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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 reestablish a health-associated microbiota, a high-throughput in vitro multispecies 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 rRNA gene amplicon sequencing as well as measurement of 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 principal-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 to 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 a 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 a 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.

SUBMITTER: Jiang Y

PROVIDER: S-EPMC7848911 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Screening for microbiome modulators requires availability of a high-throughput in vitro model that replicates subgingival dysbiosis and normobiosis, with a tool to measure microbial dysbiosis. Here, we tested various formulations to grow health- and periodontitis-associated subgingival microbiomes in parallel, and we describe a new subgingival dysbiosis index. Subgingival plaque samples pooled from 5 healthy subjects and, separately, 5 subjects with periodontitis were used to inoculate a Calgary Biofilm Device containing saliva-conditioned, hydroxyapatite-coated pegs. Microbiomes were grown for 7 d on either nutrient-rich media-including a modification of SHI medium, brain-heart infusion (BHI) supplemented with hemin and vitamin K, and a blend of SHI and BHI, each at 3 sucrose concentrations (0%, 0.05% and 0.1%)-or nutrient-limited media (saliva with 5%, 10%, or 20% inactivated human serum). The microbiomes were assessed for biomass, viability, and 16S rRNA profiles. In addition to richness and diversity, a dysbiosis index was calculated as the ratio of the sum of relative abundances of disease-associated species to that of health-associated species. The supplemented BHI and blend of SHI and BHI resulted in the highest biomass, whereas saliva-serum maximized viability. Distinct groups of bacteria were enriched in the different media. Regardless of medium type, the periodontitis-derived microbiomes showed higher species richness and alpha diversity and clustered with their inoculum separate from the health-derived microbiomes. Microbiomes grown in saliva-serum showed the highest species richness and the highest similarity to the clinical inocula in both health and disease. However, inclusion of serum reduced alpha diversity and increased dysbiosis in healthy microbiomes in a dose-dependent manner, mainly due to overenrichment of Porphyromonas species. The modification of SHI stood second in terms of species richness and diversity but resulted in low biomass and viability and significantly worsened dysbiosis in the periodontitis-derived microbiomes. Overall, saliva with 5% human serum was optimal for replicating subgingival microbiomes from health and disease.

Project description:In vitro models that closely mimic human host-microbiome interactions can be a powerful screening tool for antimicrobials and will hold great potential for drug validation and discovery. The aim of this study was to develop an organotypic oral mucosa model that could be exposed to in vitro cultured commensal and pathogenic biofilms in a standardized and scalable manner. The oral mucosa model consisted of a tissue-engineered human gingiva equivalent containing a multilayered differentiated gingiva epithelium (keratinocytes) grown on a collagen hydrogel, containing gingiva fibroblasts, which represented the lamina propria. Keratinocyte and fibroblast telomerase reverse transcriptase-immortalized cell lines were used to overcome the limitations of isolating cells from small biopsies when scalable culture experiments were required. The oral biofilms were grown under defined conditions from human saliva to represent 3 distinct phenotypes: commensal, gingivitis, and cariogenic. The in vitro grown biofilms contained physiologic numbers of bacterial species, averaging >70 operational taxonomic units, including 20 differentiating operational taxonomic units. When the biofilms were applied topically to the gingiva equivalents for 24 h, the gingiva epithelium increased its expression of elafin, a protease inhibitor and antimicrobial protein. This increased elafin expression was observed as a response to all 3 biofilm types, commensal as well as pathogenic (gingivitis and cariogenic). Biofilm exposure also increased secretion of the antimicrobial cytokine CCL20 and inflammatory cytokines IL-6, CXCL8, and CCL2 from gingiva equivalents. This inflammatory response was far greater after commensal biofilm exposure than after pathogenic biofilm exposure. These results show that pathogenic oral biofilms have early immune evasion properties as compared with commensal oral biofilms. The novel host-microbiome model provides an ideal tool for future investigations of gingiva responses to commensal and pathogenic biofilms and for testing novel therapeutics.

Project description:Ammonia, an atmospheric pollutant in the air, jeopardizes immune function, and perturbs metabolism, especially lipid metabolism, in human and animals. The roles of intestinal microbiota and its metabolites in maintaining or regulating immune function and metabolism are irreplaceable. Therefore, this study aimed to investigate how aerial ammonia exposure influences hindgut microbiota and its metabolites in a pig model. Twelve growing pigs were treated with or without aerial ammonia (35 mg/m3) for 25 days, and then microbial diversity and microbiota-derived metabolites were measured. The results demonstrated a decreasing trend in leptin (p = 0.0898) and reduced high-density lipoprotein cholesterol (HDL-C, p = 0.0006) in serum after ammonia exposure. Besides, an upward trend in hyocholic acid (HCA), lithocholic acid (LCA), hyodeoxycholic acid (HDCA) (p < 0.1); a downward trend in tauro-deoxycholic acid (TDCA, p < 0.1); and a reduced tauro-HDCA (THDCA, p < 0.05) level were found in the serum bile acid (BA) profiles after ammonia exposure. Ammonia exposure notably raised microbial alpha-diversity with higher Sobs, Shannon, or ACE index in the cecum or colon and the Chao index in the cecum (p < 0.05) and clearly exhibited a distinct microbial cluster in hindgut indicated by principal coordinate analysis (p < 0.01), indicating that ammonia exposure induced alterations of microbial community structure and composition in the hindgut. Further analysis displayed that ammonia exposure increased the number of potentially harmful bacteria, such as Negativibacillus, Alloprevotella, or Lachnospira, and decreased the number of beneficial bacteria, such as Akkermansia or Clostridium_sensu_stricto_1, in the hindgut (FDR < 0.05). Analysis of microbiota-derived metabolites in the hindgut showed that ammonia exposure increased acetate and decreased isobutyrate or isovalerate in the cecum or colon, respectively (p < 0.05). Unlike the alteration of serum BA profiles, cecal BA data showed that high ammonia exposure had a downward trend in cholic acid (CA), HCA, and LCA (p < 0.1); a downward trend in deoxycholic acid (DCA) and HDCA (p < 0.05); and an upward trend in glycol-chenodeoxycholic acid (GCDCA, p < 0.05). Mantel test and correlation analysis revealed associations between microbiota-derived metabolites and ammonia exposure-responsive cecal bacteria. Collectively, the findings illustrated that high ammonia exposure induced the dysbiotic microbiota in the hindgut, thereby affecting the production of microbiota-derived short-chain fatty acids and BAs, which play a pivotal role in the modulation of host systematic metabolism.

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

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