Journal of Oral Microbiology最新文献

Background: Low-abundance bacterial (LAB) species, despite their low prevalence, may contribute to oral inflammatory diseases by triggering host immune responses. The NLRP3 inflammasome plays a key role in inflammation, but its activation by LAB species remains unclear.

Aim: This study examined whether selected LAB species and their biofilm-secreted components induce cytokine production and inflammasome activation in human peripheral blood mononuclear cells (PBMCs).

Methods: Biofilms of selected LAB species were established, and supernatants were collected. PBMCs were stimulated with biofilms or supernatants, and cytokine levels were quantified using ELISA. The expression of NLRP3 and Caspase-1 genes was analyzed through real-time PCR.

Results: Biofilms induced significantly higher levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-18) compared to supernatants, with C. hominis, N. flavescens, and D. pneumosintes being the most potent inducers. Biofilms also led to a marked increase in NLRP3 expression, while supernatants primarily activated Caspase-1 expression, indicating distinct inflammasome activation pathways.

Conclusions: These findings highlight the immunostimulatory potential of LAB species, particularly their ability to activate NLRP3 and drive inflammation. The differential activation of NLRP3/Caspase-1 by biofilms and supernatants suggests distinct pathogenic mechanisms. Targeting such mechanisms/pathways could offer new therapeutic strategies to mitigate inflammation linked to oral infections.

Objective: In periodontal research, subgingival biofilm samples are typically collected using sterile paper points and pooled for molecular analyses. Streamlining this process by using a single paper point for molecular analysis could simplify sample collection and allow additional paper points to be used for other investigations. This pilot study evaluated the performance of three commercial DNA extraction kits for analysing small sample volumes (<10 µL).

Methods: Samples were collected from six participants, each contributing 18 paper points from both healthy and periodontitis-affected sites. Bacterial and human DNA yields were quantified using fluorometric measurements combined with qPCR, employing universal 16S primers for bacterial DNA and human-specific GAPDH primers.

Results: Among the tested kits, the DNeasy Blood and Tissue Kit demonstrated the highest efficiency, yielding significantly more total dsDNA in samples from healthy sites compared to both other kits and in samples from periodontitis-affected sites compared to one kit. Bacterial DNA yields were also significantly higher with the DNeasy Kit compared to one of the other kits in both health conditions.

Conclusion: These results suggest that one paper point is sufficient to extract DNA for subsequent bacterial analyses and that the DNeasy Blood and Tissue Kit appears to be the most efficient among the three tested kits.

Backgrounds: Periodontitis-induced alveolar bone loss is a primary cause of tooth loss. Porphyromonas gingivalis (P. gingivalis) is the primary pathogenic bacterium of periodontitis. Outer membrane vesicles (OMVs) derived from P. gingivalis (P.g-OMVs) contain various bioactive molecules, and several studies have suggested that P.g-OMVs may participate in alveolar bone loss caused by periodontitis.

Materials and methods: P.g-OMVs were isolated and characterized. The effect of P.g-OMVs on BMSCs proliferation and osteogenic differentiation was analyzed. High-throughput sequencing, RT-qPCR, and Western blot analysis were performed in BMSCs to unravel the underlying molecular mechanism.

Results: P.g-OMVs promoted proliferation but inhibited osteogenic differentiation of BMSCs. High-throughput sequencing results showed that serum amyloid A (SAA), especially SAA3, was robustly upregulated in P.g-OMVs-treated BMSCs. Upregulated SAA3 promoted TLR4, MyD88, and NF-κB p65 and inhibited osteogenic differentiation of P.g-OMVs-treated BMSCs. The knockdown of SAA3 in BMSCs downregulated P.g-OMVs-induced TLR4, MyD88, and NF-κB p65 and rescued P.g-OMVs-inhibited osteogenic differentiation.

Conclusions: Our results indicate that P.g-OMVs inhibit osteogenic differentiation of BMSCs via the SAA3-mediated TLR4/MyD88/NF-κB axis, providing novel targets for the treatment of periodontitis-induced alveolar bone loss.

Fusobacterium nucleatum, a microorganism ordinarily detected in the oral cavity, is considered as a pathobiont related to periodontitis and a range of human diseases, including colorectal cancer (CRC). The dynamics of how F. nucleatum encourages CRC tumorigenesis and progression has been well-investigated. Recently, mechanisms by which F. nucleatum regulates the tumor immune microenvironment (TiME) and subsequently alters CRC oncogenesis and advancement have drawn more and more attention. The TiME consists of immune cells and non-cellular components like cytokines in the tumor microenvironment. By contacting immune cells in the TiME, F. nucleatum fosters an immunosuppressive TiME, diminishes anti-tumor immunity and promotes CRC development. This also allows F. nucleatum to interfere with immunotherapy process and efficacy. In this review, we present a summary of how F. nucleatum interacts with immune cells within the TiME, thereby promoting CRC progression and influencing CRC immunotherapy effectiveness. This review also integrates insights from molecular pathological epidemiology (MPE) to contextualize host-microbe-environment interactions in CRC. We identify gaps in current knowledge and outline possible future research paths. These findings may offer valuable insights for future mechanistic research and the development of novel therapeutic strategies.

Introduction: Emerging research underscores the gut-brain axis in mental disorder pathophysiology, yet the oral microbiome's contribution to mental health remains underexplored. Elucidating oral microbial signatures in mental and neurological disorders may reveal novel pathobiological mechanisms and advance biomarker discovery for precision diagnostics and microbiota-targeted interventions.

Methods: This systematic review and meta-analysis investigates oral microbiota alterations across 6 different mental disorders, by synthesizing data from 20 case-control studies retrieved from PubMed, Embase, and Cochrane Library. Relative microbial abundance and beta diversity indices were extracted from between-group comparisons. Random-effects meta-analyses were conducted for alpha diversity to characterize microbiota differences between patients and controls.

Results: Key findings included a significantly higher Simpson Index in patients (SMD = 0.42; 95% CI, 0.25 to 0.60) compared to controls. Beta diversity varied significantly only in SZ and MDD. Condition-specific variations in microbial abundance were observed: Rothia enrichment in ASD, overrepresentation of H₂S-producing genera in SZ, and reduced Solobacterium and Leptotrichia in MDD.

Conclusion: Collectively, the meta-analytical synthesis suggests alterations in oral microbiota diversity across mental disorders. Disease-associated microbial shifts highlight the oral microbiome as a candidate factor warranting further investigation for potential diagnostic applications and microbial-targeted therapeutic strategies.

Background: Porphyromonas gingivalis is a keystone pathogen in periodontitis, associated with dysbiosis and chronic inflammation. While its virulence mechanisms are well characterized, its transcriptional heterogeneity at the single-cell level remains unexplored.

Materials and methods: We applied split-pool barcoding-based single-cell RNA sequencing to profile gene expression in 1,942 individual P. gingivalis W83 cells cultured under anaerobic conditions. Clustering and differential expression analyses were conducted to identify distinct transcriptional subpopulations.

Results: We identified six transcriptionally distinct clusters, with the two largest accounting for 72.7% of the population. Minor clusters exhibited signatures related to stress responses, metabolism, membrane transport, and DNA regulation. Sub-clustering of major populations revealed rare subgroups, including one enriched for genes involved in iron acquisition, proteolysis, and transport.

Conclusions: This study presents the first single-cell transcriptomic map of P. gingivalis, revealing rare but functionally significant subpopulations. Such diversity may support bacterial adaptability, virulence, and immune evasion, informing future strategies for targeted periodontal therapy.

Background: This study comparatively evaluated the analytical and diagnostic performance of a multiplex digital polymerase-chain reaction (dPCR) assay and a quantitative real-time PCR (qPCR) for the simultaneous detection and quantification of periodontal pathobionts: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum.

Materials and methods: Subgingival plaque samples from 20 periodontitis patients and 20 periodontally healthy controls were analyzed. Several analytical parameters of the dPCR assay, optimized using DNA standards, were assessed versus qPCR: dynamic range linearity, precision, accuracy, prevalence, sensitivity, specificity, and concordance. The statistical analyses included Mann-Whitney U test, Wilcoxon test, McNemar's test, and Bland-Altman plots.

Results: dPCR showed high linearity (R² > 0.99) and lower intra-assay variability (median CV%: 4.5%) than qPCR (p = 0.020), with comparable accuracy and agreement. dPCR demonstrated superior sensitivity, detecting lower bacterial loads, particularly for P. gingivalis and A. actinomycetemcomitans. The Bland-Altman plots highlighted good agreement at medium/high loads but discrepancies at low concentrations (< 3 log₁₀Geq/mL), resulting in qPCR false negatives and a 5-fold underestimation of the prevalence of A. actinomycetemcomitans in periodontitis patients. High concordance between the assays was observed for F. nucleatum across both study groups.

Conclusions: dPCR outperformed qPCR for quantifying periodontal pathobionts and had superior sensitivity and precision, making it particularly effective in detecting low-level bacterial loads.

Background: Dental caries is a bacterial-mediated infectious disease that affects the hard tissues of the tooth, with Streptococcus mutans being the primary cariogenic pathogen due to its robust biofilm-forming ability. Controlling biofilm formation is essential for caries prevention. Recent studies have indicated that D-amino acids, which are not incorporated into proteins, play regulatory roles in bacterial processes such as growth inhibition and biofilm dispersal. However, whether D-amino acids can inhibit the growth of S. mutans remains controversial. This study aimed to investigate the effects of D-amino acids on S. mutans growth and biofilm formation in vitro, as well as their anti-caries efficacy in a rat caries model.

Materials and methods: This study utilized Streptococcus mutans UA159 to screen 15 D-amino acids for growth inhibition, identifying D-histidine (D-His) as the most effective. Minimum inhibitory concentration, growth curves, biofilm assays, and transcriptomic analysis were performed in vitro. Anti-caries efficacy was evaluated in a rat model using Micro-CT and Keyes scoring.

Results: D-His significantly inhibits the planktonic growth of S. mutans and delays biofilm formation, particularly in the early stages. Furthermore, RNA sequencing revealed 417 upregulated genes and 394 downregulated genes in D-His-treated S. mutans, with significant alterations in pathways related to carbohydrate utilization, protein biosynthesis, and transmembrane transport. Moreover, D-His exhibited effective caries prevention in an in vivo rat model.

Conclusion: These findings suggest that D-His has potential as an anti-caries agent by targeting S. mutans growth and biofilm dynamics.

Background/objective: Cariogenic biofilms possess a rich extracellular polysaccharide (EPS) matrix, which can reduce the penetration of anticaries agents such as nanoparticle-based technologies. The aim of this study was to assess the potential of dextranase, an EPS-degrading enzyme, to enhance nanoparticle penetration into Streptococcus mutans in vitro biofilms.

Methods: Commercially available fluorescent nanoparticles (nanospheres, average diameter around 200 nm) were used as a proxy for nanoparticle treatments. Biofilms of fluorescent S. mutans 3209/pVMCherry were developed over 48 h in 24-well glass bottom microplates, simulating daily feast (tryptic soy broth (TSB) supplemented with 1% sucrose) and famine periods (TSB supplemented with 0.1 mM glucose). Nanoparticles were co-administered to biofilms with either dextranase (10 U/mL) or pH 6.5 phosphate buffer (placebo). Time-lapse confocal laser scanning microscopy was used to capture six image stacks over approximately 60 minutes of nanoparticle movement through the biofilm. In-house-developed quantitative image analysis methods assessed nanoparticle penetration.

Results: Nanoparticle signal intensity and overlapping signal with cells increased in the presence of dextranase, being significantly higher in the last two CLSM scans compared with the initial one (p < 0.05). Biofilm architecture changed under dextranase, increasing the interaction of nanoparticles with biofilm components.

Conclusion: Dextranase showed potential to enhance nanoparticle-based anticaries therapies.

Key messages: Dextranase increases the penetration of nanoparticles in cariogenic, extracellular polysaccharide-rich dental biofilms.

Background: Hexylresorcinol (HR) lozenges provide symptomatic relief for sore throats. Despite its recognised anaesthetic and antiseptic properties, evidence of HR bactericidal activity in these formulations is limited, being only recently described in planktonic bacteria. We defined antimicrobial/antiviral activity in planktonic and biofilm models and characterised the pharmacokinetics of HR release from lozenges.

Methods: Antimicrobial activity (purified or released from lozenges) was determined against oropharyngeal pathogens using minimum inhibitory concentration (MIC) and Log₁₀ reduction assays. Antiviral activity was determined by suspension test (EN14476). Antibiofilm effects employed minimum biofilm eradication concentration assays and confocal laser scanning microscopy. HR release from lozenges was studied in vitro and in vivo using HPLC.

Results: HR exhibited MICs ≤ 16 µg/mL against 19/25 strains including: Streptococcus, Staphylococcus and Candida spp. Marked bactericidal activity (>3_log10; >99.9% reduction) occurred within 10 minutes. Significant anti-biofilm activity was evident in streptococcal and candidal biofilms (p < 0.05). Log₁₀ reduction in virucidal infectivity of HR in lozenges ranged from 1-log₁₀ to 3.5-log₁₀. In vivo, HR exhibited rapid release (within 1 minute) from lozenges into saliva.

Conclusion: Rapid release and antimicrobial activity of HR against oropharyngeal pathogens was evident, occurring at concentrations ≥ 2-fold lower than present in saliva, highlighting the potential application of HR in the treatment of oropharyngeal infections.