Person: Sanz Alonso, Mariano
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First Name
Mariano
Last Name
Sanz Alonso
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Odontología
Department
Especialidades Clínicas Odontológicas
Area
Estomatología
Identifiers
6 results
Search Results
Now showing 1 - 6 of 6
Item Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota(Journal of Periodontal Research, 2011) Sánchez Beltrán, María Del Carmen; Llama Palacios, María Arantxazu; Blanc, Vanesa; León, Rubén; Herrera González, David; Sanz Alonso, MarianoBackground 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.Item Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states(Plos one, 2017) Romero-Lastra, Patricia; Sánchez Beltrán, María Del Carmen; Ribeiro-Vidal, Honorato; Llama Palacios, María Arantxazu; Figuero Ruiz, Elena; Herrera González, David; Sanz Alonso, MarianoBackground and objective Porphyromonas gingivalis is a keystone pathogen in the onset and progression of periodontitis. Its pathogenicity has been related to its presence and survival within the subgingival biofilm. The aim of the present study was to compare the genome-wide transcription activities of P. gingivalis in biofilm and in planktonic growth, using microarray technology. Material and methods P. gingivalis ATCC 33277 was incubated in multi-well culture plates at 37˚C for 96 hours under anaerobic conditions using an in vitro static model to develop both the planktonic and biofilm states (the latter over sterile ceramic calcium hydroxyapatite discs). The biofilm development was monitored by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). After incubation, the bacterial cells were harvested and total RNA was extracted and purified. Three biological replicates for each cell state were independently hybridized for transcriptomic comparisons. A linear model was used for determining differentially expressed genes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm differential expression. The filtering criteria of ±2 change in gene expression and significance p-values of <0.05 were selected. Results A total of 92 out of 1,909 genes (4.8%) were differentially expressed by P. gingivalis growing in biofilm compared to planktonic. The 54 up-regulated genes in biofilm growth were mainly related to cell envelope, transport, and binding or outer membranes proteins. Thirty-eight showed decreased expression, mainly genes related to transposases or oxidative stress. Conclusion The adaptive response of P. gingivalis in biofilm growth demonstrated a differential gene expression.Item Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model(Journal of Periodontal Research, 2014) Sánchez Beltrán, María Del Carmen; Marín Cuenda, María José; Figuero Ruiz, Elena; Llama Palacios, María Arantxazu; León, Rubén; Blanc, Vanesa; Herrera González, David; Sanz Alonso, MarianoBackground 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 lM). 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.Item Antimicrobial activity of red wine and oenological extracts against periodontal pathogens in a validated oral biofilm model(BMC complementary and alternative medicine, 2019) Sánchez Beltrán, María Del Carmen; Ribeiro-Vidal, Honorato; Esteban-Fernández, Adelaida; Bartolomé, Begoña; Figuero Ruiz, Elena; Moreno-Arribas, María Victoria; Sanz Alonso, Mariano; Herrera González, DavidBackground: Previous research findings support an antimicrobial effect of polyphenols against a variety of pathogens, but there is no evidence of this effect against periodontal pathogens in complex biofilms. The purpose of this study was to evaluate the antimicrobial activity of red wine and oenological extracts, rich in polyphenols, against the periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum and total bacteria growing in an in vitro oral biofilm static model. Methods: A previously validated biofilm model, including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was developed on sterile hydroxyapatite discs. Red wine (and dealcoholized wine), and two polyphenols-rich extracts (from wine and grape seeds) were applied to 72 h biofilms by dipping the discs during 1 and 5 min in the wine solutions and during 30 s and 1 min in the oenological extracts. Resulting biofilms were analyzed by confocal laser scanning microscopy and viable bacteria (colony forming units/mL) were measured by quantitative polymerase chain reaction combined with propidium monoazide. A generalized linear model was constructed to determine the effect of the tested products on the viable bacterial counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, as well on the total number of viable bacteria. Results: The results showed that red wine and dealcoholized red wine caused reduction in viability of total bacteria within the biofilm, with statistically significant reductions in the number of viable P. gingivalis after 1 min (p = 0.008) and in A. actinomycetemcomitans after 5 min of exposure (p = 0.011) with red wine. No evidence of relevant antibacterial effect was observed with the oenological extracts, with statistically significant reductions of F. nucleatum after 30 s of exposure to both oenological extracts (p = 0.001). Conclusions: Although moderate, the antimicrobial impact observed in the total bacterial counts and counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, encourage further investigations on the potential use of these natural products in the prevention and treatment of periodontal diseases.Item Analysis of viable vs. dead Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis using selective quantitative real-time PCR with propidium monoazide(Journal of periodontal research, 2012) Sánchez Beltrán, María Del Carmen; Marín Cuenda, María José; Figuero Ruiz, Elena; Llama Palacios, María Arantxazu; Herrera González, David; Sanz Alonso, MarianoBackground and Objectives: One of the major disadvantages of DNA-based microbial diagnostics is their inability to differentiate DNA between viable and dead microorganisms, which could be important when studying etiologically relevant pathogens. The aim of this investigation was to optimize a method forthe selective detection and quantification of only viable Aggregatibacter actino-mycetemcomitans and Porphyromonas gingivalis cells by combining quantitative real-time polymerase chain reaction (qPCR) and propidium monoazide (PMA). Material and Methods: Three different concentrations of PMA (10, 50 or 100lM)were added to suspensions of 106(CFU)/mL of viable/dead A. actinomycetem-comitans and P. gingivaliscells. After DNA isolation, qPCR was carried out using specific primers and probes for the tested bacteria. PMA was further tested with different mixtures containing varying ratios of viable and dead cells. The efficacyof PMA to detect viable/dead cells was tested by analysis of variance. Results: For these specific bacterial pathogens, 100lMPMA resulted in a signif-icant reduction of qPCR amplification with dead cells (106CFU/mL), while with viable cells no significant inhibition was detected. PMA was also effectivein detecting selectively viable cells by qPCR detection, when mixtures of varying ratios of viable and dead bacteria were used. Conclusions: This study demonstrated the efficiency of PMA for differentiating viable and dea dA. actinomycetemcomitans and P. gingivaliscells. This method of PMA-qPCR may be useful for monitoring new antimicrobial strategies and for assessing the pathogenic potential ofA. actinomycetemcomitansandP. gingi-valisin different oral conditions when using molecular diagnostic methods.Item Biofilm formation on dental implants with different surface micro-topography: An in vitro study(Clinical oral implants research, 2019) Bermejo, Patricia; Sánchez Beltrán, María Del Carmen; Llama Palacios, María Arantxazu; Figuero Ruiz, Elena; Herrera González, David; Sanz Alonso, MarianoObjectives: To compare biofilm formation on whole dental titanium implants with different surface micro‐topography. Methods: A multispecies in vitro biofilm model consisting of initial (Streptococcus ora‐lis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actino‐mycetemcomitans) was grown for 96 hr on sterile titanium dental implants with either minimal (Sa: 0.5–1.0 mm) or moderate‐roughness titanium surfaces (Sa: 1.1–2.0 mm). The resulting biofilms were studied with Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscope. Concentrations (colony‐forming units per mL [CFU/ml]) of each bacterium were measured by quantitative Polymerase Chain Reaction (qPCR) and compared by Student t tests. Results: A biofilm, located mainly at the peak and lateral areas of the implant threads, was observed on both implant surfaces, with a greater biomass and a greater live/dead ratio in moderate‐ compared to minimal‐roughness surface implants. Statistically sig ‐nificant higher values of total bacteria (mean difference = 2.61 × 107 CFU/ml; 95% confidence interval — CI [1.91 × 106; 5.02 × 107]; p = 0.036), F. Nucleatum (mean difference = 4.43 × 106 CFU/ml; 95% CI [1.06 × 106; 7.80 × 106]; p = 0.013) and A. actinomycetemcomitans (mean difference = 2.55 × 107 CFU/ml; 95% CI [1.07 × 107; 4.04 × 107]; p = 0.002), were found in the moderate‐ compared to minimal‐roughness surface dental implants. Conclusions: Implants with moderate‐roughness surfaces accumulated more bacte ‐rial biomass and significant higher number of pathogenic bacteria (F. nucleatum and A. actinomycetemcomitans), when compared to implants with minimal‐roughness sur ‐faces, within a similar biofilm structure.