The influence of human plasma on Staphylococcus epidermidis virulence

EUROBIOFILMS 2017 - 5th European Congress on Microbial BiofilmsS. epidermidis is one of the most common causes of medical device-related infections, being associated with increased patients’ morbidity but also with massive additional financial burden. Hence, several efforts have been made to identify S. epidermidis virulence determinants, including the mechanisms behind biofilm formation and antibiotics tolerance. However, the great majority of these studies were performed using artificial media, which composition does not reflect in vivo conditions, and in the absence of host factors reducing the clinical relevance of those findings. Thus, the aim of this work was to evaluate the influence of host factors present in human plasma on S. epidermidis growth rate, biofilm formation capacity and tolerance to antibiotics. The presence of human plasma (5 to 20%) in the culture medium (Tryptic Soy Broth) significantly decreased both growth rate and biofilm formation ability. The greater the plasma concentration (20%), the greater the inhibitory effect observed (2 log10 reduction in growth and 3-fold less biofilm biomass). Interestingly, different results were obtained when the brand of the medium was changed. Thus, due to the inherent variability of complex media, chemically defined medium (CDM) was used instead. Respecting the bacterium growth rate, once more, the greater the plasma concentration, the greater the inhibition observed although with CDM the impact was less pronounced (1 log10 reduction in the presence of 20% of plasma). However, different from what was observed with TSB, no differences were found on biofilm formation capacity. In addition, our data showed that human plasma decreased the effect of vancomycin as 1.3 log10 reduction was observed in the absence of plasma and only 0.5 in the presence of 20% of plasma. Overall, our results highlight the importance of introducing host factors in in vitro assays in order to better mimic in vivo conditions consequently increasing the clinical significance of the results obtained.info:eu-repo/semantics/publishedVersio

The role of the gene codY in S. epidermidis biofilm formation

Background: The development of viable but non-culturable (VBNC) cells within biofilms is a major clinical concern, as it contributes to the recurrence of biofilm-related infections. CodY, a global transcriptional regulator of metabolism and virulence genes, was previously found upregulated in Staphylococcus epidermidis 9142 biofilms with higher proportions of VBNC cells, suggesting a potential role in the mediation of the VBNC state. Objective: Due to strain-to-strain variability, we aimed to assess the expression of the gene codY in other S. epidermidis strains, including the strain 1457, which is amenable to mutagenesis. Additionally, to further study the function of CodY in the development of VBNC cells, a codY deletion mutant was constructed in the1457 background. Results: Under VBNC-inducing conditions, codY expression was significantly increased in all strains tested (2.5- to 4.4-fold). Furthermore, the deletion of codY gene in the strain 1457 resulted in a reduced growth rate and, more importantly, in a reduced biofilm biomass production when compared with the wild type (35% of reduction). Conclusion and significance: Our findings suggest an important role of codY gene in S. epidermidis virulence, as biofilm formation, one of the most important virulence factors of this bacterium, was affected by codY deletion. Moreover, to date, this is the first study assessing the role of codY gene in S. epidermidis.info:eu-repo/semantics/publishedVersio

Staphylococcus epidermidis is largely dependent on iron availability to form biofilms

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2017.08.009.Staphylococcus epidermidis has long been known as a major bacterial coloniser of the human skin, yet it is also a prominent nosocomial pathogen. Its remarkable ability to assemble structured biofilms has been its major known pathogenic feature to date. Notwithstanding important discoveries that have been accomplished, several questions about S. epidermidis biofilm formation still remain to be elucidated. This study aimed to assess whether iron availability modulates S. epidermidis biofilm formation and, if so, to explore how such modulation occurs. Biofilms of three S. epidermidis strains were grown under iron-enriched/-deficient conditions and several physiologic and transcriptomic changes were assessed. Our data revealed that while physiologic iron levels do not compromise biofilm formation, iron excess or deficiency is detrimental for this process. Conversely, biofilm cells were not affected in the same way when grown planktonically. By studying biofilm cells in detail we found that their viability and cultivability were seriously compromised by iron deficiency. Also, a temporal analysis of biofilm formation revealed that iron excess/deficiency: i) impaired biomass accumulation from 6 h onwards, and ii) induced changes in the biofilm structure, indicating that iron availability plays a pivotal role from an early biofilm development stage. The expression of several putative iron-related genes, namely encoding siderophore biosynthesis/transport-related proteins, was found to be modulated by iron availability, providing a biological validation of their function on S. epidermidis iron metabolism. This study therefore provides evidence that iron plays a pivotal role on S. epidermidis biofilm formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684) and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124FEDER-027462). FO and AF acknowledges the Portuguese Foundation for Science and Tecnology for the financial support through the individual grants SFRH/BD/101399/2014 and SFRH/BPD/99961/2014. NC is an Investigador FCT.info:eu-repo/semantics/publishedVersio

Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis

<p>Abstract</p> <p>Background</p> <p>Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. <it>Staphylococcus epidermidis </it>is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct <it>S. epidermidis </it>bacterial biofilms.</p> <p>Results</p> <p>RNA extracted using the different kits was evaluated for quantity, purity, integrity, and functionally. All kits were able to extract intact and functional total RNA from the biofilms generated from each <it>S. epidermidis </it>strain. The results demonstrated that the kit based on mechanical lysis and organic extraction (FastRNA^®Pro Blue) was the only one that was able to isolate pure and large quantities of RNA. Normalized expression of the <it>icaA </it>virulence gene showed that RNA extracted with PureLink™ had a significant lower concentration of <it>icaA </it>mRNA transcripts than the other kits tested.</p> <p>Conclusions</p> <p>When working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Among the RNA extraction kits tested, FastRNA^®Pro Blue was the best option for both <it>S. epidermidis </it>biofilms used.</p

Leituras e compreensões de cerrado pela comunidade do Assentamento de Rio Bonito, em Cavalcante, Goiás

Monografia (graduação)—Universidade de Brasília, Faculdade de Planaltina, Curso de Graduação em Gestão Ambiental, 2014.Este trabalho é um mergulho no universo dos moradores do Assentamento Rural Rio Bonito/Órfãos, no Norte de Goiás, para entender as percepções e interrelações deles com o lugar e com o Cerrado. Os conceitos utilizados foram os de Pertencimento, Topofilia e Representação Social. Na pesquisa, foram feitas entrevistas semi-estruturadas, com abordagem direta e coleta de dados por meio de interações e descrições de pessoas e lugares. A técnica para análise do material é a do Discurso do Sujeito Coletivo, que tem como foco captar as representações sociais, com fundamento na teoria da Representação Social. Consiste na sistematização de dados qualitativos por meio da tabulação dos dados. Para isso, foi empregada a ferramenta Qualiquantisoft, que auxilia na identificação das representações sociais. O trabalho identificou de que forma os moradores se apropriam do ambiente e interagem com ele e em que medida sua relação com o lugar lhes é relevante ou indiferente. Viu-se que essa tarefa, ao mesmo tempo em que é facilitada pelo fato de 70% dos entrevistados terem afeição pelo lugar e de 47% se sentirem pertencentes a ele, é dificultada pela precariedade da infraestrutura e pela carência de serviços públicos básicos no assentamento. Mas o senso de pertencimento e as representações sociais que as pessoas têm sobre o Cerrado podem ajudar a impedir a degradação ambiental. Ao mesmo tempo em que 29% demonstram, em suas respostas, manter com o Cerrado uma relação sustentável, e 30%, uma relação de fruição, é expressiva (32%) a parcela dos que não expressam uma compreensão conceitual do Cerrado. Para esses a interação com o bioma se dá puramente pela vivência. Quanto ao significado do Cerrado, as visões dos moradores são muito diversificadas – a maioria o vê como reserva ambiental (22%) ou tem dele uma visão utilitarista (22%). Outras visões são a beleza cênica (12%), o aspecto lúdico (6%). Chega a 13% o total dos moradores que vêem o Cerrado como área a ser desmatada para produção. As múltiplas formas de representação dos moradores com o Cerrado evidenciam que, na maioria, eles estabelecem uma relação de fruição com o ambiente e têm dele uma visão utilitarista, de aproveitamento do solo para agricultura e pecuária e de extração de madeira para construção. Na prática, aceitam que uma parte do Cerrado seja removida para o plantio e para a criação de gado. Outros têm o Cerrado como lugar de coleta de frutas e plantas medicinais. Ou seja, o bioma é de suma relevância para as pessoas, pois podem usufruir dos recursos naturais. Uma parcela mínima da comunidade considera o Cerrado irrelevante para reproduzir modos de vida. E alguns até vendem madeira, retirada da reserva legal, sem se preocupar com o replantio, como forma de conseguir renda para amenizar a situação precária em que vivem. _____________________________________________________________________________ ABSTRACTThis paper explores the universe of rural settlers of Rio Bonito-Órfãos, Northern Goiás, in order to understand their perceptions and interrelations to the place and the Cerrado vegetation. The concepts used were that of belonging, topophilia and social representation. Semi-structured interviews were conducted during the research, with a direct approach, and data collected through interactions and descriptions of individuals and places. The technique to analyse the material is the Collective Subject Discourse, targeted to seize social representations, based on the Social Representation Theory. It consists on systematizing qualitative data by data charting. For this reason, the tool Qualiquantisoft was used in order to assist on identifying social representations. The research identified the way in which the settlers avail of, and interact with the environment and to what extent their relation with the place is either relevant or indifferent to them. It became clear that the task, while facilitated by the fact that 70% of the respondents have affection for the place and 47% feel belonging to it, is hampered by poor infrastructure and want of basic public services in the settlement. However, the sense of belonging and social representation that the individuals have towards the Cerrado may help preventing environmental degradation. While 29% displayed, in their responses, keeping a sustainable relation with the Cerrado, and 30% a relation of fruition, a large part of settlers fail to express a conceptual understanding of the Cerrado. For them, interaction with the biome takes place by experience only. Regarding the Cerrado significance, the perception of settlers are widely scattered – the majority of them sees it as an environmental reserve (22%) or favour an utilitarian approach (22%). Other views are that of scenic beauty (12%) and playfulness (6%). The percentage of settlers that see the Cerrado as an area to be deforested for production reaches 13%. The inhabitants’ multiple ways of representation within the Cerrado highlight that most of them set up a fruition relation with the environment and have a utilitarian view about the use of land for agriculture, livestock and logging for constructions. In practice, they accept that part of the Cerrado be removed for planting and cattle farming. Some of them see the Cerrado as a place for gathering fruit and medicinal plants. In other words, the biome is extremely important for the people because they can take advantage of natural resources. A small part of the community considers the Cerrado irrelevant for reproducing ways of life. And some of them even sell wood from the legal forest reserve, not taking into account the reforestation as a way of profiting to soften their precarious situation

Relacionamentos y conocimientos en lo proceso de internacionalización de la firma

Based on the variables relationship and knowledge, this article aimed at analyzing how a multinational enterprise selects an entry mode to operate in a particular international market and how this initial choice evolves over time. We devised a rather new theoretical framework to address it by combining three theoretical approaches that have dealt with the firm internationalization: the Uppsala model, the relational approach, and the subsidiary development literature. We constructed a qualitative backward-looking longitudinal case study of the internationalization process of a North-American multinational enterprise in the Brazilian market. Results show that four types of relationships and three types of knowledge played the role in the events that characterized the internationalization of this firm. Based on these results, five new hypotheses concerning the interplay between relationships and knowledge in the internationalization process of the firm are suggested for future empirical tests.A partir das variáveis relacionamento e conhecimento, o objetivo neste artigo foi analisar como a multinacional seleciona um modo de entrada para operar em um determinado mercado internacional e como essa escolha inicial evoluiu ao longo do tempo. Para tanto, elaborou-se um quadro teórico que combina três abordagens teóricas de internacionalização da firma: o modelo de Uppsala, a abordagem relacional e a literatura de desenvolvimento de subsidiárias. O método de pesquisa utilizado foi o estudo de caso de natureza qualitativa e perspectiva longitudinal do processo de internacionalização de uma multinacional estadunidense no mercado brasileiro. Os resultados mostram que quatro tipos de relacionamentos e três de conhecimentos se fizeram presentes nos eventos que caracterizaram a internacionalização dessa firma. Com base nesses resultados, foram sugeridas cinco novas hipóteses para futuros testes empíricos, as quais versam, em geral, sobre a interação entre relacionamentos e conhecimentos no processo de internacionalização da firma.Desde las variables relacionamiento y conocimiento, el objetivo de este trabajo fue analizar como la multinacional elige un modo de entrada para actuar en un mercado internacional y como esta elección inicial evoluciona a lo largo del tiempo. Por lo tanto, elaboramos un cuadro teórico que combina tres enfoques teóricos de internacionalización de la firma: el de Uppsala, el relacional y la literatura de desarrollo de subsidiarias. El método de investigación usado fue el estudio de caso de naturaleza cualitativa y perspectiva longitudinal del proceso de internacionalización de una multinacional estadunidense en el mercado brasileño. Los resultados muestran que cuatro tipos de relacionamientos y tres de conocimientos se hicieron presentes en los eventos que caracterizan la internacionalización de tal firma. Con base a los resultados obtenidos, fueron sugeridas cinco nuevas hipótesis para futuros testes empíricos, las cuales, en general, tratan acerca de la interacción entre los relacionamientos y conocimientos en el proceso de internacionalización de la firma

Assessing and reducing sources of gene expression variability in Staphylococcus epidermidis biofilms

Gene expression quantification can be a useful tool in studying the properties of bacterial biofilms. Unfortunately, techniques such as RNA extraction, cDNA synthesis, and quantitative PCR (qPCR) can introduce variability into mRNA transcript measurements, obscuring biologically relevant results. Here we sought to identify the steps that impair accurate gene expression quantification from Staphylococcus epidermidis biofilm samples. We devised an experimental setup that could be used to determine the contribution of each experimental step to the variability of mRNA transcript measurement. Among factors tested, biofilm growth contributed the most bias to gene expression quantification. Additional experiments demonstrated that pooling biofilms together reduced this variability, resulting in more accurate gene expression analysis results. We therefore recommend pooling in order to reduce the variability associated with gene expression quantification from biofilm samples.We would like to thank Kimberly K. Jefferson at Virginia Commonwealth University for reviewing the manuscript. This work was co-funded by Fundacao para a Ciencia e a Tecno logia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309, FCT Strategic Project PEst-OE/EQB/LA0023/2013, and FCT project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and by OREN, FEDER, ON2 project. NORTE-07-0124-FEDER-000027. N.C. is an Investigador FCT

Novos cenários tecnológicos para gestores de bibliotecas universitárias públicas

Nos últimos anos, surgiram diversas reflexões relacionadas às rápidas mudanças, nas áreas científica, política, educacional e social, resultantes das inovações tecnológicas. A finalidade deste artigo é apresentar o resultado da pesquisa de mestrado que teve como objetivo estudar, analisar e sistematizar as principais ideias que norteiam e configuram a gestão de bibliotecas universitárias públicas, especificamente no que se refere às inovações tecnológicas. Para tanto, foi utilizado o método comparativo, elaborado a partir de quatro categorias de pesquisa, fundamentados na base teórica referente à gestão de bibliotecas universitárias, visando a sua avaliação. Foram focos da pesquisa as questões que provavelmente teriam maiores impactos nas bibliotecas universitárias, além dos principais desafios enfrentados: planejamento e avaliação; pessoal; usuários; produtos e serviços. Identificou-se a evolução e a adaptação das bibliotecas universitárias em relação ao uso das Tecnologias da Informação e Comunicação, a fim de melhor atender as necessidades informacionais de seus usuários. Planejar a unidade informacional e visualizar o futuro são possibilidades advindas da gestão do conhecimento, que podem instrumentalizar os gestores a posicionarem as bibliotecas no contexto das universidades, de forma que sejam prioritárias na alocação de recursos orçamentários e que se consolidem como corresponsáveis pelo processo de geração do conhecimento na universidade

Comparative transcriptomic analysis of Gardnerella vaginalis biofilms vs. planktonic cultures using RNA-seq

Bacterial vaginosis is the most common gynecological disorder affecting women of reproductive age. Bacterial vaginosis is frequently associated with the development of a Gardnerella vaginalis biofilm. Recent data indicates that G. vaginalis biofilms are more tolerant to antibiotics and are able to incorporate other bacterial vaginosis -associated species, yielding a multi-species biofilm. However, despite its apparent role in bacterial vaginosis, little is known regarding the molecular determinants involved in biofilm formation by G. vaginalis. To gain insight into the role of G. vaginalis in the pathogenesis of bacterial vaginosis, we carried out comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression levels of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. Similar to other bacterial species, expression of genes involved in antimicrobial resistance were elevated in biofilm cells. In addition, our data indicate that G. vaginalis biofilms assume a characteristic response to stress and starvation conditions. The abundance of transcripts encoding proteins involved in glucose and carbon metabolism was reduced in biofilms. Surprisingly, transcript levels of vaginolysin were reduced in biofilms relative to planktonic cultures. Overall, our data revealed that gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity. This phenotype may contribute towards the chronic and recurrent nature of bacterial vaginosis. This suggests that G. vaginalis is capable of drastically adjusting its phenotype through an extensive change of gene expressionThis work was presented at Biofilms7 meeting (26–28 June 2016, Porto, Portugal) and was awarded the Biofilms7—SPM Young Researcher Award. N.C. is an Investigador FCT. This work was funded by Fundação para a Ciência e a Tecnologia (FCT) by the strategic project of UID/BIO/04469/2013 unit and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and by the National Institutes of Health; P60 MD002256 “VCU NIMHD Comprehensive Center of Excellence”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The following authors had an individual FCT fellowship: J.C. (SFRH/BD/93963/2013) and A.F. (SFRH/BPD/99961/2014)