Manipulation of Saliva-Derived Microcosm Biofilms To Resemble Dysbiotic Subgingival Microbiota

Abstract

Periodontitis is a highly prevalent oral inflammatory disease triggered by dysbiotic subgingival microbiota. For the development of microbiome modulators that can reverse the dysbiotic state and re-establish a health-related microbiota, a high-throughput in vitro multi-species biofilm model is needed. Our aim is to establish a model that resembles a dysbiotic subgingival microbial biofilm by incorporating the major periodontal pathogen Porphyromonas gingivalis into microcosm biofilms cultured from pooled saliva of healthy volunteers. The biofilms were grown for 3, 7, and 10 days and analyzed for their microbial composition by 16S rDNA amplicon sequencing as well as dipeptidyl peptidase IV (DPP4) activity and butyric acid production. The addition of P. gingivalis increased its abundance in saliva-derived microcosm biofilms from 2.7% on day 3 to >50% on day 10, which significantly reduced the Shannon diversity, but did not affect the total number of operational taxonomic units (OTUs). The P. gingivalis-enriched biofilms displayed altered microbial composition as revealed by principle component analysis and reduced interactions among microbial species. Moreover, these biofilms exhibited enhanced DPP4 activity and butyric acid production. In conclusion, by adding P. gingivalis into saliva-derived microcosm biofilms, we established an in vitro pathogen-enriched dysbiotic microbiota, which resembles periodontitis-associated subgingival microbiota in terms of increased P. gingivalis abundance and higher DPP4 activity and butyric acid production. This model may allow for investigating factors that accelerate or hinder microbial shift from symbiosis to dysbiosis and for developing microbiome modulation strategies. IMPORTANCE In line with the new paradigm of the etiology of periodontitis, an inflammatory disorder initiated by dysbiotic subgingival microbiota, novel therapeutic strategies have been proposed, targeting reversing dysbiosis and restoring host-compatible microbiota, rather than eliminating the biofilms unselectively. Thus, appropriate laboratory models are required to evaluate the efficacy of potential microbiome modulators. In the present study, we used the easily obtainable saliva as an inoculum, spiked the microcosm biofilms with the periodontal pathogen Porphyromonas gingivalis, and obtained a P. gingivalis-enriched microbiota, which resembles the in vivo pathogen-enriched subgingival microbiota in severe periodontitis. This biofilm model circumvents the difficulties encountered when using subgingival plaque as the inoculum and achieves microbiota in dysbiotic state in a controlled and reproducible manner, which is required for high-throughput and large scale evaluation of strategies that can potentially modulate microbial ecology.

Porphyromonas gingivalis Outer Membrane Vesicles as the Major Driver of and Explanation for Neuropathogenesis, the Cholinergic Hypothesis, Iron Dyshomeostasis, and Salivary Lactoferrin in Alzheimer’s Disease

“…In vivo concentrations of free heme have been found to be too low (10-24 M) to support bacterial growth without the help of specialized heme acquisition systems produced by the bacteria themselves. Normal human saliva, however, has been demonstrated to support the complex poly-microbial biofilms associated with Pg [131]. Thus at least initially it seems that Pg in the oral cavity would exist in a heme normo/excess environment.…”

Hydrogel-mediated tumor T cell infiltration and immune evasion to reinforce cancer immunotherapy

“…In particular, the fluorescence intensity of AMC is positively correlated with the activity of DPP4. 26 Fig. 2A shows that S@LB hydrogel could significantly inhibit the DPP4 activity in tumors in vivo , thanks to the linagliptin in the hydrogel.…”

Dipeptidyl-Peptidase-4 and Glucagon-like-Peptide-1, a Link in the Connection between Periodontitis and Diabetes Mellitus—What Do We Know So Far?—A Scoping Review

“…In all biofilms, he discovered that DPP-4 activity significantly increased on day 10 in comparison to day 3. Nonetheless, compared to biofilms without the bacterium, those containing P. gingivalis exhibited much greater activity [25]. DPP4 activity may be seen in a variety of bacterial species.…”

“…A dysbiotic state may have been induced in the microcosm biofilm composition by the growing condition (serum in the growth medium, for example), which encouraged the development of DPP4-producing bacteria, thus promoting the development of biofilms and the colonization of mixed species in subgingival plaque. Because of this, these two biomarkers may be utilized to identify the dysbiotic condition of the microbiota in vitro, but not the quantity of P. gingivalis in biofilms [12,25].…”