RESUMO

AIM: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Assuntos

Aggregatibacter actinomycetemcomitans , Perda do Osso Alveolar , Colo , Disbiose , Microbioma Gastrointestinal , Camundongos Endogâmicos C57BL , Animais , Masculino , Camundongos , Colo/microbiologia , Perda do Osso Alveolar/microbiologia , Disbiose/microbiologia , Mucosa Intestinal/microbiologia , Leucócitos , Interleucina-6/sangue , Interleucina-6/análise , Gengiva/microbiologia , Peroxidase , Lactobacillus , Clostridiales , Fezes/microbiologia , Infecções por Pasteurellaceae/microbiologia , Baço

RESUMO

The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1ß and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes.

RESUMO

Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin-triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N-methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA-expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.

RESUMO

INTRODUCTION: The role of suppressor of cytokine signaling 2 (SOCS2) in Aggregatibacter actinomycetemcomitans (Aa)-induced alveolar bone loss is unknown; thus, it was investigated in this study. METHODS: Alveolar bone loss was induced by infecting C57BL/6 wild-type (WT) and Socs2-knockout (Socs2-/-) mice with Aa. Bone parameters, bone loss, bone cell counts, the expression of bone remodeling markers, and cytokine profile were evaluated by microtomography, histology, qPCR, and/or ELISA. Bone marrow cells (BMC) from WT and Socs2-/- mice were differentiated in osteoblasts or osteoclasts for analysis of the expression of specific markers. RESULTS: Socs2-/- mice intrinsically exhibited irregular phenotypes in the maxillary bone and an increased number of osteoclasts. Upon Aa infection, SOCS2 deficiency resulted in the increased alveolar bone loss, despite decreased proinflammatory cytokine production, in comparison to the WT mice. In vitro, SOCS2 deficiency resulted in the increased osteoclasts formation, decreased expression of bone remodeling markers, and proinflammatory cytokines after Aa-LPS stimulus. CONCLUSIONS: Collectively, data suggest that SOCS2 is a regulator of Aa-induced alveolar bone loss by controlling the differentiation and activity of bone cells, and proinflammatory cytokines availability in the periodontal microenvironment and an important target for new therapeutic strategies. Thus, it can be helpful in preventing alveolar bone loss in periodontal inflammatory conditions.

Assuntos

Perda do Osso Alveolar , Doenças Periodontais , Camundongos , Animais , Perda do Osso Alveolar/genética , Aggregatibacter actinomycetemcomitans/metabolismo , Camundongos Endogâmicos C57BL , Doenças Periodontais/metabolismo , Osteoclastos/metabolismo , Citocinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo

RESUMO

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-1ß, 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-1ß, 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-1ß. 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.

RESUMO

Aggregatibacter actinomycetemcomitans (Aa) is abundant within the microbial dysbiotic community of some patients with periodontitis. Aa outer membrane protein 29 (OMP29), a member of the OMPA family, mediates the invasion of Aa to gingival epithelial cells (GECs). This study evaluated the effect of OMP29 and its paralogue OMP29par on the response of GECs to Aa. The omp29 or/and omp29 par deletion mutants AaΔ29, AaΔ29P, and AaΔ29Δ29P were constructed, and recombinant Aa OMP29His was obtained. Microarray analysis and the evaluation of cxcl-8 gene expression were performed to examine the response of GECs line OBA-09 to Aa and its mutants. The expression of cxcl-8 and its product CXCL-8 was examined in LPS-stimulated OBA-09 cells with Aa OMP29His. Proteomics analysis showed that the deletion of omp29 led to overexpression of both OMP29par and another membrane protein OMP39, the expression of which was further increased in AaΔ29Δ29P. OBA-09 cells challenged with AaΔ29Δ29P exhibited a higher expression of cxcl-8 in comparison to wildtype Aa strain AaD7S or single-deletion mutants AaΔ29 or AaΔ29P. LPS-stimulated OBA-09 cells challenged with Aa OMP29His showed reduced expressions of cxcl-8 and its product CXCL-8. OBA-09 cells challenged with AaΔ29Δ29P in comparison to Aa strain AaD7S resulted in higher expressions of genes involved in apoptosis and inflammatory response such as bcl2, birc3, casp3, c3, ep300, fas, fosb, grb2, il-1α, il-1ß, il-6, cxcl-8, nr3c1, prkcq, socs3, and tnfrsf1ß and reduced expressions of cd74, crp, faslg, tlr1, and vcam1. The results suggested a novel strategy of Aa, mediated by OMP29 and OMP29par, to evade host immune response by inhibiting CXCL-8 expression and modulating the genes involved in apoptosis and inflammatory response in GECs. Pending further confirmation, the strategy might interfere with the recruitment of neutrophils and dampen the host inflammatory response, leading to a more permissive subgingival niche for bacterial growth.

RESUMO

Abstract Bacteria are related do different oral diseases, such as dental caries and periodontal disease. Therefore, the control or/and eradication of microorganisms and their by-products is primordial for the success of their treatment. An alternative for decrease bacterial load is the use of plant extracts used in popular medicine. The cytotoxicity and antimicrobial action of extracts of Cariniana rubra Gardiner ex Miers, Senna martiniana, Anadenanthera colubrina (Vell.) Brenan and Spiranthera odoratissima St. Hil. against strains of Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Aggregatibacter actinomyces- tencomitans and Candida albicans were investigated. Cytotoxicity was assessed at concentrations of 1, 10, 40, 80, 100 and 1000 μg/mL by means of the MTT test and compared to a control group with untreated cells. Those with acceptable cytotoxicity had the antimicrobial action measured by the XTT test. As a positive control, sodium hypochlorite was used. Cariniana rubra Gardiner ex Miers had the highest citototoxicity results while Spiranthera odoratissima St. Hil. had the best results, but all extracts showed acceptable cytotoxicity at different concentrations. The plant extracts showed higher activity against A. actinomycetencomitans: Anadenanthera columbrina (Vell.) Brenan (80.52%) at 40 μg/mL, Spiranthera odoratissima St. Hil (78.48%) in 1 μg/mL, Senna martiniana (73.28%) in the concentration of 40 μg/mL and Cariniana rubra Gardiner ex Miers (70.50%) in 10 μg/mL. All extracts analyzed showed acceptable cytotoxicity at different concentrations and were promising for inhibition of the pathogenic microorganisms studied.

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Resumo Bactérias estão relacionadas a diferentes doenças bucais, como a cárie dentária e a doença periodontal. Assim, o controle e/ou erradicação de microrganismos e seus subprodutos é primordial para o sucesso dos tratamentos. Uma alternativa para diminuir a carga bacteriana é a utilização de extratos vegetais utilizados na medicina popular. A citotoxicidade e ação antimicrobiana de extratos de Cariniana rubra Gardinerex Miers, Senna martiniana H.S. Irwin & Barneby, Anadenanthera colubrina (Vell.) Brenan e Spiranthera odoratissima St. Hil. contra cepas de Streptococcus mutans, Enterococcusfaecalis, Staphylococcus aureus, Escherichia coli, Agartibacter actinomycetencomitans e Candida albicans foram investigados. A citotoxicidade foi avaliada nas concentrações de 1, 10, 40, 80, 100 e 1000 μg/mL por meio do teste MTT. Aqueles com citotoxicidade aceitável tiveram a ação antimicrobiana medida pelo teste XTT. Cariniana rubra Gardinerex Miers apresentou os maiores resultados de citototoxicidade, enquanto Spiranthera odoratissima St. Hil. obteve os melhores resultados, mas todos os extratos apresentaram citotoxicidade aceitável em diferentes concentrações. Os extratos vegetais apresentaram maior atividade contra A. actinomycetencomitans: Anadenanthera columbrina (Vell.) Brenan (80,52%) a 40 μg/mL, Spiranthera odoratissima St. Hil (78,48%) em 1 μg/mL, Senna martiniana H.S. Irwin & Barneby (73,28%) na concentração de 40 μg/mL e Cariniana rubra Gardinerex Miers (70,50%) em 10 μg/mL. Todos os extratos analisados apresentaram citotoxicidade aceitável em diferentes concentrações e foram promissores na inibição dos microrganismos patogênicos estudados.

RESUMO

OBJECTIVES: The present study aims to evaluate the antimicrobial property of Casiopeinas® copper- and ruthenium-based compounds against Aggregatibacter actinomycetemcomitans serotype b (ATCC® 43,718™), as well as the cytotoxicity on an osteoblasts cell line of both compounds. MATERIAL AND METHODS: The antibacterial effect of the copper-based compounds (CasII-gly, CasIII-ia) and the ruthenium-based compound (RuN-6) at four different concentrations was evaluated as the inhibition ratio of the bacterial growth after 48 h under anaerobic conditions, and the cell viability was measured through resazurin assay. RESULTS: The copper- and ruthenium-based compounds used for this assay were (CasII-gly, CasIII-ia, and RuN-6), showing inhibitory activity between 39 and 62% compared to the antibiotic employed as control 66%. Cell viability was established between 61 and 96%. CONCLUSIONS: Casiopeinas® and ruthenium showed dose and time dependent, inhibitory activity on A. actinomycetemcomitans, and low toxicity on cells (osteoblast) underexposure. The compound CasII-gly showed the best antimicrobial effect, and it could be considered a possible antimicrobial agent in periodontal therapy.

Assuntos

Aggregatibacter actinomycetemcomitans , Rutênio , Sobrevivência Celular , Cobre/farmacologia , Osteoblastos , Rutênio/farmacologia , Compostos de Rutênio/farmacologia

RESUMO

Periodontitis is an infectious disease characterized by the destruction of supporting tissues. Antimicrobial photodynamic therapy (aPDT) has been proposed as an improved method for eliminating microorganisms. Its efficiency depends on the correct use of physical and chemical parameters. Thus, these parameters and their relations were evaluated in this study with the purpose of establishing lethal conditions for combating bacterial agents. Diode lasers and light-emitting diodes (LEDs) were characterized to evaluate the absorption profile and resonance of methylene blue (MB) and toluidine blue O (TBO). The relations between light energy density and photosensitizer absorption were determined. Two methodologies were used to evaluate the effects of aPDT against Aggregatibacter actinomycetemcomitans. LED light exhibited a broad emission spectrum with a peak light wavelength of 637 nm and 99% purity. The resonance intensity of MB was higher with diode laser irradiation, and TBO showed higher resonance intensity with LED irradiation. There was no difference in the absorption profile of photosensitizers using diode lasers or LEDs, and variations in power density did not result in an increasing or decrease in light absorption. A. actinomycetemcomitans was susceptible to photodynamic processes. Emission spectra and peak light wavelengths of light sources combined with the absorption profiles of photosensitizers were the main parameters involved in determining the efficiency of photodynamic effects. Power density did not alter the light absorption of photosensitizers. The association between adequate irradiation characteristics and photosensitizer absorption results in complete inactivation of A. actinomycetemcomitans. In addition, the bactericidal effect was not altered by an increase in energy densities.

Assuntos

Anti-Infecciosos , Fotoquimioterapia , Aggregatibacter actinomycetemcomitans , Fármacos Fotossensibilizantes/farmacologia , Cloreto de Tolônio

RESUMO

Leukocyte- and platelet-rich fibrin is a widely used platelet concentrate for periodontal surgery procedures. Many benefits are described regarding its use, such as antimicrobial properties. The objective of this study was to evaluate the antimicrobial effects of the different zones of this platelet concentrated against the most prevalent serotypes of Aggregatibacter actinomycetemcomitans in an in vitro mono-multiserotype model. Three patients who were treated at a School of Dentistry in the city where the researchers reside, were included. Modified direct contact method tests and results were analyzed using multivariate logistic regression analysis. In the modified direct contact method test, a decrease in bacterial count was found at time 1, but at time 2, the count increased for all serotypes and zones of L-PRF. It can be noted that the areas with more cellular content in leukocytes and platelet-rich fibrin are the areas with the most antimicrobial powe r. This platelet concentrate would have better results with serotype c. At time point 2, it is likely to act as a growth promoter of A. actinomycetemcomitans.

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La Fibrina rica en Leucocitos y Plaquetas es un concentrado plaquetario ampliamente utilizado en procedimientos quirúrgicos periodontales. Muchos beneficios se describen con respecto a su uso, tales como propiedades antimicrobianas. El objetivo de este estudio fue evaluar los efectos antimicrobianos de las diferentes zonas de este concentrado plaquetario frente a los serotipos más prevalentes de Aggregatibacter actinomycetemcomitans en un modelo mono-multi serotipo in vitro. Se incluyeron tres pacientes que fueron tratados en la Facultad de Odontología de la ciudad donde residen los investigadores. Se utilizó para su análisis una prueba de contacto directo modificado. En la prueba de contacto directo modificado, se encontró una disminución en el recuento bacteriano en el tiempo 1, pero en el tiempo 2, el recuento aumentó para todos los serotipos y zonas de L-PRF. Se puede observar que las áreas con mayor contenido celular en la Fibrina rica en Leucocitos y Plaquetas son las áreas con mayor poder antimicrobiano. Este concentrado de plaquetas tendría mejores resultados con el serotipo c. En el tiempo 2, es probable que actúe como un promotor del crecimiento de A. actinomycetemcomitans.

Assuntos

Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Aggregatibacter actinomycetemcomitans/patogenicidade , Células-Tronco , Implantes Dentários , Crescimento Bacteriano , Fibrina Rica em Plaquetas , Antibacterianos/farmacologia

RESUMO

BACKGROUND: Periodontitis is a chronic inflammatory disease associated with a dysbiotic biofilm. Many pathogens have been related with its progression and severity, one of which is Aggregatibacter actinomycetemcomitans, a Gram-negative bacteria with seven serotypes (a-g) according with the structure of its LPS, with serotype b defined as the most virulent compared with the other serotypes. The aim of this study was to evaluate the response of oral keratinocytes and macrophages to A. actinomycetemcomitans. METHODS: Oral keratinocytes (OKF6/TERT2) and macrophages (THP-1) were infected with A. actinomycetemcomitans serotypes a, b and c. The expression of IL-1ß, IL-6, IL-8, IL-18, TNF-α, MMP-9, RANKL, TLR-2, TLR-4, TLR-6, thymic stromal lymphopoietin (TSLP), and ICAM-1 was evaluated by qPCR at 2 and 24 h after infection. RESULTS: An increase in the expression of these molecules was induced by all serotypes at both times of infection, with macrophages showing higher levels of expression at 24 h compared to epithelial cells in which the highest levels were observed in the first hours after infection. CONCLUSIONS: Keratinocytes and macrophages contribute to the inflammation in periodontitis from the early stages of infection, producing the first waves of cytokines, acting as the first signal for professional immune cell recruitment and modulation of more specific immune responses.

RESUMO

Periodontitis is characterized by a dysbiotic microbial community and treatment strategies include the reestablishment of symbiosis by reducing pathogens abundance. Aggregatibacter actinomycetemcomitans (Aa) is frequently associated with rapidly progressing periodontitis. Since the oral ecosystem may be affected by metabolic end-products of bacteria, we evaluated the effect of soluble compounds released by probiotic lactobacilli, known as postbiotics, on Aa biofilm and expression of virulence-associated genes. Cell-free pH-neutralized supernatants (CFS) of Lactobacillus rhamnosus Lr32, L. rhamnosus HN001, Lactobacillus acidophilus LA5, and L. acidophilus NCFM were tested against a fimbriated clinical isolate of Aa JP2 genotype (1 × 107  CFU/well) on biofilm formation for 24 hr, and early and mature preformed biofilms (2 and 24 hr). Lactobacilli CFS partially reduced Aa viable counts and biofilms biomass, but did not affect the number of viable non-adherent bacteria, except for LA5 CFS. Furthermore, LA5 CFS and, in a lesser extent HN001 CFS, influenced Aa preformed biofilms. Lactobacilli postbiotics altered expression profile of Aa in a strain-specific fashion. Transcription of cytolethal distending toxin (cdtB) and leukotoxin (ltxA) was downregulated by CFS of LA5 and LR32 CFS. Although all probiotics produced detectable peroxide, transcription of katA was downregulated by lactobacilli CFS. Transcription of dspB was abrogated by LR32 and NCFM CFS, but increased by HN001, whereas expression of pgA was not affected by any postbiotic. Our data indicated the potential of postbiotics from lactobacilli, especially LA5, to reduce colonization levels of Aa and to modulate the expression of virulence factors implicated in evasion of host defenses.

Assuntos

Lactobacillus , Probióticos , Aggregatibacter actinomycetemcomitans/genética , Biofilmes , Ecossistema , Lactobacillus/genética , Virulência

RESUMO

A etiopatogenia da doença periodontal é complexa e exige estudos constantes em alternativas terapêuticas que possibilitem seu controle. Aggregatibacter actinomycetemcomitans está associado fortemente à periodontite crônica e à periodontite agressiva. A tecnologia do plasma frio sob pressão atmosférica tem potencial para uso na odontologia, mas sua aplicação na periodontia ainda é pouco explorada. Este estudo teve como objetivo avaliar os efeitos do plasma de baixa temperatura sob pressão atmosférica (PBTPA) como adjuvante no controle da doença periodontal experimental induzida por ligadura, inoculada com A. actinomycetemcomitans, na região cervical do primeiro molar inferior em modelo murino. Inicialmente, foram determinados os parâmetros físicos efetivos para inibição de biofilmes de A. actinomycetemcomitans utilizando cepa padrão American Type Culture Collection ATCC 29523. Foram realizados testes de screening inicial com a verificação da inibição do crescimento por exposição ao PBTPA de A. actinomycetemcomitans semeados em placas de Petri. Posteriormente, foram formados biofilmes em placas de microtitulação e foram determinados os parâmetros físicos efetivos do PBTPA. Foram realizados experimentos in vivo de indução de doença periodontal por A. actinomycetemcomitans em modelo utilizando ligaduras em ratos. A tomografia computadorizada de feixe cônico foi utilizada para avaliar o nível de perda óssea após período experimental. Os resultados mostraram que o PBTPA foi efetivo na eliminação do micro-organismo observado através da formação de halos de inibição nos tempos de 5 e 7, 5 minutos e sobre biofilmes de A. actinomycetemcomitans expostos ao PBTPA no tempo de 5 minutos. Os resultados exibiram diferença estatística significativa (p <0,001) na distância entre a junção esmalte-cemento e a crista óssea alveolar, do grupo tratado com PBTPA em relação ao grupo raspagem e ao grupo controle. Os resultados do presente estudo sugerem o potencial do PBTPA no controle da periodontite induzida, contudo análises adicionais são necessárias(AU)

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The etiopathogenesis of periodontal disease is complex requiring constant studies of therapeutic alternatives that enable its control. Aggregatibacter actinomycetemcomitans is strongly associated with chronic periodontitis and aggressive periodontitis. Cold plasma technology under atmospheric pressure has potential for use in dentistry, but its application in periodontics is still not well explored. This study aimed to evaluate the effects of low temperature atmospheric pressure plasma (PBTPA) as an adjuvant in the control of experimental periodontal disease induced by ligature inoculated with A. actinomycetemcomitans in the cervical region of the lower 1st molar in a murine model. Initially, effective physical parameters for inhibition of A. actinomycetemcomitans biofilms were determined using the standard strain American Type Culture Collection ATCC 29523. Initial screening tests were performed to verify growth inhibition after exposure of A. actinomycetemcomitans seeded in Petri dishes to the PBTPA. Subsequently, biofilms were formed on microtiter plates and the effective physical parameters of the PBTPA were determined. In vivo experiments were carried out to induce A. actinomycetemcomitans periodontal disease induced by ligature in a rat model. Cone beam computed tomography was used to assess the level of bone loss after the experimental period. The results showed that PBTPA was effective in eliminating the microorganism observed through the formation of halos inhibition during 5 and 7.5 minutes and in A. actinomycetemcomitans biofilms exposed to PBTPA at 5 minutes. The previous study using PBTPA in 5 minutes did not show cytotoxicity in Vero cells. The results showed a statistically significant difference (p <0.0001) in the distance between the enamel-cement joint and the alveolar bone crest, of the group treated with PBTPA in relation to the scalling group and the control group, confirming the potential of the technique in dentistry(AU)

Assuntos

Periodontite/complicações , Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Gases em Plasma/efeitos adversos

RESUMO

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.

Assuntos

Aggregatibacter actinomycetemcomitans/imunologia , Aggregatibacter actinomycetemcomitans/patogenicidade , Periodontite/imunologia , Periodontite/microbiologia , Polissacarídeos Bacterianos/imunologia , Virulência/imunologia , Aggregatibacter actinomycetemcomitans/genética , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Animais , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Mutação , Periodontite/complicações , Periodontite/diagnóstico , Sorogrupo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Virulência , Microtomografia por Raio-X

RESUMO

In order to improve our understanding on the microbial complexity associated with Grade C/molar-incisor pattern periodontitis (GC/MIP), we surveyed the oral and fecal microbiomes of GC/MIP and compared to non-affected individuals (Control). Seven Afro-descendants with GC/MIP and seven age/race/gender-matched controls were evaluated. Biofilms from supra/subgingival sites (OB) and feces were collected and submitted to 16S rRNA sequencing. Aggregatibacter actinomycetemcomitans (Aa) JP2 clone genotyping and salivary nitrite levels were determined. Supragingival biofilm of GC/MIP presented greater abundance of opportunistic bacteria. Selenomonas was increased in subgingival healthy sites of GC/MIP compared to Control. Synergistetes and Spirochaetae were more abundant whereas Actinobacteria was reduced in OB of GC/MIP compared to controls. Aa abundance was 50 times higher in periodontal sites with PD≥ 4 mm of GC/MIP than in controls. GC/MIP oral microbiome was characterized by a reduction in commensals such as Kingella, Granulicatella, Haemophilus, Bergeyella, and Streptococcus and enrichment in periodontopathogens, especially Aa and sulfate reducing Deltaproteobacteria. The oral microbiome of the Aa JP2-like+ patient was phylogenetically distant from other GC/MIP individuals. GC/MIP presented a higher abundance of sulfidogenic bacteria in the feces, such as Desulfovibrio fairfieldensis, Erysipelothrix tonsillarum, and Peptostreptococcus anaerobius than controls. These preliminary data show that the dysbiosis of the microbiome in Afro-descendants with GC/MIP was not restricted to affected sites, but was also observed in supragingival and subgingival healthy sites, as well as in the feces. The understanding on differences of the microbiome between healthy and GC/MIP patients will help in developing strategies to improve and monitor periodontal treatment.

Assuntos

Microbiota , Periodontite , Aggregatibacter actinomycetemcomitans , Desulfovibrio , Erysipelothrix , Fezes , Humanos , Incisivo , Dente Molar , Peptostreptococcus , RNA Ribossômico 16S/genética

RESUMO

BACKGROUND AND OBJECTIVE: Several studies have demonstrated that mast cells are equipped with versatile tools to combat and kill bacteria. Additionally, mast cells produce and secrete a variety of mediators, which either regulate the host's immune system or directly attack bacteria. In this study, the intracellular microbicidal capacity of mast cells against Aggregatibacter actinomycetemcomitans was evaluated. METHODS: Murine mast cells were challenged in vitro with A actinomycetemcomitans for 3, 5, 10, and 24 hours. Subsequently, the colony-forming units were counted. Additionally, the production and release of nitric oxide and hydrogen peroxide were analyzed by DAF-FM diacetate, the Griess reaction, and the Amplex Red kit, respectively. Cell death was evaluated using FITC Annexin V and propidium iodide staining. RESULTS: Mast cells are able to efficiently eliminate periodontopathogen, with best results after 10 hours of intracellular challenge. The production/release of nitric oxide-and to a lesser extent of hydrogen peroxide-by mast cells was in agreement with its microbicidal capacity. Ninety percent of the mast cells  maintained their cellular viability even after 24 hours of bacterial challenge. CONCLUSIONS: This is-to the best of our knowledge-the first report to describe the intracellular microbicidal activity of mast cells against A actinomycetemcomitans, concerning the production and release of potentially bactericidal substances. Further, the low number of cell deaths confirms that the decreased number of colony-forming units was due to the higher antimicrobial activity of mast cells. The results highlight the importance of these cells in the defense mechanisms of biofilm-induced periodontal disease.

Assuntos

Aggregatibacter actinomycetemcomitans , Mastócitos , Doenças Periodontais , Animais , Biofilmes , Camundongos , Óxido Nítrico , Doenças Periodontais/microbiologia

RESUMO

INTRODUCTION: Periodontitis affects more than 20% of the Latin American population. Oxidative markers are associated with greater progression of periodontitis; therefore, its role in pathogenesis should be studied. OBJECTIVE: To determine the prevalence of the main oral bacteria and viruses associated with periodontitis and estimate the total antioxidant capacity and lipid peroxidation in saliva from patients with periodontitis. MATERIALS AND METHODS: We conducted systemically a cross-sectional study in 101 healthy subjects, 87 of whom had been diagnosed with periodontitis (P), according to the criteria of the Centers of Disease Control and Prevention and the American Academy of Periodontology, and 14 without periodontal pockets as controls (C). In subgingival samples, major viruses and dental pathogenic bacteria were identified using PCR techniques. The levels of total antioxidant capacity and malon-di-aldehyde (MDA) were determined by spectrophotometry in samples of unstimulated saliva. RESULTS: The mean of periodontal depth pocket and clinical attachment loss in patients with periodontitis was 5.6 ± 1.7 and 6.1 ± 3.1 mm, respectively. The most prevalent microorganisms were Aggregatibacter actinomycetemcomitans (32.5%) and Porphyromonas gingivalis (18.6%). The patients from rural areas showed a higher percentage of A. actinomycetemcomitans (urban: 17.9% vs. rural: 48.9%, p=0.0018). In patients with periodontitis, the frequency of EBV, HSV1 and 2, and HCMV genes was 2.3%. Periodontitis patients had higher levels of MDA (P: 2.1 ± 1.5; C: 0.46 ± 0.3 µmol/g protein; p=0.0001) and total antioxidant capacity (P: 0.32 ± 0.2; C: 0.15 ± 0.1 mM; p< 0.0036). Oxidative markers showed no modifications due to the presence of periodontopathic bacteria. CONCLUSIONS: Aggregatibacter actinomycetemcomitans was the most prevalent bacteria; its presence did not modify the levels of oxidative markers in the saliva of patients with periodontitis.

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Introducción. La periodontitis afecta a más del 20 % de la población latinoamericana. La presencia de marcadores de estrés oxidativo se asocia con una mayor progresión de periodontitis, por lo que su rol en la patogenia debe estudiarse. Objetivo. Determinar la prevalencia de las principales bacterias y virus asociados con la periodontitis y estimar la capacidad antioxidante total y la peroxidación de lípidos en la saliva de los pacientes con periodontitis. Materiales y métodos. Se hizo un estudio transversal en 87 sujetos sanos diagnosticados con periodontitis (P) según los criterios de los Centers of Disease Control and Prevention y la American Academy of Periodontology y 14 sujetos sin enfermedad periodontal como grupo control (C). En las muestras subgingivales se identificaron los principales virus y bacterias mediante técnicas de PCR. Los niveles de capacidad antioxidante total y malon-di-aldehído (MDA) se establecieron mediante espectrofotometría en muestras de saliva no estimulada. Resultados. Las medias de profundidad del sondaje y del nivel de inserción clínico periodontal en pacientes con periodontitis fueron 5,6 ± 1,7 y 6,1 ± 3,1 mm, respectivamente. Los microorganismos más prevalentes fueron Aggregatibacter actinomycetemcomitans (32,5 %) y Porphyromonas gingivalis (18,6 %). Los pacientes de áreas rurales registraron un mayor porcentaje de A. actinomycetemcomitans (urbano: 17,9 % Vs. rural: 48,9 %; p=0,0018). La frecuencia de los genes EBV, HSV1 y 2, y HCMV fue de 2,3 %. En pacientes con periodontitis se evidenciaron mayores niveles de MDA (P: 2,1 ± 1,5; C: 0,46 ± 0,3 µmol/g proteína; p=0,0001) y capacidad antioxidante total (P: 0,32 ± 0,2; C: 0,15 ± 0,1 mM; p<0,0036). La presencia de bacterias periodontales patógenas no modificó los marcadores oxidativos. Conclusiones. Aggregatibacter actinomycetemcomitans fue el agente patógeno mas prevalente. Su presencia no modificó los niveles de marcadores oxidativos en la saliva de los pacientes con periodontitis.

Assuntos

Estresse Oxidativo , Periodontite/metabolismo , Periodontite/microbiologia , Saliva/química , Adulto , Biomarcadores/análise , Colômbia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Periodontite/virologia , Adulto Jovem

RESUMO

Introduction: Periodontitis affects more than 20% of the Latin American population. Oxidative markers are associated with greater progression of periodontitis; therefore, its role in pathogenesis should be studied. Objective: To determine the prevalence of the main oral bacteria and viruses associated with periodontitis and estimate the total antioxidant capacity and lipid peroxidation in saliva from patients with periodontitis. Materials and methods: We conducted systemically a cross-sectional study in 101 healthy subjects, 87 of whom had been diagnosed with periodontitis (P), according to the criteria of the Centers of Disease Control and Prevention and the American Academy of Periodontology, and 14 without periodontal pockets as controls (C). In subgingival samples, major viruses and dental pathogenic bacteria were identified using PCR techniques. The levels of total antioxidant capacity and malon-di-aldehyde (MDA) were determined by spectrophotometry in samples of unstimulated saliva. Results: The mean of periodontal depth pocket and clinical attachment loss in patients with periodontitis was 5.6 ± 1.7 and 6.1 ± 3.1 mm, respectively. The most prevalent microorganisms were Aggregatibacter actinomycetemcomitans (32.5%) and Porphyromonas gingivalis (18.6%). The patients from rural areas showed a higher percentage of A . actinomycetemcomitans (urban: 17.9% vs. rural: 48.9%, p=0.0018). In patients with periodontitis, the frequency of EBV, HSV1 & 2, and HCMV genes was 2.3%. Periodontitis patients had higher levels of MDA (P: 2.1 ± 1.5; C: 0.46 ± 0.3 µmol/g protein; p=0.0001) and total antioxidant capacity (P: 0.32 ± 0.2; C: 0.15 ± 0.1 mM; p< 0.0036). Oxidative markers showed no modifications due to the presence of periodontopathic bacteria. Conclusions: Aggregatibacter actinomycetemcomitans was the most prevalent bacteria; its presence did not modify the levels of oxidative markers in the saliva of patients with periodontitis.

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Introducción. La periodontitis afecta a más del 20 % de la población latinoamericana. La presencia de marcadores de estrés oxidativo se asocia con una mayor progresión de periodontitis, por lo que su rol en la patogenia debe estudiarse. Objetivo. Determinar la prevalencia de las principales bacterias y virus asociados con la periodontitis y estimar la capacidad antioxidante total y la peroxidación de lípidos en la saliva de los pacientes con periodontitis. Materiales y métodos. Se hizo un estudio transversal en 87 sujetos sanos diagnosticados con periodontitis (P) según los criterios de los Centers of Disease Control and Prevention y la American Academy of Periodontology y 14 sujetos sin enfermedad periodontal como grupo control (C). En las muestras subgingivales se identificaron los principales virus y bacterias mediante técnicas de PCR. Los niveles de capacidad antioxidante total y malon-di-aldehído (MDA) se establecieron mediante espectrofotometría en muestras de saliva no estimulada. Resultados. Las medias de profundidad del sondaje y del nivel de inserción clínico periodontal en pacientes con periodontitis fueron 5,6 ± 1,7 y 6,1 ± 3,1 mm, respectivamente. Los microorganismos más prevalentes fueron Aggregatibacter actinomycetemcomitans (32,5 %) y Porphyromonas gingivalis (18,6 %). Los pacientes de áreas rurales registraron un mayor porcentaje de A. actinomycetemcomitans (urbano: 17,9 % Vs. rural: 48,9 %; p=0,0018). La frecuencia de los genes EBV, HSV1 y 2, y HCMV fue de 2,3 %. En pacientes con periodontitis se evidenciaron mayores niveles de MDA (P: 2,1 ± 1,5; C: 0,46 ± 0,3 µmol/g proteína; p=0,0001) y capacidad antioxidante total (P: 0,32 ± 0,2; C: 0,15 ± 0,1 mM; p<0,0036). La presencia de bacterias periodontales patógenas no modificó los marcadores oxidativos. Conclusiones.Aggregatibacter actinomycetemcomitans fue el agente patógeno mas prevalente. Su presencia no modificó los niveles de marcadores oxidativos en la saliva de los pacientes con periodontitis.

Assuntos

Periodontite , Saliva , Vírus , Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis , Estresse Oxidativo

RESUMO

OBJECTIVES: Studies have demonstrated that children from aggressive periodontitis (AgP) parents presented precocious alterations in their periodontal condition, and the use of chemical agents in association to plaque control could be useful to control these alterations. This study aimed to evaluate the effect of Triclosan toothpaste to modulate the clinical and subgingival condition in children from AgP parents. METHODS: Fifteen children from AgP parents and 15 from periodontally healthy parents were included in this crossover placebo study. Children were randomly allocated into triclosan or placebo therapy, using selected toothpaste for 45 days. After 15 days of wash-out, groups were crossed, changing the used toothpaste. Clinical examination and saliva, crevicular gingival fluid (GCF), and subgingival biofilm collection were performed at baseline and 45 days of each phase. GCF cytokines' levels were analyzed by Luminex/MAGpix platform and subgingival and salivary periodontal pathogens' levels by qPCR. RESULTS: At baseline, AgP group presented higher plaque index (PI), gingival index (GI), and bleeding on probing (BoP), higher Aggregatibacter actinomycetemcomitans (Aa) abundance in saliva and subgingival biofilm, and lower levels of INF-É£, IL-4, and IL-17 in GCF. Placebo therapy only reduced PI in both groups. Triclosan toothpaste reduced PI and GI in both groups. Triclosan promoted reduction of BoP and probing depth (PD), Aa salivary, and IL-1ß levels in AgP group. In health group, triclosan reduced INF-É£ and IL-4 concentration. CONCLUSION: Triclosan toothpaste demonstrated to be more effective than placebo toothpaste to control the periodontal condition in children from AgP parents, by reducing the BoP, PD, salivary Aa, and IL-1ß. CLINICAL RELEVANCE: Triclosan toothpaste can improve oral conditions in higher-risk population for AgP. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov with the identifier NCT03642353.

Assuntos

Placa Dentária/prevenção & controle , Cremes Dentais/uso terapêutico , Triclosan/uso terapêutico , Aggregatibacter actinomycetemcomitans , Periodontite Agressiva , Biofilmes , Criança , Estudos Cross-Over , Citocinas , Índice de Placa Dentária , Feminino , Líquido do Sulco Gengival/química , Humanos , Masculino , Índice Periodontal , Saliva

RESUMO

In the present study, Galleria mellonella was evaluated as a potential infection model for periodontal bacteria, more specifically, Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. All the bacteria evaluated were pathogenic to G. mellonella, causing their death in a concentration-dependent manner, and a decrease in their hemocyte count. Moreover, it was possible to recover the bacteria from the larvae hemolymph and determine the colony-forming units per larvae. G. mellonella is an effective model that may help to better understand the host-microbe interactions in periodontics.