Abstract
BackgroundAnalysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF.Methodology/Principal FindingsWe 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 were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, 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/SignificanceAmong 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.
Highlights
Periodontitis remains widespread in populations aged 40 years or older [1] and represents one of the most common chronic bacterial infections in man
Specified bacterial species but few host-associated variables contributed significantly to the species diversity in gingival crevicular fluid samples A phylogenetic analysis of the bacterial species identified in GCF samples revealed 8 bacterial phyla/candidate phyla, Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, Actinobacteria, Spirochaetes, Synergistetes and TM7 (Figure 1)
We found a wide diversity of different species in GCF samples despite the small sample amounts, from 4 to 12 ug per patient, and low bacterial concentrations, which could have challenged the yield of representative bacterial DNA for processing and DNA microarray analysis
Summary
Periodontitis remains widespread in populations aged 40 years or older [1] and represents one of the most common chronic bacterial infections in man. Bacterial colonization and growth on supra- and subgingival tooth surfaces causes chronic inflammation in periodontal tissues. Increased flow of gingival crevicular fluid (GCF) [7], a serum transudate or inflammatory exudate, washes periodontal pockets and thereby provides host-derived substances that shape subgingival bacterial populations. Enriched with bacterial material from subgingival space GCF enters the oral cavity at the periodontal pocket orifice. In contrast to well-studied hostassociated constituents of GCF [8,9], no previous knowledge was found of its bacterial composition. Analysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF
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