In Vitro Selection of Lactobacillus and Bifidobacterium Probiotic Strains for the Management of Oral Pathobiont Infections Associated to Systemic Diseases

Abstract

The human oral pathobionts Aggregatibacter actinomycetemcomitans, Streptococcus mitis and Streptococcus mutans, in dysbiosis-promoting conditions, lead to oral infections, which also represent a threat to human systemic health. This scenario may be worsened by antibiotic misuse, which favours multi-drug resistance, making the research on pathogen containment strategies more than crucial. Therefore, we aimed to in vitro select the most promising probiotic strains against oral pathogen growth, viability, biofilm formation, and co-aggregation capacity, employing both the viable probiotics and their cell-free supernatants (CFSs). Interestingly, we also assessed probiotic efficacy against the three-pathogen co-culture, mimicking an environment similar to that in vivo. Overall, the results showed that Lactobacillus CFSs performed better than the Bifidobacterium, highlighting Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC04, and Limosilactobacillus fermentum LF26 as the most effective strains, opening the chance to deeper investigation of their action and CFS composition. Altogether, the methodologies presented in this study can be used for probiotic efficacy screenings, in order to better focus the research on a viable probiotic, or on its postbiotics, suitable in case of infections.

Growth Conditions Influence Lactobacillus Cell-Free Supernatant Impact on Viability, Biofilm Formation, and Co-Aggregation of the Oral Periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis

“…Interestingly, in this assay, only LBR01 and LS03 CFSs in MRS were able to significantly inhibit the two-pathogen co-aggregation, with LBR01 CFS showing a specific inhibition since the co-aggregation percentage was lower than the negative control. A similar result was also obtained in our previous work against the oral pathogens Aggregatibacter actinomycetemcomitans , Streptococcus mitis , and S. mutans [ 13 ]. However, when these CFSs were TIL-produced, no one was able to significantly reduce the two-pathogen co-aggregation.…”

“…Probiotic cell-free supernatants (CFSs) were produced as previously described [ 13 ]. Fresh probiotic cultures were inoculated with an optical density at 600 nm (OD 600 ) = 0.05 into MRS or TIL and grown ON.…”

“…Pathogen viability after CFS treatment was assessed as previously described, with few modifications [ 13 ]. Pathogens were plated independently at OD 600 = 0.05 in BHI (approximately 5 × 10 7 CFU/mL) into a 96-well plate, CFS treatment was immediately added to the bacterial culture to obtain a final volume/volume ratio of 50%, and then the plate was incubated in anaerobic conditions at 37 °C.…”

“…The biofilm formation of CFS-treated pathogens was determined through crystal violet (CV) staining, as previously described, with few modifications [ 13 ]. Pathogens were plated independently at OD 600 = 0.05 into a 48-well plate and immediately treated with probiotic CFSs (50% v / v ).…”

“…Auto- and co-aggregation assays were used to study the bacterial interactions, as previously described, with few modifications [ 13 ]. Fresh ON pathogen and probiotic cultures were centrifuged at 3000× g for 15 min at RT (Heraeus Megafuge 16R).…”