Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

Background  

Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles.     

Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

Background  

Porphyromonas gingivalis is the foremost oral pathogen of adult periodontitis in humans. However, the mechanisms of bacterial invasion and the resultant destruction of the gingival tissue remain largely undefined.     

Species specificity,surface exposure,protein expression,immunogenicity, and participation in biofilm formation of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> HmuY

Background  

Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY.     

Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells

Background  

Porphyromonas gingivalis in subgingival dental plaque, as part of a mature biofilm, has been strongly implicated in the onset and progression of chronic periodontitis. In this study using DNA microarray we compared the global gene expression of a P. gingivalis biofilm with that of its planktonic counterpart grown in the same continuous culture.     

Periodontitis in Cardiovascular Disease Patients with or without Marfan Syndrome -A Possible Role of Prevotella intermedia-

Background

Although periodontitis is a risk factor for cardiovascular disease (CVD), the influence of periodontitis on Marfan syndrome (MFS) with CVD is unclear. The aim of this study was to assess the relationship between periodontal bacterial burden and MSF with CVD.

Methods and Results

The subjects were patients with MFS with CVD (n = 47); age and gender matched non-MFS CVD patients (n = 48) were employed as controls. Full-mouth clinical measurements, including number of teeth, probing of pocket depth (PD), bleeding on probing (BOP) and community periodontal index (CPI) were recorded. We also evaluated the existence of three periodontal pathogens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia using polymerase chain reaction assays. Serum antibody titers against the pathogens were also measured. We revealed that MFS with CVD patients had periodontitis more frequently than the age and gender matched non-MFS CVD control subjects. MFS with CVD patients had significantly severer periodontitis, fewer remaining teeth and deeper PD compared to the non-MFS CVD controls. Furthermore, the serum antibody titer level against Prevotella intermedia was significantly lower in MFS plus CVD patients compared to the non-MFS CVD patients.

Conclusion

Periodontitis may influence the pathophysiology of cardiovascular complications in MFS patients. A specific periodontal pathogen might be a crucial therapeutic target to prevent CVD development.     

Setup of an In Vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens

Background

Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible.

Methods

Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups.

Findings

S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability.

Conclusions

This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.     

Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with l-N⁶-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A₂ (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. l-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA₂ but not PI-3K-dependent fashion.     

Detection rates of periodontal bacteria and herpesviruses in different forms of periodontal disease

The aim was to investigate the detection rates of periodontal bacteria (Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans) and herpesviruses (herpes simplex virus-1 [HSV-1], cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) in different forms and severity of periodontal disease, and to compare them with those in periodontally healthy subjects. One hundred and twenty-nine patients participated in the study: 39 diagnosed with periodontal abscess (PA), 33 with necrotizing ulcerative periodontitis (NUP), 27 with chronic periodontitis (CP), and 30 participants with healthy periodontal tissue represented a healthy control group. All patients with periodontal disease (PA, NUP, and CP) were also divided into two groups according to the severity of their disease: moderate and severe periodontitis. The subgingival samples were collected from the periodontitis active sites and the detection of microorganisms was performed by end-point polymerase chain reaction analyses. The results revealed significantly higher detection rates of P. gingivalis, T. forsythia, and P. intermedia in all three groups of patients with periodontitis than in healthy participants. The highest detection rate of A. actinomycetemcomitans was noticed in CP, which was significantly higher than that in PA, NUP, and healthy control. The occurrence of EBV was significantly higher in NUP than in CP and healthy participants. CMV was detected significantly more frequently in PA and NUP than in CP and healthy participants. Comparisons among healthy participants and patients with moderate and severe periodontitis showed significantly higher detection rates of EBV and CMV in patients with severe forms of periodontitis than in healthy participants and those with moderate periodontitis.     

Cytolethal Distending Toxin in Isolates of Aggregatibacter actinomycetemcomitans from Ghanaian Adolescents and Association with Serotype and Disease Progression

Background and Objectives

The cytolethal distending toxin (Cdt) is a highly conserved exotoxin that are produced by a number of Gram negative bacteria, including Aggregatibacter actinomycetemcomitans, and affects mammalian cells by inhibiting cell division and causing apoptosis. A complete cdt-operon is present in the majority of A. actinomycetemcomitans, but the proportion of isolates that lack cdt-encoding genes (A, B and C) varies according to the population studied. The objectives of this study were to examine serotype, Cdt-genotype, and Cdt-activity in isolates of A. actinomycetemcomitans collected from an adolescent West African population and to examine the association between the carrier status of A. actinomycetemcomitans and the progression of attachment loss (AL).

Materials and Methods

A total of 249 A. actinomycetemcomitans isolates from 200 Ghanaian adolescents were examined for serotype and cdt-genotype by PCR. The activity of the Cdt-toxin was examined by DNA-staining of exposed cultured cells and documented with flow cytometry. The periodontal status of the participants was examined at baseline and at a two-year follow-up.

Results

Presence of all three cdt-encoding genes was detected in 79% of the examined A. actinomycetemcomitans isolates. All these isolates showed a substantial Cdt-activity. The two different cdt-genotypes (with and without presence of all three cdt-encoding genes) showed a serotype-dependent distribution pattern. Presence of A. actinomycetemcomitans was significantly associated with progression of AL (OR = 5.126; 95% CI = [2.994–8.779], p<0.001).

Conclusion

A. actinomycetemcomitans isolated from the Ghanaian adolescents showed a distribution of serotype and cdt-genotype in line with results based on other previously studied populations. Presence of A. actinomycetemcomitans was significantly associated with disease progression, in particular the b serotype, whereas the association with disease progression was not particularly related to cdt-genotype, and Cdt-activity.     

The core genome of the anaerobic oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Background  

The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.     

Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.     

Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis

The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35–76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants’ health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0–0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1–57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0–88.2) and body mass index (OR = 3.0; 95%CI 1.7–5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is suggested.     

Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

Cationic antimicrobial peptide: LL-37 and its role in periodontitis

Background

Periodontitis i.e. inflammation of the periodontium is a multifactorial disease. Antimicrobial peptides (AMPs) which demonstrate a broad-spectrum of activity against varied number of bacteria, fungi, viruses, and parasites, and cancerous cells have been linked to periodontitis. The AMPs even possess the caliber of immunomodulation, and are significantly responsive to innate immuno-stimulation and infections. LL-37 plays a salubrious role by preventing and in treatment of chronic forms of periodontitis.

Objective

In the present work we will review the role of antimicrobial peptide LL-37 in periodontitis.

Methods

A systematic search was carried out from the beginning till August, 2016 using the Pubmed search engine. The keywords included “LL-37,” “periodontitis,” “Papillon–Lefevre syndrome,” “Morbus Kostmann,” “Haim-Munk syndrome” along with use of Boolean operator “and.”

Results

The search resulted in identifying 67 articles which included articles linking LL-37 with periodontitis, articles on Papillon–Lefevre syndrome, Morbus Kostmann, Haim-Munk syndrome, LL-37 and periodontitis and articles on pathogenicity of periodontitis.

Conclusion

The literature search concluded that LL-37 plays a pivotal role in preventing and treatment of severe form of periodontitis.

     

Periodontitis,periodontopathic bacteria and lactoferrin

Lactoferrin (LF) is a component of saliva and is suspected to be a defense factor against oral pathogens including Streptococcus mutans and Candida albicans. Periodontitis is a very common oral disease caused by periodontopathic bacteria. Antimicrobial activities and other biological effects of LF against representative periodontopathic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, have been widely studied. Association of polymorphisms in LF with incidence of aggressive periodontitis and the role of LF in the gingival crevicular fluid as a marker of periodontitis severity have also been reported. Periodontopathic bacteria reside as a biofilm in supragingival and subgingival plaque. Our recent study indicated that LF exhibits antibacterial activity against planktonic forms of P. gingivalis and P. intermedia at higher concentrations, and furthermore, LF effectively inhibits biofilm formation and reduces the established biofilm of these bacteria at physiological concentrations. A small-scale clinical study indicated that oral administration of bovine LF reduces P. gingivalis and P. intermedia in the subgingival plaque of chronic periodontitis patients. LF seems to be a biofilm inhibitor of periodontopathic bacteria in vitro and in vivo.     

Nanoparticle-Encapsulated Chlorhexidine against Oral Bacterial Biofilms

Background

Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.

Methodology/Principal Findings

The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.

Conclusions/Significance

This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.     

Oral metagenomes from Native American Ancestors reveal distinct microbial lineages in the pre-contact era

Objectives

Limited studies have focused on how European contact and colonialism impacted Native American oral microbiomes, specifically, the diversity of commensal or opportunistically pathogenic oral microbes, which may be associated with oral diseases. Here, we studied the oral microbiomes of pre-contact Wichita Ancestors, in partnership with the Descendant community, The Wichita and Affiliated Tribes, Oklahoma, USA.

Materials and Methods

Skeletal remains of 28 Wichita Ancestors from 20 archeological sites (dating approximately to 1250–1450 CE) were paleopathologically assessed for presence of dental calculus and oral disease. DNA was extracted from calculus, and partial uracil deglycosylase-treated double-stranded DNA libraries were shotgun-sequenced using Illumina technology. DNA preservation was assessed, the microbial community was taxonomically profiled, and phylogenomic analyzes were conducted.

Results

Paleopathological analysis revealed signs of oral diseases such as caries and periodontitis. Calculus samples from 26 Ancestors yielded oral microbiomes with minimal extraneous contamination. Anaerolineaceae bacterium oral taxon 439 was found to be the most abundant bacterial species. Several Ancestors showed high abundance of bacteria typically associated with periodontitis such as Tannerella forsythia and Treponema denticola. Phylogenomic analyzes of Anaerolineaceae bacterium oral taxon 439 and T. forsythia revealed biogeographic structuring; strains present in the Wichita Ancestors clustered with strains from other pre-contact Native Americans and were distinct from European and/or post-contact American strains.

Discussion

We present the largest oral metagenome dataset from a pre-contact Native American population and demonstrate the presence of distinct lineages of oral microbes specific to the pre-contact Americas.     

Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical,microbiological and serological study

Introduction

The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.

Methods

In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.

Results

A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.

Conclusions

Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.     

Lactobacillus salivarius and L. gasseri down-regulate Aggregatibacter actinomycetemcomitans exotoxins expression

Beneficial microbes, such as lactobacilli establish a symbiosis with the host and confer health-associated effects, by limiting the growth of indigenous pathogens and challenging microbes introduced by altered foods. Nevertheless, there is scarce information on the effects of beneficial microbes on the virulence properties of bacterial species associated with oral diseases, such as periodontitis. Aggregatibacter actinomycetemcomitans is a Gram-negative species highly implicated in the etiology of localized aggressive periodontitis. The objective of this study was to investigate the effect of lactobacilli on the expression of the two major virulence factors of A. actinomycetemcomitans. Lactobacillus salivarius and L. gasseri were selected as beneficial species. The gene expressions of leukotoxin (LtxA) and cytolethal distending toxin (CdtB) by A. actinomycetemcomitans were analyzed in response to challenge by lactobacilli cell-free supernatants. Neither lactobacilli affected the growth, but strongly attenuated the expressions of both CdtB and LtxA in the two A. actinomycetemcomitans strains tested. This reduction of the expression of these two exotoxins was time-dependent. These fundamental findings may indicate that lactobacilli can reduce the virulence of putative opportunistic oral pathogens, and may provide insights to future therapeutic approaches for the respective diseases.