Abstract
BACKGROUND/PURPOSE: The extrinsic black stain (EBS) is a resistant esthetic problem in children. The aim of this study was to investigate the bactericidal effect of three probiotics strains on two types of EBS-associated bacteria in-vitro and in-vivo in children. MATERIALS AND METHODS: In the in-vitro study, two bacterial strains (Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii) were revived and incubated under anaerobic or microaerophilic conditions. Cultures were grown in Brain Heart Infusion broth, adjusted to a 0.5 McFarland standard, and diluted to 1.0 × 10⁶ CFU/mL for minimum inhibitory concentration (MIC) testing. Three Probiotic strains (Streptococcus Salivarius BLIS M18, Lactobacillus reuteri LR08, Lactobacillus paracasei Lpc- 37) were similarly cultured and processed, and the MIC was determined using a 96-well microtiter plate, with bacterial growth assessed visually and spectrophotometrically. In the in-vivo study, unstimulated saliva samples were collected from patients with EBS before and after 14 days of probiotic administration. Microbial DNA was extracted and quantified using spectrophotometry. RESULTS: In the in-vitro study, bacterial growth declined progressively with increasing probiotic concentrations, confirming a dose-dependent inhibitory effect. Statistical analysis showed significant reductions for both species (P < 0.001) compared to controls, with A. actinomycetemcomitans exhibiting a stronger response. In the in-vivo study, A.actinomycetemcomitans was fully eradicated, while A. naeslundii showed only partial suppression. CONCLUSION: Probiotics significantly inhibited the growth of EBS-associated bacteria in both in- vitro and in-vivo settings, resulting in the elimination of A. actinomycetemcomitans and a marked reduction in A. naeslundii count.