**4.1 Oral microbiota as a source of probiotics**

The oral cavity is a complex habitat of a great diversity of microbial species.

Recently, it has been estimated that over 1000 bacterial species are present in it. The most commonly used probiotic bacterial strains belong to the genera Lactobacillus and Bifidobacteria. So, there is of special interest to realize whether such microbes naturally inhabit the oral cavity. In the oral cavity, lactobacilli usually comprise 1% of the total cultivable bacteria; commonly isolated species include *L .paracaseie*, *L.plantarum, L.rhamnosus, L.salivarius*. Bifidobacterial species isolated from oral samples include *B.bifidum, B.dentium and B.longum*.

A promising finding was that lactobacilli population differed in healthy and individuals with periodontal disease. In another study it is observed that healthy persons are populated by *L. gasseri*and *L. fermentum*, whereas the predominant species in periodontitis patients was *L. plantarum* while the first two were undetectable (Koll Kalis et al., 2005). Observations also showed that microorganisms with probiotic properties may really exist and inhabit in the oral cavity. Though, the complexity of biofilm development and interspecies interactions require more detailed investigations in order to state true probiotic candidates with activity in the oral cavity (Stamatova & Meurman, 2009).

### **4.2 Probiotics and resistance to oral defense mechanisms**

At first, ingested probiotics are exposed to saliva. During this first step of contact, survival and resistance to oral environmental factors are very important. Salivary proteins such as lysozyme, lactoferrine, salivary peroxidase, and secretory IgA can collectively affect viability or cell surface morphology of probiotic species. The adhesion and metabolic activity of them is then affected. Saliva role on microbial establishment can be contradictory. In one hand, saliva can inhibit colonization of probiotics (by growth inhibition, killing, or prevention of adherence to host tissues), and on the other hand, it can promote microbial colonization. It has been observed that, Lysozyme pretreatment could significantly reduce the adhesion of *L. rhamnosus* GG, *L. rhamnosus* Lc705 and *L. casei* Shirota. However, the adhesive properties of *L. johnsonii* La1 and *B. lactis* Bb12 remained unaffected. These results highlight the strainspecific response to proteolytic enzymes and this feature needs to be considered when selecting probiotics for the oral cavity.

Probiotics and the Reduction of Dental Caries Risk 279

From a view point, probiotics (lactobacilli) could hydrolyse proteins, stimulate growth of streptococci: the streptococci are acidogenic bacteria and produce low pH conditions in the oral environment (Robinson and Tamine, 1981). Also untreated caries cavities should also be questioned at this point. On the other hand, in recent studies, it was stated that probiotic might decrease the risk of the highest level of Streptococcus mutans (Ahola et al, 2002) or might increases salivary counts of lactobacilli while *S. mutans* levels were not modified

To have a beneficial effect in limiting or preventing dental caries, a probiotic must be able to adhere to dental surfaces and integrate into the bacterial communities making up the dental biofilm. Such a biofilm holds pathogens off oral tissues by filling a space which in future, could have served as a niche for pathogens, and it should also compete with and antagonize the cariogenic bacteria and thus prevent their proliferation (Caglar et al., 2005; Sheikh et al., 2011). According to our researches, it is cleared that the presence of Lactobacillus Sp. Such as *L. acidophilus DSM 20079, L. fermentum ATCC 9338* and *L.rhamnosus ATCC 7469* can cause reduction in the adherence of Streptococcal strains that it is probably related to interaction between bacteria. The mutans streptococci adherence reduction was significantly stronger in the case of *L. acidophilus* and *L. rhamnosus* while in the other study showed that *L. fermentum* reduced the adherence of non mutans Streptococci more than mutans Streptococci (figure 6). In general, Inoculation of probiotic strain before Streptoccocal isolates to in vitro system showed more effect on adherence reduction (about 25% reduction in adherence) with significant difference (Pvalue< 0.05) especially in the case of *L. rhamnosus*. It is thought that adhesion reduction is likely due to bacterial interactions and colonization of adhesion sites

**Production of antimicrobial substances** 

• Compete with pathogens for adhesion sites • Involvement in metabolism of substrates

• Modulate humoral and cellular immune response

• Modification of oxidation reduction potential

Table 3. Possible mechanisms of a probiotic in oral health

• Stimulate non specific immunity

(competing with oral micro organisms for substrates available)

• Organic acids • Hydrogen peroxide • carbon peroxide,

• diacetyl • Biosurfactants • Bacteriocins **Binding in Oral Cavity** 

**Immuno modulatory** 

**Modify oral conditions** 

(Montalto et al, 2004).

• Modulating pH

**4.4 Probiotics and dental caries** 

Other studies have also shown that lysozyme pretreatment of lactobacilli can slightly increase their adhesiveness to saliva coated surfaces. Lysozyme pretreatment could not significantly reduce the viability of lactobacilli but cell surface alterations might have contributed to the increased adhesion. Further studies on the mechanism whereby lysozyme affects adhesion are necessitated (Stamatova & Meurman, 2009).

Another aspect in oral establishment of probiotics is saliva-mediated aggregation.This ability is related to cell adherence properties. The adhesion mechanisms of lactobacilli involve hydrophobicity and surface charge, as well as specific carbohydrate and/or proteinaceous components. Organisms able to co-aggregate with other bacteria may have superior advantages over non-coaggregating organisms which are easily removed from the oral cavity. Recently, results have shown that *L. salivarius* was not able to form a biofilm in monoculture (in a microplate model), whereas when the species was added simultaneously with the inoculum of other commensal oral microorganisms, it established itself irrespective of pH. Similar findings were observed with *L. plantarum* SA-1 and *L. rhamnosus* that failed to form substantial biofilms in mono-culture but biofilm mass increased when cocultured with *A. naeslundii* (Filoche et al., 2004)*.* 

#### **4.3 Probiotics and oral health**

Several authors have suggested that probiotic bacteria could also be beneficial to oral health. Species of Lactobacillus and Bifidobacteria may exert beneficial effects in the oral cavity by inhibiting cariogenic Streptococci and Candida spp (Bhardwaj, 2010).

The mechanisms of probiotic action in the oral cavity could be similar to those described for the intestine. The mechanisms by which probiotics exert their effects are largely unknown, but may involve modifying pH, antagonizing pathogens through production of antimicrobial compounds, competing for pathogen binding and receptor sites, stimulating immune modulatory cells and producing lactase. It is also showed that they have influence to the immune system through several molecular mechanisms (Bhushan & Chachra, 2010).

To have a beneficial effect in oral cavity, a probiotic should have a tendency to form a biofilm that acts as a protective lining for oral tissues against oral diseases. Probiotics strains have been shown to vary broadly in their adhesiveness to saliva-coated HA and so in biofilm formation ability. Among probiotics strains *L. rhamnosus* GG exhibited the maximum values of adhesion, comparable to those of the early tooth colonizer *S. sanguinis*. Dairy starter *L. bulgaricus* strains adhered poorly to sHA.

Probiotic bacteria adhesion to oral soft tissues is another aspect that promotes their health effect to the host. Cell adhesion is a complex process involving contact between the bacterial cell and interaction with surfaces. The epithelial lining of the oral cavity despite its function as a physical barrier, actively participates in immune response. It has been shown that probiotic bacteria can stimulate local immunity and modulate the inflammatory response. Lactobacilli as well as other gram positive bacteria express ligands for toll-like receptors (TLRs) which initiate immune responses enabling detection of both pathogens and indigenous microbiota by epithelial cells. Recognition of commensal bacteria by these receptors (TLRs) is necessary for homeostasis, epithelial cells protection from injury and repair stimulation (Stamatova & Meurman, 2009).

#### **Production of antimicrobial substances**


278 Contemporary Approach to Dental Caries

Other studies have also shown that lysozyme pretreatment of lactobacilli can slightly increase their adhesiveness to saliva coated surfaces. Lysozyme pretreatment could not significantly reduce the viability of lactobacilli but cell surface alterations might have contributed to the increased adhesion. Further studies on the mechanism whereby lysozyme

Another aspect in oral establishment of probiotics is saliva-mediated aggregation.This ability is related to cell adherence properties. The adhesion mechanisms of lactobacilli involve hydrophobicity and surface charge, as well as specific carbohydrate and/or proteinaceous components. Organisms able to co-aggregate with other bacteria may have superior advantages over non-coaggregating organisms which are easily removed from the oral cavity. Recently, results have shown that *L. salivarius* was not able to form a biofilm in monoculture (in a microplate model), whereas when the species was added simultaneously with the inoculum of other commensal oral microorganisms, it established itself irrespective of pH. Similar findings were observed with *L. plantarum* SA-1 and *L. rhamnosus* that failed to form substantial biofilms in mono-culture but biofilm mass increased when cocultured with

Several authors have suggested that probiotic bacteria could also be beneficial to oral health. Species of Lactobacillus and Bifidobacteria may exert beneficial effects in the oral cavity by

The mechanisms of probiotic action in the oral cavity could be similar to those described for the intestine. The mechanisms by which probiotics exert their effects are largely unknown, but may involve modifying pH, antagonizing pathogens through production of antimicrobial compounds, competing for pathogen binding and receptor sites, stimulating immune modulatory cells and producing lactase. It is also showed that they have influence to the immune system through several molecular mechanisms (Bhushan & Chachra, 2010). To have a beneficial effect in oral cavity, a probiotic should have a tendency to form a biofilm that acts as a protective lining for oral tissues against oral diseases. Probiotics strains have been shown to vary broadly in their adhesiveness to saliva-coated HA and so in biofilm formation ability. Among probiotics strains *L. rhamnosus* GG exhibited the maximum values of adhesion, comparable to those of the early tooth colonizer *S. sanguinis*.

Probiotic bacteria adhesion to oral soft tissues is another aspect that promotes their health effect to the host. Cell adhesion is a complex process involving contact between the bacterial cell and interaction with surfaces. The epithelial lining of the oral cavity despite its function as a physical barrier, actively participates in immune response. It has been shown that probiotic bacteria can stimulate local immunity and modulate the inflammatory response. Lactobacilli as well as other gram positive bacteria express ligands for toll-like receptors (TLRs) which initiate immune responses enabling detection of both pathogens and indigenous microbiota by epithelial cells. Recognition of commensal bacteria by these receptors (TLRs) is necessary for homeostasis, epithelial cells protection from injury and

affects adhesion are necessitated (Stamatova & Meurman, 2009).

inhibiting cariogenic Streptococci and Candida spp (Bhardwaj, 2010).

Dairy starter *L. bulgaricus* strains adhered poorly to sHA.

repair stimulation (Stamatova & Meurman, 2009).

*A. naeslundii* (Filoche et al., 2004)*.* 

**4.3 Probiotics and oral health** 


#### **Binding in Oral Cavity**


#### **Immuno modulatory**


#### **Modify oral conditions**


Table 3. Possible mechanisms of a probiotic in oral health

#### **4.4 Probiotics and dental caries**

From a view point, probiotics (lactobacilli) could hydrolyse proteins, stimulate growth of streptococci: the streptococci are acidogenic bacteria and produce low pH conditions in the oral environment (Robinson and Tamine, 1981). Also untreated caries cavities should also be questioned at this point. On the other hand, in recent studies, it was stated that probiotic might decrease the risk of the highest level of Streptococcus mutans (Ahola et al, 2002) or might increases salivary counts of lactobacilli while *S. mutans* levels were not modified (Montalto et al, 2004).

To have a beneficial effect in limiting or preventing dental caries, a probiotic must be able to adhere to dental surfaces and integrate into the bacterial communities making up the dental biofilm. Such a biofilm holds pathogens off oral tissues by filling a space which in future, could have served as a niche for pathogens, and it should also compete with and antagonize the cariogenic bacteria and thus prevent their proliferation (Caglar et al., 2005; Sheikh et al., 2011). According to our researches, it is cleared that the presence of Lactobacillus Sp. Such as *L. acidophilus DSM 20079, L. fermentum ATCC 9338* and *L.rhamnosus ATCC 7469* can cause reduction in the adherence of Streptococcal strains that it is probably related to interaction between bacteria. The mutans streptococci adherence reduction was significantly stronger in the case of *L. acidophilus* and *L. rhamnosus* while in the other study showed that *L. fermentum* reduced the adherence of non mutans Streptococci more than mutans Streptococci (figure 6).

In general, Inoculation of probiotic strain before Streptoccocal isolates to in vitro system showed more effect on adherence reduction (about 25% reduction in adherence) with significant difference (Pvalue< 0.05) especially in the case of *L. rhamnosus*. It is thought that adhesion reduction is likely due to bacterial interactions and colonization of adhesion sites

Probiotics and the Reduction of Dental Caries Risk 281

streptococcal antigen I/II (*S. mutans* adhesion molecules) recognition were constructed and expressed in a strain of Lactobacilli. After the administration of such Lactobacilli to a rat model of dental caries development, *S. mutans* counts and caries scores were reduced obviously (Kruger et al., 2002). The above studies also suggest that consumption of products containing probiotic Lactobacilli or Bifidobacteria could reduce the number of mutans Streptococci in saliva. Oral probiotics may help fight tooth decay, since acid production from sugar is detrimental to teeth, care must be taken not to select strains with high

However, according to the researches, it is cleared that, there are some attractive vehicles for probiotic intake such as using fermented dairy products containing probiotic bacteria (milk, cheese, yogurt and ice cream) and also chewing gum, candies, tablets and water containing

Lactobacilli, as a probiotic (because of it`s known probiotic potential and it`s acid resistance and bile salt`s tolerance), are believed to interfere with pathogens by different mechanisms

As it is mentioned before, lactobacilli have been recognized for their antimicrobial activity and ability to interfere with the adhesion of pathogens on epithelial cells and for their antibiofilm production on catheter devices and voice prostheses. The mechanisms of this interfering have been demonstrated to include, among others, the release of biosurfactants. Biosurfactants, a structurally diverse group of surface active molecules synthesized by microorganisms, have recently attracted attentions in biotechnology for industrial and medical applications. Because the reason, they had several advantages on synthetic surfactants, such as low toxicity, inherent good biodegradability and ecological acceptability. Biosurfactants include unique amphipathic properties derived from their complex structures, which include a hydrophilic moiety and a hydrophobic portion (Vater et al. 2002). The use of biosurfactants from probiotic bacteria as antimicrobial and/or antiadhesive agents has been studied before and their ability to inhibit adhesion of various micro organisms isolated from explanted voice prostheses has been demonstrated (Rodrigues et al. 2004). Biosurfactants adsorption to a surface modifies its hydrophobicity, interfering in the microbial adhesion and desorption processes; so, the release of biosurfactants by probiotic bacteria in vivo can be considered as a defence weapon against other colonizing strains (van Hoogmoed et al., 2004; Rodrigues et al., 2006). Consequently, previous adsorption of biosurfactants can be used as a preventive strategy to delay the onset

of pathogenic biofilm growth, reducing the use of synthetic drugs and chemicals.

In a study, we showed that the biosurfactant derived from probiotic bacteria (*L.acidophilus, L. fermentum* and *L. rhamnosus*) could reduce the adhesion of *S. mutans* to the surfaces (fig 7) (Glass slide or Polystyrene micro titer plates). They also could make streptococcal chains

Other researchers demonstrated that, the biosurfactants from *L. acidophilus* RC14 and *L. fermentum* B54 could interfere in the adhesion and biofilm formation of the *S. mutans*. Also, it is reported that, the release of biosurfactant from *S. mitis BMS* could interfere in the

(table 3) and one of their mechanisms is biosurfactant production.

fermentation capacity.

**4.5 Probiotics-derived biosurfactant** 

probiotics.

shorter.

with probiotic strain before the presence of streptococci. Also, the probiotic strains were able to modify the proportion of the oral species within the biofilm (Tahmourespour & Kermanshahi, 2011).

Fig. 6. The percentage of streptococcal adherence reduction in the presence of probiotic strains

Nikawa et al. (2004) also reported that consumption of yoghurt containing l*actobacillus reuteri (L. reuteri)* over a period of 2 weeks reduced the concentration of *S. mutans* in the saliva by up to 80%. Comparable results were obtained by incorporating probiotics into chewing gum or lozenges. Comelli et al (2002) reported that inoculation of dairy strains before adding the oral bacteria did not increase their colonization. They also found that dairy strains and particularly *L. lactis NCC2211* were able to modify the extent of oral species within the biofilm and also able to reduce cariogenic bacteria levels. They suggest that the reduction of these strains can be explained either by competition for adhesion sites or growth factors. Miller et al., in their study about the effect of microbial interaction on In Vitro plaque formation by *Streptococcus mutans* found that microbial interaction may have the potential to affect the amount and type of plaque formed, depending upon the kinds of organisms involved. They also reported that the addition of the lactobacilli to cultures of *S. sanguis* resulted in more inhibition of plaque formation when compared with pure cultures of *S. sanguis*. A 34% inhibition of plaque formation was observed when *L. casei* interacted with *S. mutans NCTC 10449*. Furthermore Simark-Mattsson et al. (2007) have shown the interference capacities of lactobacilli against strains of *Streptococcus mutans* and *Streptococcus sobrinus*. Meurman (2005) showed the inhibitory activity of *Lactobacillus rhamnosus GG* against *Streptococcus mutans* in low pH and it can be useful for preventing the cariogenic effects of oral streptococci. In vivo studies have also confirmed the effects of probiotic bacteria consumption on decreasing the risk of dental caries and mutans Streptococcus counts. Nase *et al., (2001)* reported long term consumption of milk containing the probiotic *Lactobacillus rhamnosus* CG strain reduced caries in kindergarten children. In one of the earlier studies, Marquis *et al*, demonstrated a potential probiotic approach for reducing dental caries by using oral Streptococci that are able to metabolize arginine or urea to ammonia. Cagler *et al* have showed a reduced *S. mutans* level in patients receiving fluid or tablet probiotic forms. In another study by Cagler *et al* a significantly reduced level was observed for *S.mutans* not for Lactobacillus in an ice-cream containing *Bifidobacterium lactis*  (Caglar et al., 2005; Kargul et al., 2003). Lactobacilli have been used to deliver vaccine components for active immunization in vivo. In this way, the vectors, with the ability of the

with probiotic strain before the presence of streptococci. Also, the probiotic strains were able to modify the proportion of the oral species within the biofilm (Tahmourespour &

Fig. 6. The percentage of streptococcal adherence reduction in the presence of probiotic

Nikawa et al. (2004) also reported that consumption of yoghurt containing l*actobacillus reuteri (L. reuteri)* over a period of 2 weeks reduced the concentration of *S. mutans* in the saliva by up to 80%. Comparable results were obtained by incorporating probiotics into chewing gum or lozenges. Comelli et al (2002) reported that inoculation of dairy strains before adding the oral bacteria did not increase their colonization. They also found that dairy strains and particularly *L. lactis NCC2211* were able to modify the extent of oral species within the biofilm and also able to reduce cariogenic bacteria levels. They suggest that the reduction of these strains can be explained either by competition for adhesion sites or growth factors. Miller et al., in their study about the effect of microbial interaction on In Vitro plaque formation by *Streptococcus mutans* found that microbial interaction may have the potential to affect the amount and type of plaque formed, depending upon the kinds of organisms involved. They also reported that the addition of the lactobacilli to cultures of *S. sanguis* resulted in more inhibition of plaque formation when compared with pure cultures of *S. sanguis*. A 34% inhibition of plaque formation was observed when *L. casei* interacted with *S. mutans NCTC 10449*. Furthermore Simark-Mattsson et al. (2007) have shown the interference capacities of lactobacilli against strains of *Streptococcus mutans* and *Streptococcus sobrinus*. Meurman (2005) showed the inhibitory activity of *Lactobacillus rhamnosus GG* against *Streptococcus mutans* in low pH and it can be useful for preventing the cariogenic effects of oral streptococci. In vivo studies have also confirmed the effects of probiotic bacteria consumption on decreasing the risk of dental caries and mutans Streptococcus counts. Nase *et al., (2001)* reported long term consumption of milk containing the probiotic *Lactobacillus rhamnosus* CG strain reduced caries in kindergarten children. In one of the earlier studies, Marquis *et al*, demonstrated a potential probiotic approach for reducing dental caries by using oral Streptococci that are able to metabolize arginine or urea to ammonia. Cagler *et al* have showed a reduced *S. mutans* level in patients receiving fluid or tablet probiotic forms. In another study by Cagler *et al* a significantly reduced level was observed for *S.mutans* not for Lactobacillus in an ice-cream containing *Bifidobacterium lactis*  (Caglar et al., 2005; Kargul et al., 2003). Lactobacilli have been used to deliver vaccine components for active immunization in vivo. In this way, the vectors, with the ability of the

Kermanshahi, 2011).

strains

streptococcal antigen I/II (*S. mutans* adhesion molecules) recognition were constructed and expressed in a strain of Lactobacilli. After the administration of such Lactobacilli to a rat model of dental caries development, *S. mutans* counts and caries scores were reduced obviously (Kruger et al., 2002). The above studies also suggest that consumption of products containing probiotic Lactobacilli or Bifidobacteria could reduce the number of mutans Streptococci in saliva. Oral probiotics may help fight tooth decay, since acid production from sugar is detrimental to teeth, care must be taken not to select strains with high fermentation capacity.

However, according to the researches, it is cleared that, there are some attractive vehicles for probiotic intake such as using fermented dairy products containing probiotic bacteria (milk, cheese, yogurt and ice cream) and also chewing gum, candies, tablets and water containing probiotics.

#### **4.5 Probiotics-derived biosurfactant**

Lactobacilli, as a probiotic (because of it`s known probiotic potential and it`s acid resistance and bile salt`s tolerance), are believed to interfere with pathogens by different mechanisms (table 3) and one of their mechanisms is biosurfactant production.

As it is mentioned before, lactobacilli have been recognized for their antimicrobial activity and ability to interfere with the adhesion of pathogens on epithelial cells and for their antibiofilm production on catheter devices and voice prostheses. The mechanisms of this interfering have been demonstrated to include, among others, the release of biosurfactants. Biosurfactants, a structurally diverse group of surface active molecules synthesized by microorganisms, have recently attracted attentions in biotechnology for industrial and medical applications. Because the reason, they had several advantages on synthetic surfactants, such as low toxicity, inherent good biodegradability and ecological acceptability. Biosurfactants include unique amphipathic properties derived from their complex structures, which include a hydrophilic moiety and a hydrophobic portion (Vater et al. 2002). The use of biosurfactants from probiotic bacteria as antimicrobial and/or antiadhesive agents has been studied before and their ability to inhibit adhesion of various micro organisms isolated from explanted voice prostheses has been demonstrated (Rodrigues et al. 2004). Biosurfactants adsorption to a surface modifies its hydrophobicity, interfering in the microbial adhesion and desorption processes; so, the release of biosurfactants by probiotic bacteria in vivo can be considered as a defence weapon against other colonizing strains (van Hoogmoed et al., 2004; Rodrigues et al., 2006). Consequently, previous adsorption of biosurfactants can be used as a preventive strategy to delay the onset of pathogenic biofilm growth, reducing the use of synthetic drugs and chemicals.

In a study, we showed that the biosurfactant derived from probiotic bacteria (*L.acidophilus, L. fermentum* and *L. rhamnosus*) could reduce the adhesion of *S. mutans* to the surfaces (fig 7) (Glass slide or Polystyrene micro titer plates). They also could make streptococcal chains shorter.

Other researchers demonstrated that, the biosurfactants from *L. acidophilus* RC14 and *L. fermentum* B54 could interfere in the adhesion and biofilm formation of the *S. mutans*. Also, it is reported that, the release of biosurfactant from *S. mitis BMS* could interfere in the

Probiotics and the Reduction of Dental Caries Risk 283

factors associated with the pathogenesis of dental caries and a high content of insoluble glucans in dental plaque, which is related to an elevated risk of biofilm cariogenicity in humans. Several environmental factors can influence the expression and activity of the gtf enzymes. The existence of various enzymes in the process of carbohydrate metabolism and transport, glucan synthesis and secretion and degradation in the oral streptococci, in addition to factors that involve Post-translational modifications of the gtf enzymes, have

Our results (figure 8 & 9) suggest that either the *L .fermentum* or *L. acidophilus* derived biosurfactants themselves or a putative signaling molecule in the extract down-regulated the expression level of genes that play an important role in the process of *S. mutans* attachment and biofilm formation. In addition to down regulating gtfB and gtfC (genes involved in insoluble glucan production), it may also have an effect on converting gtf activity from producing insoluble glucans to water-soluble glucans, hence accounting for reduced *S.* 

Fig. 8. The effect of *L.fermentum*.-derived biosurfactant on gtfB/C in immobilized biofilm of *S. mutans ATCC 35668;* The mRNA expression levels were calibrated relative to the control

Fig. 9. The effect of *L.acidophilus*-derived biosurfactant on gtfB/C in immobilized biofilm of *S. mutans ATCC 35668;* The mRNA expression levels were calibrated relative to the control

group (in the absence of biosurfactant) (Tahmourespour et al.,2011, Brazillian J of

Microbiology).

group (in the absence of biosurfactant)( Tahmourespour et al.,2011, Biofouling) .

traditionally complicated the understanding of regulatory studies (Wen et al. 2010).

*mutans* biofilm adherence, and this should be studied in the future.

adhesion of the cariogenic *S. mutans* to glass in the presence and absence of a salivary conditioning film. Others also confirmed that biosurfactants had inhibitory effect on bacterial adhesion and also biofilm formation. However; the precise mechanisms of such effects have not yet been explained. It seems to be highly dependent on biosurfactant type and the properties of the target bacteria. The simplest way to explain biosurfactant antiadhesion and antibiofilm activities would be their direct antimicrobial action. However, the antimicrobial activity of biosurfactants has not been observed in all cases (Tahmourespour et al., 2011 & vater et al., 2002). Thus, it is reported that the way in which surfactants influenced bacterial surface interactions appeared to be more closely related to the changes in surface tension and bacterial cell-wall charge. These factors are very important in overcoming the initial electrostatic repulsion barrier between the microorganism cell surface and its substrate. Surfactants may affect both cell-to-cell and cellto-surface interactions. Their results support the idea that lactobacilli-derived agents remarkably have an effect on these interactions.

Fig. 7. The mean of adherence reduction percentage of mutans streptococci in presence of biosurfactants derived from *L. acidophilus, L. rhamnosus* and *L.fermentum* (Unpublished data).

As it is clear, colonization of the teeth by mutans streptococci has been associated with the etiology and pathogenesis of dental caries in humans. The ability of these organisms, particularly *Streptococcus mutans*, to synthesize extracellular glucans from sucrose using glucosyltransferases (Gtfs) is a major virulence factor of this bacterium.

The Gtfs secreted by *S. mutans* (particularly GtfB and GtfC) provide specific binding sites for either bacterial colonization of the tooth surface or attachment of bacteria to each other, modulating the formation of tightly adherent biofilms, the precursor of dental caries (Koo et al. 2010; Murata et al. 2010). However, the ability of *S. mutans* to adhere to the tooth surface is vital for the initiation and progression of dental caries. α-(1-3)- and α-(1-6)-linked glucan polymers are encoded by the genes gtfB, gtfC, and gtfD. In vitro studies have indicated that gtfB and gtfC are essential for the sucrose-dependent attachment of *S. mutans* cells to hard surfaces, but gtfD is dispensable (Yoshida et al. 2005). Therefore, these genes have become a potential target for protection against dental caries.

The effect of *L. fermentum* and *L. acidophilus* biosurfactant on gtfB and gtfC gene expression levels was also investigated in our other studies. The expression of these genes and the production of insoluble extracellular glucans mediate the attachment of *S. mutans* not only to surfaces but also to other active types of bacteria that are favorable to the organisms for the persistent colonization of tooth surfaces. Additionally, gtf genes are known virulence

adhesion of the cariogenic *S. mutans* to glass in the presence and absence of a salivary conditioning film. Others also confirmed that biosurfactants had inhibitory effect on bacterial adhesion and also biofilm formation. However; the precise mechanisms of such effects have not yet been explained. It seems to be highly dependent on biosurfactant type and the properties of the target bacteria. The simplest way to explain biosurfactant antiadhesion and antibiofilm activities would be their direct antimicrobial action. However, the antimicrobial activity of biosurfactants has not been observed in all cases (Tahmourespour et al., 2011 & vater et al., 2002). Thus, it is reported that the way in which surfactants influenced bacterial surface interactions appeared to be more closely related to the changes in surface tension and bacterial cell-wall charge. These factors are very important in overcoming the initial electrostatic repulsion barrier between the microorganism cell surface and its substrate. Surfactants may affect both cell-to-cell and cellto-surface interactions. Their results support the idea that lactobacilli-derived agents

Fig. 7. The mean of adherence reduction percentage of mutans streptococci in presence of biosurfactants derived from *L. acidophilus, L. rhamnosus* and *L.fermentum* (Unpublished data).

glucosyltransferases (Gtfs) is a major virulence factor of this bacterium.

potential target for protection against dental caries.

As it is clear, colonization of the teeth by mutans streptococci has been associated with the etiology and pathogenesis of dental caries in humans. The ability of these organisms, particularly *Streptococcus mutans*, to synthesize extracellular glucans from sucrose using

The Gtfs secreted by *S. mutans* (particularly GtfB and GtfC) provide specific binding sites for either bacterial colonization of the tooth surface or attachment of bacteria to each other, modulating the formation of tightly adherent biofilms, the precursor of dental caries (Koo et al. 2010; Murata et al. 2010). However, the ability of *S. mutans* to adhere to the tooth surface is vital for the initiation and progression of dental caries. α-(1-3)- and α-(1-6)-linked glucan polymers are encoded by the genes gtfB, gtfC, and gtfD. In vitro studies have indicated that gtfB and gtfC are essential for the sucrose-dependent attachment of *S. mutans* cells to hard surfaces, but gtfD is dispensable (Yoshida et al. 2005). Therefore, these genes have become a

The effect of *L. fermentum* and *L. acidophilus* biosurfactant on gtfB and gtfC gene expression levels was also investigated in our other studies. The expression of these genes and the production of insoluble extracellular glucans mediate the attachment of *S. mutans* not only to surfaces but also to other active types of bacteria that are favorable to the organisms for the persistent colonization of tooth surfaces. Additionally, gtf genes are known virulence

remarkably have an effect on these interactions.

factors associated with the pathogenesis of dental caries and a high content of insoluble glucans in dental plaque, which is related to an elevated risk of biofilm cariogenicity in humans. Several environmental factors can influence the expression and activity of the gtf enzymes. The existence of various enzymes in the process of carbohydrate metabolism and transport, glucan synthesis and secretion and degradation in the oral streptococci, in addition to factors that involve Post-translational modifications of the gtf enzymes, have traditionally complicated the understanding of regulatory studies (Wen et al. 2010).

Our results (figure 8 & 9) suggest that either the *L .fermentum* or *L. acidophilus* derived biosurfactants themselves or a putative signaling molecule in the extract down-regulated the expression level of genes that play an important role in the process of *S. mutans* attachment and biofilm formation. In addition to down regulating gtfB and gtfC (genes involved in insoluble glucan production), it may also have an effect on converting gtf activity from producing insoluble glucans to water-soluble glucans, hence accounting for reduced *S. mutans* biofilm adherence, and this should be studied in the future.

Fig. 8. The effect of *L.fermentum*.-derived biosurfactant on gtfB/C in immobilized biofilm of *S. mutans ATCC 35668;* The mRNA expression levels were calibrated relative to the control group (in the absence of biosurfactant)( Tahmourespour et al.,2011, Biofouling) .

Fig. 9. The effect of *L.acidophilus*-derived biosurfactant on gtfB/C in immobilized biofilm of *S. mutans ATCC 35668;* The mRNA expression levels were calibrated relative to the control group (in the absence of biosurfactant) (Tahmourespour et al.,2011, Brazillian J of Microbiology).

Probiotics and the Reduction of Dental Caries Risk 285

naturally colonizing the oral cavity. The modified strain could then be used to replace the original pathogen. They also could be used to increase the properties of a potentially beneficial strain. In field of oral immunology, probiotics are being used as passive local immunization vehicles against dental caries. Bacteriophages, have also been detected in oral

 The selection of the best probiotic for oral health and investigation the effect of other probiotic's metabolites on virulence genes and other traits of *S. mutans* are also issues that calls for further studies. It is possible that the administration way of probiotics might positively affect the effects observed as related to mutans streptococci reduction. So, further studies regarding the selection of best way for probiotic administration are necessitated.

Furthermore, the dosage of probiotic administration in each indication should be defined. Probiotics should be administered carefully and cautiously, and only on the basis of strong scientific evidence. Such evidence should direct the cautious, deliberate addition of clinically proven probiotics to commonly consumed food products to allow consumers to conveniently benefit from these organisms. Finally, safety issues are very important with

Consequently, future studies should be conducted to investigate if phage therapy might be applied for oral and dental diseases in the same way as has been attempted for systemic

Adhesion reduction can be an effective way on decreasing cariogenic potential of oral streptococci and all of the evidence has shown that probiotic bacteria such as Lactobacillus spp. can affect the oral ecology. In general, the above promising results suggest a potentially beneficial application of probiotics for the prevention of dental caries. These data also suggest that biosurfactant treatment can provide an option for controlling biofilm

The author would like to thanks Dr. Ahmad Ali ForoughiAbari, the chancellor of Islamic Azad University Khorasgan (Isfahan), branch and Dr Mehran Hoodaji for their supports

Ahola AJ., Yli-Knuuttila H. & Suomalainen T. (2002). Short term consumption of probiotic-

Anderson MH. & Shi W. (2006). A probiotic approach to caries management. *Ped Dent*, 28

Bhardwaj SB. (2010). Probiotics and oral health: an update. *Int J Contemporary Dent,* 1 (3):

containing cheese and its effect on dental caries risk factors. *Arch Oral Biol,* 47: 799–

development and also influence the adhesive ability of bacterial pathogens

and Biotechnology Research Center of this University.

pathogens, such as *Actinobacillus Actinomycetemcomitans* (Sheikh et al., 2011).

any kind of bacteriotherapy.

infections.

**6. Conclusion** 

**7. Acknowledgment** 

**8. References** 

804.

(2): 151-153.

116-119.

Other studies have focused on the production and gene regulation of virulence factors, such as gtfs, which play an important role in biofilm formation by *S. mutans*, for controlling dental caries (Tamwsada and Kawabata 2004; Huang et al. 2008). The ability of *S. mutans* to produce extracellular polysaccharides from dietary carbohydrates has been demonstrated to significantly enhance its cariogenicity. Thus, the less extracellular polysaccharide produced, the lower the cariogenicity of *S. mutans*. Also it is demonstrated that chemical surfactants exerted different effects on the synthesis of glucosyltransferases in *S. mutans*; Tween 80 sig nificantly increased the level of gtfs, while Triton X-100 decreased gtf levels. So, It is proposed that the secondary metabolite of the probiotic bacteria (*L.fermentum and L.acidophilus*) decreases the expression level of gtf genes and therefore may be useful for the control of *S. mutans* and possibly other species.
