Person:
Sanz Alonso, Mariano

First Name

Mariano

Last Name

Sanz Alonso

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Odontología

Department

Especialidades Clínicas Odontológicas

Area

Estomatología

Identifiers

Full item page

Search Results

Now showing 1 - 10 of 15

Antimicrobial effects of a new brushing solution concept on a multispecies in vitro biofilm model growing on titanium surfaces
(John Wiley & Sons, Inc, 2022-02-06) Virto Ruiz, Leire; Simões Martins ,David; Sánchez Beltrán, María Del Carmen; Encinas, Ana; Sanz Alonso, Mariano; Herrera González, David
Objectives To evaluate the antibiofilm and antibacterial effects of a new brushing solution concept, in a validated peri-implant biofilm model. Materials and Methods A multispecies in vitro biofilm model, including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum, was used. To evaluate the antibiofilm capacity, titanium discs (Ti-SLA) were immersed in 1 ml of the tested solution (one tablet dissolved in warm water) for 2 min, prior and every 24 h during a 3-day biofilm development. Negative (water) and positive (0.12% chlorhexidine/0.05% cetylpyridinium chloride mouth rinse) controls treated discs were run in parallel. To evaluate the antibacterial effects, planktonic cells and 72-h biofilms on sterile Ti-SLA discs were exposed (2 min) to the mentioned treatments. Biofilm structure was analysed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Bacterial load was measured by quantitative polymerase chain reaction and by culture in planktonic cells. Results The tested product showed antibiofilm effects, impacting on the 48-h and 72-h biofilm thickness and significantly reducing viability of all bacterial species, except A. actinomycetemcomitans. Antibacterial effects were observed against the six bacterial species in planktonic state and in 72-h biofilms, especially for F. nucleatum and A. actinomycetemcomitans. Conclusion The tested brushing solution demonstrated antibacterial and antibiofilm properties, mainly against the peri-implant pathogens included in the validated in vitro biofilm model used.
In Vitro Anti-Biofilm and Antibacterial Properties of Streptococcus downii sp. nov.
(MDPI, 2021-02-22) Cuenca, Maigualida; Sánchez Beltrán, María Del Carmen; Diz, Pedro; Martínez-Lamas, Lucía; Álvarez, Maximiliano; Limeres, Jacobo; Sanz Alonso, Mariano; Herrera González, David
The aim of this study was to evaluate the potential anti-biofilm and antibacterial activities of Streptococcus downii sp. nov. To test anti-biofilm properties, Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were grown in a biofilm model in the presence or not of S. downii sp. nov. for up to 120 h. For the potential antibacterial activity, 24 h-biofilms were exposed to S. downii sp. nov for 24 and 48 h. Biofilms structures and bacterial viability were studied by microscopy, and the effect in bacterial load by quantitative polymerase chain reaction. A generalized linear model was constructed, and results were considered as statistically significant at p < 0.05. The presence of S. downii sp. nov. during biofilm development did not affect the structure of the community, but an anti-biofilm effect against S. mutans was observed (p < 0.001, after 96 and 120 h). For antibacterial activity, after 24 h of exposure to S. downii sp. nov., counts of S. mutans (p = 0.019) and A. actinomycetemcomitans (p = 0.020) were significantly reduced in well-structured biofilms. Although moderate, anti-biofilm and antibacterial activities of S. downii sp. nov. against oral bacteria, including some periodontal pathogens, were demonstrated in an in vitro biofilm model.
Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota
(Willey, 2011-01-25) Sánchez Beltrán, María Del Carmen; Llama Palacios, María Arantxazu; Blanc, Vanesa; León, Rubén; Herrera González, David; Sanz Alonso, Mariano
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.
Detection of specific periodontal microorganisms from bacteraemia samples after periodontal therapy using molecular-based diagnostics
(Wiley, 2011-03-11) Castillo, Diana Marcela; Sánchez Beltrán, María Del Carmen; Castellanos, Jaime Eduardo; Sanz Sánchez, Ignacio; Mayorga-Fayad, Isabel; Sanz Alonso, Mariano; Lafaurie, Gloria Inés
Aim: The aim of this study was to assess the presence of subgingival pathogens in peripheral blood samples from periodontitis patients before and after scaling and root planing (Sc/RP) using nested polymerase chain reaction (nested PCR). Materials and methods: Peripheral blood samples were obtained from 42 patients with severe generalized chronic or aggressive periodontitis. In each patient, four samples of peripheral blood were drawn at different times: immediately before the Sc/RP procedure; immediately after Sc/RP; 15 and 30 min. post-Sc/RP. Blood samples were analysed for bacteraemia with anaerobic culturing and nested PCR, using universal bacterial primers that target the 16S-rRNA gene of most bacteria, subsequently re-amplified with specific primers to Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Eikenella corrodens, Campylobacter rectus and Prevotella intermedia, using a modified phenol-chloroform method for DNA extraction. Results: Presence of specific periodontal pathogens in peripheral blood after treatment was detected in 54.8% of the patients, in 47.6% with anaerobic culturing and in 19% with nested PCR. In 16.6%, the periodontal pathogens were detected before Sc/RP. P. gingivalis and A. actynomicetemcomitans were the pathogens most frequently detected in the bloodstream before and after Sc/RP. Conclusions: Nested PCR demonstrated the presence of DNA from periodontal pathogens in blood samples in severe periodontitis patients before, during and after periodontal therapy. The use of these molecular-based techniques may improve the accuracy from the results obtained by haemoculture.
Probiotic effects of orally administered Lactobacillus reuteri-containing tablets on the subgingival and salivary microbiota in patients with gingivitis. A randomized clinical trial
(Willey, 2012-05-18) Iniesta Albentosa, Margarita Isabel; Herrera González, David; Montero Solís, Eduardo; Zurbriggen, Milena; Matos, Ana R; Marín Cuenda, María José; Sánchez Beltrán, María Del Carmen; Llama Palacios, María Arantxazu; Sanz Alonso, Mariano
Objective: To investigate the effects of an orally administered probiotic on the oral microbiota. Methods: A placebo-controlled, parallel study was conducted in 40 gingivitis subjects during 8 weeks. Treatment consisted on the administration of a daily tablet, either containing Lactobacillus reuteri or placebo. Unstimulated saliva and subgingival samples were collected and analysed by culture and PCR. Clinical and microbiological outcome variables were compared between and within groups. Results: There were no significant changes between and within the groups in the clinical variables. In saliva, total anaerobic counts after 4 weeks (p = 0.021) and counts of Prevotella intermedia after 8 weeks (p = 0.030), showed reductions in the test group. In subgingival samples, significant reductions in the changes baseline to 4 weeks were observed for P. gingivalis counts (p = 0.008). With PCR, L. reuteri ATCC-PTA-5289 was more frequently detected than L. reuteri DSM-17938. Conclusions: The effect of L. reuteri administered in tablets resulted in a reduction in the number of selected periodontal pathogens in the subgingival microbiota, without an associated clinical impact.
Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states
(Public Library of Science, 2017-04-03) Romero-Lastra, Patricia; Sánchez Beltrán, María Del Carmen; Ribeiro-Vidal, Honorato; Llama Palacios, María Arantxazu; Figuero Ruiz, Elena; Herrera González, David; Sanz Alonso, Mariano
Background 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.
Analysis of viable vs. dead Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis using selective quantitative real-time PCR with propidium monoazide
(Willey, 2012-09-07) Sánchez Beltrán, María Del Carmen; Marín Cuenda, María José; Figuero Ruiz, Elena; Llama Palacios, María Arantxazu; Herrera González, David; Sanz Alonso, Mariano
Background 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.
An in vitro biofilm model associated to dental implants: Structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces
(Elsevier, 2014-10) Sánchez Beltrán, María Del Carmen; Llama-Palacios, Arancha; Fernández, Eva; Figuero Ruiz, Elena; Marín Cuenda, María José; León, Rubén; Blanc, Vanesa; Herrera González, David; Sanz Alonso, Mariano
Objectives: The impact of implant surfaces in dental biofilm development is presently unknown. The aim of this investigation was to assess in vitro the development of a complex biofilm model on titanium and zirconium implant surfaces, and to compare it with the same biofilm formed on hydroxyapatite surface. Methods: Six standard reference strains were used to develop an in vitro biofilm over sterile titanium, zirconium and hydroxyapatite discs, coated with saliva within the wells of pre-sterilized polystyrene tissue culture plates. The selected species used represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The developed biofilms (growth time 1 to 120h) were studied with confocal laser scanning microscopy using a vital fluorescence technique and with low-temperature scanning electron microscopy. The number (colony forming units/biofilm) and kinetics of the bacteria within the biofilm were studied with quantitative PCR (qPCR). As outcome variables, the biofilm thickness, the percentage of cell vitality and the number of bacteria were compared using the analysis of variance. Results: The bacteria adhered and matured within the biofilm over the three surfaces with similar dynamics. Different surfaces, however, demonstrated differences both in the thickness, deposition of the extracellular polysaccharide matrix as well as in the organization of the bacterial cells. Significance: While the formation and dynamics of an in vitro biofilm model was similar irrespective of the surface of inoculation (hydroxyapatite, titanium or zirconium), there were significant differences in regards to the biofilm thickness and three-dimensional structure.
Comparative gene expression analysis of planktonic Porphyromonas gingivalis ATCC 33277 in the presence of a growing biofilm versus planktonic cells
(BMC: BioMed Central, 2019-05-12) Sánchez Beltrán, María Del Carmen; Romero-Lastra, Patricia; Ribeiro-Vidal, Honorato; Llama-Palacios, Arancha; Figuero Ruiz, Elena; Herrera González, David; Sanz Alonso, Mariano
Background: Porphyromonas gingivalis, a microorganism residing in the oral cavity within complex multispecies biofilms, is one of the keystone pathogens in the onset and progression of periodontitis. In this in vitro study, using DNA microarray, we investigate the differential gene expression of Porphyromonas gingivalis ATCC 33277 when growing in the presence or in absence of its own monospecies biofilm. Results: Approximately 1.5% of genes (28 out of 1909 genes, at 1.5 fold change or more, p-value < 0.05) were differentially expressed by P. gingivalis cells when in the presence of a biofilm. These genes were predominantly related to the metabolism of iron, bacterial adhesion, invasion, virulence and quorum-sensing system. The results from microarrays were consistent with those obtained by RT-qPCR. Conclusion: This study provides insight on the transcriptional changes of planktonic P. gingivalis cells when growing in the presence of a biofilm. The resulting phenotypes provide information on changes occurring in the gene expression of this pathogen.
Antibacterial effects of polymeric PolymP-n Active nanoparticles. An in vitro biofilm study
(Elsevier, 2019-01) Sánchez Beltrán, María Del Carmen; Toledano-Osorio, Manuel; Bueno, Jaime; Figuero Ruiz, Elena; Toledano, Manuel; Medina-Castillo, A.L.; Osorio, Raquel; Herrera González, David; Sanz Alonso, Mariano
Objective: to study the antibacterial effect of polymeric PolymP-n Active nanoparticles using an in vitro subgingival biofilm model. Methods: Hydroxyapatite discs coated with five modalities of nanoparticles (NPs): NPs, NPs doped with zinc, calcium, silver and doxycycline, PBS as control, and Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied in a static in vitro biofilm model (12, 24, 48, and 72h). Nano-roughness of the different disc surfaces (SRa, in nm) and morphological characteristic of the biofilms (thickness (μm) and bacterial viability) were studied by different microscopy modalities. Quantitative Polymerase Chain Reaction was used to assess the effect of the nanoparticles on the bacterial load (colony forming unit per milliliter) (CFUmL-1). Analysis of variance and post-hoc testing with T3 Dunnett́s, and Student Newman Keuls correction was used. Results were considered statistically significant at p<0.05. Results: Surfaces containing the different nanoparticles showed significant increments in roughness when compared to controls (p<0.05). A similar biofilm formation and dynamics was observed, although reductions in bacterial viability were detected in biofilms in contact with the different nanoparticles, more pronounced with silver and doxycycline NPs. Doxycycline-NPs biofilms resulted in unstructured biofilm formation and significantly lower number of the six species when compared with the other nanoparticles specimens and controls (p<0.001 in all cases). Significance: Polymeric PolymP-n Active nanoparticles when combined with silver and doxycycline showed a significant antibacterial effect when tested in an in vitro subgingival biofilm model.