). In this context, due to the limitations of traditional strategies in the management of dental caries, a "probiotic" approach of the disease is necessary. The term "probiotic" used here means that mechanisms are used to selectively remove only the pathogen responsible for disease in an attempt to keep the oral ecosystem intact. Most efforts in this sense are derived from studies that have attempted to genetically modify strains of *Streptococcus mutans*, turning them into strains that in addition to not producing acids, still competing for the same ecological niche that wild strain of *S. mutans* 

In theory and experimentally in laboratory animals, when this substitute organism is introduced, it completely shifts the wild *S. mutans* causing the disease. This action stops the decay process and also prevents the re-emergence of disease-causing organisms, eliminating the possibility of re-infection, since the "normal microbiota is complete." Another way to remove pathogens is developing specific antimicrobials for certain targets (Eckert et al., 2006). The basic principle is developing a cheap molecule that targets only the organism of

In the case of the oral cavity and tooth decay, this system is attractive from the perspective of eliminating all pathogens, thus preventing the re-growth of the original infection. There are also laboratory and clinical evidence demonstrating that when the biofilm's bacterial ecosystem is free of *S. mutans*, this bacterium finds it difficult to be reintroduced due to competitive inhibition with other microorganisms (Keene & Shklair, 1974; Shi, 2005). A criticism to probiotic approaches is that they target only one of the pathogens involved with the disease, being not directed at other pathogens that may be involved with the beginning

A better understanding of bacterial communities found in biofilms, such as its diversity and interactions among cells, provides opportunities for new methods to control biofilm formation (Wade, 2010). It has been shown that blocking communication mechanisms between cells in biofilms (quorum-sensing) can partially restore their susceptibility to antimicrobial agents (Bjarnsholt et al., 2005). Other benefits may include reduction of pathogenic microorganisms due to reduction in the virulence mechanism in the microorganism of interest. In the particular case of dental caries, blocking or reducing the activity of glycosyltransferase in *S. mutans* would be interesting, since these enzymes are

In addition, probiotic approaches for oral use are being developed: such as the development of s *Lactobacillus paracasei* trains which maintain its co-aggregation activity with *S. mutans*  even when dead (Lang et al., 2010), or *Lactobacillus reuteri* strains that are able to reduce the

interest, in this case *S. mutans, S sobrinus*, or other pathogens.

of the process, as the case of dental caries.

**6. The role of antimicrobials in the future** 

implicated in the ability of this cariogenic bacterium.

(Hillman, 2002).


Microbial Dynamics and Caries: The Role of Antimicrobials 217

Hense BA et al. (2007). Does efficiency sensing unify diffusion and quorum sensing? *Nat Rev* 

Herles S, Olsen S, Afflitto J, Gaffar A. (1994). Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents. *J Dent Res*, Vol. 73, No. 11, pp. 1748-55. Hillman JD. (2002). Genetically modied *Streptococcus mutans* for the prevention of dental

Hillman JD, Brooks TA, Michalek SM, Harmon CC, Snoep JL, van Der Weijden CC (2000).

replacement therapy of dental caries. *Infect Immun,* Vol. 68, pp. 543-549. Huang Y, Hajishengallis G, Michalek SM (2001). Induction of protective immunity against

Construction and characterization of an effector strain of *Streptococcus mutans* for

*Streptococcus mutans* colonization after mucosal immunization with attenuated *Salmonella enterica* serovar typhimurium expressing an *S. mutans* adhesin under the control of in vivo-inducible nirB promoter. *Infect Immun,* Vol. 69, pp. 2154-2161. Kazemzadeh-Narbat M et al. (2010). Antimicrobial peptides on calcium phosphate-coated

titanium for the prevention of implant-associated infections. *Biomaterials,* Vol. 31,

development of carious lesions in initially caries free recruits. *J Dent Res*, Vol. 53,

coadhesion in oral biofilms, In: *Community Structure and Co-Operation in Biofilms*, Allison, DG, Gilbert, P, Lappin- Scott, HM, Wilson, M, pp. 65–85, Society for General Microbiology Symposium 59, Cambridge University Press, Cambridge. Kolenbrander, PE, Palmer, RJ. (2004). Human Oral Bacterial Biofilms, In: *Microbial Biofilms*,

Ghannoum, M, O'Toole, G, pp. 85-117, American Society for Microbiology,

Communication among oral bacteria. *Microbiol Mol Biol Rev*, Vol. 66, No. 3, pp. 486-

damage in micro-organisms. *Biochemical and Biophysical Research Communications*,

In: *Oral Bacterial Ecology: The Molecular Basis*, Kuramitsu HK, Ellen RP, pp. 131–168,

and the oropharyngeal ecosystem of tube-fed patients. *Emerg Infect Dis*, Vol. 9, No.

Keene HJ, Shklair IL. (1974). Relationship of *Streptococcus mutans* carrier status to the

Koga T, Oho T, Shimazaki Y, Nakano Y (2002). Immunization against dental caries. *Vaccine,* 

Kolenbrander, PE, Andersen, RN, Kazmerak, KM, Palmer, RJ. (2000). Coaggregation and

Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJ, Jr. (2002).

Lambert PA, Hammond SM. (1973). Potassium uxes. First indications of membrane

Lamont, RJ, Jenkinson HF. (2000). Adhesion as an ecological determinant in the oral cavity,

Lang C, Bottner M, Holz C, Veen M, Ryser M, Reindl A et al. (2010). Specic *Lactobacillus* / *Streptococcus mutans* coaggregation. *Journal of Dental Research*, Vol. 33, in press. Larsen T, Fiehn NE (1995). Development of a flow method for susceptibility testing of oral

Leibovitz A, Dan M, Zinger J, Carmeli Y, Habot B, Segal R. (2003). Pseudomonas aeruginosa

Li YH et al. (2001). Natural genetic transformation of *Streptococcus mutans* growing in

caries. *Antonie Van Leeuwenhoek*, Vol. 82, pp. 361-366.

*Microbiol,* Vol*.* 5, pp. 230–39.

pp. 9519–9526.

Vol. 20, pp. 2027-2044.

pp. 1295.

Washington.

8, pp. 956-9.

Vol. 54, pp. 796–799.

Horizon Scientific Press, Wymondham.

biofilms. *J Bacteriol*, Vol. 183, pp. 897–908.

biofilms in vitro. *APMIS*, Vol. 103, No. 5, pp. 339-44.

505.


Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. (2006). A preliminary study of the

Caglar E, Kuscu OO, Cildir SK, Kuvvetli SS, Sandalli N. (2008). A probiotic lozenge

Clancy KA, Pearson S, Bowen WH, Burne RA (2000). Characterization of recombinant,

Cummins D (199lb). Zinc citrate/Triclosan: a new antiplaque system for the control of

Cummins D, Creeth JE. (1992). Delivery of Antiplaque Agents from Dentifrices, Gels, and

Cvitkovitch DG et al. (2003). Quorum sensing and biofilm formation in streptococcal

Easton DM et al. (2009). Potential of immunomodulatory host defense peptides as novel

Eckert R, Qi F, Yarbrough k, He J, Anderson MH, Shi W. (2006). Adding selectivity to

Gibbons RJ, van Houte J. (1973). On the formation of dental plaques. *J Periodontol*, Vol. 44,

Gilbert P, Moore LE. (2005). Cationic antiseptics: diversity of action under a common

Guggenheim B, Guggenheim M, Gmur R, Giertsen E, Thurnheer T. (2004). Application of

Hamill P et al. (2008). Novel anti-infectives: is host defence the answer? *Curr Opin Biotechnol*,

Hancock RE (1999). Host defence (cationic) peptides: what is their future clinical potential?

Hancock RE, Sahl HG. (2006). Antimicrobial and host-defense peptides as new anti-infective

therapeutic strategies. *Nat Biotechnol*, Vol. 24, pp. 1551–1557.

the Zurich biofilm model to problems of cariology. *Caries Res*, Vol. 38, No. 3, pp.

antimicrobial peptides: Rational design of a multi-domain peptide against Pseudomonas spp. *Antimicrobial Agents Chemother,* Vol. 50, No. 4, pp. 1480-1488. Filoche SK, Zhu M, Wu CD. (2004). In situ biofilm formation by multi-species oral bacteria under flowing and anaerobic conditions. *J Dent Res*, Vol. 83, No. 10, pp. 802-6. Fuqua WC et al. (1994). Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators. *J Bacteriol, Vol.* 176, pp. 269-275. Gardy JL et al. (2009). Enabling a systems biology approach to immunology: focus on innate

the customized microniche. *J Bacteriol*, Vol. 176, No. 8, pp. 2137-42.

*of Applied Microbiology*, Vol. 100, No. 4, pp. 754−64.

studies. *J Clin Periodontol*, Vol. 18, pp. 455-461.

infections. *J Clin Investig*, Vol. 112, p.1626– 1632.

immunity. *Trends Immunol,* Vol. 30, pp. 249–262.

epithet. *J Appl Microbiol*, Vol. 99, pp. 703-715.

No. 6, pp. 347-60.

Vol. 19, pp. 628–636.

*Drugs,* Vol. 57, pp. 469-473.

212-22.

Mouthwashes. *J Dent Res*, Vol. 71, No. 7, pp. 1439-1449.

anti-infectives. *Trends Biotechnol,* Vol. 27, pp. 582–590.

Demegen Pharmaceuticals. (2010). *Demegen Pharmaceticals Candidiasis Wedsite*.

*Dent Res*, Vol. 95, No. 2, pp. 151-8.

effect of probiotic *Streptococcus salivarius* K12 on oral malodour parameters. *Journal* 

administered medical device and its effect on salivary mutans streptococci and lactobacilli. *International Journal of Paediatric Dentistry,* Vol. 18. No. 1, pp. 35−9. Christersson CE, Fornalik MS, Baier RE, Glantz PO. (1987). In vitro attachment of oral

microorganisms to solid surfaces: evaluation of a controlled flow method. *Scand J* 

ureolytic *Streptococcus mutans* demonstrates an inverse relationship between dental plaque ureolytic capacity and cariogenicity. *Infect Immun,* Vol. 68, pp. 2621-2629. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. (1994). Biofilms,

plaque and the prevention ofgingivitis: short term clinical and mode of action


Microbial Dynamics and Caries: The Role of Antimicrobials 219

Palmer RJ, Jr. (1999). Microscopy flowcells: perfusion chambers for real-time study of

Payne DJ, Warren PV, Holmes DJ, Ji Y, Lonsdale JT (2001). Bacterial fatty-acid biosynthesis:

Petersen FC, Scheie AAa (1998). Chemical plaque control: a comparison of oral health care

Redfield RJ. (2002). Is quorum sensing a side effect of diffusion sensing? *Trends in* 

Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS. (2003). Bacterial

Rocha-Estrada J et al. (2010). The RNPP family of quorum-sensing proteins in Gram-positive

Rosan B, Lamont RJ. (2000). Dental plaque formation. *Microbes Infect*, Vol. 2, No. 13, pp.

Russell AD, Hugo WB. (1994). Antimicrobial activity and action of silver. *Progress in Medical* 

Shai Y. (2002). Mode of action of membrane active antimicrobial peptides. *Biopolymers,* Vol.

Shapiro JA. (1998). Thinking about bacterial populations as multicellular organisms. *Annual* 

Scheie AAa (2003). The role of antimicrobials. In: *Dental caries. The disease and its clinical* 

Sharma A, Honma K, Evans RT, Hruby DE, Genco RJ (2001). Oral immunization with

Sheie AA, Petersen FC. (2004). The biofilm concept: consequences for future prophylaxis of

Shi W. (2005). Oral biolm resistance to reinfection by *S. mutans*. In: Anderson M, ed.

Shimazaki Y, Mitoma M, Oho T, Nakano Y, Yamashita Y, Okano K, *et al.* (2001). Passive

Shiner EK et al. (2005). Inter-kingdom signaling: deciphering the language of acyl

Silver SD. (1967). Acridine dye action at cellular and molecular levels. *Experimental* 

Smith DJ (2002). Dental caries vaccines: prospects and concerns. *Crit Rev Oral Biol Med,* Vol.

Socransky S (2002). Dental biofilms: difficult therapeutic targets. *Periodontol 2000* 28:12-15.

oral diseases? *Crit Rev Oral Biol Med,* Vol. 15, No. 1, pp. 4-12.

homoserine lactones. *FEMS Microbiol Rev*, Vol. 29, pp. 935-947.

*management.* Fejerskov O, Kidd E, editors. Oxford: Blackwell Munksgaard, pp. 179-

recombinant *Streptococcus gordonii* expressing *Porphyromonas gingivalis* FimA

Selective removal of a specic microbe nearly precludes its reentry into the oral

immunization with milk produced from an immunized cow prevents oral recolonization by *Streptococcus mutans*. *Clin Diagn Lab Immunol,* Vol. 8, pp. 1136-

a genomics-driven target for antibacterial drug discovery. *Drug Discov Today,* Vol.

products. In: Oral biofilms and plaque control. Busscher HJ, Evans LV, editors.

coaggregation: an integral process in the development of multi-species biofilms.

biofilms. *Methods Enzymol*, Vol. 310, pp. 160-6.

*Microbiology*, Vol. 10, No. 8, pp. 365-370.

*Trends Microbiol*, Vol. 11, No. 2, pp. 94-100.

*Review of Microbiology*, Vol. 52, pp. 81–104.

domains. *Infect Immun,* Vol. 69, pp. 2928-2934.

biolm by competitive inhibition. Los Angeles.

*Chemotherapy*, Vol. 4, pp. 505–511.

*Chemistry*, Vol. 31, pp. 351–371.

Amsterdam: Harwood Academic Publisher, pp. 277-293.

bacteria. *Appl Microbiol Biotechnol,* Vol. 87, No. 3, pp. 913-923.

6, pp. 537-544.

1599-607.

189.

1139.

13, pp. 335-349.

66, pp. 236–248.


Luoma H et al. (1978). A simultaneous reduction of caries and gingivitis in a group of

Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, *et al.* (1998). Characterization

Mah TF, O'Toole GA. (2001). Mechanisms of biofilm resistance to antimicrobial agents.

Maillard J-Y. (2002). Bacterial target sites for biocide action. *Journal of Applied Microbiology*.

Marsh PD (1994). Microbial ecology of dental plaque and its significance in health and

Marsh, PD. (2000). Oral ecology and its impact on oral microbial diversity, In: *Oral Bacterial* 

Marsh PD. (2003). Plaque as a biofilm: pharmacological principles of drug delivery and

Marsh PD. (2004). Dental plaque as a microbial biofilm. *Caries Res*, Vol. 38. No. 3, pp. 204-11. Marsh PD. (2005). Dental plaque: biological significance of a biofilm and community life-

Marsh PD. (2010). Controlling the oral biofilm with antimicrobials. *Journal of Dentistry,* Vol.

Marsh PD, Bowden GHW. (2000). Microbial community interactions in biofilms. In: Allison

Marsh PD, Bradshaw DJ. (1995). Dental plaque as a biofilm. *J Ind Microbiol*, Vol. 15, No. 3,

Marsh PD, Martin MV. (2009). Oral Microbiology,5th edn. Edinburgh: Churchill

Melo MN, Dugourd D, Castanho MA. (2006). Omiganan pentahydrochloride in the front

Miller MB, Bassler BL. (2001). Quorum sensing in bacteria. *Annu rev microbiol*, Vol. 55, pp.

Nakamura K, Tamaoki T. (1968). Reversible dissociation of *Escherichia coli* ribosomes by hydrogen peroxide. *Biochemica and Biophysica Acta*, Vol. 161, 368–376. Nealson KH et al. (1970). Cellular control of the synthesis and activity of the bacterial

Nyvad B, Fejerskov O. (1989). Structure of dental plaque and the plaque-enamel interface in

Olsson J (1998). Inhibition of dental plaque by chemical surface modification. In: *Oral* 

*biofilms and plaque control.* Busscher HJ, Evans LV, editors. Amsterdam: Harwood

human experimental caries. *Caries Res*, Vol. 23, No. 3, pp. 151-8.

luminescent system. *J Bacteriol*, Vol. 104, pp. 313–322.

*Ecology: The Molecular Basis*, Kuramitsu HK, Ellen RP, pp. 11–65, Horizon Scientific

action in the sub- and supragingival environment. *Oral Dis*, Vol. 9, Suppl. 1, pp. 16-

DG et al. *Community Structure and Co-operation in Biofilms.* Society for General Microbiology Symposium Cambridge: Cambridge University Press, No. 59, pp.

line of clinical applications of antimicrobial peptides. *Recent Pat Antiinfect Drug* 

immunotherapy in humans. *Nat Med,* Vol. 4, pp. 601-606.

*Caries Res,* Vol. 12, pp. 290-298.

*Trends Microbiol*, Vol. 9, No. 1, pp. 34-9.

Symposium Supplement, Vol. 92, 16S–27S.

disease. *Adv Dent Res, Vol.* 8, pp. 263-271.

style. *J Clin Periodontol*, Vol. 32, Suppl 6, pp. 7-15.

Press, Wymondham.

38, S1; S11-S15.

167–198.

pp. 169-75.

Livingstone.

165-99.

*Discov*, Vol. 1, pp. 201–207.

Academic Publisher, pp. 295-309.

22.

schoolchildren receiving chlorhexidine-uoride applications. Results after 2 years.

of a recombinant plant monoclonal secretory antibody and preventive


**12** 

*Japan* 

**Inhibitory Effects of the Phytochemicals** 

Tsuneyuki Oku, Michiru Hashiguchi and Sadako Nakamura

**of** *Morus alba* **and** *Salacia* **Extracts** 

**on Dental Caries** 

*University of Nagasaki Siebold,* 

**Partially Hydrolyzed Alginate, Leaf Extracts** 

Many studies on natural materials with anticariogenic effects have been carried out. Anticariogenic materials, such as polyphenols from oolong tea (Nakahara et al, 1993) and polyphenols from cacao (Ito et al, 2003), are known to be inhibitors of glucosyltransferases (GTases). These compounds have been used in foods to prevent or reduce dental caries. Sugar alcohols and oligosaccharides, which are not utilized as the substrate of GTase, are known as alternative sweeteners to sucrose (Kawanabe et al, 1992; Makinen et al, 1995;

We investigated the potentiality of inhibitory effects of some phytochemicals on dental caries, because it is very interesting that phytochemical components inhibit the activity of not only GTase but also α-glucosidase. We have clarified that some phytochemicals such as partially decomposed alginate (Alg53), extractives from the leaves of *Morus alba* (ELM) and extractives from *Salacia chinensis* (ES) have inhibitory effects on disaccharidases such as

Alginate is a polyuronic saccharide that is isolated from the cell walls of a number of brown seaweed species around the world, and produced as an extracellular matrix by certain bacteria (Draget et al, 2003). It has a gelling ability, stabilizing properties and high viscosity. Alginate and its decomposed derivatives are widely used in foods, cosmetics and pharmaceutical industries (Ci et al, 1999; Johnson et al, 1997). Alginate hydrolysates exhibit many bioactivities, such as stimulating human keratinocytes, accelerating plant root growth and enhancing penicillin production from cultures of *Penicillium chrysogenum* (Ariyo et al, 1998; Kawada et al, 1997; Natsume et al, 1994). We have clarified that alginic acid with lowered molecule (mean molecular weight about 55,000) has suppressive effects on the elevation of blood glucose and insulin secretion (Oku et al, 1997) and improves defecation

*Morus alba* has traditionally been cultivated in China, Korea and Japan to use its leaves to feed silkworms. Recently, health benefits of *Morus alba* have been clarified and naturally occurring 1-deoxynojirimycin (DNJ) was isolated from its roots (Yagi et al, 1976). DNJ is glucose analogue with a secondary amine group instead of an oxygen atom in the pyranose

**1. Introduction** 

Ooshima et al, 1992; Van Loveren, 2004).

ant the fecal conditions (Oku et al, 1998).

maltase, sucrase and trehalase.

