Actinomycetemcomitans Strains Research Articles

Periodontal pathogen Aggregatibacter actinomycetemcomitans JP2 correlates with colonic leukocytes decrease and gut microbiome imbalance in mice.

Evidence suggests that translocation of oral pathogens through the oral-gut axis may induce intestinal dysbiosis. This study aimed to evaluate the impact of a highly leukotoxic Aggregatibacter actinomycetemcomitans (Aa) strain on the gut microbiota, intestinal mucosal integrity and immune system in healthy mice. Eight-week-old male C57BL6 mice were divided into control (n = 16) and JP2 groups (n = 19), which received intragastric gavage with PBS and with a suspension of Aa JP2 (HK921), respectively, twice a week for 4 weeks. Colonic lamina propria, fecal material, serum, gingival tissues, and mandibles were obtained for analyses of leukocyte populations, inflammatory mediators, mucosal integrity, alveolar bone loss, and gut microbiota. Differences between groups for these parameters were examined by non-parametric tests. The gut microbial richness and the number of colonic macrophages, neutrophils, and monocytes were significantly lower in Aa JP2-infected mice than in controls (p < .05). In contrast, infected animals showed higher abundance of Clostridiaceae, Lactobacillus taiwanensis, Helicobacter rodentium, higher levels of IL-6 expression in colonic tissues, and higher splenic MPO activity than controls (p < .05). No differences in tight junction expression, serum endotoxin levels, and colonic inflammatory cytokines were observed between groups. Infected animals presented also slightly more alveolar bone loss and gingival IL-6 levels than controls (p < .05). Based on this model, intragastric administration of Aa JP2 is associated with changes in the gut ecosystem of healthy hosts, characterized by less live/recruited myeloid cells, enrichment of the gut microbiota with pathobionts and decrease in commensals. Negligible levels of colonic pro-inflammatory cytokines, and no signs of mucosal barrier disruption were related to these changes.

Dormancy-like Phenotype of Aggregatibacter actinomycetemcomitans: Survival during Famine.

Microbes frequently experience nutrient deprivations in the natural environment and may enter dormancy. Aggregatibacter actinomycetemcomitans is known to establish long-term infections in humans. This study examined the dormancy-like phenotype of an A. actinomycetemcomitans strain D7S-1 and its isogenic smooth-colony mutant D7SS. A tissue culture medium RPMI-1640 was nutrient-deficient (ND) and unable to support A. actinomycetemcomitans growth. RPMI-1640 amended with bases was nutrient-limited (NL) and supported limited growth of A. actinomycetemcomitans less than the nutrient-enriched (NE) laboratory medium did. Strain D7S-1, after an initial 2-log reduction in viability, maintained viability from day 4 to day 15 in the NL medium. Strain D7SS, after 1-log reduction in viability, maintained viability from day 3 to day 5. In contrast, bacteria in the NE medium were either non-recoverable (D7S-1; >6-log reduction) or continued to lose viability (D7SS; 3-log reduction) on day 5 and beyond. Scanning and transmission electron microscopy showed that A. actinomycetemcomitans in the NL medium formed robust biofilms similar to those in the NE medium but with evidence of stress. A. actinomycetemcomitans in the ND medium revealed scant biofilms and extensive cellular damage. We concluded that A. actinomycetemcomitans grown in the NL medium exhibited a dormancy-like phenotype characterized by minimum growth, prolonged viability, and distinct cellular morphology.

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.

Silica-based silver nanocomposite 80S/Ag as Aggregatibacter actinomycetemcomitans inhibitor and its in vitro bioactivity

Background/purposeAs a commonly-found pathogen in periodontal disease, Aggregatibacter actinomycetemcomitans has been reported with several antibiotic resistance. Thus, to develop an alternative and protective therapy for A. actinomycetemcomitans infections is urgently needed in dentistry. In this study, we sought to synthesize a silica-based material to deliver silver nanoparticles for antibacterial purposes. Also, the bioactivities were examined via analyzing the formation of hydroxyapatite. Materials and methodsThe 80S/Ag powders were prepared by the evaporation-induced self-assembly method, with Si, Ca, P, and Ag composition ratios of 80, 15, 5, and 1/5/10 (mole percentage), respectively. The nitrogen adsorption-desorption isotherms, transmission electron microscope, selected area electron diffraction, and Fourier transform infrared spectroscopy were conducted for textural analyses. The disk diffusion test was carried out against A. actinomycetemcomitans strain ATCC 29523. In vitro bioactivity assessment involved soaking 80S/Ag membrane powders in acellular simulated body fluid. ResultsWe successfully developed a material consisting of Si, Ca, P, and Ag, namely the 80S/Ag. In the antibacterial testing, the 80S/Ag demonstrated antibacterial activity against the commonly-found oral pathogen, A. actinomycetemcomitans, with a long-lasting effect for 168h. The formation of hydroxyapatite in simulated body fluid highlighted the characteristic of dentine remineralization for the 80S/Ag. The increased pH values after immersion in simulated body fluid would help neutralize the acidic oral environment. ConclusionOur results indicate that 80S/Ag possesses remarkable antibacterial properties, hydroxyapatite formation, and increased pH values after immersion in simulated body fluid, supporting the potential therapeutic application of 80S/Ag for treating periodontal disease.

A population-based study of the salivary prevalence of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in Saudi Arabian adults with chronic periodontitis

AimsThis study was designed to investigate the incidence of subgingival Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infections and their relationship with genetic variability in Saudi Arabian adults with chronic periodontitis and to evaluate the correlation between infection frequency and other variables. MethodsMultiplex polymerase chain reaction (PCR) assays were developed to detect P. gingivalis 16 ​S rDNA, fimbria (fimA), and collagenase (prtC) genes, as well as the A. actinomycetemcomitans 16 ​S rDNA, leukotoxin (lktA), and fimbria-associated protein (fap) genes in saliva samples from 50 patients with periodontitis and 51 healthy subjects. ResultsThe incidence of both P. gingivalis and A. actinomycetemcomitans infections was higher (72% and 14%) in the “Periodontal disease” group than in the healthy subjects (P ​< ​0.001). The P. gingivalis 16 ​S rDNA, fimA, and prtC genes were detected in 43.56%, 61.36%, and 84.09% of saliva samples, respectively, while A. actinomycetemcomitans 16 ​S rDNA, lktA, and fap genes were detected in 6.93%, 71.43%, and 0%, respectively. P. gingivalis strains with the fimA+/prtC ​+ ​genotype were found in 61.11% and 25% of chronic periodontitis patients and healthy subjects, respectively. By comparison, A. actinomycetemcomitans strains with the lktA+ ​genotype were found in 71.43% of chronic periodontitis patients. ConclusionInfection with fimA+ and prtC+ ​P. gingivalis or lktA+ ​A. actinomycetemcomitans was correlated with periodontal disease occurrence in Saudi adults. Nevertheless, A. actinomycetemcomitans lktA and P. gingivalis fimA and prtC are closely linked to periodontal destruction, whereas A. actinomycetemcomitans fap is not.

Lidocaine- and chloramphenicol-loaded nanoparticles embedded in a chitosan/hyaluronic acid/glycerol matrix: Drug-eluting biomembranes with potential for guided tissue regeneration

Guided tissue regeneration (GTR) is a dentistry technique based on the use of polymeric biomembranes as physical barriers for selective cell exclusion, directing the growth of gingival tissue, bone tissue, and periodontal ligaments in a region previously affected by periodontitis. Postoperative pain and microbial infection constitute, however, two major challenges to be tackled right after implantation. To address these challenges, we prepared and characterized eight chitosan/hyaluronic acid/glycerol (CS/HA/GL) bioresorbable membranes embedded with lidocaine- and chloramphenicol-loaded polycaprolactone nanoparticles (LDNP and CHNP, respectively), combining the local anesthetic effects of lidocaine with the antibacterial effects of chloramphenicol. The formulations were prepared with varying amounts of CS, HA, GL, LDNP, and CHNP. As a plasticizing agent, GL could modulate the samples mechanical properties such as thickness, morphology, tensile strength, elongation at break, as well as swelling and degradation in simulated saliva. Two samples exhibited greater resistance to biodegradation and were selected for further studies. Their drug release profiles indicated that LDNP and CHNP first detach from the membrane matrix, and a zeroth order drug release kinetics from the detached NPs dominates the overall process thereafter, with lidocaine being released 3 times faster than chloramphenicol, in a controlled and sustained rate over time. Drug encapsulation efficiency was such that optimal samples exhibited bactericidal activity (inhibition halos) against gram-positive S. aureus and gram-negative A. actinomycetemcomitans strains similar to that observed for free chloramphenicol. Finally, one of these samples showed no intrinsic toxicity against healthy mammalian model cells (99% viability for the unloaded membrane; 80% viability for the fully LDNP- and CHNP-loaded membrane), and may now be further optimized as a drug-eluting biomembrane with potential for GTR.

Pilus PilA of the naturally competent HACEK group pathogen Aggregatibacter actinomycetemcomitans stimulates human leukocytes and interacts with both DNA and proinflammatory cytokines

Each HACEK group pathogen, which can cause infective endocarditis, expresses type IVa pili. The type IVa major pilin PilA plays a role in bacterial colonization, virulence, twitching motility, and the uptake of extracellular DNA. The type IV prepilin homolog PilA of the periodontal pathogen A. actinomycetemcomitans (AaPilA) is linked to DNA uptake and natural competence.Our aim was to investigate the virulence properties and immunogenic potential of AaPilA. Since Neisseria meningitidis PilE, which shares sequence similarity with AaPilA, participates in sequestering host cytokines, we examined the ability of AaPilA to interact with various cytokines. Moreover, we investigated the structural characteristics of AaPilA with molecular modeling.AaPilA was conserved among A. actinomycetemcomitans strains. One of the 18 different natural variants, PilAD7S, is present in naturally competent strains. This variant interacted with DNA and bound interleukin (IL)-8 and tumor necrosis factor (TNF)-α. Specific anti-AaPilA antibodies were present in A. actinomycetemcomitans-positive periodontitis patient sera, and the production of reactive oxygen species from human neutrophils was less effectively induced by the ΔpilA mutant than by the wild-type strains. However, AaPilA did not stimulate human macrophages to produce proinflammatory cytokines, nor was it cytotoxic.The results strengthen our earlier hypothesis that the DNA uptake machinery of A. actinomycetemcomitans is involved in the sequestration of inflammatory cytokines. Furthermore, AaPilA stimulates host immune cells, such as B cells and neutrophils, making it a potential virulence factor.

Genomic Islands Shape the Genetic Background of Both JP2 and Non-JP2 Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a periodontal pathogen associated with periodontitis. This species exhibits substantial variations in gene content among different isolates and has different virulence potentials. This study examined the distribution of genomic islands and their insert sites among genetically diverse A. actinomycetemcomitans strains by comparative genomic analysis. The results showed that some islands, presumably more ancient, were found across all genetic clades of A. actinomycetemcomitans. In contrast, other islands were specific to individual clades or a subset of clades and may have been acquired more recently. The islands for the biogenesis of serotype-specific antigens comprise distinct genes located in different loci for serotype a and serotype b–f strains. Islands that encode the same cytolethal distending toxins appear to have been acquired via distinct mechanisms in different loci for clade b/c and for clade a/d/e/f strains. The functions of numerous other islands remain to be elucidated. JP2 strains represent a small branch within clade b, one of the five major genetic clades of A. actinomycetemcomitans. In conclusion, the complex process of genomic island acquisition, deletion, and modification is a significant force in the genetic divergence of A. actinomycetemcomitans. Assessing the genetic distinctions between JP2 and non-JP2 strains must consider the landscape of genetic variations shaped by evolution.

Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells.

Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This in vitro study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by Aggregatibacter actinomycetemcomitans. OBA-9 GECs were infected with A. actinomycetemcomitans strain JP2 at an MOI of 1:100 and/or co-infected with Lactobacillus acidophilus La5 (La5) or Lacticaseibacillus rhamnosus Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1β, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with A. actinomycetemcomitans or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of A. actinomycetemcomitans to GECs was 1.8 × 107 (± 1.2 × 106) cells/well in the mono-infection but reduced to 1.2 × 107 (± 1.5 × 106) in the co-infection with Lr32 and to 6 × 106 (± 1 × 106) in the co-infection with La5 (p < 0.05). GECs mono-infected with A. actinomycetemcomitans produced CXCL-8, GM-CSF, and IL-1β, and the co-infection with both probiotic strains altered this profile. While the co-infection of A. actinomycetemcomitans with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1β. The probiotics upregulated the expression of TLR2 and downregulated TLR4 in cells co-infected with A. actinomycetemcomitans. A. actinomycetemcomitans-induced the upregulation of NRLP3 was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene BCL-2 was upregulated, whereas the pro-apoptotic BAX was downregulated in cells co-infected with A. actinomycetemcomitans and the probiotics. Infection with A. actinomycetemcomitans induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in A. actinomycetemcomitans colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, L. acidophilus La5 properties suggest that this strain has a higher potential to control A. actinomycetemcomitans-associated periodontitis than L. rhamnosus Lr32.

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain CU1000N.

ABSTRACTHere, we report the complete genome sequence of Aggregatibacter actinomycetemcomitans strain CU1000N. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.

Contribution of adhesion proteins to Aggregatibacter actinomycetemcomitans biofilm formation.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with periodontal disease and multiple disseminated extra-oral infections. Colonization of these distinct physiological niches is contingent on the expression of specific surface proteins during the initiation of developing biofilms. In this investigation, we studied fimbriae and three well-characterized nonfimbrial surface proteins (EmaA, Aae, and ApiA/Omp100) for their contribution to biofilm formation. Mutations of these proteins in multiple strains covering four different serotypes demonstrated variance in biofilm development that was strain dependent but independent of serotype. In a fimbriated background, only inactivation of emaA impacted biofilm mass. In contrast, inactivation of emaA and/or aae affected biofilm formation in nonfimbriated A. actinomycetemcomitans strains, whereas inactivation of apiA/omp100 had little effect on biofilm formation. When these genes were expressed individually in Escherichia coli, all transformed strains demonstrated an increase in biofilm mass compared to the parent strain. The strain expressing emaA generated the greatest mass of biofilm, whereas the strains expressing either aae or apiA/omp100 were greatly reduced and similar in mass. These data suggest a redundancy in function of these nonfimbrial adhesins, which is dependent on the genetic background of the strain investigated.

In Search of Spectroscopic Signatures of Periodontitis: A SERS-Based Magnetomicrofluidic Sensor for Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

Recently, Porphyromonas gingivalis, the keystone pathogen implicated in the development of gum disease (periodontitis), was detected in the brains of Alzheimer’s disease patients, opening up a fascinating possibility that it is also involved in the pathobiology of this neurodegenerative illness. To verify this hypothesis, an unbiased, specific, and sensitive method to detect this pathogen in biological specimens is needed. To this end, our interdisciplinary studies demonstrate that P. gingivalis can be easily identified by surface-enhanced Raman scattering (SERS). Moreover, based on SERS measurements, P. gingivalis can be distinguished from another common periodontal pathogen, Aggregatibacter actinomycetemcomitans, and also from ubiquitous oral Streptococcus spp. The results were confirmed by principal component analysis (PCA). Furthermore, we have shown that different P. gingivalis and A. actinomycetemcomitans strains can easily adsorb to silver-coated magnetic nanoparticles (Fe2O3@AgNPs). Thus, it is possible to magnetically separate investigated bacteria from other components of a specimen using the microfluidic chip. To obtain additional enhancement of the Raman signal, the NPs adsorbed to bacterial cells were magnetically attracted to the Si/Ag SERS platform. Afterward, the SERS spectra could be recorded. Such a time-saving procedure can be very helpful in rapid medical diagnostics and thus in starting the appropriate pharmacological therapy to prevent the development of periodontitis and associated comorbidities, e.g., Alzheimerʼs disease.

Etiology of aggressive periodontitis in individuals of African descent.

Aggressive periodontitis (AgP) is a form of periodontitis that affects adolescents and has a significantly higher prevalence in individuals of African descent. AgP typically shows familial aggregation, suggesting a genetic predisposition. Young age, good health status, rapid attachment loss, and familial aggregation are the primary features of this disease. AgP has been closely linked to specific bacterial strains of Aggregatibacter actinomycetemcomitans. A. actinomycetemcomitans strains isolated from patients with AgP produce leukotoxin (LtxA), which specifically affects polymorphonuclear leukocytes in primates, especially humans. High-throughput 16S rRNA gene sequencing and bioinformatics analyses revealed differences in the subgingival microbiota between patients with AgP and those with chronic periodontitis (ChP). The genera Atopobium and Prevotella show increased prevalences in AgP than in ChP. According to AgP susceptibility, several single nucleotide polymorphisms have been detected in different genes in individuals of African descent. Interleukin (IL)-1α and IL-1β genetic polymorphisms may be associated with the severity of both ChP and AgP. An elevated serum level of IL-17 produced by Th17 cells may be a characteristic of AgP. Analyses of the relationships among bacteria, host defenses, genetic predisposition, and numerous other factors are required to understand the progression of this disease.

Expression of virulence factors under different environmental conditions in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium associated with periodontal diseases, especially aggressive periodontitis. The virulence factors of this pathogen, including adhesins, exotoxins, and endotoxin, have been extensively studied. However, little is known about their gene expression mode in the host. Herein, we investigated whether culture conditions reflecting in vivo environments, including serum and saliva, alter expression levels of virulence genes in the strain HK1651, a JP2 clone. Under aerobic conditions, addition of calf serum (CS) into a general medium induced high expression of two outer membrane proteins (omp100 and omp64). The high expression of omp100 and omp64 was also induced by an iron-limited medium. RNA-seq analysis showed that the gene expressions of several factors involved in iron acquisition were increased in the CS-containing medium. When HK1651 was grown on agar plates, genes encoding many virulence factors, including the Omps, cytolethal distending toxin, and leukotoxin, were differentially expressed. Then, we investigated their expression in five other A. actinomycetemcomitans strains grown in general and CS-containing media. The expression pattern of virulence factors varied among strains. Compared with the other five strains, HK1561 showed high expression of omp29 regardless of the CS addition, while the gene expression of leukotoxin in HK1651 was higher only in the medium without CS. HK1651 showed reduced biofilm in both CS- and saliva-containing media. Coaggregation with Fusobacterium nucleatum was remarkably enhanced using HK1651 grown in the CS-containing medium. Our results indicate that the expression of virulence factors is altered by adaptation to different conditions during infection.

Li-doped bioglass® 45S5 for potential treatment of prevalent oral diseases

ObjectivesDespite the excellent properties of both pure bioglasses (BG) and BG doped with therapeutic ions (such as Li) in hard tissue applications, there is not enough information about their role in the remineralization and bacterial-growth in oral diseases. The aim of this contribution is to evaluate the effect of both pure BG and BG doped with 5-wt% of Li (BGLi) on both the remineralization of in vitro demineralized human-teeth and the antimicrobial behavior against strains from caries and periodontitis. MethodsBioglass® 45S5 (BG) and BGLi were synthesized by the sol-gel method. The remineralization tests were carried out using in vitro demineralized enamel teeth and evaluated by Electron Microscopy (SEM) and Vickers micro-hardness (HV). The antimicrobial behavior of the particles was evaluated against S. mutans, A. actinomycetemcomitans, and P. gingivalis, representing pathogens from caries and periodontitis. ResultsEnamel lesion was partially remineralized when both bioglasses (BG and BGLi) were applied on its surface with micro-hardness recoveries around 45 %. They further inhibited the growth of S. mutans and P. gingivalis, at 50 and 200 mg/mL, respectively. BGLi presented a higher toxicity against A. actinomycetemcomitans than BG, with inhibition concentrations of 20 mg/mL and 100 mg/mL, respectively. ConclusionsBioglasses could be used in the treatment of two of the most prevalent oral diseases: caries and periodontitis, promoting the remineralization of the teeth and killing the main pathogens. The presence of Li did not affect the bioactivity of the bioglass and improved the antibacterial effect over A. actinomycetemcomitans strain.

Aggregatibacter actinomycetemcomitans induces a proatherosclerotic response in human endothelial cells in a three‐dimensional collagen scaffold model

AbstractThe role of periodontopathogens in inflammatory endothelial dysfunction is not known. This study characterizes a three‐dimensional model with human coronary artery endothelial cells on three‐dimensional (HCAEC‐3D) type I collagen scaffolds to evaluate whether infection with Aggregatibacter actinomycetemcomitans induces a proinflammatory response associated with atherosclerosis. The HCAEC‐3D culture was physicochemically characterized with regard to biocompatibility and barrier function. Then, the culture was infected with A. actinomycetemcomitans strain ATCC 29522 at multiplicities of infection (MOIs) of 1:1, 1:10, and 1:100. Cultures without infection and stimulated with A. actinomycetemcomitans lipopolysaccharide were used as controls. The secretion of soluble factors (IL‐6, IL‐1β, MCP‐1, RANTES, MIP‐1, IL‐8, IL‐1α, and TNF‐α) was evaluated via flow cytometry; TGF‐β1 was evaluated via enzyme‐linked immunosorbent assay (ELISA). The adhesion and migration of fluorescent human THP‐1 monocytes was evaluated. IL‐8, MCP‐1, and IL‐6 secretion increased in a dose‐dependent manner with A. actinomycetemcomitans infection and was significantly greater than that under control treatment. The concentration of TGF‐β1 was significantly higher at MOI 1:100 than in controls. Treatment of the 3D cultures with A. actinomycetemcomitans at different MOIs induced significant differences in the adhesion of monocytes to the endothelium compared to the control without infection. Lastly, conditioned media from 3D cultures treated with A. actinomycetemcomitans induced monocyte migration. The effects of IL‐8, MCP‐1, IL‐6, and TGF‐β1 on the endothelium indicate the ability of A. actinomycetemcomitans to induce an inflammatory response through a mechanism of monocyte adhesion and migration and endothelial dysfunction.

Residual decontamination chemical agents negatively affect adhesion and proliferation of osteoblast-like cells on implant surface

PurposeTo investigate the influence of implant surface decontaminated and uncontaminated on osteoblast-like cell adhesion and proliferationMaterials and methodsCommercially available implants of different brands and surface characteristics were selected: Biomet 3i® Nanotite (NT) and Osseotite (OT), Straumann® SLActive (SLA), and Neodent® Acqua Drive (ACQ) and Neoporos Drive CM (CM). Physical and chemical properties of the implants were investigated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and wettability analysis (WETT). Implants were previously contaminated with Aggregatibacter actinomycetemcomitans strains; after that, samples were decontaminated by different chemical methods. Decontaminated (test group; n = 15/type of implant) and uncontaminated (control group; n = 5/type of implant) samples were analyzed according to the number of human osteoblastic osteosarcoma cells (Saos-2) adhered on the implant surface after 24 h and 72 h in SEM images.ResultsACQ was found to be highly hydrophilic, and NT was the most hydrophobic implant. Increased variation of Saos-2 cell adhesion and proliferation were observed on all test and control groups. Controversially, at the proliferation analysis in 72 h, CM implant was the only implant that showed no significant difference between test and group (p = 0.2833; Tukey’s multiple comparisons test). NT implants showed the greater value of cell proliferation when compared with all types of implant surface (p = 0.0002; Tukey’s multiple comparisons test).ConclusionsThese findings suggest that decontaminated surfaces were able to impair the counting of osteoblast-like cell adhesion and proliferation.

Comparison of Minimum Inhibitory Concentrations of Hypericum Perforatum L. Essential Oils, 0.2 % Chlorhexidine and 10 % Povidone-iodine Over Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis

Hypericum perforatum is a useful plant because of its biological features, such as bactericidal, virucidal, and fungicidal effects. Elimination of periodontal pathogens, including Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, from the oral environment, is important for the stability of periodontal health. Therefore, we aimed to determine the antimicrobial activity of the Hypericum perforatum L. essential oils over these pathogens using cell cultures. The standard and clinical bacterial strains were used in this study. The essential oils extracted from Hypericum perforatum L. flowers were analyzed with gaschromatography/mass spectrometry. The antimicrobial activities of the essential oils along with 0.2 % chlorhexidine, 10 % povidone-iodine, and combinations of essential oils and 0.2 % chlorhexidine, and essential oils and 10 % povidone-iodine were evaluated by measuring the minimum inhibitory concentrations. Essential oils’ minimum inhibitory concentration value for clinical A. actinomycetemcomitans strains was significantly lower than that of the povidone-iodine solution (P<0.05). However, despite the low minimum inhibitory concentration value of essential oils, when compared to 0.2 % chlorhexidine, there was no statistically significant difference between the agents (P=0.113). For clinical strains of Porphyromonas gingivalis, the essential oils showed a lower minimum inhibitory concentration value compared to chlorhexidine and povidone-iodine. The essential oils combined with chlorhexidine or povidone-iodine showed significantly lower minimum inhibitory concentration value for both strains of Aggregatibacter actinomycetemcomitans (P<0.05). With the lower minimum inhibitory concentration values, the essential oils showed strong antimicrobial activity. Moreover, when combined with chlorhexidine or the povidone-iodine, the antimicrobial effect of essential oils on Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis strains increased synergistically.

O-Polysaccharide Plays a Major Role on the Virulence and Immunostimulatory Potential of Aggregatibacter actinomycetemcomitans During Periodontal Infection.

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium with high immunostimulatory and pathogenic potential involved in the onset and progression of periodontitis, a chronic disease characterized by aberrant immune responses followed by tooth-supporting bone resorption, which eventually leads to tooth loss. While several studies have provided evidence related to the virulence factors of A. actinomycetemcomitans involved in the host cell death and immune evasion, such as its most studied primate-specific virulence factor, leukotoxin, the role of specific lipopolysaccharide (LPS) domains remain poorly understood. Here, we analyzed the role of the immunodominant domain of the LPS of A. actinomycetemcomitans termed O-polysaccharide (O-PS), which differentiates the distinct bacterial serotypes based on its antigenicity. To determine the role of the O-PS in the immunogenicity and virulence of A. actinomycetemcomitans during periodontitis, we analyzed the in vivo and in vitro effect of an O-PS-defective transposon mutant serotype b strain, characterized by the deletion of the rmlC gene encoding the α-L-rhamnose sugar biosynthetic enzyme. Induction of experimental periodontitis using the O-PS-defective rmlC mutant strain resulted in lower tooth-supporting bone resorption, infiltration of Th1, Th17, and Th22 lymphocytes, and expression of Ahr, Il1b, Il17, Il23, Tlr4, and RANKL (Tnfsf11) in the periodontal lesions as compared with the wild-type A. actinomycetemcomitans strain. In addition, the O-PS-defective rmlC mutant strain led to impaired activation of antigen-presenting cells, with less expression of the co-stimulatory molecules CD40 and CD80 in B lymphocytes and dendritic cells, and downregulated expression of Tnfa and Il1b in splenocytes. In conclusion, these data demonstrate that the O-PS from the serotype b of A. actinomycetemcomitans plays a key role in the capacity of the bacterium to prime oral innate and adaptive immune responses, by triggering the Th1 and Th17-driven tooth-supporting bone resorption during periodontitis.

Oral Lactobacillus strains reduce cytotoxicity and cytokine release from peripheral blood mononuclear cells exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro

BackgroundThis study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1β secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay.ResultsSupernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1β secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1β secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1β, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed.ConclusionsLactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.