The Effects Of Sub-MICs Of Antimicrobial Agents On Streptococcus Mutans Biofilm Formation

Many studies have demonstrated that sub-minimal inhibitory concentrations(sub-MICs) of antimicrobial agents can inhibit bacterial biofilm formation.However, the mechanisms that sub-MICs antimicrobial agents employ to inhibitbiofilm formation remain unclear. At present, most studies are focused onGram-negative bacteria; nevertheless, the effects of sub-MICs of antimicrobialagents on Gram-positive bacteria may be more complex. Streptococcus mutans isa major cariogenic bacterium. In this study, the S. mutans growth curve and theexpression of the genes related to S. mutans biofilm formation were evaluatedafter treatment with1/2of the MICs of chlorhexidine, tea polyphenols andsodium fluoride, which are common anticaries agents. The BioFlux system wasemployed to generate a biofilm under a controlled flow. The morphologicalchanges of the S. mutans biofilm were observed and analyzed using FieldEmission Scanning Electron Microscopy and Confocal Laser ScanningMicroscopy.The results indicated that these three common anticaries agents could significantly upregulate the expression of the genes related to S. mutans biofilmformation, and S. mutans exhibited a dense biofilm with an extensiveextracellular matrix after the treatment with sub-MICs of sodium fluoride andchlorhexidine. Our findings suggest that sub-MICs of anticaries agents favorS. mutans biofilm formation, which might encourage dental caries progression.1Determination the minimal inhibitory concentration(MIC)and minimum bactericidal concentration(MBC) of antimicrobialagents on S. mutans.The antimicrobial agents sodium fluoride、tea polyphenols、chlorhexidine、metronidazole and penicillin were dissolved into sterile distilled water inproportion, respectively. Filter sterilized. Resuscitate the S. mutans UA159, anddetermination the MIC and MBC of antimicrobial agents on S. mutans bytwo-fold dilution method. The results show that the antimicrobial agents had awide range of antimicrobial activities against S. mutans. The MBC was generallyhigher than the MIC for each antimicrobial, with the exception of chlorhexidinewhere it was the same.2The effects of sub-MICs antimicrobial agents on the growthcurve of S. mutans.The antimicrobial agents were blending with S. mutans in1/2MICsconcentration, the suspension were cultured at37℃under an atmosphere. Takesamples at0,2,4,6,8,10,12,24,48h and measure OD600, in the same time record theviable cell counts. The growth curve were drew with the two sets of data. The results showed that the effect of sodium fluoride was the strongest, followed bypenicillin and chlorhexidine. The effect of metronidazole and tea polyphenolswere the weakest and close to that of the control group.3The expression of genes related to biofilm formation in S.mutans with sub-MICs of the antimicrobial agentsThe real-time PCR primers were designed. The assay was divided into twoexperimental groups, the planktonic group and the biofilm group. A mixture of1/2MICs of each prepared antimicrobial agent and an S. mutans culture wastransferred into the test tubes in the planktonic group. The bilfilm group wasidentical to the planktonic group, except that it was cultured in a24-well platewith sucrose added to each well at a concentration of1%, all the cultures wereincubated at37℃under anaerobic conditions. Take samples after24h andextract the RNA. The reverse transcription and real-time PCRs were performed todetermine the expression of related genes. The results show that, almost all thegenes are significantly up-regulated. It show that the sub-MICs antimicrobialagents may induce the expression of genes related to S. mutans biofilmformation.4Morphological observation of S. mutans with sub-MICsantimicrobial agents by FE-SEMThe tooth that was cleaned, cut into2-mm-thick sections, and immersed in75%alcohol for30min.2ml of BHIS was mixed with each antimicrobial agent ata concentration of0.5×MIC and was pipetted into a24-well tissue-culture platedwith10μL of a24h S. mutans culture. Enamel sections were introduced into each well, and the plate was incubated at37℃for24h under anaerobic conditions.Thereafter, the enamel sections were taken, washed, fixed, dehydrated, dried andobserved by FE-SEM. The results show that the biofilm of S. mutans treated withsub-MICs of sodium fluoride or chlorhexidine became denser, containing moreextracellular matrix and fewer water channels. Unexpectedly, the surface of S.mutans treated with sub-MICs of tea polyphenols displayed a large number ofbacteria, and little extracellular matrix was found in the visual field. These resultssuggest that sub-MICs of sodium fluoride or chlorhexidine may reduce S. mutansbiofilm formation, but the tea polyphenols might inhibit its formation byinhibiting the synthesis of extracellular matrix.5Morphological observation of S. mutans bioflim under BioFluxsystem with sub-MICs antimicrobial agents by CLSMS. mutans culture was mixed with each antimicrobial agent at aconcentration of0.5×MIC and sucrose was added at a concentration of1%. Thenthe BioFlux device was used in cultivating the biofilm under controllable shearforces for15h. S. mutans cells were then stained using LIVE/DEAD bacterialstains. A confocal laser-scanning microscope (CLSM) was used to observe thebioflim. Many intracellular spaces were observed in the control S. mutans biofilm.Compared with the control, the biofilm of S. mutans treated with sub-MICs ofsodium fluoride or chlorhexidine with few water channels. Unexpectedly, someof the S. mutans cells treated with a sub-MICs of tea polyphenols no aggregationor bioflim formation was observed. This result is consistent with the results offront, also suggest that sub-MICs of sodium fluoride or chlorhexidine may reduceS. mutans biofilm formation, but the tea polyphenols might inhibit its formation.