Influence Of Lactobacillus Acidophilus On Streptococcus Mutans In Vitro

Dental caries,a common diseases affecting oral and systemic health, isranked as three chronic non-infection diseases with cancer andangiocardiopathy by WHO. Although it developes slowly,but it ischaracterised with high incidence、extensive popular area and irreversibledamage on teeth.As we all know,bacterias are the major factors in theformation and deveplopment of dental caries. Streptococcus mutans (Sm), anormal resident flora in oral, is recognized as an important cariogenicbacteria and its main virulence factors includes acid-production、acid-resistance、 the production of adhesin and dextran.Above all, theformation of dental plaque structure plays an essential role in caries.Plaque control is the key content in prevention and treatment of dentalcaries.At present antibiotics application and mechanical removal are widelyused in clinic to control dental plaque.However,these two methods are notenough.Long-term antibiotics application will result in bacterial drugresistance and dysbacteriosis,increasing the risk of opportunisticinfection.Besides, it is difficult to maintain the ideal level of plaque control for a long period by mechanical removal method without the strongcompliance of operators.Lactobacillus is widely used probiotics which play an important role inregulating the ecological balance of the gastrointestinal tract.Lactobacilluscan compete binding sites with pathogenic bacteria in biofilms.On the otherhand,its metabolism including organic acids、bacteriocins、class bacteriocinsand other antibacterial ingredients can significantly inhibit the growth ofpathogenci bacteria.Although some lactobacillus can coaggregate with Streptococcusmutans and some of them can significantly inhibit Streptococcusmutans in planktonic.However, bacteria physiological stage changessignificantly in dental plaque biofilm campareing with planktonic,includingstronger bacterial drug resistance and resistance to host immunedefense.So,it is meaningful to study the influence of lactobacillus onStreptococcus mutans in planktonic and biofilm and discover new methodsfor dental caries prevention on the basis of regulating the ecologicalbalance of oral.Objective:Streptococcus mutans are recognized major cariogenicbacteria and Lactobacillus are not only resident floras but also widely usedprobiotic. Lactobacillus acidophilus ATCC4356was selected to study theinfluence on Streptococcus mutans UA159in planktonic and biofilm. Wehope that it may apply useful informations for prevention and treatment of caries.Methods:(1) L.acidophilus ATCC4356and S.mutans UA159weresuspended in PBS in different ratio at room temperature,phenomenon ofcoaggregation was detected after vortexing15sec and keep standing for20min.(2) Influence factors of bacteria concentration、buffer pH、sugarfermentation substrate、saliva、Ca2+on coaggregation were detected.(3)L.acidophilus ATCC4356was inoculated in MRS liquid medium and itssterile culture supernatant was obtained and concentrated to detect theinhibition of L.acidophilus ATCC4356metabolites on S.mutans UA159.Then concentrated supernatant was treated in60℃water bath for20minutes and detected its inhibition on S.mutans UA159.And thenconcentrated supernatant were digested with trypsin、pepsin、proteinase K、papain to obtain the diversity of inhibition on Streptococcus mutans andanalysis the active ingredient.The soluble protein in concentratedsupernatant was preliminary analysis by SDS-PAGE compared toconcentrated MRS.(4) BHI liquid medium plus S.mutans UA159、supernatant of L.acidophilus ATCC4356and BHI liquid medium in1/1ratio plus S.mutans UA159、 BHI liquid medium plus L.acidophilusATCC4356and S.mutans UA159in1/1ratio was designed in experimentfor biofilm formation. And then detected the influence of L.acidophilusATCC4356and its metabolites on S.mutans UA159by silver nitratestaining and scanning eletron microscopy. Results:(1) Coaggregation of L.acidophilus ATCC4356with S.mutansUA159was observed.(2) There were differences in bacteria concentrationon coaggregation and buffer pH、sugar fermentation substrate、saliva werenot. Coaggregation disappeared when bacterias were washed by EDTAand it can be restored by resuspended in Ca2+solution.(3) L.acidophilusATCC4356metabolites resulted in apparente inhibition zones on S.mutansUA159which was used as indicator bacteria. There was no significantchange after treatment in60℃water bath for20min.However, theinhibitory effect disappeared after treatment of trypsin、pepsin、proteinaseK、 papain. The main different soluble protein strip of concentratedsupernatant was at about6.3KD in SDS-PAGE.(4) There was differencebetween L.acidophilus ATCC4356plus S.mutans UA159coculture biofilmand S.mutans UA159pure culture biofilm. Besides, the obvious differenceof S.mutans UA159biofilms in BHI plus L.acidophilus ATCC4356culturesupernatant and pure BHI was observed.Conclusions:(1) L.acidophilus ATCC4356can coaggregate withS.mutans UA159.(2) This coaggregation occurred in the presence ofdifferent sugar substitutes、 wide pH range、 saliva,but it wascalcium-dependent.(3) L.acidophilus ATCC4356metabolites restrainedS.mutans UA159and effective antibacterial substance was defined asproteins under conditions eliminating the effects of organic acids andhydrogen peroxide. It was speculated that the protein may be molecular weight of6.3KD bacteriocine according to SDS-PAGE.(4)S.mutansUA159biofilm formation was inhibited by L.acidophilus ATCC4356andits metabolites...