Lactobacillus acidophilus LA-5 Ameliorates Inflammation and Alveolar Bone Loss Promoted by A. actinomycetemcomitans and S. gordonii in Mice and Impacts Oral and Gut Microbiomes.

The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1ß and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes.

Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells.

Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This in vitro study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by Aggregatibacter actinomycetemcomitans. OBA-9 GECs were infected with A. actinomycetemcomitans strain JP2 at an MOI of 1:100 and/or co-infected with Lactobacillus acidophilus La5 (La5) or Lacticaseibacillus rhamnosus Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1ß, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with A. actinomycetemcomitans or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of A. actinomycetemcomitans to GECs was 1.8 × 107 (± 1.2 × 106) cells/well in the mono-infection but reduced to 1.2 × 107 (± 1.5 × 106) in the co-infection with Lr32 and to 6 × 106 (± 1 × 106) in the co-infection with La5 (p < 0.05). GECs mono-infected with A. actinomycetemcomitans produced CXCL-8, GM-CSF, and IL-1ß, and the co-infection with both probiotic strains altered this profile. While the co-infection of A. actinomycetemcomitans with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1ß. The probiotics upregulated the expression of TLR2 and downregulated TLR4 in cells co-infected with A. actinomycetemcomitans. A. actinomycetemcomitans-induced the upregulation of NRLP3 was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene BCL-2 was upregulated, whereas the pro-apoptotic BAX was downregulated in cells co-infected with A. actinomycetemcomitans and the probiotics. Infection with A. actinomycetemcomitans induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in A. actinomycetemcomitans colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, L. acidophilus La5 properties suggest that this strain has a higher potential to control A. actinomycetemcomitans-associated periodontitis than L. rhamnosus Lr32.

Bifidobacterium Strains Present Distinct Effects on the Control of Alveolar Bone Loss in a Periodontitis Experimental Model.

Periodontitis is an inflammatory disease induced by a dysbiotic oral microbiome. Probiotics of the genus Bifidobacterium may restore the symbiotic microbiome and modulate the immune response, leading to periodontitis control. We evaluated the effect of two strains of Bifidobacterium able to inhibit Porphyromonas gingivalis interaction with host cells and biofilm formation, but with distinct immunomodulatory properties, in a mice periodontitis model. Experimental periodontitis (P+) was induced in C57Bl/6 mice by a microbial consortium of human oral organisms. B. bifidum 1622A [B+ (1622)] and B. breve 1101A [B+ (1101)] were orally inoculated for 45 days. Alveolar bone loss and inflammatory response in gingival tissues were determined. The microbial consortium induced alveolar bone loss in positive control (P + B-), as demonstrated by microtomography analysis, although P. gingivalis was undetected in oral biofilms at the end of the experimental period. TNF-α and IL-10 serum levels, and Treg and Th17 populations in gingiva of SHAM and P + B- groups did not differ. B. bifidum 1622A, but not B. breve 1101A, controlled bone destruction in P+ mice. B. breve 1101A upregulated transcription of Il-1ß, Tnf-α, Tlr2, Tlr4, and Nlrp3 in P-B+(1101), which was attenuated by the microbial consortium [P + B+(1101)]. All treatments downregulated transcription of Il-17, although treatment with B. breve 1101A did not yield such low levels of transcripts as seen for the other groups. B. breve 1101A increased Th17 population in gingival tissues [P-B+ (1101) and P + B+ (1101)] compared to SHAM and P + B-. Administration of both bifidobacteria resulted in serum IL-10 decreased levels. Our data indicated that the beneficial effect of Bifidobacterium is not a common trait of this genus, since B. breve 1101A induced an inflammatory profile in gingival tissues and did not prevent alveolar bone loss. However, the properties of B. bifidum 1622A suggest its potential to control periodontitis.

Lactobacilli postbiotics reduce biofilm formation and alter transcription of virulence genes of Aggregatibacter actinomycetemcomitans.

Periodontitis is characterized by a dysbiotic microbial community and treatment strategies include the reestablishment of symbiosis by reducing pathogens abundance. Aggregatibacter actinomycetemcomitans (Aa) is frequently associated with rapidly progressing periodontitis. Since the oral ecosystem may be affected by metabolic end-products of bacteria, we evaluated the effect of soluble compounds released by probiotic lactobacilli, known as postbiotics, on Aa biofilm and expression of virulence-associated genes. Cell-free pH-neutralized supernatants (CFS) of Lactobacillus rhamnosus Lr32, L. rhamnosus HN001, Lactobacillus acidophilus LA5, and L. acidophilus NCFM were tested against a fimbriated clinical isolate of Aa JP2 genotype (1 × 107  CFU/well) on biofilm formation for 24 hr, and early and mature preformed biofilms (2 and 24 hr). Lactobacilli CFS partially reduced Aa viable counts and biofilms biomass, but did not affect the number of viable non-adherent bacteria, except for LA5 CFS. Furthermore, LA5 CFS and, in a lesser extent HN001 CFS, influenced Aa preformed biofilms. Lactobacilli postbiotics altered expression profile of Aa in a strain-specific fashion. Transcription of cytolethal distending toxin (cdtB) and leukotoxin (ltxA) was downregulated by CFS of LA5 and LR32 CFS. Although all probiotics produced detectable peroxide, transcription of katA was downregulated by lactobacilli CFS. Transcription of dspB was abrogated by LR32 and NCFM CFS, but increased by HN001, whereas expression of pgA was not affected by any postbiotic. Our data indicated the potential of postbiotics from lactobacilli, especially LA5, to reduce colonization levels of Aa and to modulate the expression of virulence factors implicated in evasion of host defenses.