Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis

Objective: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. Methods: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. Results: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997–0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%−72.73; kappa 0.37–0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2–100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). Conclusions: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test

Hypochlorous Acid as a Potential Postsurgical Antimicrobial Agent in Periodontitis: A Randomized, Controlled, Non-Inferiority Trial

Antecedentes: El ácido hipocloroso (HOCl) es un agente antimicrobiano con alta afinidad por las bacterias Gram negativas del biofilm subgingival. Podría tener un efecto equivalente o no inferior al de la clorhexidina (CHX) para evitar la recolonización de estos microorganismos tras el periodo posquirúrgico. Objetivo: El objetivo es comparar la reducción del índice de placa (IP), el índice gingival (IG), la profundidad de bolsa (PD), la ganancia de nivel de inserción clínica (CAL) y la recolonización bacteriana de microorganismos periodontopáticos en el biofilm subgingival a los 7, 21 y 90 días después del Desbridamiento a Colgajo Abierto (DCA) bajo dos protocolos antimicrobianos: (A) HOCl 0. 05% seguido de HOCl 0,025% y (B) CHX 0,2%/CHX 0,12% utilizados durante 21 días sin higiene bucal regular durante el periodo posquirúrgico. Material y métodos: Se realizó un ensayo controlado aleatorizado de no inferioridad. Treinta y dos pacientes fueron divididos aleatoriamente para recibir cada protocolo antiplaca tras la OFD en pacientes con periodontitis. Se evaluaron los índices clínicos y la recolonización bacteriana mediante qPCR durante 90 días. Los datos se analizaron mediante ANOVA de medidas repetidas, modelos de efectos mixtos ajustados por tratamiento, tiempo y la prueba de Chi-cuadrado/Fisher. También se realizó un análisis de no inferioridad mediante la prueba de hipótesis de no inferioridad de Hodges-Lehmann. Resultados: El HOCl no fue inferior al CHX en la reducción de la IP. Ambos grupos mostraron una reducción comparable de la recolonización de Porphyromonas gingivalis, Tannerella forsythia y Eubacterium nodatum. Sin embargo, el protocolo con HOCl no fue inferior al protocolo con CHX para Treponema denticola y Aggregatibacter actinomicetemcomitans. Conclusiones: El HOCl mejoró la cicatrización periodontal. El HOCl mostró un impacto en la reducción de la recolonización de bacterias periodontopáticas en el postoperatorio. © 2023 por los autores.Background: Hypochlorous acid (HOCl) is an antimicrobial agent with high affinity to Gram-negative bacteria of the subgingival biofilm. It could have an equivalent or no inferiority effect to chlorhexidine (CHX) to avoid recolonization of these microorganisms after the post-surgical period. Objective: The objective is to compare the reduction of plaque index (PI), gingival index (GI), pocket depth (PD), gain of clinical attachment level (CAL), and bacterial recolonization of periodontopathic microorganisms in subgingival biofilm at 7, 21, and 90 days after Open Flap Debridement (OFD) under two antimicrobial protocols: (A) HOCl 0.05% followed by HOCl 0.025% and (B) CHX 0.2%/CHX 0.12% used per 21 days without regular oral hygiene during the post-surgical period. Material and methods: A no-inferiority randomized controlled trial was carried out. Thirty-two patients were randomly divided to receive each antiplaque protocol after OFD in patients with periodontitis. Clinical indexes and bacterial recolonization were assessed using qPCR for up to 90 days. Data were analyzed using repeated measures ANOVA, mixed effects models adjusted for treatment, time, and the Chi-squared/Fisher test. A no-inferiority analysis was also performed using the Hodges–Lehmann hypothesis test for non-inferiority. Results: HOCl was not inferior to CHX in reducing PI. Both groups showed a comparable reduction of recolonization for Porphyromonas gingivalis, Tannerella forsythia, and Eubacterium nodatum. However, the HOCl protocol was non-inferior to the CHX protocol for Treponema denticola and Aggregatibacter actinomicetemcomitans. Conclusions: HOCl improved periodontal healing. HOCl showed an impact in reducing the recolonization of periodontopathic bacteria in the postoperative period. © 2023 by the authors

El efecto de interacción de los títulos de anticuerpos anti-RgpA y anti-PPAD: Un indicador para el diagnóstico de la artritis reumatoide

Porphyromonas gingivalis secreta factores de virulencia como Arg-gingipains y peptidil arginina deiminasa (PPAD), que están asociados con la patogénesis de la artritis reumatoide (AR). Sin embargo, no existe información sobre los títulos de anticuerpos frente a estas enzimas bacterianas como indicadores sistémicos o biomarcadores en la AR. En este estudio transversal se evaluó a 255 individuos: 143 con diagnóstico de AR y 112 sin AR. Se utilizaron modelos de regresión logística ajustados por edad, sexo, índice metabólico basal, tabaquismo y gravedad de la periodontitis para evaluar la asociación de la AR con el factor reumatoide (FR), los anticuerpos antiproteínas citrulinadas (ACPA), la velocidad de sedimentación globular, la proteína C reactiva de alta sensibilidad, los anti-RgpA, los anti-PPAD y los anti-RgpA/anti-PPAD doblemente positivos. Se observó que el FR (odds ratio [OR] 10,6; intervalo de confianza [IC] del 95%: 4,4-25), los ACPA (OR 13,7; IC del 95%: 5,1-35) y la doble positividad anti-RgpA/anti-PPAD (OR 6,63; IC del 95%: 1,61-27) se asociaban con el diagnóstico de AR. Los anti-RgpA también se asociaron con la AR (OR 4,09; IC 95%: 1,2-13,9). La combinación de anti-RgpA/anti-PPAD mostró una elevada especificidad del 93,7% y un VPP del 82,5% en la identificación de individuos con AR. Los anticuerpos anti-RgpA se asociaron con el índice inflamatorio periodontal en individuos con AR (p < 0,05). La doble positividad de los anticuerpos anti-RgpA/anti-PPAD mejoró el diagnóstico de AR. Por lo tanto, los anticuerpos RgpA y anti-RgpA/anti-PPAD pueden ser biomarcadores de la AR.Porphyromonas gingivalis secretes virulence factors like Arg-gingipains and peptidyl arginine deiminase (PPAD), that are associated with rheumatoid arthritis (RA) pathogenesis. However, there is no information regarding the antibody titers for these bacterial enzymes as systemic indicators or biomarkers in RA. In this cross-sectional study, 255 individuals were evaluated: 143 were diagnosed with RA, and 112 were without RA. Logistic regression models adjusted for age, sex, basal metabolic index, smoking, and periodontitis severity were used to evaluate the association of RA with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), erythrocyte sedimentation rate, high sensitivity C-reactive protein, anti-RgpA, anti-PPAD, and double positive anti-RgpA/anti-PPAD. It was found that RF (odds ratio [OR] 10.6; 95% confidence interval [CI] 4.4–25), ACPAs (OR 13.7; 95% CI 5.1–35), and anti-RgpA/anti-PPAD double positivity (OR 6.63; 95% CI 1.61–27) were associated with RA diagnoses. Anti-RgpA was also associated with RA (OR 4.09; 95% CI 1.2–13.9). The combination of anti-RgpA/anti-PPAD showed a high specificity of 93.7% and 82.5% PPV in identifying individuals with RA. RgpA antibodies were associated with the periodontal inflammatory index in RA individuals (p < 0.05). The double positivity of the anti-RgpA/anti-PPAD antibodies enhanced the diagnosis of RA. Therefore, RgpA antibodies and anti-RgpA/anti-PPAD may be biomarkers for R

Evaluación de los cambios en la expresión de mediadores inflamatorios inducidos por vesículas de membrana externa de Porphyromonas gingivalis en macrófagos humanos

Porphyromonas gingivalis es un patógeno periodontal, que secreta vesículas de membrana externa (OMV) como vehículo de factores de virulencia e incluso se ha reportado que estas pueden ser más virulentas que la propia bacteria y podrían modular la respuesta inmune. Objetivo: Determinar las variaciones en la expresión de mediadores inflamatorios en macrófagos humanos estimulados con vesículas de membrana externa de P. gingivalis. Métodos: Monocitos U937 fueron diferenciados a macrófagos y estimulados a diferentes tiempos con OMV, lisado bacteriano y bacteria viva. En experimentos independientes cada estímulo fue pre-tratado con los inhibidores KYT-1, KYT-36 y Polimixina-B para bloquear la acción de las gingipaínas R y K y además del LPS respectivamente. Se determinaron los niveles solubles de citocinas y quimiocinas y también del ARNm. Se usó ANOVA factorial para el análisis estadístico. Resultados: Las vesículas estimularon la producción de citocinas proinflamatorias y quimiocinas. Sin embargo, cuando se bloquean las gingipaínas R y K presentes en OMV, los niveles de IL-1β, IL-6, MCP-1, RANTES, MIP-1α e IL-8 aumentaron significativamente comparado con células sin estímulo y las estimuladas sin el bloqueo (p0,05). De manera contraria, se encontró una disminución en los niveles de TNF-α después de bloquear las proteasas o LPS. Conclusión: Las vesículas de membrana externa de P. gingivalis más que las bacterias vivas inducen cambios en la secreción de mediadores inflamatorios en macrófagos humanos por eventos de proteólisis dependientes de gingipaínas lo cual podría estar involucrado en el establecimiento de un estado crónico característico de la enfermedad periodontal.Abstract: Porphyromonas gingivalis is a periodontal pathogen, which secretes outer membrane vesicles (OMV) as a vehicle for virulence factors and it has even been reported that these can be more virulent than the bacterium itself and could modulate the immune respon se. Objective: To determine the variations in the expression of inflammatory mediators in human macrophages stimulated with outer membrane vesicles of P. gingivalis . Methods : U937 monocytes were differentiated into macrophages and stimulated at different t imes with OMV, bacterial lysate and live bacteria. In independent experiments each stimulus was pre - treated with inhibitors KYT - 1, KYT - 36 and Polymyxin - B to block the action of the R and K gingipains and in addition to the LPS respectively. The soluble lev els of cytokines and chemokines and also of the mRNA were determined. Factorial ANOVA was used for the statistical analysis. Results: The vesicles stimulated the production of proinflammatory cytokines and chemokines. However, when the R and K gingipains p resent in OMV are blocked, the levels of IL - 1β, IL - 6, MCP - 1, RANTES, MIP - 1α and IL - 8 increased significantly compared with cells without stimulus and those stimulated without the blockade (p 0.05). Conversely, a decrease in TNF - α levels was found after bl ocking proteases or LPS. Conclusion: The outer membrane vesicles of P. gingivalis , more than live bacteria , induce changes in the secretion of inflammatory mediators in human macrophages due to gingipain s - dependent proteolysis events , which could be involv ed in the establishment of a chronic state characteristic of periodontal disease.Maestrí

Viability and effects on bacterial proteins by oral rinses with hypochlorous acid as active ingredient

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

Antibiotic Susceptibility and Resistance Genes in Oral Clinical Isolates of Prevotella intermedia, Prevotella nigrescens, and Prevotella melaninogenica

The Prevotella genus is a normal constituent of the oral microbiota, and is commonly isolated from mechanically treated polymicrobial infections. However, antibiotic treatment is necessary for some patients. This study compared the antibiotic susceptibility and the presence of resistance genes in clinical oral isolates of P. intermedia, P. nigrescens, and P. melaninogenica. Antibiotic susceptibility was assessed using the agar dilution method. PCR confirmed the species and resistance gene frequency in the Prevotella species. The frequencies of species P. intermedia, P. nigrescens, and P. melaninogenica were 30.2%, 45.7%, and 24.1%, respectively. No isolates of P. intermedia were resistant to amoxicillin/clavulanic acid, tetracycline, or clindamycin. P. nigrescens and P. melaninogenica were resistant to amoxicillin/clavulanic acid and tetracycline at frequencies of 40% and 20%, respectively. P. intermedia was resistant to metronidazole at a frequency of 30%, P. nigrescens at 20%, and P. melaninogenica at 40%. P. nigrescens and P. melaninogenica were resistant to 50% and 10% clindamycin, respectively. The gene most frequently detected was tetQ, at 43.3%, followed by tetM at 36.6%, blaTEM at 26.6%, ermF at 20%, cfxA, cfxA2, and nimAB at 16.6%, and nimAEFI at 3.3%. P. nigrescens was the species with the highest resistance to antibiotics such as amoxicillin/clavulanic acid, amoxicillin, and clindamycin, in addition to being the species with the largest number of genes compared to P. intermedia and P. melaninogenica

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

Memorias : XX Congreso Institucional de Investigaciones

Este documento, recoge 10 resúmenes de los trabajos presentados como resultado del proyecto en investigación sobre salud oral e investigación en ingeniería, salud y medio ambiente y biología, para el XX congreso institucional de investigaciones de la Universidad del Bosque. Los productos obtenidos como parte de la investigación son: (1) Condición periodontal en pacientes colombianos con artritis idiopática juvenil (AIJ); (2) Efecto del ácido hipocloroso sobre el sistema amortiguador del pH de la saliva. Estudio in vitro e in vivo; (3) Efecto oxidativo del ácido hipocloroso sobre las proteínas salivales: estudio in vitro; (4) Evaluación del ácido hipocloroso como agente antiplaca para uso en la cavidad oral. Parte I: sustantividad, efecto antiplaca y efectos adversos; (5) Identificación de los factores de virulencia de Klebsiella pneumoniae aislada de cavidad oral asociada a fuentes de contaminación exógena; (6) Modulación de la expresión de factores de crecimiento por ácido hipocloroso sobre fibroblastos gingivales humanos; (7) Potencial de diferenciación y expansión neuronal in vitro a partir de Células Troncales de pulpa dental humana; (8) Resolvina D1 inhibe la expresión de moléculas de adhesión en células endoteliales de arteria coronaria humana estimuladas con lipopolisacárido de P. gingivalis. Estudio in vitro; (9) Viabilidad de microorganismos periodontopáticos y bacilos entéricos después del tratamiento con ácido hipocloroso en comparación con clorhexidina; (10) Modelo in vivo de regeneración ósea mandibular a partir de células troncales dentales humanas.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias1308-519-28960Inducción de disfunción endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibición de la inflamación por resolvina (rvd1) y estatina (rosuvastatina)n