Project description:Analysis of gingival crevicular fluid (GCF) samples may give information of the identity of unattached (planktonic) subgingival bacteria, the 35 forefront candidates for systemic dispersal via ulcerated periodontal pocket epithelium. Our study represents the first one targeting the identity of bacteria in gingival crevicular fluid. Methodology/Principal findings: We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla 45 were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, 46 per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance: Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms. The microbial profiles of GCF and subgingival plaque were analyzed from 17 subjects with periodontal disease.

Project description:Dysbiosis of subgingival microbiome promotes the growth of periodontopathogens and the development of periodontitis, an irreversible chronic inflammatory disease. Untreated periodontitis leads to the destruction of connective tissues, alveolar bone resorption and ultimately to tooth loss. Periodontitis has been associated with inflammatory metabolic diseases such as type 2 diabetes. While periodontitis-induced inflammation is a key player in both, the development of subgingival microbiome dysbiosis and in the host-microbiome interaction, the effects of hyperglycemia on the regulation of the host genes controlling the inflammatory response and the host-microbiome interaction are still scarce. We investigated the impacts of a hyperglycemic microenvironment on the inflammatory response and gene expression of a gingival fibroblasts-macrophages coculture model stimulated with dysbiotic subgingival microbiomes. A coculture model composed of immortalized human gingival fibroblasts overlaid with U937 macrophages-likes cells were stimulated with subgingival microbiome collected from four healthy donors and four patients with periodontitis. Pro-inflammatory cytokines and matrix metalloproteinase were measured by a Luminex assay while the coculture RNA was submitted to a microarray analysis. Subgingival microbiomes were submitted to 16s rRNA gene sequencing. Data were analyzed by using an advanced multi-omics bioinformatic data integration model. Our results showed that krt76, krt27, pnma5, mansc4, rab41, thoc6, tm6sf2, and znf506 as well as the pro-inflammatory cytokines IL-1, GM-CSF, FGF2, IL-10, the metalloproteinases MMP3 and MMP8, and bacteria from the ASV 105, ASV 211, ASV 299, Prevotella, Campylobacter and Fretibacterium genera are key correlated variables contributing to periodontitis-induced inflammatory response in a hyperglycemic microenvironment. To conclude, our multi-omics integration analysis unveiled unique differentially interrelated bacterial genera, genes and pro-inflammatory cytokines involved in the regulation of the inflammatory response in a hyperglycemic microenvironment. These data also highlight the importance of considering hyperglycemic conditions in the development of new drugs or treatments for periodontal disease in link with type 2 diabetes.

Project description:Background: The food-borne pathogen Campylobacter is one of the most important zoonotic pathogens. Compared to other zoonotic bacteria, Campylobacter species are quite susceptible to environmental or technological stressors. This might be due to the lack of many stress response mechanisms described in other bacteria. Nevertheless, Campylobacter is able to survive in the environment and food products. Although some aspects of the heat stress response in Campylobacter (C.) jejuni are already known, information about the heat stress response in the related species C. coli and C. lari are still unknown. Results: The stress response to elevated temperatures (46°C) was investigated by survival assays and whole transcriptome analyses for the strain C. jejuni NCTC11168, C. coli RM2228 and C. lari RM2100. While C. jejuni showed highest thermotolerance followed by C. lari and C. coli, none of the strains survived at this temperature for more than 24 hours. Transcriptomic analyses revealed that only 3 % of the genes in C. jejuni and approx. 20 % of the genes of C. coli and C. lari were differentially expressed after heat stress, respectively. The transcriptomic profiles showed enhanced gene expression of several chaperones like dnaK, groES, groEL and clpB in all strains, but differences in the gene expression of transcriptional regulators like hspR, perR as well as for genes involved in metabolic pathways, translation processes and membrane components. However, the function of many of the differentially expressed gene is unknown so far. Conclusion: We could demonstrate differences in the ability to survive at elevated temperatures for C. jejuni, C. coli and C. lari and showed for the first time transcriptomic analyses of the heat stress response of C. coli and C. lari. Our data suggest that the heat stress response of C. coli and C. lari are more similar to each other compared to C. jejuni, even though on genetic level a higher homology exists between C. jejuni and C. coli. This indicates that stress response mechanisms described for C. jejuni might be unique for this species and not necessarily transferable to other Campylobacter species.

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Specified Bacterial Species in Gingival Crevicular Fluid Predict Diversity of Unattached Subgingival Microbiota

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