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.

Probiotics alter the immune response of gingival epithelial cells challenged by Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Although previous studies revealed the potential use of probiotics in the control of periodontitis, little is known about their interactions with gingival epithelial cells (GECs). Since GECs comprise the first defense in the subgingival microenvironment, the aim of this study was to evaluate the effect of probiotic lactobacilli and bifidobacteria strains on OBA-9 cells challenged with Porphyromonas gingivalis. METHODS: Immortalized human GECs (OBA-9) were challenged with live P. gingivalis (strains W83 and ATCC33277) and co-infected with one of 12 tested probiotic strains at a multiplicity of infection (MOI) of 1:1000 for 2 hours. Bacterial adhesion and invasion were determined by antibiotic exclusion analysis and CFU counting. OBA-9 viability was assessed by MTT assay, and levels of inflammatory mediators (TNF-α, IL-1ß, and CXCL8) in the supernatants were determined by ELISA. The expression of genes encoding Toll-like receptors (TLR2, TLR4) was evaluated by RT-qPCR. RESULTS: Both strains of P. gingivalis were able to adhere and invade OBA-9 cells, with significant loss in cell viability, increase in the levels of TNF-α and IL-1ß, and upregulation of TLR4. However, co-infection with probiotics attenuated these effects in P. gingivalis challenged GECs. Most probiotics maintained OBA-9 viability and reduced pathogens adhesion and invasion. Furthermore, probiotics were able to adhere to GECs, which was enhanced for most strains in the presence of P. gingivalis. The synthesis of IL-1ß and TNF-α by P. gingivalis in challenged GECs was reduced in co-culture with most of the tested probiotics, whereas the secretion of CXCL8 increased, and TLR4 was downregulated. CONCLUSION: Probiotics can alter the interaction of GECs with P. gingivalis by modulating the pathogen's ability to adhere and invade these cells, as well as by regulating the innate immune response. Such properties are strain-specific and may indicate the most efficient probiotics to control periodontitis.

Quantification of Porphyromonas gingivalis and fimA genotypes in smoker chronic periodontitis.

AIM: Porphyromonas gingivalis fimA genotypes were associated with virulence factors in vitro, but little evidence of an association with disease severity were shown in humans. We aimed to correlate levels of P. gingivalis fimA genotypes II and IV and probing depth in smoker-chronic periodontitis subjects. MATERIAL AND METHODS: One hundred and sixty eight subgingival samples of 20 smokers non-treated chronic periodontitis subjects obtained from sites with different probing depths [shallow (< or =3 mm), intermediate (4-6 mm), deep (> or =7 mm)] were analysed by real-time PCR for P. gingivalis and genotypes fimA II and IV. RESULTS: P. gingivalis and fimA IV were detected in all subjects, whereas fimA II was detected in 18 subjects (90%). One hundred and fifty two sites (90.5%) harboured P. gingivalis. Genotypes II and IV were detected in 28% and 69.6% of sites, respectively. The proportions of genotypes II and IV in relation to P. gingivalis levels were similar in shallow, intermediate and deep probing sites (2.4%, 4.6%, 1.4% for genotype II and 15.5%, 17.7%, 11.7% for genotype IV, respectively), indicating that other non-tested genotypes were more abundant. Increased levels of genotype IV were associated with increasing probing depth, but not of genotype II. CONCLUSIONS: The data suggested an association between P. gingivalis genotype fimA IV and disease severity in smoker-chronic periodontitis subjects.