Naturally occurring mutations in regulatory proteins among streptococcus pyogenes isolates from distinct human infections

Streptococcus pyogenes (Group A Streptococcus, GAS) is among the most prevalent bacterial pathogens of humans and it is responsible for a wide range of infections, from pharyngitis and impetigo to life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. However, until today, despite many years of research, there is still no consensus regarding which are the genotypic or phenotypic characteristics that confer to an isolate a certain tissue preference or a more invasive potential. The nasopharyngeal mucosa and skin are considered the primary sources of isolates responsible for invasive infections. This suggests a further ability of these isolates to invade and survive in deeper tissues. In the last years, spontaneous mutations occurring in the covRS two-component regulatory system have been considered a possible explanation for the transition from localized to systemic infection. It is estimated that this system controls directly or indirectly the expression of 10-15% of the GAS genome. As a consequence of these mutations, among the distinct patterns of expression of several virulence factors, the downregulation of the extracellular cysteine protease SpeB, has been considered crucial for the switch to a hypervirulent phenotype. The downregulation of SpeB has also been described as a consequence of mutations in a stand-alone transcriptional regulator named RopB. However, contrasting results were reported whether covRS mutations were more prone to occur in certain lineages, namely those more frequently associated with invasive infections or if they occur in a similar proportion among invasive and non-invasive isolates. To address these questions, in the present thesis, we determined the sequence of the covRS and ropB of 191 isolates from invasive infection and pharyngitis and evaluated the production of SpeB, as well of NAD glycohydrolase (NADase) and streptolysin S (SLS), which are two virulence factors supposed to be under the influence of CovRS. Moreover, skin and soft tissue infections (SSTI) are frequently considered the focal points for the development of invasive disease. However, most of the knowledge about GAS skin isolates is from studies that intended to find differences between isolates recovered from invasive and non-invasive infections generically. The majority of these studies use isolates recovered from SSTI but mostly from pharyngitis and compared those together against the isolates recovered from invasive infections, resulting in few data regarding isolates recovered from SSTI. Therefore, we characterized by multiple typing methods a total of 320 isolates from SSTI recovered in Portugal and performed the comparison with invasive isolates recovered during the same period which were previously characterized. All SSTI isolates were also tested for SpeB activity and for those without detectable SpeB activity we determined the sequence of covRS and ropB genes. Overall, we found that isolates with null covS alleles, which are predicted to eliminate the protein function have a significant association with invasive infections comparative with isolates from pharyngitis and SSTI. Additionally, none of these isolates, as expected, had SpeB activity, and, with few exceptions, they showed an increased activity of both NADase and SLS that could explain their potentially higher invasiveness. Even so, this mechanism was found to be uncommon, corresponding to only 10% of invasive isolates, which could be due to an overall fitness cost of these mutations. Moreover, null covS alleles were not more prevalent among isolates from clones frequently associated with invasive infection such as emm1 and emm64 and instead they were distributed throughout diverse genetic backgrounds. The few exceptions regarded the levels of NADase and SLS points to the complexity of the regulatory networks among distinct GAS lineages. Additionally, no null covR alleles were detected in our isolates and ropB null alleles were found in a low fraction of GAS isolates and were not associated with any infection type. Regarding SpeB activity, it was detected in a similar proportion in isolates recovered from the different sources, and therefore its absence was not associated to any type of infection, suggesting that its abrogation cannot by itself explain the higher ability of certain clones to cause invasive disease. Among SSTI isolates, we found that emm89 type isolates were the most prevalent and were significantly associated with these infections when compared with invasive isolates. In contrast, emm1, emm3, and emm64 isolates were associated with invasive infections. Within emm89 isolates, SSTI were only associated to those that lack the hasABC locus, corresponding to a recently emerged acapsular clade (clade 3) that also carries a variant of the ngs-ifs-slo locus. These results suggest that for some unknown reason these isolates may have an increased potential to cause SSTI. As a consequence of known differences in the emm-type between isolates causing these two types of infections, we also found significant associations between the ability to bind to different host proteins. This ability was presumed by inferring the emm-cluster through the emm-type results. The emm-cluster is a recent classification based on the entire sequence of the emm gene (M protein) where each cluster shares binding motifs to host proteins and other structural properties. Therefore, the ability to bind fibrinogen and albumin were significantly associated with invasive isolates, whereas the ability to bind to C4BP and IgG were associated with SSTI isolates. Differences in the presence of superantigen (SAg) genes, SAg profiles and in the distribution of sequence types (ST) determined by Multilocus Sequence Typing (MLST) were also noted. The possible impact of these differences in the ability of the isolates to cause these distinct infections remains to be clarified. Moreover, within each emm type the same MLST defined lineages and SAg profiles could be found in both types of infection, questioning the possibility that these characteristics dictate the tissue tropism of each isolate. In summary, the results described in this thesis indicate that isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites and while some GAS clones have more capacity to invade deeper tissues, others are more prone to cause SSTI. Moreover, the significant presence of null covS mutations among invasive isolates and the fact that no association was observed regarding the absence of SpeB activity in isolates from different types of infections, suggests that the role of spontaneous mutations impairing the CovRS activity is probably related with the regulation of others virulence factors under its control in addition to SpeB

Consequences of the variability of the CovRS and RopB regulators among Streptococcus pyogenes causing human infections

Copyright © 2015, The Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/To evaluate the importance of covRS and ropB mutations in invasive disease caused by Group A Streptococci (GAS), we determined the sequence of the covRS and ropB genes of 191 isolates from invasive infections and pharyngitis, comprising a diverse set of emm types and multilocus sequence types. The production of SpeB and the activity of NAD glycohydrolase (NADase) and streptolysin S (SLS) were evaluated. The results support the acquisition of null covS alleles (predicted to eliminate protein function), resulting in downregulation of SpeB and upregulation of NADase and SLS, as a mechanism possibly contributing to higher invasiveness. Among the isolates tested, this mechanism was found to be uncommon (10% of invasive isolates) and was not more prevalent among clones with enhanced invasiveness (including M1T1) but occurred in diverse genetic backgrounds. In lineages such as emm64, these changes did not result in upregulation of NADase and SLS, highlighting the diversity of regulatory pathways in GAS. Despite abrogating SpeB production, null alleles in ropB were not associated with invasive infection. The covRS and ropB genes are under stabilising selection and no expansion of isolates carrying null alleles has been observed, suggesting that the presence of these regulators is important for overall fitness.info:eu-repo/semantics/publishedVersio

Emergence of the same successful clade among distinct populations of emm89 Streptococcus pyogenes in multiple geographic regions

© 2015 Friães et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.The emergence of clades within emm89 Streptococcus pyogenes isolates that rapidly became the dominant lineages expressing this emm type was recently reported in the United Kingdom and in a study that included isolates from the United States, Finland, and Iceland (United States/FI/IC). In the United Kingdom, the emerging clade was associated with the absence of the hasABC locus, responsible for the synthesis of the hyaluronic acid capsule. The study from the United States/FI/IC highlighted the strict association of the emerging clade with an nga promoter variant, also found in contemporary emm1 isolates, which results in increased expression of the nga locus. The study from the United Kingdom also examined this region and found that the nga-ifs-slo locus and surrounding sequences of the emerging clade shared 99% DNA identity with that of contemporary emm1 and emm12 strains, but the authors do not offer any information on the nga promoter (1). The acquisition of this region by emm1 isolates is currently considered the major molecular event triggering the success and enhanced virulence of this clone.info:eu-repo/semantics/publishedVersio

Translation and validation of Convergence Insufficiency Symptom Survey (CISS) to Portuguese - psychometric results

Purpose: Translate and adapt the Convergence Insuficiency Symptom Survey (CISS) questionnaire to the Portuguese language and culture and assess the psychometric properties of the translated questionnaire (CISSvp). Methods: The CISS questionnaire was adapted according to the methodology recommended by some authors. The process involved two translations and back-translations performed by independent evaluators, evaluation of these versions, preparation of a synthesis version and its pre-test. The final version (CISSvp) was applied in 70 patients (21.79 ± 2.42 years) students in higher education, and at two different times, by two observers, to assess its reliability. Results: The results showed good internal consistency of the CISSvp (Cronbach's alpha - α=0.893). The test re-test revealed an average of the differences between the first and second evaluation of 0.75 points (SD ± 3.53), which indicates a minimum bias between the two administrations. The interrater reliability assessed by intraclass correlation coefficient ranged from 0.880 to 0.952, revealing that the CISSvp represents an appropriate tool for measuring the visual discomfort associated with near vision tasks with a high level of reproducibility. Conclusions: The CISS Portuguese version, showed good psychometric properties and has been sown to be applicable to the Portuguese population, to quantify the visual discomfort associated with near vision, in higher education students

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB

Streptococcus pyogenes como agente de infecção da pele e tecidos moles

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2011Em Portugal, os estudos epidemiológicos em Streptococcus pyogenes englobam sobretudo estirpes responsáveis por infecções do aparelho respiratório superior e por infecções invasivas. No entanto, não há registo de estudos focados em estirpes de S. pyogenes recolhidas de infecções de pele e tecidos moles. Este estudo baseou-se na caracterização de 320 estirpes recolhidas em vários hospitais do país, durante o período de 2003 a 2009, a partir de amostras de pus de lesões cutâneas, como feridas e abcessos. A caracterização das estirpes foi efectuada por diferentes métodos de tipagem, nomeadamente serotipagem T, tipagem emm, determinação dos perfis de toxinas e determinação dos perfis de macro-restrição obtidos por electroforese em campo pulsado (PFGE). Em todos os métodos utilizados verificou-se uma elevada diversidade da população (índice de diversidade de Simpson ≥0.86). As estirpes do tipo emm89 (n=62) foram as mais frequentes, seguidas por estirpes do tipo emm1 (n=55). Quando consideradas em conjunto, estas estirpes correspondem a 36,6% da população total e constituem os dois principais grupos definidos por comparação dos perfis de PFGE. Assim como estes, verificou-se que a maioria dos grupos definidos por PFGE são principalmente constituídos por estirpes do mesmo tipo emm. Relativamente aos perfis de toxinas, observou-se que estirpes com o mesmo tipo emm, normalmente, apresentam um perfil igual ou semelhante. Também se determinou a resistência a alguns antimicrobianos de interesse clínico ou epidemiológico e os respectivos génotipos. No total, observou-se uma resistência aos macrólidos de 10,3%, com uma dominância de estirpes de fenótipo cMLSB em relação ao fénótipo M. A resistência à tetraciclina foi observada em 47 estirpes, das quais 12 eram também resistentes aos macrólidos. Tendo em conta os diferentes tipos emm detectados e o número de estirpes correspondentes, a implementação da vacina experimental 26-valente em todas as faixas etárias poderia prevenir 67,8% das infecções de pele e tecidos moles em Portugal.In Portugal, epidemiological studies of Streptococcus pyogenes concentrate mostly on isolates recovered from pharyngitis and invasive infections. However, there is no record of studies focused on S. pyogenes isolates recovered from skin and soft tissue infections. This work aimed to characterize 320 isolates recovered in several hospitals across the country, from skin lesions, like wounds and abscesses. The typing methods used were T typing, emm typing, exotoxin profiling and pulsed field gel electrophoresis (PFGE) profiling. In all of these, a very diverse population was detected (Simpson’s index of diversity ≥ 0.86). The most prevalent emm type was emm89 (n=62) followed by emm1 (n=55). Together, isolates of these emm types accounted 36.6% of the population and composed the two major clusters defined by comparison of PFGE profiles. Like these clusters, the majority of clusters are composed mostly of isolates with the same emm type. Toxin profiling revealed that isolates with the same emm type, usually share the same or a similar toxin profile. We also determined the resistance to a panel of antibiotics of clinical and epidemiological interest, as well as the associated resistance genotypes. Erythromycin resistance was observed in 33 isolates (10%), of which 11 presented the M phenotype, while 22 were constitutively resistant to clindamycin (cMLSB phenotype). Tetracycline resistance was detected in 47 isolates (14.7%), of which 12 were also resistance to erythromycin. According to the different emm types detected, the implementation in all age groups of the experimental 26-valent M protein-based vaccine could prevent 67.8% of skin and soft tissue infections in Portugal

How do consumers react after service failure provided by AI (vs human) agents

Dissertation presented as the partial requirement for obtaining a Master's degree in Statistics and Information Management, specialization in Marketing Research and CRMConsumers tend to react to service failures in very different ways and due to a variety of constraints. With the rapid development of new technologies like Artificial intelligence (AI), which is reshaping the way several tasks are performed, little is known about the effects on consumers’ reactions. Since people tend to prefer humans instead of artificial intelligence, we propose to study the gap in the relationship between customers and service providers, by analyzing how consumers react after a service failure when the service provider is an AI or a human agent. This study (N=248) demonstrates that people perceive human (vs. AI) agents as more empathetic. In addition, when the failure occurs with a premium service, consumers are more satisfied and prefer a human (vs. AI) agent while with a basic service, people choose the AI agent. Nevertheless, the tendency is for non-human agents start to execute some tasks to accelerate and reduce costs of the whole process of the service industries and others. However, human agents show better results in the service premium category while those effects don’t show relevance in a basic service context. These findings suggest that it is critical for people to trust Artificial Intelligence mainly when the investment in the service is high, due to the cost-effectiveness of the service. Furthermore, the empathy between customer and employee is still a key factor for consumers to choose any kind of service, thus the preference for humans

Rastreio pré-natal não invasivo: o futuro da obstetrícia

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de MedicinaNoninvasive prenatal testing (NIPT) brought into obstetrics the unprecedented ability to access the fetal genome with a noninvasive technique. Currently, this technique’s most reliable application is in prenatal screening for trisomy 21. Several studies comparing its performance with traditional maternal serum screening have been published in the last decade, showing its potential in improving overall aneuploidy detection rate, reducing the need for invasive diagnostic procedures (IDP) and broadening our ability to explore fetal genetic features. Its reliable performance and noninvasive nature have resulted in its inclusion in many different prenatal screening models worldwide. However, the use of NIPT in prenatal care does not obviate the need for a confirmatory IDP after a positive test result. The existence of a significant number of discordant and “no call” test results justifies the need for a confirmatory IDP. Pregnancy, maternal and fetal-related conditions may be the reason for these discordant results. In addition, it is important to highlight its limitations regarding congenital conditions other than the most common trisomies, where ultrasound, traditional serum screening and IDP still play an irreplaceable role. Health care providers are thus responsible for recognizing the test’s limitations and interpreting its results, as well as defining situations where NIPT may not be the most adequate screening test..........................................................................................................O teste pré-natal não invasivo trouxe à obstetrícia uma capacidade única de aceder ao genoma fetal de forma não invasiva. Atualmente, a aplicação mais bem aceite é o rastreio pré-natal da trissomia 21. Vários estudos comparando o seu desempenho com o tradicional rastreio bioquímico no sangue materno têm sido publicados na última década, mostrando o seu contributo superior na taxa de deteção das aneuploidias mais comuns, reduzindo o número de procedimentos invasivos de diagnóstico realizados e alargando o nosso espectro de análise do genoma fetal. O seu desempenho promissor e natureza não invasiva têm levado à sua introdução em diferentes modelos de rastreio pré-natal por todo o mundo. Contudo, o recurso ao teste pré-natal não invasivo não descarta a necessidade subsequente de realização de um procedimento invasivo de diagnóstico confirmatório, após a obtenção de um resultado positivo no teste não invasivo. Este facto é justificado pela existência de um número significativo de resultados discordantes e 'no-call' results. Características próprias da gravidez, maternas ou fetais podem ser a causa desses erros. É importante sublinhar a sua limitação no rastreio de condições congénitas que vão além das trissomias mais comuns, onde os métodos de rastreio e diagnóstico tradicionais ainda desempenham um papel preponderante. Os profissionais de saúde são, por isso, responsáveis por reconhecer as limitações deste teste e interpretar os seus possíveis resultados, assim como definir situações em que o teste pré-natal não invasivo pode não ser o método de rastreio mais adequado a ser aplicado

Aumento da biodisponibilidade oral de fármacos peptídicos recorrendo à encapsulação com alginato e quitosano.

Aguarda-se o resumo do autor.Financiado pela Fundação para a Ciência e a Tecnologia - Bolsa de doutoramento SFRH/BD/5085/200