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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

summary Noon stomatal conductance of lethal-yellowing (LY) affected coconut palms (Cocos nucifera L. Atlantic Tall ecotype) decreased progressively as the disease developed. In the early stages of the disease and before leaf yellowing started, stomatal conductance decreased to a minimum in leaves at both the top, the middle and the base of the crown. Since altered stomatal behaviour might affect gas exchange and related processes in the plant, such as water movement and photosynthesis, these results support the hypothesis that LY-induced stomatal closure is central to the development of LY symptoms in coconut palms, and therefore to the mode of pathogenicity of the LY mycoplasma-like organism. Leaf yellowing occurred simultaneously with a decrease in photosynthetic rates and a decrease in protein, chlorophyll and carotenoid contents. Based on these biochemical changes a second hypothesis is proposed, that LY-associated leaf yellowing is part of a leaf senescence process. The concentration of abscisic acid in the leaves and the capacity of leaf tissue to form ethylene increased in diseased palms, suggesting that a hormonal imbalance occurs in LY-affected palms. Furthermore, treatment of symptomless palms with an ethylene-releasing agent (ethephon) resulted in symptoms that mimicked some of the LY symptoms. A third hypothesis is therefore proposed, that a hormonal imbalance might be related to LY symptom development, at least with respect to nutfall and leaf senescence.

7 páginas, 5 figuras, 1 tabla -- PAGS nros. 823-829, Aim: Capsular polysaccharides play an important role in the virulence of Gram-positive and Gram-negative bacteria. In Porphyromonas gingivalis, six serotypes have been described based on capsular antigenicity and its pathogenicity has been correlated both in vitro and in animal models. This study aimed to investigate the differential response of human dendritic cells (DCs) when stimulated with different P. gingivalis capsular serotypes. Materials and Methods: Using different multiplicity of infection (MOI) of the encapsulated strains K1–K6 and the non-encapsulated K− strain of P. gingivalis, the mRNA expression levels for interleukin (IL)-1β, IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and TNF-β in stimulated DCs were quantified by real-time reverse transcription-polymerase chain reaction. Results: All P. gingivalis capsular serotypes induced a T-helper type 1 (Th1) pattern of cytokine expression. K1- and K2-stimulated DCs expressed higher levels of IL-1β, IL-6, IL-12p35, IL-12p40, and IFN-γ and at lower MOI than DCs stimulated with the other strains. Conclusions: These results demonstrate a differential potential of P. gingivalis capsular serotypes to induce DC responses and a higher capacity of strains K1 W83 and K2 HG184 than other K serotypes to trigger cytokine expression

9 páginas, 4 figuras, 1 tabla -- PAGS nros. 689-697, Background and Objective: Dendritic cells are able to prime and polarize naïve T cells towards either a T helper 1 or a T helper 2 response, depending on the antigen type and concentration, the costimulatory signals and the local cytokine millieu. In this investigation, we analyzed the response of human dendritic cells to stimulation with different concentrations of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans. Material and Methods: Using different concentrations of P. gingivalis ATCC 33277 and A. actinomycetemcomitans ATCC 33384, we determined the expression of the maturation markers CD80 and CD86 from purified human dendritic cells by flow cytometry. We also evaluated the mRNA expression levels for the cytokines interleukin-1β, interleukin-2, interleukin-5, interleukin-6, interleukin-10, interleukin-12, interleukin-13, interferon-γ, tumor necrosis factor-α and tumor necrosis factor-β by quantitative real-time reverse transcription–polymerase chain reaction. Results: Both P. gingivalis and A. actinomycetemcomitans led to dendritic cell maturation, but the expression of CD80 was higher when the dendritic cells were stimulated with A. actinomycetemcomitans. Although both pathogens induced a T helper 1 pattern of cytokine expression, A. actinomycetemcomitans-stimulated dendritic cells expressed interleukin-1β, interleukin-12, interferon-γ, tumor necrosis factor-α and tumor necrosis factor-β at lower bacterial concentrations than P. gingivalis. While 106 bacteria/mL of P. gingivalis or 104 bacteria/mL of A. actinomycetemcomitans induced expression of interleukin-12p40, the expression of other cytokines required 10 to 100-fold higher concentrations of bacteria. Conclusion: These results demonstrate that A. actinomycetemcomitans is a more potent immunogen than P. gingivalis because, at least in vitro, it induces stronger differentiation and activation of dendritic cells. In addition, our data also show that for a given strain, the bacterial load determines the pattern of cytokines that are expressed, The work in the author’s laboratories has been supported by grant from the Spanish Ministry of Health (FIS-060181 to MS), Spanish Ministry of Science and Education (SAF2006-4826 and SAF2006-04903 to AS and JAGS, respectively) and Chilean Government (CONICYT-26080046 to RV)

ResumenPropósitoDeterminar la presencia de Aggregatibacter actinomycetemcomitans (Aa) en sujetos con diagnóstico de periodontitis agresiva (PAg) y en los individuos pertenecientes a su núcleo familiar.MétodoSe tomaron muestras microbiológicas subgingivales a 30 pacientes con P. Agresiva, 21 tenían periodontitis agresiva generalizada (PAgG) y 9 con periodontitis agresiva localizada (PAgL), para determinar la presencia de Aa. Las muestras fueron cultivadas en medios selectivos (Dentaid - 1) y Aa fue identificado por la morfología de sus colonias y por test de la catalasa. A los familiares más cercanos (30 familiares) de los probandos, también se les realizaron test microbiológicos.ResultadosEl 70% de los sujetos diagnosticados con PAg presentaban Aa. El promedio de edad de los sujetos positivos para el microorganismo fue de 30,9+7,73 años, de ellos el 33,3% presentaban PAgL. En 14 de los 21 probandos positivos para Aa se determinó la presencia de Aa, en al menos un miembro del grupo familiar. Se examinaron 30 individuos pertenecientes al núcleo familiar y el 43,3% eran positivos para Aa.ConclusionesUn poco más de 2/3 de los sujetos diagnosticados con PAg eran portadores de Aa y menos de la mitad de los familiares de los probandos examinados eran positivos para este microorganismo.AbstractAimDetermine the presence of Aggregatibacter actinomycetemcomitans (Aa) in subjects with aggressive periodontitis (AgP) and individuals as part of their family group.MethodSubgingival plaque samples were collected from 30 aggressive, 9 localized and 21 generalized periodontitis patients, to determinate the presence of Aa. The samples were cultured on selective media (Dentaid - 1) and Aa was identified by its colony morphology and catalase test. The closest relatives (30 families) of probandos, were also conducted microbiological tests.Results70% of subjects were diagnosed with AgP harbored Aa. The average age of subjects positive for the pathogen was 30.9 +7.73 years, of which 33.3% had localized aggressive periodontitis. In 14 of 21 probandos found positive for Aa of at least one member of the family group. We examined 30 individuals belonging to the family and 43.3% were positive for Aa.ConclusionsMore than half of the subjects with AgP harbored Aa and less than half of the families of probandos examined were positive for this pathogen.

ResumenPorphyromonas gingivalis es un microorganismo fuertemente asociado con la etiología de la periodontitis. Esta bacteria posee varios factores de virulencia, dentro de los que destacan las gingipaínas, debido a sus múltiples acciones relacionadas con la destrucción de la matriz extracelular del tejido conectivo periodontal, la modulación del sistema inmune del hospedero y la estimulación de la expresión de citoquinas pro-inflamatorias. Estas proteinasas tienen afinidades específicas siendo Arg-gingipaínas (RgpA y RgpB, codificadas por los genes rgpA y rgpB, respectivamente) y Lys-gingipaínas (Kgp, codificada por el gen kgp). Se ha descrito que existen polimorfismos en los genes que codifican para esta proteinasas. El objetivo del presente estudio fue describir la frecuencia de los genotipos identificados para los genes rgpA y kgp en aislados clínicos de P. gingivalis, obtenidos desde pacientes con periodontitis. Para ello se utilizó amplificación por PCR de los genes rgpA y kgp, seguido de análisis de restricción. De un total de 47 aislados provenientes de 4 individuos con periodontitis crónica y 2 con periodontitis agresiva, se genotipificaron 38 aislados para el gen rgpA, exhibiendo la totalidad de éstos el patrón electroforético A (100%). Para el gen kgp se genotipificaron 43 aislados, presentando 28 de ellos (65.2%) el perfil electroforético kgp-I y 15 aislados (34.8%) el perfil kgp-II. En los aislados provenientes de un individuo fue posible apreciar ambos genotipos descritos para el gen kgp. Los resultados indican un predominio del patrón electroforético A (rgpA) y que el genotipo kgp-I fue el más frecuentemente encontrado de los genotipos kgp.AbstractPorphyromonas gingivalis is a microorganism strongly associated with the etiology of periodontitis. This periodontal bacterium possesses an array of virulence factors, among which gingipains have a key importance, being involved with extracellular matrix destruction of periodontal tissues, modulation of host immune response and stimulation in the production of pro-inflammatory cytokines by different types of cells. These proteinases have specific affinities, being Arg-gingipains (RgpA and RgpB, encoded by rgpA and rgpB genes, respectively) and Lys-gingipains (Kgp, encoded by the kgp gene). It has been described that there are polymorphisms in the genes encoding for gingipains. Therefore, the aim of the present study was to describe the frequency of rgpA and kgp genotypes in clinical isolates of P. gingivalis obtained from periodontitis patients. For determining the rgpA and kgp genotypes, we used PCR amplification and restriction analysis. From 47 isolates obtained from 4 individuals with chronic periodontitis and 2 subjects with aggressive periodontitis, 38 were typified for rgpA gene and all exhibited the electrophoretic pattern A (100%). For kgp gene, we characterized 43 isolates, 28 of them (65.2%) with the kgp-I electrophoretic profile and 15 isolates (34.8%) with the kgp-II profile. In the isolates belonging to one individual, we found both genotypes of kgp gene. The results indicate a clear predominance of the electrophoretic pattern A (for rgpA gene) and kgp-I genotype was the most frequently found of the kgp genotypes.