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Toothbrushes are colonised by microorganisms, implying a risk of infection. That risk can be reduced by decreasing the microbial contamination of the filaments. Therefore, this study aimed to determine the antiseptic efficacy of a 0.05% chlorhexidine + 0.05% cetylpyridinium chloride mouthwash on toothbrushes.Twelve toothbrushes used 3 times/day for 14 days by orally and systemically healthy people were randomly split into two groups, and their heads were immersed for 2h in PBS (control) or Perio·Aid Active Control (treatment). The microorganisms were recovered, and their number was calculated by culture, quantitative PCR and viability PCR. Statistical differences were first assessed with a Two-Way Mixed ANOVA, and subsequently with the Student's t test.The results showed no statistical differences in the total number of cells for the treatment (mean ± CI95% of 7.27 ± 1.09 logBased on our findings, sanitisation of toothbrushes with this mouthwash reduces the number of live microorganisms adhered to the filaments. Such decrease of the bacterial load could include bacteria from the oral cavity, from the environment and from nearby toothbrushes since the quantification was not limited to any bacterial taxon.

There is scarce knowledge regarding the antimicrobial resistance profile of F. alocis. Therefore, the objective of this research was to assess antimicrobial resistance in recently obtained F. alocis clinical isolates and to identify the presence of antimicrobial resistance genes. Isolates were obtained from patients with periodontal or peri-implant diseases and confirmed by sequencing their 16S rRNA gene. Confirmed isolates had their genome sequenced by whole genome sequencing and their phenotypical resistance to nine antibiotics (amoxicillin clavulanate, amoxicillin, azithromycin, clindamycin, ciprofloxacin, doxycycline, minocycline, metronidazole, and tetracycline) tested by E-test strips. Antimicrobial resistance genes were detected in six of the eight isolates analyzed, of which five carried tet(32) and one erm(B). Overall, susceptibility to the nine antibiotics tested was high except for azithromycin in the isolate that carried erm(B). Moreover, susceptibility to tetracycline, doxycycline, and minocycline was lower in those isolates that carried tet(32). The genetic surroundings of the detected genes suggested their inclusion in mobile genetic elements that might be transferrable to other bacteria. These findings suggest that, despite showing high susceptibility to several antibiotics, F. alocis might obtain new antimicrobial resistance traits due to its acceptance of mobile genetic elements with antibiotic resistance genes in their genome.

Ultraviolet-B radiation (UV-B) and cold limit the growth and development of plants, which generates changes in gene expression. This allows plants to respond to stress through regulatory proteins, such as transcription factors, that activate or repress the expression of stress-response genes. RNA-Seq data and WGCNA analyses were utilized to identify the hub genes. Our study found a total of 25, 24, and 29 transcription factors at different time points T1, T2, and T3, respectively, under combined stress (ultraviolet-B radiation and cold). RVE1 (MYB-related), COL2 (CO-like), and DBB1b (DBB) were identified as candidate hub genes. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that RVE1, DBB1b, and COL2 were mostly involved in energy production, the antioxidant system (enzymatic and non-enzymatic), signaling through abscisic acid and CA2+, response to light stimulus, and cellular homeostasis. These findings provide the basis for further investigation related to UV-B radiation and cold stress response mechanisms in plants.

Aerosols released from the oral cavity help spread the SARS-CoV-2 virus. The use of a mouthwash formulated with an antiviral agent could reduce the viral load in saliva, helping to lower the spread of the virus. The aim of this study was to assess the efficacy of a mouthwash with 0.07% cetylpyridinium chloride (CPC) to reduce the viral load in the saliva of Coronavirus disease 2019 (COVID-19) patients.In this multi-centre, single-blind, randomized, parallel group clinical trial, 80 COVID-19 patients were enrolled and randomized to two groups, namely test (n = 40) and placebo (n = 40). Saliva samples were collected at baseline and 2 h after rinsing. The samples were analysed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and an enzyme-linked immunosorbent assay test specific for the nucleocapsid (N) protein of SARS-CoV-2.With RT-qPCR, no significant differences were observed between the placebo group and the test group. However, 2 h after a single rinse, N protein concentration in saliva was significantly higher in the test group, indicating an increase in lysed virus.The use of 0.07% CPC mouthwash induced a significant increase in N protein detection in the saliva of COVID-19 patients. Lysis of the virus in the mouth could help reduce the transmission of SARS-CoV-2. However, more studies are required to prove this.

The aim of this study was to analyze the distribution of β-lactamase genes and the multidrug resistance profiles in β-lactam-resistant subgingival bacteria from patients with periodontitis. Subgingival samples were obtained from 130 Spanish patients with generalized periodontitis stage III or IV. Samples were grown on agar plates with amoxicillin or cefotaxime and incubated in anaerobic and microaerophilic conditions. Isolates were identified to the species level by the sequencing of their 16S rRNA gene. A screening for the following β-lactamase genes was performed by the polymerase chain reaction (PCR) technique: blaTEM, blaSHV, blaCTX-M, blaCfxA, blaCepA, blaCblA, and blaampC. Additionally, multidrug resistance to tetracycline, chloramphenicol, streptomycin, erythromycin, and kanamycin was assessed, growing the isolates on agar plates with breakpoint concentrations of each antimicrobial. β-lactam-resistant isolates were found in 83% of the patients. Seven hundred and thirty-seven isolates from 35 different genera were obtained, with Prevotella and Streptococcus being the most identified genera. blaCfxA was the gene most detected, being observed in 24.8% of the isolates, followed by blaTEM (12.9%). Most of the isolates (81.3%) were multidrug-resistant. This study shows that β-lactam resistance is widespread among Spanish patients with periodontitis. Furthermore, it suggests that the subgingival commensal microbiota might be a reservoir of multidrug resistance and β-lactamase genes. Most of the samples yielded β-lactam-resistant isolates, and 4 different groups of bla genes were detected among the isolates. Most of the isolates were also multidrug-resistant. The results show that, although β-lactams may still be effective, their future might be hindered by the presence of β-lactam-resistant bacteria and the presence of transferable bla genes.

Esta investigación está estructurada para que puedan comprender en forma clara sobre la dispersión del riesgo y como debe ser tratado mediante el uso del seguro. En los países de américa latina en la cual la actividad aseguradora cobra día a día mayor importancia como formas de desarrollo personales e institucionales, sean éstas de derecho público o privado, se avizora una clara tendencia a profundizar la necesidad de cobertura de los riesgos, mediante un seguro que permita salvaguardar sus intereses personales y patrimoniales de los usuarios, clientes, empresas o corporaciones que lo posean. Al seguro se lo debe analizar no como un gasto sino como una inversión que proteja su patrimonio y cediendo su riesgo a una aseguradora; no asuma su riesgo, porque puede perder capitales importantes que han sido fruto de su esfuerzo. No obstante, lo que se trata es de crear una conciencia preventiva y una acertada cultura del seguro, enfocado a compartir con los asegurados y no asegurados del país información básica que ayude a comprender lo que significa este servicio tan importante, además sus características, principios, y demás beneficios que permita a las personas naturales o jurídicas, hacer uso responsable y oportuno para proteger su vida, sus seres queridos, bienes más preciados y todo aquello que puede ser objeto de asegurabilidad.

El presente artículo de investigación nos permitirá aprender por completo un programa de educación financiera aplicada para las compañías de seguros en donde se van aplicar diferentes codificaciones y resoluciones que se resuelven por los organismos de control financiero. En los contenidos de esta investigación podremos determinar las aplicaciones adecuadas en el sistema financiero y se podrán aplicar el impacto del código de transparencia y derechos del usuario. Esta normativa de educación financiera tiene como objetivo de la resolución es de que los ciudadanos puedan disponer de servicios de óptima calidad y que mejor hacerlo a través de la capacitación y las compañías de seguros permitan conocer de manera transparente los servicios y productos que ofrecen de su información administrativa, técnica, contable, económica y financiera. El propósito permitirá conocer como proyectar varias alternativas de educar a los usuarios, consumidores y público en general sobre el seguro y el porqué de su importancia de obtener los productos que están en el mercado financiero, este asesoramiento es en base a los muchos años de estar en el mercado de los seguros. Al realizar consultas en el rango del 2012 al 2018 en Google Académico se encuentra: “Seguros”, cuenta con 5 citaciones, con un promedio de 12 papers relacionados al tema, un índice H de 1 a nivel mundial, “empresas comerciales” con 320, citaciones, con un promedio de 8 papers relacionados al tema, un índice H de 2 a nivel mundial.

espanolLa tecnica de microscopia electronica de barrido (SEM) fue utilizada para estudiar la estructura externa corionica de los huevos de la polilla de la papa: Symmetrischema tangolias y Tecia solanivora (Lepidoptera: Gelechiidae). Mediante este estudio, se observo diferencias marcadas en la reticulacion de los coriones de ambas especies; se reporto el numero de aeropilos y micropilos por huevo, se describio y diferencio las caracteristicas superficiales de estas estructuras. Con el uso del microscopio confocal se determino el tamano de los ejes mayor y menor, y se propuso un modelo matematico estocastico que identifico a los huevos de cada una de estas especies; usando estos valores se calculo la superficie y el volumen de los huevos de T. solanivora, los cuales resultaron ser mas grandes que los de S. tangolias. EnglishScanning electron microscopy (SEM), was used to study the external chorionic structure ofpotato moth eggs: Symmetrischema tangolias and Tecia solanivora (Lepidoptera: Gelechiidae). Differenceswere observed between egg chorium reticulations for each moth species. The number of aeropyls andmicropyls per egg were reported and their superficial characteristics were differentiated and described inthis study. A confocal microscope was used to determine the size of the major and minor axes of the eggs.These values were used to propose a stochastic mathematical model that identifies each species’ eggs, andto calculate their surface area and volume. Results indicate eggs of T. solanivora to be larger than those ofS. tangolias.

Background Peri-implant mucositis and peri-implantitis are the main biological complications associated with dental implants. Since most authors agree that bacteria play a major etiological role, the main aims of this study were to determine if a formulation of erythritol and chlorhexidine applied with an air polishing system inhibits biofilm regrowth over dental implants and to compare the decontamination capacity of this therapy with that of mechanical removal by saline and gauze. Material and Methods A multispecies biofilm (P. gingivalis, A. actinomycetemcomitans, F. nucleatum, A. naeslundii, V. parvula and S. oralis) was grown for 14 days on 52 dental implants in an artificial mouth. These implants were divided into three groups according to the applied treatment: 14 negative control (CON), 19 erythritol-chlorhexidine (ERY) and 19 gauze with saline (GAU) samples. Twelve dental implants from the ERY and GAU groups and 8 implants from the CON group were re-incubated for 7 additional days after treatment. The bacterial count was performed by quantitative polymerase chain reaction (qPCR) using propidium monoazide (PMA). A descriptive and bivariate analysis of the data was performed. Results The erythritol and chlorhexidine formulation significantly inhibited biofilm regrowth in comparison with the mechanical treatment (GAU), since a significant decrease in all the species was observed in the ERY group (except for Aggregatibacter actinomycetemcomitans). The antibiofilm and antibacterial capacity of the two active treatment groups (ERY and GAU) was similar for a 14 days multispecies in vitro biofilm, except for the lower count of A. naeslundii in the GAU group. Conclusions The use of erythritol powder with chlorhexidine applied with an air polishing system reduces biofilm regrowth over dental implants when compared with mechanical removal by saline and gauze. This effect might be beneficial for patients included in peri-implant maintenance programs. Key words:Dental implants, biofilms, peri-implantitis, erythritol, chlorhexidine.

Aim To investigate microbial profiles in placentas from a population of East African mothers with and without adverse pregnancy outcomes and with regard to their periodontal status. Material and methods Thirty-six placentas from pregnant women from Tanzania were classified into three groups according to both pregnancy outcome and the mother's periodontal health. The microbial composition in each group was then compared using 16S rRNA metagenomics. Additionally, placenta specimens were analyzed histologically for chorioamnionitis by a single pathologist blinded to the clinical data. Results The greatest differences were observed in the group of mothers with periodontitis. The microbial load was low in all three groups of mothers. Periodontitis had a notable influence on the structure of the placental microbiota. Three phyla and 44 genera were associated with periodontitis, whereas only the Tenericutes phylum was associated with the adverse pregnancy variable. Streptococcaceae and Mycoplasmataceae families were associated with both periodontitis and adverse pregnancy outcomes. Finally, although the differences for chorioamnionitis were not significant, this intra-amniotic infection was more frequent in the placentas from mothers with periodontitis. Conclusions Our findings suggest that bacteria from the oral cavity may involve the feto-placental unit, and that periodontitis may be a modulating factor of the microbial community present in this niche.

Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity in these oral rinses and their exact mechanism of action remain unknown. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium compound in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting the viral fusion step with target cells after disrupting the integrity of the viral envelope. We also found that CPC-containing mouth rinses decreased more than a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. This activity was effective for different SARS-CoV-2 variants, including the B.1.1.7 or Alpha variant originally identified in United Kingdom, and in the presence of sterilized saliva. CPC-containing mouth rinses could therefore represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with., This research was funded by Dentaid SL. The authors also acknowledge the crowdfunding initiative #Yomecorono. N. I-U. is supported by grant PID2020-117145RB-I00 from the Spanish Ministry of Science and Innovation. E. Pradenas was supported by a doctoral grant from the National Agency for Research and Development of Chile (ANID 72180406).

In this study we developed a novel anti-fraud and anti-corruption control model during the public procurement process considering the following processes: i) compliance, ii) risk management and iii) ISO 37001 anti-bribery. This exploratory research included participation from expert auditors in public procurement was requested, who provided criteria that were quantified. We identified concepts and reporting that made it possible to reveal acts of corruption and fraud. Then, specialized bibliography was reviewed to decipher theories and methodologies that are applied in risk control processes. Audit reports were also studied to identify the causes and consequences of the application of public procurement processes. In contracting, risks were identified in all components of the model, which were made visible to make corrections or store a risk. In practice, this model can aid in decreasing corruption and fraud in the contracting process.

Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity present in these oral rinses and their exact mechanism of action remain unknown. Here we show that Cetylpyridinium chloride (CPC), a quaternary ammonium compound present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting the viral fusion step with target cells after disrupting the integrity of the viral envelope. We also found that CPC-containing mouth rinses decreased more than a thousand times the infectivity of SARS-CoV-2in vitro, while the corresponding vehicles had no effect. This activity was effective for different SARS-CoV-2 variants, including the B.1.1.7 variant, predominant in UK, also in the presence of sterilized saliva. CPC-containing mouth rinses could therefore represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

Introduction: Antibiotic resistance is widely found even among bacterial populations not having been exposed to selective pressure by antibiotics, such as tetracycline. In this study we analyzed the tetracycline-resistant subgingival microbiota of healthy subjects and of patients with periodontitis, comparing the prevalence of tet genes and their multidrug resistance profiles. Methods: Samples from 259 volunteers were analyzed, obtaining 813 tetracycline-resistant isolates. The prevalence of 12 antibiotic resistance genes was assessed, and multidrug profiles were built. Each isolate was identified by 16S rRNA sequencing. Differences in qualitative data and quantitative data were evaluated using the chi-square test and the Mann-Whitney-U test, respectively. Results: tet(M) was the most frequently detected tet gene (52.03%). We observed significant differences between the prevalence of tet(M), tet(W), tet(O), tet(32) and tet(L) in both populations studied. Multidrug resistance was largely observed, with resistance to kanamycin being the most detected (83.64%). There were significant differences between the populations in the prevalence of kanamycin, chloramphenicol, and cefotaxime resistance. Resistant isolates showed significantly different prevalence between the two studied groups. Conclusion: The high prevalence of multidrug resistance and tetracycline resistance genes found in the subgingival microbiota, highlights the importance of performing wider and more in-depth analysis of antibiotic resistance in the oral microbiota. info:eu-repo/semantics/publishedVersion

Background To determine whether an experimental abutment mimicking the macro- and microstructure of a dental implant is a suitable method for recovering biofilm, and to describe the features of biofilms formed around such abutments on healthy implants. Material and Methods Experimental abutments were used in 15 patients without peri-implant diseases. After 14 days’ absence of dental hygiene in this area, the abutments were retrieved and analyzed through confocal laser scanning microscopy and scanning electron microscopy. The biofilm formation on the surface of the first 5 abutments was determined by a fluorescence-staining method using SYTO9 nucleic acid stain. In order to study the biofilm’s coverage and vitality, 10 additional abutments were assessed using live & dead bacterial viability. Descriptive and bivariate analyses of the data were performed. Results A global plaque coverage of the abutments was observed in all cases. The submucosal area of the abutment was mostly covered with biofilm (over 21%). Moreover, significant differences between supra- and subgingival locations were detected. Conclusions This in vivo experimental model allows detailed observation of the extensive plaque growth found on exposed experimental abutments mimicking dental implants when hygiene measures are absent. The biofilm coverage is significantly higher in the supragingival zone than in the subgingival portion. Key words:Dental implants, biofilm, peri-implant diseases.

Tetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one. This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm. info:eu-repo/semantics/publishedVersion

Objectives To study oral Prevotella spp. isolated from patients with chronic periodontitis, to determine their susceptibility to azithromycin and erythromycin and to screen the presence of macrolide resistance genes therein. Material and methods Isolates with a Prevotella-like morphology were obtained from subgingival samples of 52 patients with chronic periodontitis. Each isolate was identified to the species level by sequencing of the 16S rRNA gene. In 100 Prevotella spp. isolates, azithromycin and erythromycin susceptibility was determined using the E test method, and the screening of erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q) and mef(A) genes was done by PCR. Results Prevotella intermedia and Prevotella nigrescens were the most identified species (33% each). Minimum inhibitory concentrations (MICs) ranges for both antibiotics were 0.016/0.032 to >256 μg/ml. MIC50 values for azithromycin and erythromycin were 1.5 and 1 μg/ml, respectively, and MIC90 values were >256 μg/ml for both antibiotics. Nineteen per cent of the isolates carried erm(B), and 51% carried erm(F). Conclusions The MIC values found were high compared to previous studies. erm(F) was greatly prevalent, and we describe for the first time the erm(B) gene in Prevotella spp. The presence of either of the genes seems to be associated with a higher degree of resistance to azithromycin and erythromycin.

Phytochemical analysis of Azorella compacta resulted in the isolation of six known diterpenes and one unknown norditerpene using chromatographic meth­ods. The structure of this new compound was established by spectroscopic analysis. The bioactivity of the new compound was assessed against six oral bacteria using a drop test method. A high level of growth inhibition was observed against F. nucleatum and P. gingivalis ; however, other species such as V. parvula and A. actinomycetemcomitans were not affected.

It is estimated that six million perinatal deaths occur every year worldwide, with premature birth being the main cause. Scientific evidence has shown that there is an association between periodontal health during pregnancy and adverse outcomes of labor, although interventional studies based on the treatment of periodontitis have failed to document an impact on reducing the incidence of preterm birth (PB) or low birth weight (LBW). Two pathogenic mechanisms have been proposed to explain this association. The direct pathway is based on the presence of gram-negative anaerobic bacteremia originating in the gingival biofilm, whereas the indirect pathway involves the production of pro-inflammatory markers which enter the bloodstream from the gingival submucosa. The result is the same: the development of an immune inflammatory response and/or the local suppression of growth factors in the fetal-placental unit, which in turn triggers labor. In the present review, we describe current concepts pertinent to PB and LBW, chronic and aggressive periodontitis, and the most frequent aspects of periodontal pathology during pregnancy. We evaluate the scientific evidence available to date, and offer a detailed description of the two pathways proposed to explain the association of maternal periodontitis with preterm and LBW delivery.

Background and Objective Bacteria in the oral cavity grow in the form of biofilms; these structures are subject to constant saliva or gingival crevicular fluid flow conditions. The aims of this study were: (i) to develop and to characterize an in-vitro biofilm model with oral bacteria growing under flow and shear conditions; and (ii) to demonstrate the usefulness of the model for evaluating the activity of three antiplaque agents. Material and Methods We used a bioreactor to grow the oral bacteria Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis under planktonic conditions. Biofilms were established using a modified Robbins device on hydroxyapatite (HAP) discs. Three- to 7-d-old biofilms were analysed using culture methods, scanning electron microscopy, Live/Dead staining and fluorescence in-situ hybridization (confocal laser scanning microscopy). Finally, we assessed the antimicrobial activity of three mouthrinses [0.12% chlorhexidine (CHX), 0.12% chlorhexidine and sodium fluoride (CHX+NaF) and 0.12% chlorhexidine and 0.05% cetylpyridinium chloride (CHX+CPC)] using a planktonic test (short interval–killing test) and in our 4-d biofilm model. Results The viable cell counts showed that each species was consistently found in the biofilms throughout the study. The architecture and cell distribution were similar to those described for biofilms in situ, with the exception of a thin layer of living cells that was found close to the HAP. The effectiveness test of the mouthwashes demonstrated that cells in biofilms showed more tolerance compared with planktonic cells. Moreover, it was observed that in 4-d biofilm formed in vitro, CHX+CPC caused significantly higher mortality compared with CHX (p = 0.003) and CHX+NaF (p

Background and Objectives Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. Material and Methods PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. Results When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10. P. gingivalis showed a vitality reduction in the biofilm of 3 log10, while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. Conclusion These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.

Objectives. The aim of this investigation was to evaluate whether the adenosine triphosphate (ATP) bioluminescence method is an appropriate tool to assess the efficacy of antiseptic mouthrinses in terms of quantitative reductions of total viable microbial counts in mixed biofilm populations in vitro. Study Design. Three mouthrinses, containing respectively, chlorhexidine and cetylpyridinium chloride (CHX/CPC), essential oils (EO) and amine fluoride/stannous fluoride (AFSF), as well as Phosphate Buffered Saline (PBS) used as control, were tested in an in vitro static biofilm model by ATP bioluminescence and compared to culture method. Biofilms were grown on saliva-coated hydroxyapatite disks for 72 hours and then exposed for 1 minute to the mouthrinse or control by immersion. The antibacterial effect of the rinses was tested by analysis of variance. The reliability of the ATP bioluminescence method was assessed by calculating the Pearson correlation coefficients when compared to the viable cell counts obtained by culture. Results. Using ATP bioluminescence, the antimicrobial activity of the tested mouthrinses was demonstrated when compared to the PBS control. The ATP bioluminescence values were significantly correlated (0.769, p

Background Periodontal disease in cats is highly prevalent, and its aetiology is associated to bacteria located in the subgingival microbiota, being Porphyromonas sp. the most prevalent genus. The conventional technique to sample the subgingival microbiota is the use of cotton swabs over the mucosa and teeth; however the use of subgingival paper points could improve the bacterial recovery. Aim The objective was to compare two microbial sampling approaches for the evaluation of the periodontal disease-associated microflora in cats. Methods The study was designed as a pilot study. Ten cats were clinically evaluated and sampled under sedation. Subgingival pooled samples were collected from four sites. In parallel, samples were obtained with a cotton swab, by striking over the gingival margin and surface of the upper right canine. Samples were cultured on blood agar (aerobic and anaerobic incubation), Dentaid-1 (for Aggregatibacter actinomycetemcomitans and enterics), and a specific medium for Bartonella henselae. Results For total anaerobic counts, paper point samples (6.59 ± 0.5) demonstrated significantly higher counts (p = 0.03) than cotton swab samples (5.54 ± 1.1). Moreover, the use of paper points increased the frequency detection of most pathogens thus reducing false negatives for Porphyromonas gulae (100% with paper points samples and 80% with cotton swab samples). Conclusions Significant higher recoveries of anaerobic bacteria and more frequent detection of putative periodontal pathogens was observed when microbiological sampling was performed with paper points, in cats with periodontal disease.

Sanchez MC, Llama-Palacios A, Blanc V, Leon R, Herrera D, Sanz M. Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota. J Periodont Res 2011; 46: 252–260. © 2011 John Wiley & Sons A/S Background and Objective: There are few in vitro models available in the scientific literature for study of the structure, formation and development of the subgingival biofilm. The purpose of this study was to develop and validate an in vitro biofilm model, using representative selected bacteria from the subgingival microbiota. Material and Methods: Six standard reference strains were used to develop biofilms over sterile ceramic calcium hydroxyapatite discs coated with saliva within the wells of presterilized polystyrene tissue culture plates. The selected species represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The structure of the biofilm obtained was studied using a vital fluorescence technique in conjunction with confocal laser scanning microscopy. The biofilm bacterial kinetics were studied by terminal restriction fragment length polymorphism analysis. Results: After 12 h, initial and early colonizers were the first microorganisms detected adhering to the calcium hydroxyapatite discs. The intermediate colonizer F. nucleatum was not detected in the model until 24 h of incubation. Late colonizers A. actinomycetemcomitans and P. gingivalis could be measured inside the biofilm after 48 h. The biofilm reached its steady state between 72 and 96 h after inoculation, with bacterial vitality increasing from the hydroxyapatite surface to the central part of the biofilm. Conclusion: An in vitro biofilm model was developed and validated, demonstrating a pattern of bacterial colonization and maturation similar to the in vivo development of the subgingival biofilm.

ResumenLas células dendríticas son células presentadoras de antígeno capaces de inducir la activación y maduración de linfocitos T CD4+ vírgenes hacia un fenotipo efector especifico Th1 o Th2, dependiendo del tipo de antigeno presentado, las senales co-estimuladoras que expresan y el ambiente de citoquinas en el cual se produce la interaccion celula-celula. El objetivo del presente trabajo es analizar la respuesta de las celulas dendriticas estimuladas in vitro con distintas concentraciones de las bacterias periodontopatógenas Pg y Aa. En celulas dendriticas derivadas de monocitos de sangre periferica estimuladas con 101 a 109 bacterias/mL de Pg y Aa se evaluo la expresion del marcador de maduracion CD80 mediante citometria de flujo y de las citoquinas IL1β, IL2, IL5, IL6, IL10, IL12, IL13, IFNγ, TNFα y TNFβ mediante RT-PCR cuantitativa. Aa y Pg indujeron maduracion de las celulas dendriticas, detectandose significativamente mayor expresion de CD80 con la estimulacion de Aa, e indujeron predominantemente la expresion de citoquinas propias de una respuesta Th1. Dependiendo de la carga bacteriana, fueron detectados distintos umbrales de induccion de expresion de citoquinas. Aa indujo la sintesis de IL1β, IL12, IFNγ, TNFα y TNFβ a menor carga bacteriana que Pg. Tomados en conjunto, estos datos nos permiten especular un mayor potencial antigenico y proyectar una mayor capacidad patogenica durante la infeccion periodontal de Aa en comparación a Pg.AbstractDendritic cells are potent antigen-presenting cells able to prime naive T cells and polarize them towards a Th1 or Th2 response, depending on the type of the antigen presented to the TCR, the type of costimulatory signals, and the cytokine pattern in the environment. The aim of this work was to analyze the response of dendritic cells to in vitro stimulation with Pg and Aa. In monocyte-derived dendritic cells stimulated with 101 to 109 bacteria/mL of Pg or Aa were evaluated both the expression of the maturation marker CD80 by flow cytometry and the expression of the cytokines IL1β, IL2, IL5, IL6, IL10, IL12, IL13, IFNγ, TNFα and TNFβ by quantitative RT-PCR. Both Pg and Aa led to dendritic cell maturation, detecting higher CD80 expression upon Aa-stimulation, and induced a Th1 pattern of cytokine expression. Aa-stimulated dendritic cells expressed IL1β, IL12, IFNγ, TNFα and TNFβ mRNAs with lower bacterial charge than with Pg. Furthermore, our data indicated the existence of distinct thresholds for the induction of the different cytokines analyzed. Taken together, these data allow us to speculate a higher antigenic potential and higher pathogenic capacity of Aa than Pg during periodontal infections.

Aim To investigate the subgingival microbiota of distinct periodontitis patient populations, in Chile, Colombia and Spain, using identical clinical and bacteriological methods. Material and methods In this multicentre study, 114 chronic periodontitis patients were selected. Patients were examined using an identical clinical protocol and pooled subgingival samples were obtained from each patient. Samples were processed in the three laboratories by means of culturing under identical clinical and microbiological protocols. Total anaerobic counts and frequency of detection and proportions of nine periodontal pathogens were calculated. Variables were analysed by means of anova, chi(2), Kruskal-Wallis and Dunn's multiple comparison tests. Results The Colombian population demonstrated greater severity of periodontitis, with significantly deeper mean probing pocket depth, and had a significantly lower percentage of current smokers. When comparing samples from the three patient populations, the total counts were significantly higher in the Colombian patients. The numbers of putative pathogens differed among groups. Tannerella forsythia was found less frequently in Chilean samples, while Parvimonas micra and enteric rods differed significantly among the three population groups. Conclusion Significant differences among Chile, Colombia and Spain existed regarding the frequency and proportions of specific periodontal pathogens in the subgingival microbiota of periodontitis patients.

Objective: Neutrophils play a crucial role in the defense of invading bacteria by releasing biologically active molecules. The response of peripheral blood neutrophils was studied in periodontitis-affected patients and in healthy controls towards stimulation to Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) extracts. Materials and methods: Peripheral venous blood was drawn from 23 adult patients with moderate to advanced chronic periodontitis (probing depth ≥5 mm, attachment loss ≥3 mm), and 30 healthy volunteers. Neutrophil response followed by metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) secretion was assayed by zymography and enzyme-linked immunosorbent assay, respectively, on both whole blood and purified neutrophils. In addition to periodontal pathogen extracts, known stimulating agents were tested, such as Escherichia coli-lipopolysaccharide (LPS), phytohemagglutinin, and zymosan A. Results: Neutrophil response, expressed as a secretion ratio under stimulated and non-stimulated conditions, measured in whole blood, showed no differences between periodontitis and healthy controls. Instead, in purified neutrophils from patients, MMP-9 exhibited a significantly higher secretion ratio with LPS and Pg (1.5- to 2-fold), whereas IL-8 showed a larger increase in secretion ratio (3- to 7-fold) in the presence of Pg, Aa, LPS, and zymosan A. Conclusion: Peripheral neutrophils of periodontitis-affected patients are more reactive as suggested by their significantly higher response toward periodontal pathogen extracts and other stimulating agents.

Epidemiologic and randomized controlled studies have shown that periodontal diseases may be associated with preterm labor and delivery of infants with low birth weights. The purpose of the present study was to determine the presence of microbial invasion of the amniotic cavity by periodontopathic bacteria in pregnant women with a diagnosis of threatened premature labor.A periodontal examination and collection of amniotic fluid and subgingival plaque samples were performed on women identified as having threatened premature labor (preterm premature rupture of membranes without clinical infection or labor and preterm labor with intact membranes) and a gestational age ranging between 24 and 34 weeks. Samples collected from amniotic fluid and from the four deepest periodontal pockets in each patient were pooled in prereduced transport fluid and cultured. Porphyromonas gingivalis was identified primarily by colony morphology under stereoscopic microscope and rapid biochemical tests. Amniotic fluid or plaque samples were homogenized, DNA was extracted, and polymerase chain reaction (PCR) amplification of 16S rRNA with specific and universal primers was carried out.Twenty-six women with threatened premature labor were included: eight with preterm premature rupture of membranes and 18 with preterm labor with intact membranes. Eight women presented with gingivitis, 12 with chronic periodontitis, and six without periodontal disease. Microbial invasion of the amniotic cavity as detected by P. gingivalis PCR was 30.8% (eight of 26 patients). In these eight patients, P. gingivalis was present in both the subgingival samples and the respective amniotic fluid sample.The presence of microbial invasion of the amniotic cavity by P. gingivalis could indicate a role for periodontal pathogenic bacteria in pregnant women with a diagnosis of threatened premature labor.

Objectives The objective of this study was to identify the DNA of oral bacteria in placental samples from women with and without periodontitis who had or had not had preterm births and/or low birthweight (PB/LBW) neonates. Methods Data were gathered from 57 puerperal women in relation to socio-demographic, gynaecological, and periodontal variables and to placental histomorphology. Fifty-seven biopsies, 28 from mothers with periodontitis, were taken aseptically from preterm placentas (n = 36) and from full-term placentas (n = 21). Total DNA was extracted, and the presence of 15 oral bacteria was assessed using Nested-PCR. Results The placentas from women with periodontitis showed a higher prevalence of periodontopathogens compared to those from women without periodontitis (P = 0.009). Samples showed low prevalences of Actinomyces israelii, Parvimonas micra and Tannerella forsythia. An association was found between Eikenella corrodens in placenta and periodontitis (P = 0.002). The most ubiquitous bacterium, Fusobacterium nucleatum, was more prevalent in mothers with periodontitis and PB/LBW (P = 0.033). Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were not detected. Conclusions These results, along with previous findings, show that oral bacteria may be normally present in the placenta, however, the levels of certain oral pathogens in the placenta would highly depend on the mother's periodontal state.

Chronic periodontitis is an infectious disease characterized by alveolar bone destruction and teeth loss. Receptor activator of nuclear factor-kappa B ligand (RANKL) is an osteoclastogenic cytokine, a central regulatory factor in the osteoclast's lifespan, and a participant in physiological and pathological bone resorption. Gingival T cells synthesize RANKL, contributing to molecular local imbalance that entails the alveolar bone resorption seen in periodontitis. Our study was aimed at associating the levels of RANKL with the CD4(+) T-cell activity present in gingival tissues of chronic periodontitis patients.Gingival biopsies were obtained from 33 chronic periodontitis patients and 20 healthy controls. Specimens were either formalin fixed and paraffin embedded for real-time reverse transcription-polymerase chain reaction (RT-PCR) and histologic analysis or tissue digestion processed for cell culture and flow-cytometry analysis. RANKL mRNA and protein levels were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) in gingival-cell culture supernatants. Gingival leukocytes were quantified by flow cytometry. RANKL and CD4 immunoreactivity were analyzed by flow cytometry and confocal microscopy.RANKL mRNA levels were higher in patients with periodontitis than in healthy subjects, and spontaneous and lipopolysaccharide (LPS)- and phytohemagglutinin (PHA)-stimulated RANKL synthesis were higher also in patients than controls. CD4(+) T lymphocytes were the predominant infiltrate cell subset present in gingival tissues of periodontitis patients. Furthermore, an association between RANKL and CD4(+) T cells was determined by double-staining flow cytometry and confocal microscopy.Taken together, these data demonstrate that gingival CD4(+) T cells are the main cells responsible for higher levels of RANKL observed in human chronic periodontitis patients.

Background: Actinobacillus actinomycetemcomitans is considered a major etiologic agent of aggressive periodontitis (AgP). Other periodontopathic bacteria such as Porphyromonas gingivalis are also suspected of participating in aggressive periodontitis although the evidence to support this is controversial. The aim of the present study was to determine the prevalence of eight periodontopathic bacteria in Chilean patients with AgP. Methods: Subgingival plaque samples were collected from 36 aggressive, 30 localized, and six generalized periodontitis patients. Samples from 17 advanced chronic periodontitis (CP) patients were taken as controls. Samples collected from the four deepest periodontal pockets in each patient were pooled in prereduced transport fluid (RTF) and cultured. Periodontal bacteria were primarily identified by colony morphology under stereoscopic microscope and rapid biochemical tests. The identity of some bacterial isolates was confirmed by colony polymerase chain reaction (PCR). Results: AgP showed a significatively higher prevalence of C. rectus than CP (P = 0.036). The only statistical difference found was for C. rectus. Patients with AgP showed a higher, but not statistically significant, prevalence of P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. A similar prevalence in both groups of patients was observed for F. nucleatum and P. intermedia/nigrescens, and A. actinomycetemcomitans was less prevalent in AgP than CP patients. In localized AgP, P. intermedia/nigrescens, E. corrodens, F. nucleatum, and P. micros were the more prevalent pathogens in contrast to generalized AgP patients who harbored A. actinomycetemcomitans, P. gingivalis, and Capnocytophaga sp. as the most prevalent bacteria. Conclusions: C. rectus, P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. were the most predominant periodontopathic bacteria of AgP in this Chilean population, but the only statistical difference found here between AgP and CP was for C. rectus, suggesting that the differences in clinical appearance may be caused by factors other than the microbiological composition of the subgingival plaque of these patients. In this study, the prevalence of A. actinomycetemcomitans was much lower than that of P. gingivalis. J Periodontol 2005;76:289-294.

Xanthophyllomyces dendrorhous (formely Phaffia rhodozyma) is a basidiomycetous yeast-like fungus that produces carotenoids useful for the food industry. Recently, its sexual cycle was reported but little is known about its genetic constitution. To inquire into the ploidy state of X. dendrorhous, biased mutant spectrum, genetic complementation and mitotic recombination analysis were used. A wild-type strain was subjected to N-methyl-N'-nitro-N-nitrosoguanidine mutagenic treatment. Auxotrophic and carotene mutants were forced to revert to the wild-type phenotype. Pigment producing and prototroph revertants behaved as diploid except for adenine less mutants. These results are in agreement with the limited spectrum of auxotrophs obtained in this strain for the ADE1 locus. To analyze the genetic characteristic of the adenine genetic marker of X. dendrorhous, protoplast fusion experiments with several adenine less mutants were performed. The experiments presented in this work suggest that the ATCC 2430 (UDC 67-385) strain of X. dendrorhous is diploid and a heterozygous constitution is proposed for the ADE1 locus.

Conditions inducing the development of holobasidia with terminal basidiospores in wild-type and astaxanthin mutant strains of Xanthophyllomyces dendrorhous were reexamined. Important factors for the development of holobasidia were the incubation temperature and the medium composition. A temperature of 9 degrees C was demonstrated to enhance holobasidia formation. Minimal growth medium with glucose as sole carbon source at concentrations between 80 and 120 mM, and ammonium nitrate with concentrations of 28 mM gave optimal results. A period of 20 or more days was needed for the formation of holobasidia with basidiospores. Additionally, mutant strains of X. dendrorhous were observed to have different abilities to produce holobasidia and strains obtained after protoplast fusion, which have been called fusant in this study, to have an increased capacity to form holobasidia.

The purpose of this study was to evaluate the subgingival microbiota and determine the most prevalent periodontal pathogens implicated in feline periodontal disease and to correlate these findings with the clinical periodontal status. Subgingival microbiological samples were taken under sedation from 50 cats with clinical signs of periodontal disease. Pooled paper point samples from 4 selected subgingival sites were cultured on blood agar and on Dentaid-1 medium. Suspected pathogens were identified, subcultured, and preserved. The association between the microbiological findings and the clinical status was studied using correlation coefficients (CC). In addition, cats were stratified in subgroups according to presence of putative pathogens, and comparisons were carried out using unpaired t-test. Three bacterial species were frequently detected including Porphyromonas gulae (86 %), Porphyromonas circumdentaria (70 %) and Fusobacterium nucleatum (90 %). The mean proportion of total flora was high for P. gulae (32.54 %), moderate for P. circundentaria (8.82 %), and low for F. nucleatum (3.96 %). Among the clinical variables, tooth mobility was correlated (CC > 0.50, p < 0.001) with recession, pocket depth, attachment level, gingival index, and calculus index (CC = 0.29, p = 0.04) as well as with total bacterial counts (CC = 0.38, p = 0.006). Cats with more than 10 % of P. gulae showed significantly more mobility (p = 0.014) and recession (p = 0.038), and a tendency for deeper probing pocket depths (p = 0.084) and attachment loss (p = 0.087). The results from this cross-sectional study confirmed that P. gulae is the most relevant pathogen in periodontal disease in cats.

Ácido linoleico conjugado (ALC) es el nombre con el que se designa a una mezcla de isómeros posicionales, conjugados con isomería cis-trans del ácido linoleico. Hay evidencia de que estos compuestos presentan propiedades bioactivas benéficas para la salud, tales como efecto inmunomodulador, anticancerígeno, antidiabético, regulador del peso, antiarterioesclerótico, entre otros. El ALC puede ser obtenido de la leche y sus derivados, así como de productos cárnicos de mamíferos rumiantes. Sin embargo, científicos alrededor del mundo se han esforzado en la búsqueda de procesos biotecnológicos para la obtención de este bioactivo, así como de incrementar su presencia en fuentes tradicionales. Por tanto, el objetivo del presente trabajo es proporcionar un panorama general de la naturaleza del ALC, sus fuentes, propiedades biológicas y beneficios saludables debido al consumo, así como su obtención en condiciones in vitro a partir del uso de herramientas biotecnológicas tales como microrganismos y enzimas más allá de fuentes convencionales.

Objective: There is no study characterizing the variability of Aggregatibacter actinomycetemcomitans isolates in periodontitis patients in Spain. It is therefore the aim of this investigation to study the serotype distribution of A. actinomycetemcomitans strains isolated from periodontitis patients in spain. The polymorphism of the genes that codifies the leukotoxin and the operon of the cytolethal-distending toxin (cdt) will also be investigated. Design.: From a total of 701 patients samples, 40 A. actinomycetemcomitans-positive periodontitis patients were s included in the study (mean age 45.3, 62,5% females) and their clinical periodontal status was assessed. On average, 1-3 isolates from each patient were subcultured and characterized by PCR. Results: Using culture the prevalence of A. actinomycetemcomitans was 5.7%. The most frequent serotype was "b", being 30 patients infected by a unique serotype, while 7 patients showed co-colonization, mostly with serotypes "a" and "b". From the 79 pure isolates obtained, 24 were from serotype "a", 30 from serotype "b", 12 from serotype "c" and 4 from serotype "d". Further characterization of these samples showed that none of these 79 isolates demonstrated the 530-bp detion in the leukotoxin's promoter region that characterizes the JPs strain. Conversely 65.8% of the isolates were cdt+. Conclusions: The most commonserotypes were "a" and "b", being serotype "b" the most prevalent in mono-colonization, while serotypes "e" and "f" were not detected.In the majority of samples,operon that codifies the cdt (65.8%) and the genes responsible for the codification of leukotin (100%) were found. None of the isolates were JP2 strains.

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade- and met- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.

Stable red astaxanthin-producing transformants were obtained after genetic transformation of two Phaffia rhodozyma mutants. A yellow mutant, accumulating β-carotene, and an albino mutant, accumulating phytoene, from P. rhodozyma were transformed using a genomic library of wild-type strain UCD 67-385 in the pBluescript vector. Hybridization assays, using the pBluescript DNA as a radioactive probe, indicate integration of vector sequences into the genome of the transformants. Transformants DNA was digested with restriction endonucleases, ligated with T4 DNA ligase and then used to transform E. coli. Ampicillin resistant plasmids, containing 0.1, 0.2, and 2.5 kb DNA inserts of P. rhodozyma, were rescued from the yeast red transformants. The molecular analysis indicate that transformation has occurred by an integration event of donor DNA into the genome of the host strains.