Molecular Microbial Characterization Of Dental Plaque/Biofilm And To Detect The Effect Of Lactobacillus Species On Biofilm Formation Of Streptococcus Mutans

Introduction:Oral cavity is one of the most complex microbial habitats in the human body thatcomprises of more than600diverse arrays of bacterial species. More than250oralmicrobes have been isolated and characterized by the cultivation method but over450microbial species have been identified by the culture independent molecular methods.Among many oral diseases dental caries and periodontitis are common diseases. Bothdiseases are polymicrobial disease. Polymicrobial communities of bacteria are formedin the biofilm form by the early colonizer like Streptococcus mutans. So, the completepicture of microbial communities responsible for dental caries and periodontitis isimportant to depict to find proper treatment of disease. Conventional bacterial culturalmethod failed to depict complete picture as still few oral bacterial strains uncultivated.With the emergence of molecular methods targeting16S rRNA likes polymerase chainreaction based denaturing gradient gel electrophoresis (PCR DGGE), sequencing, clonelibrary etc. Among them PCR DGGE is one is useful for analyzing the bacterialdiversity profile in different disease condition. This technique is a fingerprintingapproach and used by environmental ecologists to survey entire bacterial communitieswithout cultivation. Biofilm formation by microorganisms is a big problem andprovides shelter and resistance to bacterium inside it. Streptococcus mutans is amongthe early colonizer of oral cavity and initiator of biofilm. S. mutans in the presence ofsucrose produces acid which lower the pH and initiate caries formation. GtfB and LuxSgene is responsible for biofilm formation in the presence of sucrose. So any form oftreatment directed to reduce S. mutans count is useful for maintaining oral health. Nowa day’s consumption of probiotics containing product is increasing. Lactobacilusspecies and Bifidobacterium species are the most widely used probiotics. Despite the wide spread use of probiotics, its use in oral disease still in its infancy and limited to themanagement of dental caries, periodontal disease and halitosis Lactobacilli have gaineda great interest in dental research for several decades. It can produce bacteriocins whichinhibits wide variety of bacterial species including oral streptococci. So probiotics canbe a good candidate for oral disease treatmentObjectivesMolecular characterization of microorganisms in dental plaque/biofilm byusing PCR DGGE.Determine the Effect of Lactobacillus species on biofilm formation ofStreptococcus mutans.Determine the Effect of Lactobacillus species on Streptococcus mutans viability.Determine the Effect of Lactobacillus species on genes involved in biofilmformation.Material and Method:For microbial characterization dental plaque samples were collected from dentalcaries and periodontitis patients, DNA was extracted from plaque samples, PCR wasperformed using primer with GC clamp and run on denaturing gradient gelelectrophoresis.Biofilm formation capability of S. mutans, L. bulgaricus, L. acidophilus, L.rhamnosus and L. salivarius was assessed using crystal violet based96well platemethod at24hrs,48hrs and72hrs. To detect the effect of Lactobacillus species on S.mutans biofilm formation, they are incubated in1:1ratio and analyzed by crystal violetbased96well plate method, confocal microscopy.Effect of Lactobacillus species on viability of S. mutans is detected by PMAbased real time PCR by targeting S. mutans specific16S rRNA gene.Effect of Lactobacillus species on biofilm formation genes. GtfB and LuxS geneswere selected. RNA was extracted, cDNA synthesized and real time PCR was done forselected gene.Results:Each band in DGGE profile showed the presence of single or more than onebacterial species in sample. It is clearly observed that number of bands detected inperiodontitis groups (10.36±3.5) were less than dental caries (12.73±3.2) and normalgroups (17.06±4.34) with P values0.002and0.001among disease and normal group.The three groups failed to form significant cluster. Sequencing results showed the presence of23genera. Mostly Gram negative bacteria in periodontitis group, whiledental caries group contain approximate equal numbers of Gram positive and negativebacteria.S. mutans used in this study is a good biofilm former and its ability increased in thepresence of sucrose and with increasing time. All the four lactobacillus strain used arenot a good biofilm former as compared to S. mutans. All the lactobacillus strain reducedbiofilm formation by S. mutans. But L. acidophilus and L. salivarius reduced to a greatextent. These two strains reduce the depth of biofilm almost half. Most of the results arestatistically significant wit P value≤0.005except few have greater than0.005. Thesetwo strains also reduced the viable count of S. mutans from1.70x105to3.08x103and3.13x102respectively with P value0.002.Gene expression of GtfB and LuxS genes involved in biofilm formation wasreduced in the presence of L. acidophilus and L. salivarius. The expression of GtfB inthe presence of both lactobacillus strains reduced to80%and90%respectively, whilethe expression of LuxS was reduced to56%and83%. Results were found statisticallysignificant with P value≤0.005.Conclusion:Oral cavity harbors diverse array of microorganisms which are present inequilibrium. Once the equilibrium is destroyed it leads to disease state. Dental cariesand periodontitis are the common polymicrobial oral cavity disease. This needs to bedissected to establish good remedies to fight with disease. PCR DGGE is one of theuseful techniques used to detect many bacteria in any environment so we used thistechnique. Advantage of it is visual detection of profile; bands of interest can be cut,cloned and sequenced to get phylogenetic identity. Less diversity diseased group mightbe due to the presence of only acid producing bacteria or bacteria which survives hostdefense.Biofilm formation in the oral cavity is a big concern as it provides microbe’sshelter and more resistance to antimicrobial compounds. So any remedies to reducebiofilm formation might be important. Due to emergence of antibiotic resistancebacteriotherapy is becoming famous in the light of probiotics lactobacillus andbifidobacterium species. We also detect the effect of four lactobacillus strains onbiofilm formation of S. mutans. All of them reduce biofilm but L. salivarius and Lacidophilus reduced the biofilm greatly as well as bacterial count. This action might bedue to the presence of bacteriocins responsible to kill S. mutans, it might also interfere with the adherence of S. mutans and restrict biofilm formation. so probiotic approachmight be useful and as an alternate to antibiotics. Still further research is needed indifferent aspect to assure this phenomenon.