Probioticsderived 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 anti-biofilm 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 anti-adhesive 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 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 cell-to-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).

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. a-(1-3)- and a-(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 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.

□ biofilm S.mutans ATCC3566S

□ biof. S.mutans 3E668+L.f biosurfactant_

T T

ib

J

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) .

H biofilm S.mutans ATCC3566B

■ biof. S.mutans 35668+L.a biosurfactant

H biofilm S.mutans ATCC3566B

■ biof. S.mutans 35668+L.a biosurfactant

gtfB gtfC

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).

gtfB gtfC

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).

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.

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