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Streptococcus mitis is found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study, in silico analyses indicated the presence of an Rgg/SHP cell-to-cell communication system in S. mitis. Although Rgg presented higher similarity to a repressor in Streptococcus pyogenes, auto-inducing assays and an rgg deletion mutant revealed that in S. mitis Rgg acts as an activator. Transcriptome analysis showed that in addition to shp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster in Streptococcus pneumoniae indicated that S. mitis lacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of auto induction, growth was not significantly affected for the rgg mutant under normal or antibiotic stress conditions. The S. mitis SHP was, however, fully functional in promoting cross-species communication and increasing S. pneumoniae surface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in co-cultures using a S. mitis reporter strain. In competitive assays, a slight advantage was observed for the rgg mutants. We conclude that the Rgg/SHP system in S. mitis regulates the expression of its own shp, and activates an Rgg/SHP system in S. pneumoniae that regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multi-species interactions. [ABSTRACT FROM AUTHOR]

Background: Dentists, as prominent prescribers, are key stakeholders in addressing the antimicrobial resistance (AMR) crisis. Dental students' perceptions about the topic have been underexplored in the Asia-Pacific region, a key location for the development and spread of AMR. Thus, the aim of this study was to evaluate the awareness and confidence to prescribe antimicrobials amongst dental students studying in the region., Methods: Students from 15 dental schools in 4 countries were invited to participate in a cross-sectional online survey during 2022-2023. A previously validated and standardised 14-item instrument was utilised., Results: In all, 1413 responses were collected from Australia (n = 165), Sri Lanka (n = 112), Japan (n = 173), and Vietnam (n = 963). Of those, 201 were from final-year students (14.2%). On a scale from 1 to 10, awareness on AMR was placed at a mean (SEM) priority of 8.09 (0.05). With regards to target areas to address for mitigation of the AMR crisis, participants placed general public awareness at the top (mean [SEM] 8.53 [0.05]). Final-year students presented a mean (SEM) level of confidence to prescribe antibiotics of 6.01 (0.14) on a scale from 1 to 10, whilst 59.7% and 56.8% indicated feeling pressured to prescribe by patients or when lacking time, respectively. Final-year students participating in research activities assigned a higher priority to AMR compared to their peers not involved in research (mean [SEM] 8.6 [0.19] vs 7.81 [0.16]; P = .01)., Conclusions: This study highlights a need for increased awareness and confidence to prescribe amongst dental students in the Asia-Pacific region, an understudied population thus far. To mitigate this issue, the implementation (followed by assessment) of local educational and antibiotic stewardship initiatives is warranted., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Challenges from infections caused by biofilms and antimicrobial resistance highlight the need for novel antimicrobials that work in conjunction with antibiotics and minimize resistance risk. In this study we investigated the composite effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells), a human milk protein-lipid complex and amoxicillin on microbial ecology using an ex vivo oral biofilm model with pooled saliva samples. HAMLET was chosen due to its multi-targeted antimicrobial mechanism, together with its synergistic effect with antibiotics on single species pathogens, and low risk of resistance development. The combination of HAMLET and low concentrations of amoxicillin significantly reduced biofilm viability, while each of them alone had little or no impact. Using a whole metagenomics approach, we found that the combination promoted a remarkable shift in overall microbial composition compared to the untreated samples. A large proportion of the bacterial species in the combined treatment were Lactobacillus crispatus , a species with probiotic effects, whereas it was only detected in a minor fraction in untreated samples. Although resistome analysis indicated no major shifts in alpha-diversity, the results showed the presence of TEM beta-lactamase genes in low proportions in all treated samples but absence in untreated samples. Our study illustrates HAMLET's capability to alter the effects of amoxicillin on the oral microbiome and potentially favor the growth of selected probiotic bacteria when in combination. The findings extend previous knowledge on the combined effects of HAMLET and antibiotics against target pathogens to include potential modulatory effects on polymicrobial biofilms of human origin., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Brar, Dhariwal, Shekhar, Junges, Hakansson and Petersen.)

Background: Torquetenovirus (TTV) is a small DNA virus constituting the human virome. High levels of TTV-DNA have been shown to be associated with immunosuppression and inflammatory chronic disorders., Aim: To assess the possible association between the salivary viral load of TTV-DNA in patients hospitalised due to COVID-19 and disease severity., Methods: Saliva samples collected from 176 patients infected with SARS-CoV-2 were used to investigate the presence of SARS-CoV-2 and TTV-DNA by use of real-time RT-PCR., Results: The majority of patients were male with severe COVID-19. Presence of SARS-CoV-2 was observed in the saliva of 64.77% of patients, showing TTV-DNA in 55.68% of them. Patients with impaired clinical conditions ( p < 0.001), which evolved to death ( p = 0.003), showed a higher prevalence of TTV-DNA. The median viral load in patients with severe condition was 4.99 log 10 copies/mL, in which those who were discharged and those evolving to death had values of 3.96 log 10 copies/mL and 6.27 log 10 copies/mL, respectively. A statistically significant association was found between the distribution of TTV-DNA viral load in saliva samples and severity of COVID-19 ( p = 0.004) and disease outcomes ( p < 0.001)., Conclusions: These results indicate that TTV-DNA in saliva could be a useful biomarker of COVID-19 severity and prognosis.

Structural health monitoring (SHM) has become paramount for developing cheaper and more reliable maintenance policies. The advantages coming from adopting such process have turned out to be particularly evident when dealing with plated structures. In this context, state-of-the-art methods are based on exciting and acquiring ultrasonic-guided waves through a permanently installed sensor network. A baseline is registered when the structure is healthy, and newly acquired signals are compared to it to detect, localize, and quantify damage. To this purpose, the performance of traditional methods has been overcome by data-driven approaches, which allow processing a larger amount of data without losing diagnostic information. However, to date, no diagnostic method can deal with varying environmental and operational conditions (EOCs). This work aims to present a proof-of-concept that state-of-the-art machine learning methods can be used for reducing the impact of EOCs on the performance of damage diagnosis methods. Generative artificial intelligence was leveraged to mitigate the impact of temperature variations on ultrasonic guided wave-based SHM. Specifically, variational autoencoders and singular value decomposition were combined to learn the influence of temperature on guided waves. After training, the generative part of the algorithm was used to reconstruct signals at new unseen temperatures. Moreover, a refined version of the algorithm called forced variational autoencoder was introduced to further improve the reconstruction capabilities. The accuracy of the proposed framework was demonstrated against real measurements on a composite plate.

Antifungal agents are widely used to specifically eliminate infections by fungal pathogens. However, the specificity of antifungal agents has been challenged by a few studies demonstrating antibacterial inhibitory effects against Mycobacteria and Streptomyces species. Here, we evaluated for the first time the potential effect of fluconazole, the most clinically used antifungal agent, on a human oral microbiota biofilm model. The results showed that biofilm viability on blood and mitis salivarius agar media was increased over time in the presence of fluconazole at clinically relevant concentrations, despite a reduction in biomass. Targeted PCR revealed a higher abundance of Veillonella atypica , Veillonella dispar, and Lactobacillus spp. in the fluconazole-treated samples compared to the control, while Fusobacterium nucleatum was reduced and Streptococcus spp were not significantly affected. Further, we tested the potential impact of fluconazole using single-species models. Our results, using Streptococcus mutans and Streptococcus mitis luciferase reporters, showed that S. mutans planktonic growth was not significantly affected by fluconazole, whereas for S. mitis , planktonic growth, but not biofilm viability, was inhibited at the highest concentration. Fluconazole's effects on S. mitis biofilm biomass were concentration and time dependent. Exposure for 48 h to the highest concentration of fluconazole was associated with S. mitis biofilms with the most increased biomass. Potential growth inhibitory effects were further tested using four non-streptococcal species. Among these, the planktonic growth of both Escherichia coli and Granulicatella adiacens was inhibited by fluconazole. The data indicate bacterial responses to fluconazole that extend to a broader range of bacterial species than previously anticipated from the literature, with the potential to disturb biofilm communities.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The collateral impact of antibiotics on the microbiome has attained increasing attention. However, the ecological consequences of long-term antibiotic exposure on the gut microbiome, including antibiotic resistance, are still limited. Here, we investigated long-term exposure effects to amoxicillin on the human gut microbiome and resistome. Fecal samples were collected from 20 patients receiving 3-months of amoxicillin or placebo treatment as part of a Norwegian multicenter clinical trial on chronic low back pain (AIM study). Samples were collected at baseline, last day of treatment, and 9 months after antibiotic cessation. The abundance and diversity of microbial and resistome composition were characterized using whole shotgun and functional metagenomic sequencing data. While the microbiome profiles of placebo subjects were stable over time, discernible changes in diversity and overall microbiome composition were observed after amoxicillin treatment. In particular, health-associated short-chain fatty acid producing species significantly decreased in proportion. However, these changes were short-lived as the microbiome showed overall recovery 9 months post-treatment. On the other hand, exposure to long-term amoxicillin was associated with an increase in total antimicrobial resistance gene load and diversity of antimicrobial resistance genes, with persistent changes even at 9 months post-treatment. Additionally, beta-lactam resistance was the most affected antibiotic class, suggesting a targeted response to amoxicillin, although changes at the gene level varied across individuals. Overall, our results suggest that the impact of prolonged amoxicillin exposure was more explicit and long-lasting in the fecal resistome than in microbiome composition. Such information is relevant for designing rational administration guidelines for antibiotic therapies.

Introduction: Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization., Methods: Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples., Results: The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol&#95;MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol&#95;MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness., Discussion: Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rajar, Dhariwal, Salvadori, Junges, Åmdal, Berild, Fugelseth, Saugstad, Lausten-Thomsen, Greisen, Haaland and Petersen.)

Background: The antimicrobial resistance (AMR) crisis is a major global threat and one of its biggest drivers is the overuse of antibiotics in humans. Dentists are responsible for 5-10% antibiotic prescriptions worldwide and recent data suggest that knowledge and prescribing practices need improvement., Methods: A cross-sectional web-survey was sent to dental students from six universities in Norway, Canada, and Brazil. Topics addressed covered awareness, confidence to prescribe antibiotics, and education needs. Data were presented descriptively and statistical testing was employed to compare group means when applicable., Results: In total, 562 responses were collected across the three countries with a response rate of 28.6%. 'Antibiotic resistance' was among the highest priorities (scale 1-10) with an average of 8.86 (SEM ± 0.05), together with 'Gender inequality' (8.68 ± 0.07) and 'Climate change' (8.68 ± 0.07). Only 28.8% thought that Dentistry was engaged in national/international campaigns promoting awareness on the topic and 8.9% stated to have heard about the 'One Health' concept. Final year dental students showed an average confidence to prescribe antibiotics of 7.59 (± 0.14). Most students demonstrated interest in receiving additional education on all topics listed, with the three most pressing being 'antibiotic prescription for treatment of infections' (82.9%), 'drug interactions' (80.9%), and 'spread of antibiotic resistance' (79.6%). A trend was observed between higher awareness regarding the topic and higher confidence to prescribe., Conclusions: There is a need to revisit dental education on antibiotic resistance with a global perspective and to create more stewardship initiatives that promote awareness on the topic., (© 2022. The Author(s).)

Background: COVID-19 is a disease affecting various human organs and systems, in which the virus seeks to interact with angiotensin-converting enzyme 2 receptors. These receptors are present in the oral cavity, but the direct relationship between such an interaction and possible oral manifestations of COVID-19 is still unclear., Aim: The present study evaluated oral manifestations in a cohort of COVID-19 patients during the period of hospitalisation., Methods: In total, 154 patients presenting moderate-to-severe forms of COVID-19 had their oral mucosa examined twice a week until the final outcome, either discharge or death. The oral alterations observed in the patients were grouped into Group 1 (pre-existing conditions and opportunistic oral lesions) and Group 2 (oral mucosal changes related to hospitalization)., Results: Oral lesions found in the patients of Group 1 are not suggestive of SARS-CoV-2 infection as they are mainly caused by opportunistic infections. On the other hand, oral alterations found in the patients of Group 2 were statistically ( P < 0.001) related to intubation and longer period of hospitalisation., Conclusion: It is unlikely that ulcerative lesions in the oral cavity are a direct manifestation of SARS-CoV-2 or a marker of COVID-19 progression., Competing Interests: No potential conflict of interest was reported by the authors., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The study of resistomes using whole metagenomic sequencing enables high-throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules-the 'Antimicrobial Resistance Gene Table' module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the 'Integration' module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the 'Antimicrobial Resistance Gene List' module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

The mitis group of streptococci comprises species that are common colonizers of the naso-oral-pharyngeal tract of humans. Streptococcus pneumoniae and Streptococcus mitis are close relatives and share ~60-80% of orthologous genes, but still present striking differences in pathogenic potential toward the human host. S. mitis has long been recognized as a reservoir of antibiotic resistance genes for S. pneumoniae , as well as a source for capsule polysaccharide variation, leading to resistance and vaccine escape. Both species share the ability to become naturally competent, and in this context, competence-associated killing mechanisms such as fratricide are thought to play an important role in interspecies gene exchange. Here, we explore the general mechanism of natural genetic transformation in the two species and touch upon the fundamental clinical and evolutionary implications of sharing similar competence, fratricide mechanisms, and a large fraction of their genomic DNA.

Objectives: The aims of this study were to explore the shape of the relationship of income and education with periodontal health, and to assess the interactions between them and race/ethnicity., Method: Individual level data from the Brazilian National Oral Health Survey in 2010 (Pesquisa Nacional de Saúde Bucal-SB Brasil 2010) were obtained for 9,779 subjects. Relations between per capita income and education with periodontal health were smoothed using Locally Weighted Scatter-plot Smoother (Lowess) technique. Multivariable logistic regression was used to assess independent effects of income, education, race/ethnicity adjusted for age, sex and time since last dental appointment., Results: Prevalence of adults with moderate to severe and severe periodontitis was 17.6 and 6.5%, respectively. The relationship between periodontal health and income was curvilinear, showing a threshold of no relationship for income levels higher than US$ 600/month. In multivariable analysis, after controlling for covariates, only income was significantly associated with periodontal health. There was no significant interaction of income with race or education, neither between race and education., Conclusion: The relation between periodontal health and income was curvilinear and indicated the presence of a threshold, supporting income transfer programs. Beyond the threshold, only education presented a negative linear relationship with moderate to severe periodontitis.

Natural transformation is regarded as an important mechanism in bacteria that allows for adaptation to different environmental stressors by ensuring genome plasticity. Since the discovery of this phenomenon in Streptococcus pneumoniae , remarkable progress has been made in the understanding of the molecular mechanisms and pathways coordinating this process. Recently, the advent of high-throughput sequencing allows the posing of questions that address the system at a larger scale but also allow for the creation of high-resolution maps of transcription. Thus, while much is already known about genetic competence in streptococci, recent studies continue to reveal intricate novel regulation pathways and components. In this perspective article, we highlight the use of transcriptional profiling and mapping as a valuable resource in the identification and characterization of "hidden gems" pertinent to the natural transformation system. Such strategies have recently been employed in a variety of different species. In S. mutans , for example, genome editing combined with the power of promoter mapping and RNA-Seq allowed for the identification of a link between the ComCDE and the ComRS systems, a ComR positive feedback loop mediated by SigX, and the XrpA peptide, encoded within sigX , which inhibits competence. In S. pneumoniae , a novel member of the competence regulon termed BriC was found to be directly under control of ComE and to promote biofilm formation and nasopharyngeal colonization but not competence. Together these new technologies enable us to discover new links and to revisit old pathways in the compelling study of natural genetic transformation.

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact., Competing Interests: The authors have declared that no competing interests exist.

Despite vaccines, Streptococcus pneumoniae kills more than a million people yearly. Thus, understanding how pneumococci transition from commensals to pathogens is particularly relevant. Quorum sensing regulates collective behaviors and thus represents a potential driver of commensal-to-pathogen transitions. Rgg/small hydrophobic peptide (SHP) quorum-sensing systems are widespread in streptococci, yet they remain largely uncharacterized in S. pneumoniae . Using directional transcriptome sequencing, we show that the S. pneumoniae D39 Rgg0939/SHP system induces the transcription of a single gene cluster including shp and capsule gene homologs. Capsule size measurements determined by fluorescein isothiocyanate-dextran exclusion allowed assignment of the system to the regulation of surface polysaccharide expression. We found that the SHP pheromone induced exopolysaccharide expression in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the Rgg system resulted in a mutant with increased capsule size. In line with previous studies showing that capsule expression is inversely associated with biofilm formation, we found that biofilm formed on lung epithelial cells was decreased in the overexpression strain and increased in an rgg deletion mutant. Although no significant differences were observed between D39 and the rgg deletion mutant in a mouse model of lung infection, in competitive assays, overexpression reduced fitness. This is the first study to reveal a quorum-sensing system in streptococci that regulates exopolysaccharide synthesis from a site distinct from the original capsule locus. IMPORTANCE Quorum sensing regulates bacterial social behaviors by production, secretion, and sensing of pheromones. In this study, we characterized a new quorum-sensing system of the Rgg/SHP class in S. pneumoniae D39. The system was found to directly induce the expression of a single gene cluster comprising the gene for the SHP pheromone and genes with putative functions in capsule synthesis. Capsule size, as measured by dextran exclusion, was increased by SHP exposure in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the gene cluster increased capsule size, supporting the role of Rgg/SHP in the synthesis of surface polysaccharides. Further, we found that biofilm formation on epithelial cells was reduced by overexpression of the system and increased in a mutant with an rgg deletion. Placing surface polysaccharide expression under quorum-sensing regulation may enable S. pneumoniae to tune interactions with the host and other bacteria in accordance with environmental and cell density conditions.

Natural transformation is used by bacteria to take up DNA from their surroundings and incorporate it into their genomes. Streptococci do so during a transient period of competence, triggered by pheromones that they produce, secrete and sense under conditions influenced by the environment. In Streptococcus mutans, Streptococcus suis, and species of the bovis, salivarius and pyogenic groups of streptococci, the pheromone XIP is sensed by the intra-cellular regulator ComR, that in turn activates the transcription of comS, encoding the XIP precursor, and of sigX, encoding the only known alternative sigma factor in streptococci. Although induction of comR during competence has been known for more than fifteen years, the mechanism regulating its expression remains unidentified. By a combination of directional RNA-sequencing, optimal competence conditions, stepwise deletions and marker-less genome editing, we found that SigX is the missing link in overproduction of ComR. In the absence of comR induction, both sigX expression and transformation were significantly reduced. Placing comR and comS transcripts under the control of different regulators so as to form two interlocked positive feedback circuits may enable S. mutans to fine-tune the kinetics and magnitude of the competence response according to their need.

The discovery that Streptococcus pneumoniae uses a competence-stimulating peptide (CSP) to induce competence for natural transformation, and that other species of the mitis and the anginosus streptococcal groups use a similar system, has expanded the tools to explore gene function and regulatory pathways in streptococci. Two other classes of pheromones have been discovered since then, comprising the bacteriocin-inducing peptide class found in Streptococcus mutans (also named CSP, although different from the former) and the SigX-inducing peptides (XIP), in the mutans, salivarius, bovis, and pyogenes groups of streptococci. The three classes of peptide pheromones can be ordered from peptide synthesis services at affordable prices, and used in transformation assays to obtain competent cultures consistently at levels usually higher than those achieved during spontaneous competence. In this chapter, we present protocols for natural transformation of oral streptococci that are based on the use of synthetic pheromones, with examples of conditions optimized for transformation of S. mutans and Streptococcus mitis.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

The aim of this study was to assess the accuracy of clinical diagnosis for lip lesions based on sensitivity and specificity. The retrospective analysis focused on the detection of lesions caused by potentially malignant disorders (PMDs) and malignant lesions (n = 1195). All cases were classified as benign, PMD, and malignant lesions. Concordance between diagnoses based on clinical examination and those based on histopathological analysis was assessed, and accuracy for the identification of PMD and malignant lesions was calculated. Histopathological analysis revealed 44 lesion types; PMD and malignant lesions comprised 8.3% of all cases. Compared with histopathological analysis, clinical examination showed 97.4% accuracy for the identification of non-malignant and potentially malignant/malignant cases. Degrees of specific sensitivity ranged from 34% to 77% for different lesions, and were highest for autoimmune (77%) and reactive (72%) lesions. Positive and negative predictive values for the identification of PMD and malignant lesions were 81.9% and 98.9%, respectively. Clinical examination showed a high degree of accuracy for the detection of PMD and malignant lip lesions, indicating good reliability.

Objective: Streptococcus mitis is a predominant oral colonizer, but difficulties in genetic manipulation of this species have hampered our understanding of the mechanisms it uses for colonization of oral surfaces. The aim of this study was to reveal optimal conditions for natural genetic transformation in S. mitis and illustrate its application in direct genome editing., Methods: Luciferase reporter assays were used to assess gene expression of the alternative sigma factor (σ(X)) in combination with natural transformation experiments to evaluate the efficiency by which S. mitis activates the competence system and incorporates exogenous DNA. Optimal amounts and sources of donor DNA (chromosomal, amplicon, or replicative plasmid), concentrations of synthetic competence-stimulating peptide, and transformation media were assessed., Results: A semi-defined medium showed much improved results for response to the competence stimulating peptide when compared to rich media. The use of a donor amplicon with large homology flanking regions also provided higher transformation rates. Overall, an increase of transformation efficiencies from 0.001% or less to over 30% was achieved with the developed protocol. We further describe the construction of a markerless mutant based on this high efficiency strategy., Conclusion: We optimized competence development in S. mitis, by use of semi-defined medium and appropriate concentrations of synthetic competence factor. Combined with the use of a large amplicon of donor DNA, this method allowed easy and direct editing of the S. mitis genome, broadening the spectrum of possible downstream applications of natural transformation in this species.

Purpose: To evaluate elementary schoolteachers' knowledge and decision making regarding dental trauma in Porto Alegre, Brazil., Materials and Methods: A questionnaire-based survey was conducted among elementary schoolteachers (n=442). Questions encompassed sociodemographic characteristics and decision making regarding dental trauma. Data on the appropriate answer regarding what to do with a traumatised or avulsed tooth were analysed with multiple logistic regressions, adjusting for age, gender, work experience and previous training., Results: The study population consisted largely of women (90%), was 40 to 49 years old (44.3%) and had more than 15 years of work experience (56.6%). Women (OR=2.68/p=0.041), teachers under 30 years old (OR=4.95/p=0.041), those with more than 15 years of work experience (OR=8.95/p<0.001) or those who had already received previous dental trauma instructions (OR=1.95/ p=0.119) were more likely to choose the appropriate answer for an avulsion situation. Teachers under 30 years old (OR=2.88/ p=0.279), those with more than 15 years of work experience (OR=4.55/p=0.001) or those who had received previous training (OR=3.39/p=0.009) presented higher probabilites of choosing the appropriate answer for a crown fracture event., Conclusion: Greater work experience or previous instructions on how to approach dental trauma in the school environment were found to be major factors in schoolteachers' decision making regarding dental trauma.

Background and Objective: The number of elderly living in long-term care (LTC) institutions has been increasing in many countries. The objective of this study was to evaluate the caregivers' attitudes and considerations regarding oral health and oral health practices in an LTC institution in Porto Alegre, Brazil., Materials and Methods: The population comprised the caregivers (n = 27) at an LTC in Porto Alegre, Brazil. Participants answered a questionnaire assessing the time of work at the LTC, number of elderly under their responsibility, their previous training to perform general and oral healthcare, availability of oral hygiene supplies and oral hygiene tasks they performed., Results: A total of eighteen (75%) and 23 (95.8%) of the caregivers answered they performed teeth and denture brushing as part of their routine. When asked about performing hygiene of the mucosa with gauze, 21 (87.5%) participants stated that they did so. However, when asked if they would motivate the independent elderly to perform oral hygiene, only 10 (41.7%) of the caregivers responded positively., Conclusion: Caregivers' oral health practices are in accordance with good oral health methods and are not associated with difficulties in performing oral hygiene. However, the high number of elderly people in their care could possibly overwhelm caregivers' practices. Future qualitative assessment of these issues is needed., (© 2012 The Gerodontology Society and John Wiley & Sons A/S.)

The aim of this study was to measure the publication rate of editorial board members in their board journals and to evaluate associated variables. We evaluated the ten highest-ranked journals according to the 5-year impact factor under 'Dentistry, Oral Surgery & Medicine' subject category for 2010, 2011, and 2012. All original research papers with at least one member of the editorial board as author were counted. Final analyses assessed associated variables such as size of the editorial board, number of papers published each year, and each journal's impact factor. Overall, there was an increase in the average number of articles published from 2010 (115.2 ± 52.2) to 2012 (134.7 ± 47.4). The number and percentage of articles published with editorial board members as authors over the three years did not follow the same pattern, with a slight decrease from 2010 to 2011 and an increase in 2012. The number of articles with editorial board members as authors was significantly higher for journals with impact factors ≥4.0. Journals with a higher impact factor and larger editorial board were associated with higher chances of editorial board members publishing in their respective journals. Participation of editorial board members as authors in publishing varies significantly among journals.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are critical for tissue remodeling during wound repair. Psychological stress has been found to impair wound healing in humans and animals. The objective of this study was to assess MMP and TIMP gene expression during stress-impaired healing. Female SKH-1 mice (n=299) were divided into control and stress groups (13h restraint/day for 3days prior to and 5days post-wounding). Two 3.5mm cutaneous full-thickness wounds were placed on the dorsum of each mouse and wound measurements were performed daily. RT-PCR for gene expression of MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 was performed at days 1, 3 and 5. Immunohistochemical analyses of the healed wounds were performed at days 15 and 28. As expected, wounds healed more slowly in restraint-stressed mice compared to controls. Stressed mice exhibited MMP-8 overexpression and lower TIMP-1 levels during healing, and poorer collagen organization once healed. MMP-8 overexpression may have stemmed from a higher level of neutrophils, observed in wound tissue on days 3 and 5. These findings implicate higher neutrophil numbers, MMP-8 overexpression, and TIMP-1 under-expression, as mechanisms that may compromise wound outcomes such as scarring under conditions of stress., (Copyright © 2012. Published by Elsevier Inc.)

The aim of the present study was to evaluate two dental curricula at a school of dentistry in southern Brazil. The study population included dentists trained in the last two classes of the institution's old curriculum (n = 98) and graduates of the first two classes of the new curriculum (n = 56). A questionnaire with open and closed questions was used for an overall evaluation of different aspects of the curricula, such as study methods, importance given to basic sciences, quality of theoretical and clinical guidance, perception about skills needed to perform different dental procedures, professional goals and an overall assessment. Students in the new curriculum reported more frequent use of the internet (69.6%) and scientific articles (50.0%). More importance was given to the basic sciences in the new curriculum. Graduates of the old curriculum alleged themselves to be more capable of both performing conventional (99%) and complex amalgam restorations (68.4%), as well as three-unit fixed prostheses (62.2%). Graduates of the new curriculum alleged higher capability with periodontal surgeries (48.2%), treatment of temporomandibular joint (TMJ) disorders (58.1%) and public health planning (78.6%). Regarding professional goals, the new curriculum was associated with an increase in the graduates' willingness to work in the public health system and to pursue an academic career. New curriculum graduates reported higher overall assessments regarding their educational, as well as theoretical and clinical, outcomes. A new curricular approach was associated with several changes from the perspective of the students.

As shown in 150 patients, myelography and computed tomography (CT) lead to identical results in the investigation of vertebral disc lesions of the lower lumbar spine. In less severe disc changes, where myelography is not indicated, CT findings largely correspond with clinical findings. CT investigations are risk free, without side effects, painless, fast and economical.

The intensity fluctuations of the radiation from a beating heart filled with radioactively labeled blood is analysed in such a way that the stroke volume and thus the cardiac output can be determined. The method is independent of any background radiation and irregularities of the heart rate. Further features are the speed of the method and the immediate imaging of the kinetics of the heart wall.

The concept of renal clearance measured during steady-state conditions includes the effect of filtration and secretion as well as urine flow. On the other hand, renal clearance, as used in nuclear medicine, only describes the effect of filtration and secretion. A way for determining steady-state renal clearance with a bolus injection technique is described. Steady-state renal clearance reflects the efficiency of filtration and secretion, urine flow, and the steady-state chemical potentials in the kidneys.