Mutans Streptococci Extracellular Polysaccharide Synthesis and Biofilm Formation in Children

Objective: To measure and compare the extracellular water-soluble and water-insoluble polysaccharide production ability and biofilm formation ability of mutans stretococci (MS) strains isolated from 20 caries free (CF) children and 20 children with early childhood caries (ECC)(2-6 years old). Methods: Fifty-six MS strains from ECC children and eight strains from CF children were confirmed by fermentation tests and AP-PCR. These MS strains were inoculated in BHI broth with 5% sucrose to produce extracellular polysaccharides. After a 48-hour incubation the cultures were centrifuged (10,000g) and supernatants ethanol extracted for water-soluble polysaccharide (WSP). The pellets were dissolved and centrifuged in 0.5N NaOH, and the supernatants ethanol-extracted for water-insoluble polysaccharide (WISP). WSP and WISP quantities from each strain were measured by an anthrone-sulfuric acid colorimetric microassay. The same strains were again inoculated in BHI broth with 5% sucrose for biofilm formation during 48-hours. Biofilm was stained with 3% crystal violet dye for 5 minutes, then rinsed and dried. The biofilm was then destained with 2% acid-alcohol for 48 hours and the solutions measured for optical density. Results: CF MS strains produced significantly more WISP with mean±SD as 0.67±0.22 (CF) and 0.45±0.25 (ECC) mg/ml (Mann-Whitney test, P<0.05). No statistical difference was found in WSP production or biofilm formation ability between the two groups. All 20 ECC children harbored multiple MS strains whereas only 6 of the 20 CF children had MS. Conclusion: WSP and biofilm formation was not correlated to caries status in children while MS from ECC children produce less WISP as compared to MS from CF children, with a high proportion of CF children harboring no MS. Supported by: NIH/NIDCR T32DE07306 through the COHORT grant