Jingping Liang

Prevalence and molecular diversity of Archaea in subgingival pockets of periodontitis patients

INTRODUCTION The aim of this study was to investigate the prevalence and molecular diversity of Archaea in the subgingival crevices of patients with chronic periodontitis. METHODS Subgingival plaque was collected from 41 patients with chronic periodontitis and 15 healthy subjects. The prevalence of Archaea in those plaque samples was tested by polymerase chain reaction with two broad-range archaeal primer sets. Amplicons from eight Archaea-positive plaque samples were cloned and sequenced for molecular diversity analysis using one of these two primer sets and a novel third primer set. RESULTS Archaea were detected in the subgingival plaque of patients with chronic periodontitis at a prevalence of 70.7-73.2%, but were not detected in healthy subjects. Using one primer set, all sequences of the archaeal amplicons were identified as Methanobrevibacter oralis-like species. With another primer set, the amplicons were also found to be identical to the uncultured M. oralis-like species except one phylotype was found to belong to the class Thermoplasmata. CONCLUSION Archaea might be correlated with periodontal diseases. The diversity of Archaea associated with periodontitis was limited. Almost all sequenced amplicons fell into the genus Methanobrevibacter of the Euryarcheota phylum. M. oralis-like species was the predominant but non-exclusive archaeon in the subgingival dental plaque of patients with periodontitis.

Use of the quorum sensing inhibitor furanone C-30 to interfere with biofilm formation by Streptococcus mutans and its luxS mutant strain.

Streptococcus mutans is recognised as a major aetiological agent of dental caries. One of its important virulence factors is its ability to form biofilms on tooth surfaces. The aim of this study was to evaluate the effects of the quorum sensing inhibitor furanone C-30 on biofilm formation by S. mutans and its luxS mutant strain. The effects of furanone C-30 on biofilms of both strains formed on 96-well microtitre plates at 37 °C were determined by a colorimetric technique (MTT assay). Different concentrations of furanone C-30 (0.0, 2.0 and 4.0 μg/mL) and different time points of biofilm formation (4, 14 and 24 h) were investigated. The structures and thickness of the biofilms were observed by confocal laser scanning microscopy (CLSM). Quorum sensing-related gene expression (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time polymerase chain reaction (RT-PCR). The results showed that synthetic furanone C-30 can inhibit biofilm formation by S. mutans and its luxS mutant strain, although it does not affect the bacterial growth rate itself. The quantities of biofilm formed by both strains significantly decreased (P<0.05) and the biofilms became thinner and looser as revealed by CLSM with increasing concentrations of furanone C-30. Expression of the genes tested was downregulated in the biofilms by the addition of furanone C-30. These results revealed that synthetic furanone C-30 can effectively inhibit biofilm formation by S. mutans and its luxS mutant strain.

Exploring the Dynamic Core Microbiome of Plaque Microbiota during Head-and-Neck Radiotherapy Using Pyrosequencing

Radiotherapy is the primary treatment modality used for patients with head-and-neck cancers, but inevitably causes microorganism-related oral complications. This study aims to explore the dynamic core microbiome of oral microbiota in supragingival plaque during the course of head-and-neck radiotherapy. Eight subjects aged 26 to 70 were recruited. Dental plaque samples were collected (over seven sampling time points for each patient) before and during radiotherapy. The V1–V3 hypervariable regions of bacterial 16S rRNA genes were amplified, and the high-throughput pyrosequencing was performed. A total of 140 genera belonging to 13 phyla were found. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) and 11 genera (Streptococcus, Actinomyces, Veillonella, Capnocytophaga, Derxia, Neisseria, Rothia, Prevotella, Granulicatella, Luteococcus, and Gemella) were found in all subjects, supporting the concept of a core microbiome. Temporal variation of these major cores in relative abundance were observed, as well as a negative correlation between the number of OTUs and radiation dose. Moreover, an optimized conceptual framework was proposed for defining a dynamic core microbiome in extreme conditions such as radiotherapy. This study presents a theoretical foundation for exploring a core microbiome of communities from time series data, and may help predict community responses to perturbation as caused by exposure to ionizing radiation.

Investigation of Supragingival Plaque Microbiota in Different Caries Status of Chinese Preschool Children by Denaturing Gradient Gel Electrophoresis

This study aimed to detect differences in the richness of total supragingival plaque microbiota as well as the species composition of oral streptococci involved in the different stages of dental caries. Forty-five plaque samples were collected from caries-moderate (CM, 4 ≤ dmfs ≤ 6), caries-susceptible (CS, dmfs ≥ 10), and age-matched caries-free children separately. Total DNA was isolated directly from each sample, and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analyses using universal and primers specific for oral streptococci were carried out. Using 16S rDNA PCR-DGGE, 34 different species of bacteria were identified in a culture-independent manner and classified into 11 genera according to phylogenetic analysis. Among them, Mitis group streptococci and Campylobacter, which were present in health status, no longer appeared in caries-susceptible samples. In addition, Capnocytophaga, Burkholderia, and Prevotella were found significantly less frequently in the CS group samples (P < 0.05), while there were no significant differences among the prevalence of Neisseria, Leptotrichia, Haemophilus, Mutans group streptococci, Corynebacterium, and Actinomyces in the three groups. Further DGGE analysis of rnpB gene amplicons obtained with oral streptococci species-specific primers showed that a total of 23 species of oral streptococci were identified. Streptococcus sanguinis, Streptococcus mitis, and Streptococcus oralis showed a significantly higher prevalence in healthy children (P < 0.05), while that of Streptococcus mutans and Streptococcus sobrinus did not vary among the three groups. Overall, these results suggest that supragingival plaque microbiota as a whole undergoes a more complicated shift in the caries process.

Effects of Wnt/β-catenin signalling on proliferation and differentiation of apical papilla stem cells.

Objectives:  The Wnt signalling pathway has been shown to play an important role in tooth development, however its effects with stem cells from the apical papilla (SCAP) have remained unclear. The purpose of this study was to determine effects of Wnt/β‐catenin on proliferation and differentiation of SCAP in vitro.

Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing

Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700) clustered into 10,365 operational taxonomic units (OTUs; 97% identity), representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in healthy plaques exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed.

Preliminary study of the presence and association of bacteria and archaea in teeth with apical periodontitis

AIM To investigate, by reverse transcription polymerase chain reaction (RT-PCR), the presence and association of bacteria and archaea in primary and secondary root canal infections. METHODOLOGY A total of 77 root canal samples from 77 Chinese patients, 42 with necrotic pulp tissues (primary infection) and 35 with failed prior conventional root canal treatment (secondary infection), aseptically exposed at the first patient visit, were studied. Total RNA was isolated directly from each sample, and 16S rRNA gene-based RT-PCR assays were used to determine the presence of bacteria and archaea, respectively. RESULTS Bacteria were detected in 39/42 (93%) of root canal samples from teeth with primary infections, and archaea in 16/42 (38%). In the cases diagnosed as secondary root-infected canals, bacteria were detected in 30/35 (86%), whilst archaea were detected in 6/35 (17%) of cases. Amongst the canals, which were positive for bacteria, archaea were always found in combination with bacteria. The incidence of symptomatic cases positive for both bacteria and archaea (16/22, 73%) were significantly higher than those positive for bacteria alone (21/47, 45%) (P < 0.05). CONCLUSIONS This study confirms the presence of archaea in root canal infections and further implicates them in an association with clinical symptoms. The nature of this association requires further study.

Characterization of oral bacterial diversity of irradiated patients by high-throughput sequencing

The objective of this study was to investigate the compositional profiles and microbial shifts of oral microbiota during head-and-neck radiotherapy. Bioinformatic analysis based on 16S rRNA gene pyrosequencing was performed to assess the diversity and variation of oral microbiota of irradiated patients. Eight patients with head and neck cancers were involved in this study. For each patient, supragingival plaque samples were collected at seven time points before and during radiotherapy. A total of 147 232 qualified sequences were obtained through pyrosequencing and bioinformatic analysis, representing 3 460 species level operational taxonomic units (OTUs) and 140 genus level taxa. Temporal variations were observed across different time points and supported by cluster analysis based on weighted UniFrac metrics. Moreover, the low evenness of oral microbial communities in relative abundance was revealed by Lorenz curves. This study contributed to a better understanding of the detailed characterization of oral bacterial diversity of irradiated patients.

Role of p38 mitogen-activated protein kinase pathway in Porphyromonas gingivalis lipopolysaccharide-induced VCAM-1 expression in human aortic endothelial cells.

BACKGROUND Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) has been reported to induce the expression of vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelial cells. This finding suggests the potential roles for Pg in the pathogenesis of atherosclerosis. However, the mechanism involved in Pg LPS-induced VCAM-1 production in endothelial cells remains unclear. METHODS Quantitative real-time polymerase chain reaction and Western blotting were used, respectively, to investigate the mRNA expression and protein production of VCAM-1 in human aortic endothelial cells (HAECs) induced by Pg LPS. The involvement of the p38 mitogen-activated protein kinase (p38 MAPK) cell signaling pathway in VCAM-1 expression was investigated by assays with specific inhibitors. RESULTS Pg LPS-induced expression in HAECs of VCAM-1 occurred in a dose- and time-dependent manner. In addition, the p38 MAPK inhibitor (SB 203580) significantly attenuated Pg LPS-induced VCAM-1 expression. CONCLUSION Activation of p38 MAPK is at least partially involved in Pg LPS-induced VCAM-1 expression in HAECs, which may contribute to the acceleration of atherosclerosis.

Role of the luxS Gene in Initial Biofilm Formation by Streptococcus mutans

Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS luxS gene on initial biofilm formation by Streptococcus mutans. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The luxS mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the luxS mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the luxS mutant strain, except the gtfB gene. QS luxS gene can affect the initial biofilm formation by S. mutans.