Bifidobacterium animalis subsp lactis HN019 presents antimicrobial potential against periodontopathogens and modulates the immunological response of oral mucosa in periodontitis patients

Abstract

Objective: To evaluate the effects of Bifidobacterium animalis subsp. lactis HN019 (HN019) on clinical periodontal parameters (plaque accumulation and gingival bleeding), on immunocompetence of gingival tissues [expression of beta-defensin (BD)-3, toll-like receptor 4 (TLR4), cluster of differentiation(CD)-57 and CD-4], and on immunological properties of saliva (IgA levels) in non-surgical periodontal therapy in generalized chronic periodontitis (GCP) patients. Adhesion to buccal epithelial cells (BEC) and the antimicrobial properties of HN019 were also investigated.

Materials and methods: Thirty patients were recruited and monitored clinically at baseline (before scaling and root planing-SRP) and after 30 and 90 days. Patients were randomly assigned to Test (SRP+Probiotic, n = 15) or Control (SRP+Placebo, n = 15) group. Probiotic lozenges were used for 30 days. Gingival tissues and saliva were immunologically analyzed. The adhesion of HN019 with or without Porphyromonas gingivalis in BEC and its antimicrobial properties were investigated in in vitro assays. Data were statistically analyzed (p<0.05).

Results: Test group presented lower plaque index (30 days) and lower marginal gingival bleeding (90 days) when compared with Control group. Higher BD-3, TLR4 and CD-4 expressions were observed in gingival tissues in Test group than in Control group. HN019 reduced the adhesion of P. gingivalis to BEC and showed antimicrobial potential against periodontopathogens.

Conclusion: Immunological and antimicrobial properties of B. lactis HN019 make it a potential probiotic to be used in non-surgical periodontal therapy of patients with GCP.

Clinical relevance: B. lactis HN019 may be a potential probiotic to improve the effects of non-surgical periodontal therapy. Name of the registry and registration number (ClinicalTrials.gov): "Effects of probiotic therapy in the treatment of periodontitis"-NCT03408548.

Conflict of interest statement

E.I DuPont de Nemours & Co. Danisco Sweeteners Oy provided financial support in the form of a salary for ACO and donated probiotics. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Flowchart of the study design.

Fig 2

IgA levels (A) of Test and Control groups at baseline and at 30 and 90 postoperative days. Mean changes in IgA levels relative to baseline values obtained for the Test and Control groups are shown in (B). Ratio of IgA levels = changes in IgA levels in relation to baseline values. n.s* No significant changes were observed in IgA levels at both 30 and 90 days, compared to baseline, for Test and Control groups. n.s** No significant differences between Test and Control groups at both 30 and 90 days.

Fig 3. Relative frequency values for BD-3 and TLR4 immunolabeling scores in Test and Control groups at healthy and diseased sites at baseline and at 30 postoperative days, as well as within-group and between-group comparison results.

*Significant difference (Kruskal-Wallis, Dunn, p<0.05). *Significant difference when compared to Test baseline and Control baseline. **Significant difference when compared to Test baseline. §Significant difference when compared to Control baseline. ¶Significant difference when compared to Test baseline. #Significant difference when compared to Control 30 days.

Fig 4. Relative frequency values for CD-57 and CD-4 immunolabeling scores for test and control groups at healthy and diseased sites at baseline and at 30 postoperative days, as well as within-group and between-group comparison results.

*Significant difference (Kruskal-Wallis, Dunn, p

Fig 5. Photomicrographs showing BD-3 (A-H) and TLR4 (I-P) immunolabeling in gingival biopsies at baseline (A, C, E, G, I, K, M, O) and 30 days (B, D, F, H, J, L, N, P) from healthy (A-D, I-L) and diseased (E-H, M-P) sites in Test (A, B, E, F, I, J, M, N) and Control (C, D, G, H, K, L, O, P) groups.

Arrows = BD-3 and TLR4-positive cells; ep = Epithelial tissue; ct = Connective tissue. Scale = 25 μm. Counterstaining: Hematoxylin.

Fig 6. Photomicrographs showing CD-57 (A-H) and CD-4 (I-P) immunolabeling in gingival biopsies (connective tissue) at baseline (A, C, E, G, I, K, M, O) and 30 days (B, D, F, H, J, L, N, P) from healthy (A-D, I-L) and diseased (E-H, M-P) sites in Test (A, B, E, F, I, J, M, N) and Control (C, D, G, H, K, L, O, P) groups.

Arrows = CD-57 and CD-4 positive cells. Scale = 25 μm. Counterstaining: Hematoxylin.

Fig 7. Photomicrographs showing buccal epithelial cells in adhesion assays using crystal violet and safranin.

(A) P. gingivalis alone attached in a buccal cell (original magnification x100). (B)–B. lactis HN019 alone attached in a buccal cell (original magnification x100). (C)–Mixture of B. lactis HN019 with P. gingivalis attached in a buccal cell (original magnification x100). Red arrows indicate P. gingivalis (Gram-negative bacteria with shorter rods or coccobacilli morphology). Blue arrows indicate Bifidobacterium (Gram-positive bacteria with long bacilli morphology).

Fig 8. Growth inhibition of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum by Bifidobacterium animalis subs. Lactis HN019 (agar diffusion method).

(A) Clear areas around the wells indicate zones of inhibition. (B) Means and standard deviations of the zone of inhibition observed in sensitivity of different periodontopathogens to B. lactis HN019.