Determining Selenomonas Species Subclusters in Periodontal Samples by FISH

Abstract

The highly destructive pathogenic processes in patients with periodontitis are attributed to the presence of subgingival biofilms comprising key periodontal pathogens, such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, as well as other periodontopathogens including Fusobacterium nucleatum, Selenomonas spp., Centipeda spp., and Campylobacter spp. Considering the vast microbial diversity in periodontitis, we aimed to analyze the presence of various bacterial species in subgingival dental plaque, with a special focus on Selenomonas spp. We first developed a phylogenetic tree for Selenomonas–Veillonella clusters, using in silico analysis followed by fluorescence in situ hybridization (FISH) on subgingival dental plaque samples from 22 patients with a history of chronic periodontitis, by using specific 16S rRNA oligonucleotide probes. These oligonucleotide probes’ specificity and hybridization conditions were determined on previously characterized bacterial strains. Qualitative and quantitative analysis of FISH slides was carried out by using an epifluorescence microscope. The majority of the patient samples showed high fluorescence signals with the oligonucleotide probes SEL1150, Sspu439, and SEL1469 (specific for Selenomonas spp.), ACI623 (identifying Selenomonas, Veillonella, and Dialister spp.), Tfor127 (T. forsythia) and L-Pgin1006-23 (P. gingivalis). SEL1150 showed specificity for bacterial species in the subclusters A, B, and C, namely S. dianae, S. infelix, S. flueggei, C. periodontii, S. artemidis, S. noxia, and S. sputigena; Sspu439 for S. sputigena; SEL1469 for subclusters A and B, and for S. sputigena; ACI623 for bacterial species in subclusters C and F, namely the S. sputigena and Veillonella species. The experimentally observed specificities of the oligonucleotide probes corresponded with our in silico analysis. Selenomonas spp. may play a role in the subgingival microbiome of periodontitis and contribute to the disease process. Targeting Selenomonas spp. with specific therapeutic strategies could offer new insights into the management of periodontitis. However, further studies are needed to determine a definite functional significance.