Table 1.
List of bacterial strains used in this study.
Fig 1.
Biofilm values of A. actinomycetemcomitans strains with addition of probiotic bacteria on pre-formed biofilm.
(A) represents Y4 strain (serotype b). Whereas (B and C) represents SUNY75 strain (serotype a) and OMZ534 strain (serotype e) respectively. Probiotic species and controls were labelled with number as follows; 1: L. fermentum JCM 1137, 2: L. acidophilus JCM 1021, 3: L. fermentum NBRC 15885, 4: L. fructosum NBRC 3516, 5: L. plantarum NBRC 15891, 6: L. casei subsp. rhamnosus NBRC 3831, 7: L. johnsonii NBRC 13952. Positive controls (8) are A. actinomycetemcomitans biofilm without probiotic addition, and A. naeslundii JCM 8349 was used as a negative control (9). Bars represent the mean, error bars represent standard deviation and significance was measured using paired T-test (* = P< 0.05, ** = P< 0.001).
Table 2.
Degradation percentage after the addition of probiotic cells on pre-formed A. actinomycetemcomitans strains.
Fig 2.
Percentage of biofilm degradation by probiotic cells or cell-free supernatant (concentrated) against A. actinomycetemcomitans Y4 biofilm.
Black bars represent biofilm degradation by cells and white columns represent biofilm degradation by a cell-free supernatant. Bars represent the mean, error bars represent standard deviation.
Fig 3.
Influence of culture medium nutrient and cell viability on biofilm degradation activity.
Fig 3A represents a comparison of biofilm degradation by probiotic cells in a nutrient rich medium and a co-aggregation buffer. The nutrient rich medium contained a low density of probiotic cells (OD 0.05 at 600 nm absorbance). Washed cell pellets from an overnight culture were co-incubated with pre-formed A. actinomycetemcomitans Y4 biofilm to form the co-aggregation buffer. Fig 3B represents biofilm degradation by dead probiotic cells (autoclaved). Bars represent the mean, error bars represent standard deviation.
Fig 4.
Co-incubation of pre-formed A. actinomycetemcomitans Y4 biofilms with probiotic cells leading to biofilm degradation.
A. actinomycetemcomitans Y4 biofilm was allowed to form under static anaerobic conditions for 72 h before a further 24 h co-culture with probiotic cells in a co-aggregation buffer. Biofilm with the addition of a co-aggregation buffer was used as a control. Grey columns represent biofilm formation and empty circles represent viable A. actinomycetemcomitans Y4 from the biofilm supernatant. Bars and circle represent the mean, error bars represent standard deviation.
Fig 5.
Influence of probiotic bacteria supernatant and lactic acid on biofilm growth.
Percent biofilm degradation following (A) the addition of an untreated cell-free supernatant (black column) or an adjusted pH (6.5) cell-free supernatant (gray column). (B) Biofilm growth of A. actinomycetemcomitans Y4 with lactic acid at various concentrations compared with the addition of probiotic cells. Biofilm of A. actinomycetemcomitans Y4 was allowed to pre-form under static anaerobic conditions for 72 h prior to the addition of lactic acid or probiotic cells. Bars represent the mean, error bars represent standard deviation and significance was measured using paired T-test (* = P< 0.05, ** = P< 0.001).
Fig 6.
Effect of enzymes and influence of lipase inhibitor on the formed biofilm (A, C and E) show degradation activity by the presence of proteinase K, amylase, lipase, or a combination of all enzymes against Y4, SUNY 75, and OMZ 534 respectively. (B, D and F) are the influence of lipase inhibitor on biofilm degradation against Y4, SUNY 75 and OMZ 534 respectively. Probiotic strains and controls were labelled as follows. 1: Positive control (A. actinomycetemcomitans biofilm without the addition of probiotic cells), 2: L. fermentum NBRC 15885, 3: L. casei subsp. rhamnosus NBRC 3831, 4: L. fructosum NBRC 3516, 5: L. acidophilus JCM 1021, 6: L. johnsonii NBRC 13952, 7: L. plantarum NBRC 15891, 8: A. naeslundii JCM 8439 (negative control species). Bars represent the mean, error bars represent standard deviation and significance was measured using paired T-test (* = P< 0.05, ** = P< 0.001).
Fig 7.
Profile of lipase activity from an overnight culture in a supernatant of probiotic bacteria.
Probiotic strains and controls were labelled was follows. 1: Positive control (lipase enzyme with final concentration 0.01 mg/mL), 2: L. fermentum NBRC 15885, 3: L. casei subsp. rhamnosus NBRC 3831, 4: L. fructosum NBRC 3516, 5: L. acidophilus JCM 1021, 6: L. johnsonii NBRC 13952, 7: L. plantarum NBRC 15891, 8: A. naeslundii JCM 8439 and 9: negative control (sterilized distilled water). Bars represent the mean, error bars represent standard deviation.