EFFECT OF LACTOBACILLUS ACIDOPHILUS AND STREPTOCOCCUS SALIVARIUS ON GROWTH OF PERIODONTAL PATHOGENS – AN IN-VITRO STUDY.

Background: Probiotics are defined as “live bacteria that naturally occur in certain foods as live micro-organisms which, when administered in adequate amount, confer a health benefit on the host.” They are safe for human consumption only when ingested in certain quantity, have beneficial effects on human health. Literature pertaining to the antimicrobial activity of probiotics is very limited. So this study was conceptualized to comprehensively report the antimicrobial potential of probiotics against periodontal pathogens. Objectives: To determine the effect of Lactobacillus Acidophilus and Streptococcus Salivarius probiotics on periodontal pathogens, (Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia) in-vitro using well diffusion method. Methods: Actinobacillus actinomycetemcomitans was cultured using tryptic Porphyromonas gingivalis was cultured using modified Wilkins-Chalgren (MWC) medium and Prevotella intermedia was cultured using brain heart infusion agar and incubated in Brewer-like anaerobic jars. Plates were inoculated with the probiotic strain of the lactobacillus acidophilus and Salivarius. Zone of inhibition was measured and compared with unaided eye using a Vernier caliper measuring the zone sizes. Results: Comparison of Zone of Inhibition after 24 hours and 48 hours using one way ANOVA test showed statistically significant results with inoculation of probiotics. Post hoc Tukey test compared within groups that showed the statistically significant difference between all organisms except for within A. actinomycetumcomitans and Prevotella intermedia. Conclusions: Lactobacillus and S. salivarius had inhibitory effect on periodontal pathogens in the oral microflora.

Antimicrobial Tests:-About 15-20 ml Mueller-Hinton agar was poured and allowed to solidify on glass petro plates of same size. All plates had agar surfaces, which was streaked by a sterile cotton swab with the known bacterial strain. Plates were punched with a sterile cork borer of 4 mm size. About 100 μL of each sample were poured with micropipette into the bore. For 30 min plates were kept. Then plates were incubated at 37°C for 48 hours. That's how Lawn cultures were made for these pathogens. Three culture plates were done per organism, resulting in a total of 12 plates for the four pathogens. The first plate was inoculated with the strain of the probiotic lactobacillus acidophilus, the second plate was inoculated with the strain of the probiotic S. salivarius and the third plate was kept as the control. This procedure was carried out for all the four pathogens. They were then incubated for 24 and 48 hours. After incubation, the zone of inhibition was measured and compared with unaided eye using a Vernier caliper measuring the zone sizes to the nearest millimeter.

Results:-
When lactobacillus was inoculate, comparison of Zone of Inhibition (ZOI) was done after 24 hours using one way ANOVA test showed that the mean of Zone of Inhibition value is highest in P. gingivalis (1.224) than A. actinomycetumcomitans (1.188) and P. intermedia (table no 1). This difference is statistically significant with a test value of 59.13(p <0.001). Post hoc Tukey test showed that the difference between P. gingivalis and A. actinomycetumcomitans was statistically significant with a mean difference of 0.188 (p <0.001). When Zone of Inhibition of P. intermedia and P. gingivalis was compared it was found to be statistically significant with a mean difference of -.224 (p <0.001).
Comparison of ZOI was done after 48 hours using lactobacillus, one way ANOVA test showed that the mean Zone of Inhibition value is highest in A. actinomycetumcomitans (1.416) followed by P. gingivalis (1.304) least in P. intermedia (1.086). This difference in Zone of Inhibition was statistically significant with a test value of 124.578 (p <0.001). Post hoc Tukey test showed that the difference between P. gingivalis and A. actinomycetumcomitans was statistically significant with a mean difference of 0.33 (p <0.001). The difference in Zone of Inhibition between Actinomycetumcomitans and P. gingivalis was statistically significant with a mean difference of 0.112 (p <0.001). The difference between P. intermedia and P. gingivalis was also statistically significant with a mean difference of 0.218 (p <0.001).
When S. salivarius was inoculated, comparison of ZOI mm was done after 24 hours using one way ANOVA test showed that the mean Zone of Inhibition value was highest in A. actinomycetumcomitans (0.81) followed by P. gingivalis (0.714) least in P. intermedia (0.598). This difference was statistically significant with a test value of 184.8 (p <0.001). The difference between mean Zone of Inhibition of A. actinomycetumcomitans and P. gingivalis was statistically significant with a mean difference of 0.096 (p=0.003). The difference between A. actinomycetumcomitans and P. intermedia was statistically significant with a mean difference of 0.212 (p <0.001). The difference between Zone of Inhibition of P. gingivalis and P. gingivalis was statistically significant with a mean difference of 0.116 (p <0.001).
Comparison of ZOI mm 48 hours was measured for S. salivarius using one way ANOVA test showed that the mean value of Zone of Inhibition is highest in A. actinomycetumcomitans (0.97) followed by P. gingivalis (0.914) least in P. intermedia (0.826). The difference in Zone of Inhibition between P. gingivalis and A. actinomycetumcomitans was statistically significant with a mean difference of 0.056 (p <0.001). The difference between actinomycetumcomitans and P. intermedia was statistically significant with a mean difference of 0.144 (p <0.001). The difference between P. intermedia and P. gingivalis was statistically significant with a mean difference of .088 (p <0.001).

Discussion:-
Inter-microbial interactions, and colonization in the oral cavity are considered to be of major significance for probiotic actions when they are integrated into the oral biofilm in the oral cavity. 9,10 In this study, our aim was to compare zone of inhibition of the inoculated probiotics in vitro and to compare their effects on the growth of other oral bacteria. Lactobacillus acidophilus and S. salivarius are important members of a healthy oral microflora bacteria as shown by previous study. 13, Therefore, the role of probiotics bacteria in the microbiota is important for the oral colonization of lactobacillus acidophilus and S. salivarius, and our study supports the fact that the colonization of oral indigenous bacteria is affected by probiotic bacteria.
In our study, the growth of all pathogens P. gingivalis, P. intermedia and A. actinomycetumcomitans were reduced significantly, this was probably due to a combination of competition for nutrients and result of the acidic conditions generated by lactobacillus acidophilus. The growth inhibition of P. gingivalis, P. intermedia, A. actinomycetumcomitans by oral lactobacillus was related to the ability of lactobacillus to reduce the mutual growth factor vitamin K. 17 Zhu et al in 2010 performed growth-inhibition study 13 showed probiotic Bifidobacterium inhibited the growth of P. gingivalis when inoculated first there was inhibition initially, but later no inhibition was seen when both Bifidobacterium and P. gingivalis were inoculated together at the same time. 13 The reason for inhibition was the result of the of inhibitory substances produced by probiotic bacteria or competition for nutrients. 13 In our study, the increased zone of P. gingivalis in culture containing lactobacillus might be due to a result of the increased level of probiotics. 18 Piwat et al 19 conducted an in-vitro study which explains how adhesion, aggregation and co-aggregation mechanisms of probiotic bacteria can influence the growth of other periodontal pathogens. These organisms attach to the oral hard tissues more strongly than pathogens, by competing for the adhesion surfaces. In our study zone of inhibition may have formed because the probiotics have inhibited the periodontal pathogen's active multiplication the same way. 19,20 A study by Morales A, et al showed the additional use of L. Rhamnosussachets provided similar results when compared with scaling and root planning, thus showing benefit of probiotics. 21 One of the suggested mechanism stated by Hojo et al says that lactobacillus acidophilus and P. gingivalis compete with each other for vitamin K and inhibits the growth of latter by possibly competing for the growth factor in a co-culture. 17 Gruner D, et al 22 included clinical trials which measured the influence of Lactobacilli for probiotic therapy in periodontal pathogens and there was no significant differences when compared with the control group. 22 This shows the need of further studies to establish our positive findings.