The potential bactericidal effect of oral probiotics on extrinsic black stain-associated bacteria in children: An in-vitro and in-vivo study.

2025-10
Journal of dental sciences 20(4)

PubMed: 41040574
DOI: 10.1016/j.jds.2025.04.032

Study Design

Population: children with extrinsic black stain (EBS)
Methods: In-vitro testing of three probiotic strains against Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii using minimum inhibitory concentration (MIC) testing; in-vivo collection of unstimulated saliva samples from patients with EBS before and after 14 days of probiotic administration

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.

Research Insights

Supplement	Dose	Health Outcome	Effect Type	Effect Size	Source
Streptococcus salivarius BLIS M18	—	Reduced Bacterial Load	Beneficial	Moderate	View source 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.