Analyzing GTF Activity Of Different Genotype Streptococcus Mutans Isolated From Children With Different Caries Experience

Objectives:Caries is a frequent disease in deciduous teeth, the inciden ce of dental caries related primarily to Streptococcus mutans. The glucosylt ransferase, produced by Streptococcus mutans, plays a very important role in the oral bacterial adhesion and plaque formation, is to be considered on e of the most important cariogenical factors.Based on the pre-test, this stud y is to explore the different glucosyltransferase activity of different genoty pe Streptococcus mutans isolated from children with different caries in diff erent pH values. The results can provide evidence of the methods for impr oving the risk prediction of dental caries in deciduous teeth. Methods:Th e isolated strains of Streptococcus mutans with different genotypes were us ed, chosen 66 strains with 23 different genotypes from the 111 strains whi ch are obtained by the preliminary experiments. The strains were reanimate d in BHI plates, Typical bacteria were inoculated in TYC liquid and cultiv ated in anaerobic condition(80%N2,10%H2 and 10%CO2) at 37℃for 2 4h. Prepared same concentration of suspension (A540nm=1.0), and then i noculated 100μl of suspension to 10ml TYC at different pH ranging from 5.0 to 7.0 which contains 0.25%(w/v) glucose and 0.05%(w/v)Tween8 0 and cultured for 18h at 37℃. The cells were subsequently removed by centrifugation at 3300rpm for 30min and the supernatant was added with solid ammonium sulfate to give 60% saturation at 4℃for 12h, the precipi tates formed were then collected by centrifugation and dissolved with 0.01 % NaNO3 of 0.01mol/L phosphate buffer pH6.4, after being exhaustively dialyzed against the same buffer at 4癈, the sediment was loaded on to he dialysis bag that had been previously equilibrated with phosphate buffer pH6.4 for 48h, the Condensed liquid is crude enzyme preparation. Then mixed 0.1ml 0.5mol/L phosphate buffer,0.1ml 0.5mol/L sucrose buffer and 0.1ml distilled water to 1.0ml enzyme solution and incubated for 1h. The reaction was terminated by 0.1ml 0.24mol/L HCl. After mixed 2.5ml distilled water, the mixture was centrifuged at the rate of 2000 rpm for 5 minutes. Reducing sugar was measured by the Somogyi-Nelson method us ing glucose as a standard.Read the absorbance at 620nm against a blank. One unit of GTF was defined as the amount of enzyme required to incorp orate 1μmol of glucose into glucan polysaccharide per minute under stand ard assay conditions. Results:There were significant difference in glucosyl transferase activity among the strains isolated from children with different caries susceptibility:high caries-susceptible group>middle caries-susceptibl e group>caries-free group,Statistically significant difference(p<0.05). In the same group, the more genotypes the strain has,the more glucosyltransf erase activity it produced, but there was no obvious correlation between th e numbers of genotypes and the glucosyltransferase activity (p>0.05). Th ere were significant differences in glucosyltransferase activity with different pH values among the different genotypes of strains isolated from children with different caries susceptibility. In the same group, the glucosyltransfer ase activity decreased gradually with pH decreasing. The glucosyltransferas e activity is generally reduced when at pH 5.0, but still have active which derived from the more genotype Streptococcus mutans isolated from childr en with high caries. Conclusion:In the neutral condition, there were signi ficant differences in the glucosyltransferase activity among the different gen otypes of strains isolated from children with different caries, The higher ca ries susceptibility the strain has, the more glycosyltransferase activity it pro duced.The glucosyltransferase activity with different genotypes of strains is related to culture conditions, the glucosyltransferase activity decreased gra dually with pH decreasing. In the different pH condition,the more genotype s the strain has, the more glucosyltransferase activity it produced, so there was a certain relevance between the incidence of high caries and the gen otypes.