| [Objective]Streptococcus mutans (MS) is the most important cariogenic bacteria in the mouth, lactobacilli (LB) and bifidobacterium are also considerated as the pathogens which lead to caries. Few research has been done on their effect against MS, their mechanism of inhibition effect on MS is not yet clear. The aim of this study was to compare the effects of five probiotic LB strains on growth and biofilm formation of standard strains and clinical isolates of MS, to explore the mechanism of its antibacterial effects and discuss its application value for caries prevention.[Methods]1. Inhibitory effects of LB on MSLB was adjusted to1×106CFU/ml,1×108CFU/ml and3×108CFU/ml, MS was adjusted to1.5×108CFU/ml,0.25ml of the MS and LB supensions were combined with1.5ml BHI broth and incubated microaerophilicly at37℃for24h. The S. aureus ATCC25923culture was used as the negative control. To determine the antibacterial activity of the LB supernatants, we used the same method described above, but LB supensions were replaced by LB supernatants. After24h viable number of MS were counted on MSB agar.2. Biofilm formationMS was adjusted with saline to1.5×108CFU/ml. Individual, sterile petri dishes were filled with9.5ml of BHI broth, and a sterile coverglass was added to petri dish.100ul of the MS suspension was mixed in the dishes with400μl of the untreated and treated LB supernatants. As a control, MRS broth was used. All petri dishes were incubated microaerophilicly at37℃for24h.After24h,the coverglasses were stained with a0.01%acridine orange solution. Biofilm was observed with fluorescence microscope.3. SDS-PAGEThe supernatants following ultrafiltration were concentrated by freeze-drying and suspended in100μl of Milli-Q water and were subjected to15%Tris-HCL gel electrophoresis at120V for90min. Proteins were visualized after electrophoresis by staining the gel with Coomassie Brilliant-Blue R250solution for1.5h and decolorizing for4h.4.20caries-active children (DMFS≥6)were selected as experimental group, MS were isolated from saliva, after identification one strain was selected and was mixed with lactobacilli at37℃, after incubation, viable bactrial number of MS was counted. At the same time dental plaques from caries-active children were mixed with lactobacilli in vitro to form biofilm, after24h, the population of MS, oral Streptococcus and lactobacilli in the biofilm were enumerated.[Result]1. An LB concentration of1×106CFU/ml showed no antimicrobial activity against MS, but LB at concentration of1×108CFU/ml and3×108CFU/ml showed strong inhibitory effects (P<0.05). 2. The untreated supernatants of L. casei Shirota, L. casei LC01, L. plantarum ST-Ⅲ, L. paracasei Lpc-37and L. rhamnosus HN001all exerted a strong inhibitory effect on the biofilm formation of MS, but only L. casei Shirota and L. rhamnosus HN001had an inhibitory effect after treatment (P<0.05).3. We found additional enhanced bands for L. casei Shirota and L. rhamnosus HN001at approximately6.5kDa. The extra two protein of6.5kDa correlated with the observation that, after pH adjustment and ultrafiltration, supernatants of L. casei Shirota and L. rhamnosus HN001still had an inhibitory effect on MS.4. The inhibition rate of Z. casei Shirota, L. casei LC01, L. plantarum ST-III and L. paracasei Lpc-37on Streptococcus mutans were72.27%,62.82%,64.42%,68.58%, they all showed strong inhibition on Streptococcus mutans (P<0.05). L. casei Shirota, L. casei LC01, L. plantarum ST-III and L. paracasei Lpc-37can reduce the number of MS and oral Streptococcus in the biofilm (P<0.05), meanwhile the adhesive capacity of lactobacilli and pH of biofilm had no significant change (P>0.05).[Conclusion]All five probiotic LB strains could inhibit the growth and biofilm formation of MS, likely by producing an acid environment, bacteriocin-like polypeptides or both. Therefore, ingestion of lactobacillus products contain probiotic lactobacilli may contribute to help caries prevention. |
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