Investigation of Oral Shedding of Torquetenovirus (TTV) in Moderate-to-Severe COVID-19 Hospitalised Patients.

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 log10 copies/mL, in which those who were discharged and those evolving to death had values of 3.96 log10 copies/mL and 6.27 log10 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.

In Vitro Impact of Fluconazole on Oral Microbial Communities, Bacterial Growth, and Biofilm Formation.

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.

Differential response to prolonged amoxicillin treatment: long-term resilience of the microbiome versus long-lasting perturbations in the gut resistome.

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.

Markerless Genome Editing in Competent Streptococci.

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.

Awareness regarding antimicrobial resistance and confidence to prescribe antibiotics in dentistry: a cross-continental student survey.

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.

Lack of direct association between oral mucosal lesions and SARS-CoV- 2 in a cohort of patients hospitalised with COVID-19.

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.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

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_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_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.

Trajetória de educação na pós-graduação e atuação profissional de egressos de Odontologia: uma análise de doze anos (2007 a 2019) / Patterns of further education and employment of dental graduates: a twelve-year analysis from 2007 to 2019

Avaliar a trajetória de graduados em Odontologia é importante para garantir o alinhamento adequado dos currículos odontológicos às necessidades da sociedade. Este estudo observacional transversal descritivo teve como objetivo caracterizar o perfil sociodemográfico-familiar e a trajetória de educação na pós-graduação e de atuação profissional de cirurgiões-dentistas egressos de universidade pública do Sul do Brasil. Foram convidados a participar do estudo todos os egressos que concluíram a graduação em Odontologia nesta universidade, entre 2007 e 2019. A coleta de dados aconteceupelo autopreenchimento de instrumento semiestruturado online, pré-testado, organizado em três dimensões (perfil sociodemográfico-familiar, educação na pós-graduação e atuação profissional). Aanálise das questões objetivas foi realizada por estatísticas descritivas e as abertas pela análise temática de conteúdo. Participaram do estudo 245 cirurgiões-dentistas (percentual de resposta: 25,7%). A maioria eram mulheres (68,6%), com 26 a 32 anos (73,9%), solteiros (71,4%), sem filhos (92,7%), naturais (51,4%) e residentes (59,2%) na cidade onde cursaram Odontologia e trabalham (67,5). A renda pessoal mensal foi de 5 a 10 saláriosmínimos (36,7%). Atuam na Odontologia (97,1%) e estão satisfeitos com a profissão (79,6%). Realizaram ou estão realizando cursos de pós-graduação (94,3%), concluídos em até três anos após a graduação (58%), principalmente especialização (62,8%), nas áreas de Saúde Coletiva, Ortodontia e Implantodontia. Setor privado mostrou-seo maior empregador dos cirurgiões-dentistas (60,8%), entretanto, a atuação no serviço público cresceu entre os egressos de 2010-2019 (2,4% para 20,7%). Estudos de acompanhamento destes egressos são recomendados para avaliação do curso e das demandas/desafios contextuais que caracterizam a profissão de cirurgião-dentista no Brasil (AU).

The assessment of dental graduates' trajectory over time is important to ensure a proper alignment of dental curricula with the needs of society. This cross-sectional, observational anddescriptivestudy aimed at characterizing the sociodemographic profile, the postgraduateeducation choices,and the employment pattern of dental graduates from a publicuniversity in the South region of Brazil. Dental graduates from 2007 to 2019 were invited to participate via e-mail. The survey was self-administered and hosted online. The content of the surveyencompassed three main spheres of interest-sociodemographic profile, postgraduateeducation choices, and employment pattern. Atotalof245 graduates participated in the study(response rate of 25.7%). The majority were women(68.6%), aged 26 to 32 years old (73.9%), single (71.4%), without children (92.7%), born (51.4%) and residing(59.2%) inPorto Alegre, the city in which the university is located and where they work (67.5%). Monthly income reported was 5 to 10 times the minimum wage (36.7%). Most participantswere activelyworkingin Dentistry (97.1%) and weresatisfied with the dental profession (79.6%). The majority attendedor is currently enrolled in postgraduatecourses (94.3%), completedwithin three years after graduation, mainlyspecialization courses (62.8%), in the areas of Public Health, Orthodontics and Implantology. Most reported to be working in the private sector (60.8%). However, there was an increase in employmentin the public sector among those who graduatedbetween2010and 2019compared to 2007-2008(from 2.4% to 20.7%). Fundamentally, the continuing assessment of dental graduates' path and perceptions is essential to better adjust and shape dental curricula in the future (AU).

ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data.

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.

Competence in Streptococcus pneumoniae and Close Commensal Relatives: Mechanisms and Implications.

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.

Threshold-effect of income on periodontitis and interactions with race/ethnicity and education.

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.

Threshold-effect of income on periodontitis and interactions with race/ethnicity and education / Efeito limiar de renda na periodontite e interações com raça/etnia e educação

ABSTRACT: 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.

RESUMO: Objetivo: Os objetivos deste estudo foram explorar a relação entre renda e educação com doença periodontal e avaliar a interação entre eles e raça/etnia. Método: Dados individuais do inquérito epidemiológico de saúde bucal da Pesquisa Nacional de Saúde Bucal (SB Brasil 2010) foram obtidos para 9.779 indivíduos. A relação entre renda per capita e educação com saúde periodontal foi suavizada usando técnica de Locally Weighted Scatter-plot Smoother (LOWESS). Utilizou-se regressão logística multivariável para avaliar os efeitos independentes de sexo, idade, renda, educação, raça/etnia, posse de bens, última visita ao dentista e número de pessoas por dormitório. Resultados: A prevalência de adultos com doença periodontal moderada e grave foi de 17,6 e 6,5%, respectivamente. A relação entre saúde periodontal e renda foi curvilínea, com limiar de R$ 1.050/mensais, a partir do qual não havia relação entre as variáveis. Na análise multivariavel, após ajuste por covariadas, apenas renda estava associada significativamente com saúde periodontal. Não foram encontradas interações significantes entre renda e educação ou raça/etnia, nem entre educação com raça/etnia. Conclusões: A relação entre saúde periodontal e renda foi curvilínea com a presença de efeito de limar, dando suporte para programas de transferência de renda. Além do limiar, apenas educação mostrou associação linear negativa com periodontite moderada a severa.

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

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.

Hidden Gems in the Transcriptome Maps of Competent Streptococci.

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.

A Quorum-Sensing System That Regulates Streptococcus pneumoniae Biofilm Formation and Surface Polysaccharide Production.

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.

A positive feedback loop mediated by Sigma X enhances expression of the streptococcal regulator ComR.

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.

Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones.

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.

Markerless Genome Editing in Competent Streptococci.

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.

Accuracy of clinical diagnosis for the identification of potentially malignant disorders and malignant lip lesions.

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.

Overcoming the Barrier of Low Efficiency during Genetic Transformation of Streptococcus mitis.

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.