Molecular Oral Microbiology最新文献

Porphyromonas gingivalis is a keystone pathogen in periodontitis, and Streptococcus sanguinis is an abundant oral commensal bacterium associated with periodontal health. However, the interaction between P. gingivalis and S. sanguinis remains obscure. Here, we established a strategy for high-throughput measurement of the cell number of P. gingivalis in the coculture with S. sanguinis by detecting the concentration of hydrogen sulfate. The interaction between P. gingivalis and over 2000 S. sanguinis single-gene mutants was characterized using this strategy, and several interaction-associated genes in S. sanguinis were determined by detecting more P. gingivalis cells in the coculture with matched S. sanguinis mutants. Three S. sanguinis interaction-associated genes were predicted to be responsible for cysteine metabolism, and the supplementation of exogenous L-cysteine promoted the cell number of P. gingivalis in the coculture with S. sanguinis. Thus, exogenous L-cysteine and the compromised cysteine metabolism in S. sanguinis enhanced the growth of P. gingivalis in the existence of S. sanguinis. Additionally, the interaction between P. gingivalis and other Streptococcus spp. was examined, and S. pneumoniae was the only streptococci that had no inhibition on the cell number of P. gingivalis. In total, this study established a new strategy for high-throughput screening of the interaction between Streptococcus and P. gingivalis and discovered a set of genes in S. sanguinis that impacted the interaction. The influence of exogenous L-cysteine on the interaction between P. gingivalis and S. sanguinis in the oral cavity needs further investigation.

Background: Type 2 diabetes mellitus (T2DM) may affect the oral microbial community, exacerbating periodontal inflammation; however, its pathogenic mechanisms remain unclear. As nucleotide-binding oligomerization domain 2 (NOD2) plays a crucial role in the activation during periodontitis (PD), it is hypothesized that changes in the oral microbial community due to diabetes enhance periodontal inflammation through the activation of NOD2.

Methods: We collected subgingival plaque from 180 subjects who were categorized into two groups based on the presence or absence of T2DM. The composition of oral microbiota was detected by 16S rRNA high-throughput sequencing. In animal models of PD with or without T2DM, we assessed alveolar bone resorption by micro-computerized tomography and used immunohistochemistry to detect NOD2 expression in alveolar bone. Primary osteoblasts were cultured in osteogenic induction medium with high or normal glucose and treated with inactivated bacteria. After 24 h of inactivated bacteria intervention, the osteogenic differentiation ability was detected by alkaline phosphatase (ALP) staining, and the expressions of NOD2 and interleukin-12 (IL-6) were detected by western blot.

Results: The relative abundance of Parvimonas and Filifactor in the T2DM group was increased compared to the group without T2DM. In animal models, alveolar bone mass was decreased in PD, particularly in T2DM with PD (DMPD) group, compared to controls. Immunohistochemistry revealed NOD2 in osteoblasts from the alveolar bone in both the PD group and DMPD group, especially in the DMPD group. In vitro, intervention with inactivated Parvimonas significantly reduced ALP secretion of primary osteoblasts in high glucose medium, accompanied by increased expression of NOD2 and IL-6.

Conclusions: The results suggest that T2DM leading to PD may be associated with the activation of NOD2 by Parvimonas.

Background: The oral microbiome-dependent nitrate (NO₃ ^-)-nitrite (NO₂ ^-)-nitric oxide (NO) pathway may help regulate blood pressure. NO₂ ^--producing bacteria in subgingival plaque are reduced in relative abundance in patients with untreated periodontitis compared with periodontally healthy patients. In periodontitis patients, the NO₂ ^--producing bacteria increase several months after periodontal treatment. The early effects of periodontal treatment on NO₂ ^--producing bacteria and the NO₃ ^--NO₂ ^--NO pathway remain unknown. The aim of this study was to determine how periodontal treatment affects the oral NO₂ ^--producing microbiome and salivary NO₃ ^- and NO₂ ^- levels over time.

Methods: The subgingival microbiota of 38 periodontitis patients was analysed before (baseline [BL]) and 1, 7 and 90 days after periodontal treatment. Changes in NO₂ ^--producing bacteria and periodontitis-associated bacteria were determined by 16s rRNA Illumina sequencing. Saliva samples were collected at all-time points to determine NO₃ ^- and NO₂ ^- levels using gas-phase chemiluminescence.

Results: A significant increase was observed in the relative abundance of NO₂ ^--producing species between BL and all subsequent timepoints (all p < 0.001). Periodontitis-associated species decreased at all timepoints, relative to BL (all p < 0.02). NO₂ ^--producing species negatively correlated with periodontitis-associated species at all timepoints, with this relationship strongest 90 days post-treatment (ρ = -0.792, p < 0.001). Despite these findings, no significant changes were found in salivary NO₃ ^- and NO₂ ^- over time (all p > 0.05).

Conclusions: Periodontal treatment induced an immediate increase in the relative abundance of health-associated NO₂ ^--producing bacteria. This increase persisted throughout periodontal healing. Future studies should test the effect of periodontal treatment combined with NO₃ ^- intake on periodontal and cardiovascular health.

Biofilms are subjected to many environmental pressures that can influence community structure and physiology. In the oral cavity, and many other environments, biofilms are exposed to forces generated by fluid flow; however, our understanding of how oral biofilms respond to these forces remains limited. In this study, we developed a linear rocker model of fluid flow to study the impact of shear forces on Streptococcus gordonii and dental plaque-derived multispecies biofilms. We observed that as shear forces increased, S. gordonii biofilm biomass decreased. Reduced biomass was largely independent of overall bacterial growth. Transcriptome analysis of S. gordonii biofilms exposed to moderate levels of shear stress uncovered numerous genes with differential expression under shear. We also evaluated an ex vivo plaque biofilm exposed to fluid shear forces. Like S. gordonii, the plaque biofilm displayed decreased biomass as shear forces increased. Examination of plaque community composition revealed decreased diversity and compositional changes in the plaque biofilm exposed to shear. These studies help to elucidate the impact of fluid shear on oral bacteria and may be extended to other bacterial biofilm systems.

Infection is a known cause of abdominal aortic aneurysm (AAA), and matrix metalloproteases-2 (MMP-2) secreted by vascular smooth muscle cells (SMCs) plays a key role in the structural disruption of the middle layer of the arteries during AAA progression. The periodontal pathogen Porphyromonas gingivalis is highly associated with the progression of periodontitis. GroEL protein of periodontal pathogens is an important virulence factor that can invade the body through either the bloodstream or digestive tract and is associated with numerous systemic diseases. Although P. gingivalis aggravates AAA by increasing the expression of MMP-2 in animal studies, the molecular mechanism through which P. gingivalis regulates the expression of MMP-2 is still unknown and requires further investigation. In this study, we first confirmed through animal experiments that P. gingivalis GroEL promotes MMP-2 secretion from vascular SMCs, thereby aggravating Ang II-induced aortic remodeling and AAA formation. In addition, rat vascular SMCs and A7r5 cells were used to investigate the underlying mechanisms in vitro. The results demonstrated that GroEL can promote the interaction between the K639 site of MMP-2 and SUMO-1, leading to MMP-2 SUMOylation, which inhibits the reoccurrence of non-K639-mediated monoubiquitylation. Hence, the monoubiquitylation-mediated lysosomal degradation of MMP-2 is inhibited, consequently promoting MMP-2 stability and production. SUMOylation may facilitate intra-endoplasmic reticulum (ER) and Golgi trafficking of MMP-2, thereby enhancing its transport capacity. In conclusion, this is the first report demonstrating the presence of a novel posttranslational modification, SUMOylation, in the MMP family, suggesting that P. gingivalis GroEL may exacerbate AAA formation by increasing MMP-2 production through SUMOylation in vascular SMCs. This study also provides a novel perspective on the role of SUMOylation in MMP-2-induced systemic diseases.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMBs) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n = 10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the MicrobiomeAnalyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.

Numerous cellular processes are regulated in response to the metabolic state of the cell, and one such regulatory mechanism involves lysine acetylation. Lysine acetylation has been proven to play an important role in the virulence of Streptococcus mutans, a major cariogenic bacterial species. S. mutans' glucosyltransferases (Gtfs) are responsible for synthesizing extracellular polysaccharides (EPS) and contributing to biofilm formation. One of the most common nonsteroidal anti-inflammatory drugs is acetylsalicylic acid (ASA), which can acetylate proteins through a nonenzymatic transacetylation reaction. Herein, we investigated the inhibitory effects of ASA on S. mutans. ASA treatment was observed to impede the growth of S. mutans, leading to a reduction in the production of water-insoluble EPS and the formation of biofilm. Moreover, ASA decreased the enzyme activity of Gtfs while increasing the protein acetylation level. The in vivo anticaries efficacy of ASA has further been proved using the rat caries model. In conclusion, ASA as an acetylation agent attenuated the cariogenic virulence of S. mutans, suggesting the potential value of protein acetylation on antimicrobial and anti-biofilm applications to S. mutans.

Background: Considered the second largest and most diverse microbiome after the gut, the human oral ecosystem is complex with diverse and niche-specific microorganisms. Although evidence is growing for the importance of oral microbiome in supporting a healthy immune system and preventing local and systemic infections, the influence of craniomaxillofacial (CMF) trauma and routine reconstructive surgical treatments on community structure and function of oral resident microbes remains unknown. CMF injuries affect a large number of people, needing extensive rehabilitation with lasting morbidity and loss of human productivity. Treatment efficacy can be complicated by the overgrowth of opportunistic commensals or multidrug-resistant pathogens in the oral ecosystem due to weakened host immune function and reduced colonization resistance in a dysbiotic oral microbiome.

Aims: To understand the dynamics of microbiota's community structure during CMF injury and subsequent treatments, we induced supra-alveolar mandibular defect in Hanford miniature swine (n = 3) and compared therapeutic approaches of immediate mandibullar reconstructive (IMR) versus delayed mandibullar reconstructive (DMR) surgeries.

Methods: Using bacterial 16S ribosomal RNA gene marker sequencing, the composition and abundance of the bacterial community of the uninjured maxilla (control) and the injured left mandibula (lingual and buccal) treated by DMR were surveyed up to 70-day post-wounding. For the injured right mandibula receiving IMR treatment, the microbial composition and abundance were surveyed up to 14-day post-wounding. Moreover, we measured sera level of biochemical markers (e.g., osteocalcin) associated with bone regeneration and healing. Computed tomography was used to measure and compare mandibular bone characteristics such as trabecular thickness between sites receiving DMR and IMR therapeutic approaches until day 140, the end of study period.

Results: Independent of IMR versus DMR therapy, we observed similar dysbiosis and shifts of the mucosal bacteria residents after CMF injury and/or following treatment. There was an enrichment of Fusobacterium, Porphyromonadaceae, and Bacteroidales accompanied by a decline in Pasteurellaceae, Moraxella, and Neisseria relative abundance in days allotted for healing. We also observed a decline in species richness and abundance driven by reduction in temporal instability and inter-animal heterogeneity on days 0 and 56, with day 0 corresponding to injury in DMR group and day 56 corresponding to delayed treatment for DMR or injury and immediate treatment for the IMR group. Analysis of bone healing features showed comparable bone-healing profiles for IMR vs. DMR therapeutic approach.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMB) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n = 10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the Microbiome Analyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.

Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B₃ derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth-essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability.