Rubén León

Bio: Rubén León is an academic researcher from University of Chile. The author has contributed to research in topics: Periodontitis & Porphyromonas gingivalis. The author has an hindex of 21, co-authored 51 publications receiving 1871 citations. Previous affiliations of Rubén León include Complutense University of Madrid.

Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide

Abstract: Real-time PCR (qPCR) is a widely used technique in analysing environmental and clinical microbiological samples. However, its main limitation was its inability to discriminate between live and dead cells. Recently, propidium monoazide (PMA) together with qPCR has been used to overcome this problem, with good results for different bacterial species in different types of samples. Our objective was to implement this technique for analysing mortality in multi-species oral biofilms formed in vitro with five oral bacteria: Streptococcus oralis, Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum and Prevotella intermedia. We also tested its effectiveness on biofilms treated with an antiseptic solution containing 0.07% w/w cetylpyridinium chloride (CPC). Standardisation of the qPCR-PMA method was performed on pure, heat-killed planktonic cultures of each species, detecting mortality higher than 4 log in S. oralis, S. gordonii and F. nucleatum and higher than 2 for V. parvula and P. intermedia. We obtained similar results for all species when using CPC. When we analysed biofilms with qPCR-PMA, we found that the mortality in the non-CPC treated multi-species biofilms was lower than 1 log for all species. After treatment with CPC, the viability reduction was higher than 4 log in S. oralis and S. gordonii, higher than 3 log in F. nucleatum and P. intermedia and approximately 2 in V. parvula. In short, we standardised the conditions for using qPCR-PMA in 5 oral bacterial species and proved its usefulness for quantification of live and dead cells in multi-species oral biofilms formed in vitro, after use of an antiseptic.

Antimicrobial therapy in periodontitis: the use of systemic antimicrobials against the subgingival biofilm

Abstract: Objectives: The aim was to answer three relevant questions: can systemic antimicrobials be efficacious if the biofilm is not disrupted? Can the type of debridement of the subgingival biofilm impact upon the clinical outcomes of the adjunctive antimicrobial therapy? Is the efficacy of the adjunctive systemic antimicrobial therapy dependent on the quality of the debridement of the subgingival biofilm and the sequence debridement-antibiotic usage? Material and Methods: Relevant papers were searched, critically analysed and their data were extracted. Results: For the first question, studies assessing susceptibility of bacteria in biofilms, and clinical studies evaluating systemic antimicrobials as monotherapy, were reviewed. For the second question, clinical studies comparing systemic antimicrobials as adjuncts to non-surgical debridement or to periodontal surgery and clinical trials using systemic antibiotics with periodontal surgery were evaluated. For the third question, a previous systematic review was updated. Conclusion: If systemic antimicrobials are indicated in periodontal therapy, they should be adjunctive to mechanical debridement. There is not enough evidence to support their use with periodontal surgery. Indirect evidence suggests that antibiotic intake should start on the day of debridement completion, debridement should be completed within a short time (preferably < 1 week) and with an adequate quality, to optimize the results.

Detection of Porphyromonas gingivalis in the Amniotic Fluid in Pregnant Women With a Diagnosis of Threatened Premature Labor

Abstract: Background: 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.Methods: 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. Amnioti...

Prevalence of periodontopathic bacteria in aggressive periodontitis patients in a Chilean population.

Abstract: 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.

Subgingival microbial profiles in chronic periodontitis patients from Chile, Colombia and Spain

Abstract: 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.

Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action

Abstract: Biofilm refers to the complex, sessile communities of microbes found either attached to a surface or buried firmly in an extracellular matrix as aggregates. The biofilm matrix surrounding bacteria makes them tolerant to harsh conditions and resistant to antibacterial treatments. Moreover, the biofilms are responsible for causing a broad range of chronic diseases and due to the emergence of antibiotic resistance in bacteria it has really become difficult to treat them with efficacy. Furthermore, the antibiotics available till date are ineffective for treating these biofilm related infections due to their higher values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), which may result in in-vivo toxicity. Hence, it is critically important to design or screen anti-biofilm molecules that can effectively minimize and eradicate biofilm related infections. In the present article, we have highlighted the mechanism of biofilm formation with reference to different models and various methods used for biofilm detection. A major focus has been put on various anti-biofilm molecules discovered or tested till date which may include herbal active compounds, chelating agents, peptide antibiotics, lantibiotics and synthetic chemical compounds along with their structures, mechanism of action and their respective MICs, MBCs, minimum biofilm inhibitory concentrations (MBICs) as well as the half maximal inhibitory concentration (IC50) values available in the literature so far. Different mode of action of anti biofilm molecules addressed here are inhibition via interference in the quorum sensing pathways, adhesion mechanism, disruption of extracellular DNA, protein, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways. From this study, we conclude that the molecules considered here might be used to treat biofilm-associated infections after significant structural modifications, thereby investigating its effective delivery in the host. It should also be ensured that minimum effective concentration of these molecules must be capable of eradicating biofilm infections with maximum potency without posing any adverse side effects on the host.

Microbial Prevalence, Diversity and Abundance in Amniotic Fluid During Preterm Labor: A Molecular and Culture-Based Investigation

Abstract: Background Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking. Methods and Findings In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r2 = 0.42; P<0.002). Conclusions The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.

Critical review on biofilm methods

Abstract: Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Inflammation and Bone Loss in Periodontal Disease

Abstract: Inflammation and bone loss are hallmarks of periodontal disease (PD). The question is how the former leads to the latter. Accumulated evidence demonstrates that PD involves bacterially derived factors and antigens that stimulate a local inflammatory reaction and activation of the innate immune system. Proinflammatory molecules and cytokine networks play essential roles in this process. Interleukin-1 and tumor necrosis factor-alpha seem to be primary molecules that, in turn, influence cells in the lesion. Antigen-stimulated lymphocytes (B and T cells) also seem to be important. Eventually, a cascade of events leads to osteoclastogenesis and subsequent bone loss via the receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) axis. This axis and its regulation are not unique to PD but rather are critical for pathologic lesions involving chronic inflammation. Multiple lines of evidence in models of PD clearly indicate that increases in RANKL mRNA expression and protein production increase the RANKL/OPG ratio and stimulate the differentiation of macrophage precursor cells into osteoclasts. They also stimulate the maturation and survival of the osteoclast, leading to bone loss. OPG mRNA expression and protein production do not generally seem to be increased in the periodontitis lesion. Studies of RANKL and OPG transgenic and knockout animals provide further support for the involvement of these molecules in the tissue loss observed in PD. Ironically, periodontal practitioners have focused on the bacterial etiology of PD and believed that plaque removal was aimed at eliminating specific bacteria or bacterial complexes. However, it seems that the reduction of inflammation and attenuation of the host's immune reaction to the microbial plaque, eventually leading to a decrease in the ratio of RANKL/OPG and a decrease in associated bone loss, are the actual and desired outcomes of periodontal therapy. Future therapeutic options are likely to have regulation of the RANK-RANKL-OPG axis as their goal.