Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Continued Vaccine Breakthrough Cases of Serotype 3 Complicated Pneumonia in Vaccinated Children, Portugal (2016-2019).

We previously reported that despite the use of pneumococcal conjugate vaccines (PCVs), vaccine serotypes remained important causes of pneumonia with pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]). We cultured and performed PCR on 174 pleural fluid samples recovered from pediatric patients in Portugal from 2016 to 2019 to identify and serotype Streptococcus pneumoniae. Most PCPP cases (n = 87/98) were identified by PCR only. Serotypes 3 (67%), 14, and 8 (5% each) were the most frequent. Vaccine breakthrough cases were seen among age-appropriately, 13-valent, PCV vaccinated children (median: 3 years, range: 17 months to 7 years), mostly with serotype 3 (n = 27) but also with serotypes 14 and 19A (n = 2 each). One breakthrough was seen with serotype 14 in an age-appropriately, 10-valent, PCV-vaccinated child and another with serotype 3 in a child to whom the 23-valent polysaccharide vaccine was administered. While the relative risk of serotype 1 PCPP decreased almost 10-fold from the period of 2010 to 2015 to the period of 2016 to 2019 (relative risk [RR] = 0.106), that of serotype 3 PCPP almost doubled (RR = 1.835). Our data highlight the importance of molecular diagnostics in identifying PCPP and document the continued importance of serotype 3 PCPP, even when PCV13 use with almost universal coverage could be expected to reduce exposure to this serotype. IMPORTANCE The use of conjugate vaccines against Streptococcus pneumoniae in children has led to substantial reductions in pneumococcal invasive disease. However, the reductions seen in each of the 13 serotypes currently included in the highest-valency vaccine approved for use in children (PCV13), were not the same. It is becoming clear that most vaccine breakthroughs worldwide involve serotype 3 and are frequently associated with complicated pneumonia cases, often with empyema or pleural effusion. Here, we show that despite almost universal PCV13 use, which would be expected to reduce vaccine serotype circulation and further reinforce vaccine direct protection, pneumococci and serotype 3 remain the major causes of pediatric complicated pneumonia. Molecular methods are essential to identify and serotype pneumococci in these cases, which frequently reflect vaccine breakthroughs. A broader use of molecular diagnostics will be essential to determine the role of this important serotype in the context of PCV13 use in different geographic regions.

Adhesion and optics: The challenges of esthetic oral rehabilitation on varied substrates-Reflections based on a clinical report.

Patients with challenging prosthodontic conditions require rehabilitation with a biological, functional, and esthetic approach. When one or more teeth are badly discolored, their restoration is problematic because poor appearance affects not only the crown but also the periodontal tissues. This clinical report describes a complex esthetic rehabilitation with conservative tissue management and ceramic restorations. Subepithelial connective tissue graft surgery and the replacement of a cast metal post with a glass fiber post addressed the problem of a discolored maxillary central incisor. The discolored right maxillary incisor was restored with a combination of a medium-opaque, lithium-disilicate ceramic coping to mask the dark root and to approximate the color of the other incisors. Subsequently, 6 ceramic veneers were placed. A knowledge of the materials' optical properties and adhesion possibilities helped solve this complex problem.

Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells.

OBJECTIVES: To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. RESULTS: Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. CONCLUSIONS: The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.

Streptococcus canis Are a Single Population Infecting Multiple Animal Hosts Despite the Diversity of the Universally Present M-Like Protein SCM.

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence.

Pediatric Complicated Pneumonia Caused by Streptococcus pneumoniae Serotype 3 in 13-Valent Pneumococcal Conjugate Vaccinees, Portugal, 2010-2015.

Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]) is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010-2015 to identify and serotype Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.

cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

Clinical Time Required and Internal Adaptation in Cavities restored with Bulk-fill Composites.

AIM: The aim of this study was to compare the restorative time required and the internal adaptation after thermomechanical aging of class I restorations using either the conventional incremental technique or bulk-fill technique. MATERIALS AND METHODS: Cavities (Class I) were prepared on the occlusal surface of human third molars. 40 teeth were divided into four experimental groups according to the restorative technique (n = 10): G1 = 3 mm increment of Surefill SDR Flow + 1 mm Filtek P60; G2 = 3 mm increment of Filtek Bulk-Fill + 1 mm Filtek P60; G3 = Filtek P60 inserted with incremental technique; G4 = 3 mm increment of Filtek Z350 Flow XT + 1 mm increment of Filtek P60. The required restorative clinical time for each technique was marked. Specimens were submitted to thermomechanical loading (20,000 mechanical cycles-80 N/thermal cycling-5/55°C for 30 seconds). After, samples were sectioned, ratio of internal gaps to interface length (%) was recorded using dye-staining-gap technique. Data were submitted to analysis of variance (ANOVA) and Tukey's test (95% significance). RESULTS: There was no significant difference in gap formation and none of the groups was completely gap-free. However, a significant reduction on required restorative clinical time was observed for G1 (167 ± 7 seconds), G2 (169 ± 4 seconds), and G4 (169 ± 8 seconds) when compared with G3 (204 ± 8 seconds). CONCLUSION: No significant difference in gap formation was found among bulk-fill and conventional incremental restorative technique tested; however, the use of a bulk-fill composite reduced 20% of the required restorative clinical time. CLINICAL SIGNIFICANCE: None of the restorative techniques applied were able to prevent internal gap formation. The use of bulk-fill composite reduced the required clinical time to perform class I restorations.

Beta-hemolytic Streptococcus dysgalactiae strains isolated from horses are a genetically distinct population within the Streptococcus dysgalactiae taxon.

The pathogenic role of beta-hemolytic Streptococcus dysgalactiae in the equine host is increasingly recognized. A collection of 108 Lancefield group C (n = 96) or L (n = 12) horse isolates recovered in the United States and in three European countries presented multilocus sequence typing (MLST) alleles, sequence types and emm types (only 56% of the isolates could be emm typed) that were, with few exceptions, distinct from those previously found in human Streptococcus dysgalactiae subsp. equisimilis. Characterization of a subset of horse isolates by multilocus sequence analysis (MLSA) and 16S rRNA gene sequence showed that most equine isolates could also be differentiated from S. dysgalactiae strains from other animal species, supporting the existence of a horse specific genomovar. Draft genome information confirms the distinctiveness of the horse genomovar and indicates the presence of potentially horse-specific virulence factors. While this genomovar represents most of the isolates recovered from horses, a smaller MLST and MLSA defined sub-population seems to be able to cause infections in horses, other animals and humans, indicating that transmission between hosts of strains belonging to this group may occur.

Comparing Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Phenotypic and Molecular Methods for Identification of Species within the Streptococcus anginosus Group.

The heterogeneity of members of the Streptococcus anginosus group (SAG) has traditionally hampered their correct identification. Recently, the group was subdivided into 6 taxa whose prevalence among human infections is poorly described. We evaluated the accuracy of the Rapid ID32 Strep test, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and a PCR multiplex method to identify 212 SAG isolates recovered from human infections to the species and subspecies level by using multilocus sequence analysis (MLSA) as the gold standard. We also determined the antimicrobial susceptibilities of the isolates. Representatives of all SAG taxa were found among our collection. MALDI-TOF MS and the Rapid ID32 Strep test correctly identified 92% and 68% of the isolates to the species level, respectively, but showed poor performance at the subspecies level, and the latter was responsible for major identification errors. The multiplex PCR method results were in complete agreement with the MLSA identifications but failed to distinguish the subspecies Streptococcus constellatus subsp. pharyngis and S. constellatus subsp. viborgensis. A total of 145 MLSA sequence types were present in our collection, indicating that within each taxon a number of different lineages are capable of causing infection. Significant antibiotic resistance was observed only to tetracycline, erythromycin, and clindamycin and was present in most taxa. MALDI-TOF MS is a reliable method for routine SAG species identification, while the need for identification to the subspecies level is not clearly established.

Changes in gene expression profile in two multidrug resistant cell lines derived from a same drug sensitive cell line.

Resistance to chemotherapy is one of the most relevant aspects of treatment failure in cancer. Cell lines are used as models to study resistance. We analyzed the transcriptional profile of two multidrug resistant (MDR) cell lines (Lucena 1 and FEPS) derived from the same drug-sensitive cell K562. Microarray data identified 130 differentially expressed genes (DEG) between K562 vs. Lucena 1, 1932 between K562 vs. FEPS, and 1211 between Lucena 1 versus FEPS. The NOTCH pathway was affected in FEPS with overexpression of NOTCH2 and HEY1. The highly overexpressed gene in MDR cell lines was ABCB1, and both presented the ABCB1 promoter unmethylated.

Próteses parciais fixas totalmente cerâmicas: fatores que determinam sucesso e fracasso / All ceramic fixed partial dentures: factors that determine success and failure

Apesar do advento dos implantes osseointegráveis, as próteses parciais fixas continuam a ser restaurações com larga utilidade no arsenal restaurador dos cirurgiões-dentistas. No entanto, a exigência estética dos dias atuais faz com que as próteses livres de metal ocupem o lugar das consagradas próteses metalocerâmicas. Sendo assim, diversos fatores devem ser compreendidos e avaliados para se planejar a confecção de uma prótese parcial fixa totalmente cerâmica. O objetivo desta revisão é expor os fatores que podem determinar o sucesso ou fracasso de próteses parciais fixas totalmente cerâmicas.

Despite the advent of osseointegrated implants, fixed partial dentures remain to have a broad utility in the restorative arsenal of the dentists. However, modern day esthetic requirements causes metal-free prosthesis take the place once occupied in the past by the classic porcelain fused to metal FPDs. This way, several factors must be understood and assessed to plan the fabrication of an all- -ceramic fixed partial denture. The purpose of this review article is to look at the factors that can determine success or failure of all-ceramic fixed partial dentures.

Estética com implantes imediatos / Esthetics with imediate implants

Considerações estéticas em implantodontia tem sido motivo de diversos estudos e publicações nos últimos 15 anos, atentando para a existência de diversos parâmetros que devem ser criteriosamente analisados durante a etapa de planejamento para a instalação de implantes osseointegrados. Alguns fatores se destacam na busca do resultado estético ideal, como o posicionamento tridimensional adequado do implante, anatomia do rebordo alveolar, fenótipo periodontal, tipo de material restaurador, perfil emergente da prótese, dimensões do espaço edêntulo disponíveis, e o momento de instalação do implante.

Over the past 15 years, dental esthetics has been an important issue in implant dentistry, pointing out for the need of a thorough analysis of several parameters during treatment planning for implant surgery. Many factors are of importance in the search for an ideal esthetic outcome, such as tridimensional positioning of the implants, anatomy of the alveolar ridge, periodontal phenotype, restorative materials used, emergence profile, edentulous space dimensions, as well as timing of implant placement.

Reabilitação anterior com coroas Procera / Anterior rehabilitation with Procera crowns

O objetivo deste trabalho é apresentar um caso de fechamento de diastema com coroas de alumina densamente sinterizadas através da utilização do Sistema Procera®, mostrando as etapas e requisitos técnicos para o sucesso dos casos. O caso apresentado seguiu o seguinte protocolo clínico: enceramento diagnóstico, confecção de preparo com término em chanfro e desgaste de 1,5mm nas paredes axiais e 2,0mm de desgaste incisal, confecção de restaurações provisórias, moldagem com técnica de duplo fio de afastamento, enceramento das subestruturas para duplo escaneamento, aplicação cerâmica e cimentação por meio de cimento resinoso. O caso se enquadrava dentro das indicações estabelecidas pelo fabricante. Os resultados obtidos foram julgados como bastante satisfatórios pela paciente e pelo profissional responsável sob os pontos de vista estético, funcional e biológico.

This article aims to present a clinical case of a diastema closure with densely sintered alumina crowns using the Procera® systems, highlighting its sequential steps and technical prerequisites for success. The case presented followed the protocol: diagnostic waxing, chamfer preparations with a 1,5 mm axial reduction and a 2,0 mm incisal reduction, temporary restorations, impression with the double cord technique, substructure wax up for double scanning, ceramic application, and cementation with a resin cement. The case was within the ceramic system’s manufacturers indications. Results obtained were judged as satisfying esthetically, functionally and biologically by the patient and the responsible dentist.

Population genetics of Streptococcus dysgalactiae subspecies equisimilis reveals widely dispersed clones and extensive recombination.

BACKGROUND: Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America. METHODOLOGY/PRINCIPAL FINDINGS: The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred approximately 4.4 times more frequently than by point mutation. CONCLUSIONS/SIGNIFICANCE: A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population.

The Viriato study: update on antimicrobial resistance of microbial pathogens responsible for community-acquired respiratory tract infections in Portugal.

The Viriato study is a prospective, multicentre laboratory-based surveillance study of antimicrobial susceptibility in which 30 microbiology laboratories throughout Portugal are asked to isolate, identify and submit to a central laboratory for testing Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis responsible for community-acquired lower respiratory tract infections and Streptococcus pyogenes from tonsillitis. To monitor changes in antimicrobial resistance patterns of these frequent respiratory pathogens. Susceptibility was determined by disk diffusion (Kirby-Bauer) or using Etest strips following the Clinical and Laboratory Standards Institute guidelines. From 1999 to 2007 over 13 900 isolates were analysed. Among S. pneumoniae penicillin non-susceptibility decreased from 25% in 1999 to 18% in 2007 (p = 0.002) but resistance to macrolides showed a steady increase, reaching 20% in the last 6 years. Resistance to amoxicillin and the quinolones remained stable and very low (1-2%) throughout the study period. Antimicrobial resistance among H. influenzae and M. catarrhalis remained stable. The most significant resistance was to ampicillin, of 10-12% and greater than 80%, respectively, as a result of the production of beta-lactamases. Macrolide resistance among S. pyogenes was stable during 1999-2003 (20-23%) but after 2003 there was a steady decline in resistance, which in 2007 reached 10%. The Viriato surveillance study showed that penicillin remains the most active antimicrobial agent against S. pyogenes causing tonsillitis, and amoxicillin-clavulanate and the quinolones are the most active in vitro simultaneously against S. pneumoniae, H. influenzae and M. catarrhalis responsible for community-acquired lower respiratory tract infections in Portugal.

Influence of Zn-Cd substitution: spectroscopic and theoretical investigation of 8-hydroxyquinoline complexes.

It is now well known that zinc is crucial for the synthesis of nucleic acids and, consequently, for cellular division. However, (67)Zn, the NMR-detectable isotope, is one of the isotopes most poorly studied by NMR. The strategy used for NMR studies is the substitution of Zn by (113)Cd. In this work, we employed (13)C, (113)Cd NMR (CPMAS), X-ray and DFT calculation in order to evaluate the Zn-Cd substitution using 8-hydroxyquinoline like prototype compound. Our results show that there are strong structural and electronics effects are involved in the substitution.

XAF1 mRNA expression improves progression-free and overall survival for patients with advanced bladder cancer treated with neoadjuvant chemotherapy.

PURPOSE: The aim of this study was to investigate whether mRNA expression of the apoptosis-associated genes, XAF1 and XIAP, in bladder cancer patients correlates with response to neoadjuvant treatment. METHODS: Gene expression was analyzed by a real-time quantitative PCR method in paired samples from 14 bladder cancer patients treated with a combination of neoadjuvant gemcitabine and cisplatin. The prognostic significance of XAF1 and XIAP mRNA expression as well as the correlation with several clinical and pathological findings were evaluated. RESULTS: The clinical response in the XAF1-high subset (n = 5) was remarkably higher compared with the XAF1-low subset (n = 9) (100% vs. 44.4%; P = 0.038). These results translated into a notably improvement of progression-free survival (PFS) in the XAF1-high subset (log-rank P = 0.012). In addition, patients in the XAF1-high subset had a 3.9-fold decreased chance of dying from the disease (hazard ratio for death (HR), 0.257; (CI 95%), 0.043-1.536, P = 0.036). When we evaluated the expression of XIAP, although an inverse correlation was found between expression and pathological response, there were no statistically significant associations with the clinical response, the length of PFS, and OS. CONCLUSIONS: This is one of the few studies to address the role of XAF1 in a clinical setting. The data presented here identify XAF1 as a novel predictive and prognostic factor in bladder cancer patients. Furthermore, our observations are in line with previous studies, which point towards XAF1 as a tumor-suppressor gene. Nonetheless, additional studies, both mechanistic and translational, are warranted and may help not only in corroborating the role of XAF1 as a prognostic marker, but also as a potential target for anticancer therapy.