Antimicrobial potential of plant extracts from the Brazilian Cerrado

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.

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.

Antimicrobial Effect of Leukocyte- and Platelet-Rich Fibrin Against Aggregatibacter actinomycetemcomitans Mono-Multiserotypes Model

ABSTRACT: 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.

RESUMEN: 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.

Efeitos do plasma frio sob pressão atmosférica como adjuvante no controle da periodontite experimental induzida por Aggregatibacter actinomycetemcomitans / Effect of atmospheric pressure cold plasma as adjuvante in the control of experimental periodontitis induced by Aggregatibacter actinomycetemcomitans

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)

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)

Salivary markers of oxidative stress and periodontal pathogens in patients with periodontitis from Santander, Colombia / Marcadores salivales de estrés oxidativo y agentes patógenos periodontales en pacientes con periodontitis en Santander, Colombia

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.

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.

Essential Oil from Psidium cattleianum Sabine (Myrtaceae) Fresh Leaves: Chemical Characterization and in vitro Antibacterial Activity Against Endodontic Pathogens

Abstract Endodontic infections result from oral pathogenic bacteria which reach and infect dental pulp, as well as surrounding tissues, through cracks, unrepaired caries and failed caries restorations. This study aims to determine the chemical composition of essential oil from Psidium cattleianum leaves (PC-EO) and to assess its antibacterial activity against endodontic bacteria. Antibacterial activity of PC-EO was evaluated in terms of its minimum inhibitory concentration (MIC) values by the broth microdilution method on 96-well microplates. Bacteria Porphyromonas gingivalis (MIC = 20 µg/mL), Prevotella nigrescens (MIC = 62.5 µg/mL), Fusobacterium nucleatum (MIC = 12.5 µg/mL), Actinomyces naeslundii (MIC = 50 µg/mL), Bacteroides fragilis (MIC = 12.5 µg/mL), Aggregatibacter actinomycetemcomitans (MIC = 6.25 µg/mL) and Peptostreptococcus anaerobius (MIC = 62.5 µg/mL) were evaluated and compared to chlorhexidine dihydrochloride (CDH), the positive control. PC-EO was obtained by hydrodistillation with the use of a Clevenger-type apparatus whereas its chemical composition was analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Viridiflorol (17.9%), β-caryophyllene (11.8%), 1,8-cineole (10.8%) and β-selinene (8.6%) were the major constituents found in PC-EO, which exhibited high antibacterial activity against all endodontic pathogens under investigation. Therefore, PC-EO, a promising source of bioactive compounds, may provide therapeutic solutions for the field of endodontics.

Porphyromonas gingivalis ligada a enfermedad periodontal y su relación con la artritis reumatoide: identificación de nuevos mecanismos biomoleculares / Porphyromonas gingivalis linked to periodontal disease and its relationship with rheumatoid arthritis: Identification of new biomolecular mechanisms

Objective: Review the existing scientific literature regarding the pathogenicity of Porphyromonas gingivalis, linked to periodontal disease (PD) (oral dysbiosis), and its association with the activation of pathophysiological mechanisms of rheumatoid arthritis (RA), in order to expose the new mechanisms biomolecular involved. Methods: Systematic search in the MeSH, pubmed, Science Direct, Nature y Google academic database, using the keywords: Aggregatibacter actinomycetemcomitans; rheumatoid arthritis; citrullination; dysbiosis; dentistry; periodontitis; porphyromonas gingivalis; rheumatology. Out of a total of 297 publications, 52 were selected, all from 2018; based on the inclusion and exclusion criteria established by the authors. Results: Pg infection linked to periodontal disease is strongly implicated in the pathogenesis and development of RA. Their relationship is linked to the citrullination process and production of citrullinated antipeptide antibodies. Associations have been identified between the microbial virulence of this agent and the expression of multiple genes related to the activation of the immune response and the onset of the chronic inflammatory process. Conclusions: There is a high association between the pathogenesis of both diseases, where microorganisms linked to PD such as Pg have the ability to increase citrullination, galactosylation, fucosylation, as well as excessive glycosylation of fragments antigen-binding (Fab), and therefore the aggressiveness of RA.

Objetivo: revisar la literatura científica existente con respecto a la patogenicidad de Porphyromonas gingivalis, ligada a enfermedad periodontal (EP) (disbiosis oral), y su asociación con la activación de mecanismos fisiopatológicos en la artritis reumatoide (AR), a fin de exponer los nuevos mecanismos biomoleculares implicados. Métodos: búsqueda sistemática en la base de datos del Medical Subject Headings (MeSH), PubMed, Science Direct, Nature y Google académico usando las palabras clave: Aggregatibacter actinomycetemcomitans; artritis reumatoide; citrulinación; disbiosis; odontología; periodontitis; Porphyromonas gingivalis y reumatología. De un total de 297 publicaciones, se seleccionaron 52, todas a partir del año 2018; la selección fue hecha a partir de los criterios de inclusión y exclusión establecidos por los autores. Resultados: la infección por Porphyromonas gingivalis, ligada a la EP, está fuertemente implicada en la patogénesis y desarrollo de AR. Su relación se vincula con el proceso de citrulinación y producción de anticuerpos antipéptidos citrulinados. Se han identificado asociaciones entre la virulencia microbiana de dicho agente y la expresión de múltiples genes, relacionados con la activación de la respuesta inmune y el inicio del proceso inflamatorio crónico. Conclusiones: existe una alta asociación entre la patogenia de ambas enfermedades, donde microorganismos ligados a la EP, como Porphyromonas gingivalis, tienen la capacidad de aumentar la citrulinación, galactosilación, fucosilación, así como la excesiva glicosilación de Fragmentos de unión al antígeno (Fab), y por lo tanto, la agresividad de la AR.

Detection of Biofilm Proteins from Aggregatibacter actinomycetemcomitans Induced by Glucose, Lactose, Soy Protein, and Iron Along with Protein Density Analysis

@#Introduction: Aggregatibacter actinomycetemcomitans (A.a) is the main bacterium that causes aggressive periodontitis which is capable of forming biofilms on the surface of periodontal tissues. The objective of the study of the bacterial biofilm proteins is to provide an alternative to early prevention of oral infection of Periodontitis caused by A.a. Biofilms formed characterized by its molecular weight will then be used for the purpose of making Periodontal Disease Detection Kits. This project is aimed to select the molecular weight of biofilm protein of A.a exposed to 5% glucose, lactose, soy protein, and iron. Methods: Exposure to 5% glucose, lactose, soy protein, and iron is nenecessary to form biofilm of A.a. Then, the biofilm is characterized by SDS-PAGE electrophoresis to measure the molecular weight. Results: From this study A.a biofilm bands were formed with the number of protein bands that varied depending on the induction used. A.a induced by 5% glucose had one protein band (37.5 kDa), A.a induced by 5% lactose had five protein bands (77.9 kDa, 52.6 kDa, 46.8 kDa, 36.6 kDa, and 28.5 kDa), A.a induced by Soy Protein had seven protein bands (77.9 kDa, 71.3 kDa, 47.4 kDa, 40.4 kDa, 37.2 kDa, 28.8 kDa and 11.8 kDa) and A.a induced by 5% iron did not form protein band. Conclusion: Exposure to 5% glucose, lactose, soy protein, and iron to A.a resulted in band of biofilm protein. However, A.a biofilm induced by 5% iron has no protein bands.

Diagnóstico molecular de microorganismos periodontopatógenos en pacientes alcohó-licos - fumadores con periodontitis crónica de la ciudad de Loja, Ecuador / Diagnóstico molecular de microrganismos periodontopatogênicos em pacientes taba-gistas com periodontite crônica na cidade de Loja, Equador / Molecular diagnosis of microorganisms periodontopathogens in alcoholic - smoking patients with chronic periodontitis of the city of Loja, Ecuador

Los pacientes alcohólicos y fumadores presentan mayor predisposición de desarrollar enfermedad periodontal. Objetivo: Determinar la presencia de los microorganismos periodontopatógenos: Porphyromona gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum y Aggregatibacter actinomycetemcomitans mediante la Reacción en Ca-dena de la Polimerasa (PCR), en pacientes alcohólicos fumadores con periodontitis crónica. Materiales y métodos: El universo fue de 31 pacientes de 18 y 62 años de edad de sexo masculino alcohólicos y/o fumadores en los cuales fueron excluidos aquellos que consumían algún tipo de droga, dando una muestra de 23 pacientes con bolsas periodontales ≥ 6mm del Centro de Rehabilitación de la ciudad de Loja, Ecuador. Se realizó un examen periodontal completo y la toma de muestras en dos de los sitios más profundos de cada paciente. Las variables analizadas fueron: presencia de biofilm dental, sangrado al sondaje, profundidad al sondaje y nivel de inserción clínica. Los datos fueron analizados con la prueba de Kruskal Wallis con un nivel de significancia del 5%. Resultados: La periodontitis crónica estuvo en el 52,1% de los pacientes de 18 a 30 años, siendo más susceptibles los alcohólicos de riesgo y fumadores leves. El 91,04 % de alcohóli-cos y fumadores se encuentran asociados con la presencia de biofilm dental (p = 0,028) y en el diagnóstico molecular el 41,18% de los pacientes presentan más de 3 microorganismos (p = 0,039). Conclusión: Se evidenció la presencia de los periodontopatógenos estudiados en pacientes alcohólicos fumadores con periodontitis crónica.

Alcoholic and smoking patients have a greater predisposition to develop periodontal disease. Objective: To determine the presence of microorganisms periodontopathogens: Porphyromona gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans by Polymerase Chain Reaction (PCR), in alcoho-lic-smoking patients with chronic periodontitis. Materials and methods: The group was composed of 31 patients between the ages of 18 to 62-year-old, male alcoholics and/or smokers in which were excluded those who take some kind of drug, giving a sample of 23 patients with periodontal pockets ≥ 6mm from the Center of Rehabilitation of the city of Loja, Ecuador. A full periodontal examination and the sample taking were in two of the most profound sites of each patient. The variables analyzed were: the presence of dental biofilm, bleeding on probing, probing depth and clinical attachment level. The data were analyzed with the Kruskal Wallis Test with a significance level of 5%. Results: The chronic periodontitis was in the 52.1% of the patients between the ages of 18 to 30 years old, is more susceptible in risky alcoholics-smokers and low smokers. The 91.04 % of alcoholics and smokers are associated with the presence of dental biofilm (p = 0.028) and in the molecular diagnosis the 41.18% of patients exhibit more than 3 microorganisms (p = 0.039). Conclusion: That observed presence of the periodontopathogens in alcoholic-smoking patients with chronic periodontitis.

Os pacientes alcoólicos e fumantes apresentam uma possibilidade maior de desenvolver doença peridental. Objetivo: Determinar a presença do periodontopatógenos de microorganismos Porphyromona gingivalis, Treponema denticola, Fusobacterium nucleatum e Aggregatibacter actinomycetemcomitans por meio da Reação em Cadeia de Polimerasa (PCR),em pacientes alcoólicos e fumantes com periodontite crônico. Materiais e métodos: O universo pertenceu a 31 pacientes de 18 e 62 anos de idade de sexo masculino alcoólicos e/ou fumantes nos quais foram excluídos aqueles que consumiram algum tipo de droga, tendo uma amostra de 23 pacientes com bolsas peridentais. 6mm do Centro de Reabilitação da cidade de Loja, Equador. Um exame periodontal completo e a amostragem foram realizados em dois locais profundamente localizados de cada paciente. As variáveis analisadas eram: presença de biofilm dental, sangrou ao sondaje, profundidade ao sondaje e nível de suplemento clínico. Os dados foram analisados com o teste de Kruskal Wallis com um nível de significancia de 5%. Resultados: O periodontite crônico estava em 52,1% dos pacientes de 18 para 30 anos, enquanto sendo mais suscetível os alcoólicos de risco e fumantes ligeiros. 91,04% de alcoólicos e fumantes são associados com a presença de biofilm dental (p = 0028) e na diagnose 41,18% molecular dos pacientes apresenta mais de 3 microorganismos (p = 0039). Conclusões: A presença do periodontopatógenos foi comprovada nos pacientes alcoólicos e fumantes com periodontite crônico.

Effect of Various Agents on Oral Bacterial Phagocytosis in THP-1 Cells

Phagocytosis is a fundamental process in which phagocytes capture and ingest foreign particles including pathogenic bacteria. Several oral pathogens have anti-phagocytic strategies, which allow them to escape from and survive in phagocytes. Impaired bacteria phagocytosis increases inflammation and contributes to inflammatory diseases. The purpose of this study is to investigate the influences of various agents on oral pathogenic phagocytosis. To determine phagocytosis, Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were stained with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), and was measured using flowcytometery and confocal microscopy. The influencing factors on phagocytosis were evaluated through the pretreatment of ROS inhibitor (N-acetyl-L-cysteine (NAC)), lysozyme, potassium chloride (KCI) and adenosine triphosphate (ATP) in THP-1 cells. Expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). The phagocytosis of various bacteria increased in a MOI-dependent manner. Among the tested bacteria, phagocytosis of P. gingivalis showed the highest fluorescent intensity at same infection time. Among the tested inhibitors, the NAC treatment significantly inhibited phagocytosis in all tested bacteria. In addition, NAC treatment indicated a similar pattern under the confocal microscopy. Moreover, NAC treatment significantly increased the bacteria-induced secretion of IL-1β among the tested inhibitors. Taken together, we conclude that the phagocytosis occurs differently depending on each bacterium. Down-regulation by ROS production inhibited phagocytosis and lead increased of oral pathogens-associated inflammation.

Características clínicas y microbiológicas de pacientes con periodontitis agresiva generalizada / Clinical and microbiological characteristics of a patients with generalized aggressive periodontitis

RESUMEN: Objetivo: el objetivo de este estudio es describir las características clínicas y microbiológicas de una muestra de pacientes diagnosticados con periodontitis agresiva generalizada (PAgG). Materiales y métodos: En este estudio de corte transversal, 20 sujetos menores de 30 años con PAgG atendidos en las clínicas odontológicas de la Universidad de Antioquia en Medellín Colombia, fueron invitados a participar entre diciembre del 2015 y marzo del 2017, las muestras microbiológicas fueron analizadas usando técnicas de cultivo y tomadas en los seis sitios más profundos de cada paciente (≥ 5mm). Resultados: Prevotella ssp y F. nucleatum fueron detectados en altos porcentajes, Porphyromonas gingivalis (P.g) fue positivo para la mitad de los sujetos estudiados; además de los microorganismos comúnmente estudiados, el 10% de los pacientes fueron positivos para bacilos entéricos gram-negativos. Conclusiones: se observaron grandes proporciones de microorganismos que incluyeron Prevotella spss y F. nucleatum; el 10% de los pacientes fueron positivos para bacilos entéricos gram-negativos.

ABSTRACT: Aim: The aim of this study is to describe the clinical and microbiological characteristics of a sample of patients diagnosed with generalized aggressive periodontitis (PAgG). Materials and methods: In this cross-sectional study, 20 subjects under 30 years of age with PAgG treated at the dental clinics of the University of Antioquia in Medellín, Colombia were invited to participate between December 2015 and March 2017, the microbiological samples analyzed using culture techniques were taken at the six deepest sites of each patient (≥ 5mm). Results: Prevotella ssp and F. nucleatum were detected in high percentages, Porphyromonas gingivalis (P.g) was positive for half of the studied subjects; In addition to the microorganisms commonly studied, 10% of the patients were positive for gram-negative enteric bacilli. Conclusions: large proportions of microorganisms were observed, including Prevotella spss and F. nucleatum; 10% of the patients were positive for gram-negative enteric bacilli.

Susceptibilidad antimicrobiana de dos periodontopatógenos a moxifloxacina y amoxicilina ácido clavulánico / Antimicrobial susceptibility of moxifloxacin and amoxicillin clavulanic acid against two periodontopathogens

Fundamento: se ha documentado un aumento importante de la resistencia de algunos perio-dontopatógenos a los antimicrobianos usados de forma tradicional como coadyuvantes en la terapia periodontal. Objetivo: evaluar la susceptibilidad de Aggregatibacter actinomycetemcomitans y Porphyromonas gingivalis a la moxifloxacina y amoxicilina ácido clavulánico en pacientes con periodontitis crónica. Métodos: se realizó un estudio observacional de corte transversal el universo estuvo constitui-do por 30 pacientes con periodontitis crónica. La toma y procesamiento de las muestras microbiológicas se realizaron mediante protocolos descritos de forma previa. Para evaluar la susceptibilidad de A. actinomycetemcomitans y P. gingivalis frente a la moxifloxacina y amoxicilina ácido clavulánico, se usaron pruebas de difusión en disco. Resultados: P. gingivalis fue el microorganismo más prevalente en la población estudiada. Los dos periodontopatógenos cultivados fueron muy sensibles a los dos antimicrobianos evaluados. Conclusiones: A. actinomycetemcomitans y P. gingivalis son muy susceptibles a la moxifloxacina y la amoxicilina ácido clavulánico en la población estudiada, donde se convierte de esta manera en una alternativa coadyuvante para el tratamiento de la periodontitis.

Background: a significant increase in the resistance of some antimicrobial periodontopathogens traditionally used as adjuncts in periodontal therapy has been reported. Objective: to assess the susceptibility of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis to moxifloxacin and amoxicillin clavulanic acid in patients with chronic periodontitis. Methods: an observational cross-sectional study of the universe composed of 30 patients with chronic periodontitis was conducted. Microbiological samples processing were performed following protocols described previously. Disk diffusion tests were used to assess the susceptibility of A. actinomycetemcomitans and P. gingivalis against moxifloxacin and amoxicillin clavulanic acid. Results: P. gingivalis was the most prevalent microorganism in the population studied. The two periodontopathogens grown were highly sensitive to both antimicrobials tested. Conclusions: A. actinomycetemcomitans and P. gingivalis are highly susceptible to moxifloxacin and amoxicillin clavulanic acid in this population, therefore it is an alternative adjuvant for the treatment of periodontitis.

Presence of Aggregatibacter actinomycetemcomitans in saliva and cardiac tissue samples of children with congenital heart disease.

Aim: The purpose of this study was to analyze the presence of Aggregatibacter actinomycetemcomitans in saliva and cardiac tissue samples of children requiring cardiac surgery in Istanbul, Turkey. Subjects and Methods: Twenty‑five patients (mean age: 6.24 ± 2.93) undergoing surgery for congenital heart defects (CHDs) and an age/gender‑matched control group of 25 healthy children were enrolled in the study. Saliva samples were collected from all children; plaque index (PI) and gingival index (GI) were also determined. In CHD group, cardiac tissue samples were received during surgery. All samples were evaluated for the presence of A. actinomycetemcomitans and its highly leukotoxic JP2 clonal strains using polymerase chain reaction. The findings were analyzed by Mann–Whitney U, Chi‑square, and Fisher’s exact tests. Results: No significant differences were found in PI and GI values between the groups. A. actinomycetemcomitans was not detected in cardiac tissue samples. A. actinomycetemcomitans in saliva was detected in 2 (8%) of the CHD and 5 (20%) of the control children (p > 0.05). A. actinomycetemcomitans JP2 clonal strains were determined from 1 (4%) of the control group while it was not determined from the samples of the CHD group. Conclusions: Early colonization of A. actinomycetemcomitans in oral cavities could be assessed as a risk marker for periodontal disease. Periodontal pathogens may enter bloodstream through bacteremia; thus, the presence of periodontal pathogens in the oral cavity of children should be assessed as a risk marker for cardiac diseases in older ages.

Transmissão intrafamiliar de patógenos periodontais / Intrafamilial transmission of periodontal pathogens: a literature review

Objetivo: revisar quais seriam os diferentes fatores envolvidos na transmissão de periodontopatógenos entre membros de uma mesma família e quais as suas consequências. Material e métodos: uma revisão da literatura foi realizada na base de dados PubMed, utilizando os termos "vertical transmission", "periodontal pathogens", "oral colonization", e "periodontitis". Resultados: após a leitura do título e resumo, 30 artigos foram incluídos nesta revisão. A transmissão de patógenos periodontais entre indivíduos de uma mesma família está relacionada à passagem via salivar e ao compartilhamento alimentar e de higiene, aos cuidados dos filhos pelos pais ou cuidadores, e ao contato íntimo entre cônjuges. Estudos que avaliaram a transmissão do Aggregatibacter actinomycetemcomitans entre indivíduos de uma mesma família mostraram a ocorrência da transmissão vertical, embora também ocorra transmissão horizontal. Entretanto, resultados semelhantes não puderam ser observados para o Porphyromonas gingivalis. Enquanto alguns relatos indicam a ocorrência de transmissão horizontal desta bactéria, diversos outros estudos indicam características bacterianas que reduzem sua ocorrência. Conclusão: a colonização oral por microrganismos patogênicos está relacionada à transmissão vertical e horizontal de patógenos, embora a persistência dos microrganismos pareça estar relacionada a fatores individuais do hospedeiro e características dos patógenos. Além disso, atividades preventivas e terapêuticas devem ser realizadas de forma a alterar o processo de transmissão, colonização e o maior risco do desenvolvimento de problemas periodontais.

Objective: to review the different factors involved in the transmission of periodontopathogens between members of the same family and their consequences. Material and methods: an electronic literature review was conducted at the PubMed using the keywords "vertical transmission", "periodontal pathogens", "oral colonization", and "periodontitis". Results: after reading of title and abstract, 30 articles were included. The transmission of periodontal pathogens among individuals of the same family is related to the passage through salivary and food and hygiene sharing, the care of the children by parents or caregivers, and the intimate contact between individuals. Studies evaluating the transmission of Aggregatibacter actinomycetemcomitans among individuals from the same family showed the occurrence of vertical transmission and horizontal transmission. However, similar results could not be observed for Porphyromonas gingivalis. While some reports indicate the occurrence of horizontal transmission, several other studies indicate bacterial characteristics that reduce its occurrence. Conclusion: oral colonization by pathogenic microorganisms is related to its vertical and horizontal transmission, although the persistence of the microorganisms seems to be related to individual host factors and pathogen characteristics. In addition, preventive and therapeutic activities must be performed in a way that will alter the transmission, colonization and the greater risk of developing periodontal problems.

Influence of Aae Autotransporter Protein on Adhesion and Biofilm Formation by Aggregatibacter actinomycetemcomitans

Abstract The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins.

Resumo O peridontopatógeno Aggregatibacter actinomycetemcomitans coloniza a cavidade oral aderindo e invadindo as células epiteliais e participando da formação de biofilme em superfícies duras. Aae, uma proteína autotransportadora está relacionada com a adesão bacteriana às células epiteliais. Devido às múltiplas funções desempenhadas por proteínas bacterianas autotransportadoras, este estudo teve como objetivo avaliar o papel de aae de A. actinomycetemcomitans tanto na capacidade de aderir à hidroxiapatita recoberta por saliva (SHA), quanto a de formar biofilme. Um mutante nulo aae foi construído. Suas propriedades hidrofóbicas, bem como a sia capacidade para aderir às células epiteliais, à SHA e para formar biofilme foram avaliadas e comparadas com a cepa -mãe, A. Actinomycetemcomitans VT1169. O mutante nulo aae apresentou redução de hidrofobicidade, assim como diminuição da adesão à SHA e na formação de biofilme, quando comparado à cepa parental. Estes dados sugerem que aae media a adesão de A. Actinomycetemcomitans às células epiteliais e pode também estar envolvida na formação de biofilme e na interação com proteínas salivares adsorvidas.

Aggregatibacter actinomycetemcomitans en periodontitis crónica en individuos de Corrientes, Argentina: estudio preliminar / Aggregatibacter actinomycetemcomitans in chronic periodontitis of individuals from Corrientes, Argentina: a preliminary study

Objetivo: establecer la prevalencia de Aggregatibacter actinomycetemcomitans en pacientes con periodontitis crónica en distintos estadios yla distribución de serotipos, utilizando la técnica de reacción en cadena de la polimerasa. Materiales y métodos: participaron 54 sujetos, entre 35 y 65 años de edad, con diagnóstico de periodontitis crónica. La periodontitis se clasificó en leve, moderada y severa. Como grupo control se incluyeron 24 sujetos sin periodontitis, con los mismos parámetros de inclusión. Para la detección de Aggregatibacter actinomycetemcomitans se procesaron muestras de placa subgingival obtenidas con cono de papel absorbente, conservadas a -20ºC hasta su procesamiento. El ácido dexorribonucleico se extrajo por el método de bromuro de cetiltrimetilamonio y se utilizó la técnica de reacción en cadena de polimerasa para su identificación y serotipado. Resultados: los resultados de las reacciones fueron leidos por electroforesis en geles de agarosa teñidos con bromuro de etidio y la visualización fue realizada por transiluminación ultravioleta. La prevalencia de A. actinomycetemcomitans en periodontitis crónica fue del 14,91 por ciento; el serotipo más frecuente fue el b. No se obtuvieron diferencias estadísticamente significativas entre los grupos con y sin periodontitis (p=0,064), ni entre los estadios de periodontitis y el serotipo presente (p=0,2139). Conclusión: aunque los resultados coinciden con la bibliografía, sería conveniente repetir el estudio sobre una muestra mayor.

Identification of Non-Aggregatibacter actinomycetemcomitans Bacteria Grown on the Tryptic soy-Serum-Bacitracin-Vancomycin Medium

The aim of this study was to identify the non-Aggregatibacter actinomycetemcomitans bacteria grown on the tryptic soy-serum-bacitracin-vancomycin (TSBV) medium, an A. actinomycetemcomitans selective medium. A total of 82 unidentified bacterial isolates from the oral cavities of a Korean population were kindly provide by the Korean Collection for Oral Microbiology. All the clinical isolates were grown on TSBV medium and bacterial DNA purified from each isolate was subjected to PCR with universal primers specific for bacterial 16S rRNA genes (16S rDNAs) sequence. The each bacterial 16S rDNA was amplified by PCR and the nucleotide sequences of it was determined by the dideoxynucleotide chain termination method. They were identified by 16S rDNA sequence comparison method at the specie-level. The data showed that Neisseria spp. (42 strains), Fusobacterium spp. (10 strains), Capnocytophaga spp. (8 strains), Propionibacterium acnes (5 strains), Aggregatibacter aprophilus (4 strains), Campylobacter spp. (5 strains), Veillonella dispar (3 strains), Streptococcus sp. (1 strain), Haemophilus parainfluenzae (1 strain), Leptotrichia wadei (1 strain), Morococcus sp./Neisseria sp. (1 strain), and Staphylococcus sp. (1 strain) were identified. These results could be used to develop a new A. actinomycetemcomitans-selective medium which is more effective than the TSBV medium in future studies.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

ABSTRACT In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b of A. actinomycetemcomitans.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production.Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation.Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels.Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b.Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.

Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient

Abstract: This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients.

Resumo: Este estudo investigou o efeito de enxaguantes à base de ácido hipocloroso (HOCl) e clorexidina (CHX) sobre a viabilidade bacteriana de S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae e E. cloacae. O percentual de bactérias sobreviventes foi testado pelo método de fluorescência utilizando Live/Dead kit(r) e BacLight (Molecular Probes(r)), fazendo comparação entre os grupos com os testes de Kruskal-Wallis e U Mann-Whitney e correção de Bonferroni (p<0,012). O efeito de HOCl e CHX sobre P. gingivalis e S. mutans foi determinado por SDS-PAGE. O CHX mostrou eficácia superior ao HOCl contra S. mutans, A. israelii, E. corrodens e E. cloacae (p<0,001), ao passo que P. gingivalis, A. actinomycetemcomitans, C. rectus e K. oxytoca foram melhores que o CHX para o HOCl (p=0,001). O K. pneumoniae teve efeito similar para o CHX e para o HOCl. As proteínas de P. gingivalis e S. mutans foram afetadas de modo semelhante por CHX e HOCl. O HOCl reduziu a viabilidade bacterial, especialmente nas bactérias periodontopáticas, o que pode recomendar o uso no controle do biofilme subgingival em pacientes periodentais.

Antibacterial Activity of Essential Oil of Syzygium Aromaticum (L.) Merr.Perry (Cloves) Against Clinical Isolates of Aggregatibacter Actinomycetemcomitans.

Aim: Aggregatibacter actinomycetemcomitans implicated in the etiology of aggressive, chronic periodontitis and other systemic infections. The aim of this study was to investigate the antibacterial efficacy of essential oil of a traditional medicinal plant, Syzygium aromaticum (L.) Merr.perry (cloves) against the clinical isolates of A.actinomycetemcomitans obtained from the patients with orodental infections. Methodology: Essential oil of clove flower buds was distilled by Neo-Clevenger’s method and the antibacterial activity was tested by disc diffusion and agar punch well method. The minimum inhibitory concentration of clove oil was determined by micro broth dilution method. Results: 80.8% of the isolates showed sensitivity against clove oil by disc diffusion technique and the minimum inhibitory concentration of clove oil was ranged between 3.125μl/ml to 12.5μl/ml. Conclusion: Essential oil of Syzygium aromaticum showed good antibacterial activity and can be considered as alternative treatment option for control of periodontitis and other orodental infections caused by A.actinomycetemcomitans.