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: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.
Project description:Respiratory-tract infections are a predominant cause for clinical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for bacterial pathogens commonly found in respiratory tract infections: Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure-culture bacterial reference strains, reflecting the genomic variation in the four species and, furthermore in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. Peptide biomarker candidates for each species is presented. As a proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, demonstrating their potential as effective peptide biomarker candidates for diagnostics of infectious diseases.