ect of a probiotic strain (Lactobacillus acidophilus) on the plaque formation of oral Streptococci

Th e objective of this study was to investigate the ability of biofi lm formation among mutans and non mutans oral streptococci and to determine the eff ect of Lactobacillus acidophilus DSM  as a probiotic strain on the adhesion of selected streptococcal strains on the surfaces. Th e sample comprised  isolates of oral streptococci from dental plaque and caries of volunteer persons. Streptococcus mutans ATCC (no) was as an standard strain. Th e probiotic strain was Lactobacillus acidophilus DSM . Th e ability of biofi lm formation was investigated with colorimetric method and the strongest isolates were selected. Th en the eff ect of probiotic strain on the adhesion of streptococci isolates was determined in polystyrene microtiter plate simultaneously and  minutes before streptococci entrance to the system. Th e results showed that  of mutans streptococci were strongly adherent (SA) and in non mutans streptococci, only . of isolates were found strongly adherent. Th e strong biofi lm forming bacterium isolated was Streptococcus mutans strain. In the next step, in the presence of probiotic strain the streptococcal adhesion were reduced, and this reduction was non signifi cantly stronger if the probiotic strain was inoculated to the system before the oral bacteria. Th e Lactobacillus acidophilus had more eff ect on adherence of mutans streptococci than non mutans streptococci with signifi cant diff erence (p < .). Adhesion reduction is likely due to bacterial interactions and colonization of adhesion sites with probiotic strain before the presence of streptococci. Adhesion reduction can be an eff ective way on decreasing cariogenic potential of oral streptococci. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


Introduction
Dental caries and periodontal disease are major public health problems that disturb all countries in the world []. Industrialized nations have controlled the problem with fluoride enriched water and personal hygiene products since early in the s, but cariogenicity remains a crisis that economically burdens the health care system. Dental disease remains a "silent epidemic" in the world that threatens children and adults [, ]. The oral streptococci especially mutans streptococci are related with the development of caries in humans and animals []. Th e adhesion of oral streptococci such as Streptococcus mutans to tooth surfaces has the major role in their pathogenicity. Going along with the increasing antibiotic resistance of bacteria, new methods such as whole bacteria replacement therapy for decreasing of oral cavity pathogens must be investigated [, ]. In general, a probiotic, is a live microorganism which benefi cially aff ects the host animal by improving its intestinal microbial balance. It's valuable eff ects may be mediated by direct antagonistic effect against specific groups of organisms, resulting in a decrease in numbers or by an eff ect on their metabolism or by stimulation of immunity [, -]. Lactobacillus acidophilus is a widely studied probiotic bacterium. So, in this study we have determined the effect of Lactobacillus acidophilus DSM  as a probiotic strain on the adhesion of oral streptococci to the surfaces.

Strains and culture condition
In this study we isolated  strains of streptococci from dental plaque and caries of applicants with mean age of  years old with the help of dentistry sterile curettes. Th e standard strain was Streptococcus mutans ATCC . All strains were cultured on Blood agar and mitis salivarius agar media and incubated in CO  enriched, °C atmosphere. Strain's identifi cation was done with usual biochemical tests and Rapid identifi cation kit of Streptococci (Rap ID STR kit). Lactobacillus acidophilus DSM  was cultured in MRS broth or Agar.

Biofi lm production assay by microtiter plate test
To assess the biofi lm formation potential of the isolates, an overnight culture of each was grown in tryptic soy broth supplemented with  sucrose (TSB, Merck, Germany) for  - h at  °C. Th e suspensions were adjusted with TSB to . on the McFarland turbidity standard as measured by absorbance (. -. at  nm) in a spectrophotometer, corresponding to approximately   cfu/ml. Then, from each culture,  micro liter (μl) volumes were transferred into eight wells of a microtiter plate (×  cfu/well). Blank wells contained broth, only. Plates were made in duplicate, covered, and incubated for  h at °C. At  h, the planktonic suspension and nutrient solutions were aspirated and each well was washed three times with  μl of sterile physiological saline. Th e plates were vigorously shaken in order to remove all non adherent bacteria. Th e remaining attached bacteria were fi xed with  μl of  ethanol per well and, after  min, plates were emptied and left to dry. Each well was then stained for  min with . ml of  crystal violet. Excess stain was rinsed off by placing the plates under running tap water. Crystal violet (CV) stain is suitable for determining the amount of biofi lm. After drying the stained plates, biofi lms were visible as purple rings formed on the sides of each well. Th e quantitative analysis of biofi lm production was performed by adding  μl of  (v/v) glacial acetic acid (Merck, Germany) per well. Th en the optical density (OD) of the stain was measured at  nm by an ELISA reader (STAT-FAX ) as described previously []. Based on the OD of the bacterial fi lms, strains were classifi ed into the following categories, as previously described by Stepanovic et al. []: OD<ODc: not a biofilm producer (Non adherent); ODc<OD< ODc: a weak biofi lm producer (Weakly adherent); ODc < OD< ODc: a moderate biofi lm producer (Moderately adherent); ODc < OD: a strong biofilm producer (Strongly adherent). ODc and OD were defi ned as the mean OD of the blank wells and wells with biofi lm, respectively.

Th e eff ect of Lactobacillus acidophilus on biofi lm formation
To assess the eff ect of Lactobacillus acidophilus on biofi lm formation, an overnight culture of each isolate was grown in tryptic soy broth supplemented with  sucrose. An overnight culture of the probiotic strain in MRS broth was also prepared. The suspensions were adjusted with the same broth to . on the McFarland turbidity standard as measured by absorbance (. -. at  nm) in a spectrophotometer, corresponding to approximately   cfu/ml. Biofilm formation was done according to description above with and without probiotic strain. In brief, adding mixed suspension of probiotic and streptococci (:) simultaneously and alternatively a suspenssion of probiotic strain was inoculated into the wells  min before the streptococcal suspension. Blank wells contained PBS instead of probiotic strains. After  hours incubation and washing  times with PBS, staining with CV was done. Th e Optical density was read with ELISA reader [].

Statistical analysis
Th e data were analyzed using Duncan's test of SAS software (Version .).

Results
Isolated strains were tested using the PVC microtiter biofi lm assay for biofi lm production. Biofi lm formation at  h incubation varied greatly. According to the OD values, ,  and  of Mutans streptococci were Strongly Adherent (SA), Modereately Adherent (MA) and Weakly Adherent (WA), respectively. In non mutans streptococci, only . of isolates were found strongly adherent and . of isolates were weakly adherent; these data showed signifi cant diff erences (p<.) with the same Mutans streptococci group ( Figure ).
According to the OD values again, Streptococcus mutans  with highest OD were scored as highly strong adherent (strong biofilm forming bacterium) and Streptococcus mutans ATCC  (no ) was also strongly adherent. Based on the microtiter plate test results, the streptococcal isolates with the strong ability of biofi lm formation were selected. Th en the eff ect of Lactobacillus acidophilus on the adherence of streptococci to microtiter plate was assessed by  methods (: Mixed suspension of streptococci and probiotic, : Adding streptococci isolates  min after probiotic strain). Th e results showed the adherence reduction in the presence of Lactobacillus acidophilus (Figures  and ). Th e adherence reduction of Streptococcus mutans ATCC  (strain ) to poly styrene wells in the presence of probiotic by two used methods showed signifi cant diff erences (. and . respectively (p<.)). Th is adherence reduction for strong biofi lm forming bacterium (Streptococcus mutans ) was significantly (p<.) lower than about standard strain (figure  A) and there were not significant differences between  used methods in the case of this bacterium. The mean of adherence reduction in  methods was . and .  respectively. So, there is no significant difference between the  methods ( Figure A) but in the case of each bacterium it could be different. In general, the Lactobacillus acidophilus had more effect on adherence of mutans streptococci than non mutans streptococci with significant difference (p <.) ( Figure B).

Discussion
Lactobacillus acidophilus was preferred in this study as a probiotic bacterium because of it`s known probiotic potential and it's acid resistance and bile salt`s tolerance []. Th is bacterium such as other lactic acid bacteria produces various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocins or bactericidal proteins []. According to the results, it is cleared that the presence of Lactobacillus acidophilus DSM  can cause reduction in the adherence of Streptococcal strains that it is probably related to interaction between bacteria. Th is adherence reduction was signifi cantly stronger in the case of mutans streptococci while in the other study we showed that Lactobacillus fermentum reduced the adherence of non mutans streptococci more than mutans streptococci without any signifi cant diff erence []. Inoculation of Lactobacillus acidophilus before Streptoccocal isolates (method ) had more eff ect on adherence reduction without any signifi cant diff erence. But in the case of Lactobacillus fementum adherence reduction was significantly stronger (p<.) if the probiotic strain was inoculated to the system before the oral bacteria []. It is thought that adhesion reduction is likely due to bacterial interactions and colonization of adhesion sites with probiotic strain before the presence of streptococci. Also, the probiotic strains were able to modify the proportion of the oral species within the biofi lm. Comelli et al. [] reported that inoculation of dairy strains before adding the oral bacteria did not increase their colonization. Th ey also found that dairy strains and particularly Lactobacillus lactis NCC were able to modify the extent of oral species within the biofi lm. Th ey also suggest that the reduction of these strains can be explained either by competition for adhesion sites or growth factors. Miller et al. [] in their study about the eff ect of microbial interaction on in vitro plaque formation by Streptococcus mutans found that microbial interaction may have the potential to affect the amount and type of plaque formed, depending upon the kinds of organisms involved. Th ey also reported that the addition of the lactobacilli to cultures of Streptococcus sanguis resulted in more inhibition of plaque formation when compared with pure cultures of Streptococcus sanguis. A  inhibition of plaque formation was observed when Lactobacillus casei interacted with Streptococcus mutans NCTC . Furthermore Simark-Mattsson et al. [] have shown the interference capacities of lactobacilli against strains of Streptococcus mutans and Streptococcus sobrinus. Meurman [] showed the inhibitory activity of Lactobacillus rhamnosus GG against Streptococcus mutans in low pH and it can be useful for preventing the cariogenic eff ects of oral streptococci. In vivo studies have also confi rmed the effects of probiotic bacteria consumption on decreasing the risk of dental caries and mutans streptococcus counts [-].

Conclusion
Adhesion reduction can be an effective way on decreasing cariogenic potential of oral streptococci and all of the evidence has shown that probiotic bacteria such as Lactobacillus spp. can affect the oral ecology.

Declaration of Interest
Th ere is no confl ict of interest in this study.