Genetic diversity and evolutionary relationships among Legionella pneumophila clinical isolates, Portugal, 1987 to 2012

This study was supported by the Portuguese Foundation for Science and Technology (FCT) [grant number PTDC/SAU-ESA/64269/2006].The genetic diversity of 89 clinical Legionella isolates, collected between 1987 and 2012, in 22 hospitals from the five regions of Portugal, was analysed in this study using monoclonal antibodies (MAbs) of the Dresden panel and the sequence-based typing (SBT) protocol. The eBURST algorithm was used to infer levels of relatedness between isolates. All isolates collected were Legionella pneumophila, which were further characterised into four subgroups by MAbs, and 30 sequence types (STs) by SBT. Twelve of the STs were unique to Portugal; one of them (ST100) was represented by 32 epidemiologically related isolates. The ST44 was the profile with the highest number of epidemiologically unrelated isolates. The eBURST analyses indicate that, within the group formed by the 30 STs identified in this study, 17 STs were genetically close to at least another ST in the group. The comparison between the eBURST diagrams obtained with the STs from this study and the entire SBT database of the European Working Group for Legionella, showed that 24 (seven of them unique to Portugal) of our 30 STs were related with STs identified in others countries. These results suggest that the population of L. pneumophila clinical strains in Portugal includes both worldwide and local strains.publishersversionpublishe

Doença dos legionários : estudo da diversidade de isolados de legionella obtidos em Portugal, 1987-2016

RESUMO: O trabalho desenvolvido dividiu-se em duas áreas do conhecimento do género Legionella: a epidemiologia e a interação bactéria-hospedeiro natural. Estudou-se a epidemiologia da Doença dos Legionários em Portugal, no período entre 1987 e 2016, analisando 205 isolados, 178 dos quais recuperados de doentes com formas graves de pneumonia e 27 de amostras ambientais. Entre os isolados clínicos, 130 foram enviados através do Programa de Vigilância Epidemiológica Integrada da Doença dos Legionários e 48 foram recuperados num hospital da área de Lisboa, com vários casos de infeção hospitalar durante 21 anos e em cuja água do sistema de distribuição foi sistematicamente isolada a bactéria. Na tipificação destes isolados, utilizaram-se duas metodologias preconizadas pelo Grupo Europeu, anticorpos monoclonais (MAbs) do Painel de Dresden e a tipificação baseada em sequências (SBT). No grupo dos isolados provenientes de casos de infeção hospitalar foram aplicados mais dois métodos, o que avalia os polimorfismos de fragmentos amplificados por PCR (AFLP) e a sequenciação total do genoma (WGS). Os resultados da tipificação mostraram que todos os isolados pertencem à espécie Legionella pneumophila e maioritariamente ao serogrupo 1, e que todos, à exceção de um, reagem com o monoclonal MAb3/1. Na tipificação baseada em sequências, identificaram-se 39 perfis diferentes, 16 dos quais são novos, isto é, nunca antes identificados. Na sequenciação total do genoma, dos 48 isolados provenientes de infeção hospitalar, foi possível agrupá-los num mesmo clone, com uma microevolução marcada essencialmente pela fixação de mutações pontuais. Entre os isolados foi possível identificar três sub-linhagens, com base no número de diferenças nucleotídicas. A caracterização feita diretamente nas amostras clínicas por uma técnica de nested PCR permitiu a identificação de alguns alelos, verificando-se que só em amostras provenientes de sobrenadantes de culturas de amibas foi detetado o perfil alélico completo. Foi ainda efetuado o estudo da relação filogenética entre os perfis alélicos identificados em Portugal e os reportados à Base de dados Europeia por outros países. A população de Legionella responsável por casos de doença em Portugal é constituída por uma mistura de perfis específicos (exclusivos de Portugal) e de perfis comuns a outros países, tendo-se verificado nesta avaliação que 34 dos perfis têm relação com, pelo menos, um perfil dos existentes na referida Base. Na segunda parte desta tese, desenvolveu-se o estudo da interação Legionella-hospedeiro natural, tendo-se utilizado a espécie Acanthamoeba castellanii. Avaliaram-se as taxas de internalização e multiplicação às 4, 14 e 22h, a sensibilidade ao sódio e ao choque osmótico com potássio, e ainda, o transcritoma da bactéria, 22 horas após o início da co-cultura. Os resultados mostraram especificidade em relação às duas estirpes utilizadas e, embora estas apresentem neste momento do seu ciclo de vida um fenótipo característico de fase transmissível, verificou-se que o padrão de expressão génica é semelhante ao evidenciado por outras estirpes, na fase replicativa, sugerindo que a Legionella na fase final do seu ciclo de multiplicação intracelular, já está a preparar a próxima fase replicativa.ABSTRACT: The work developed was divided into two different areas of knowledge of the genus Legionella: epidemiology and natural bacterial-host interaction. The epidemiology of Legionnaires‘ disease was studied in Portugal between 1987 and 2016, analyzing 205 isolates, 178 of which recovered from patients with severe forms of disease and 27 from environmental samples. Among the clinical isolates, 130 were sent by the Program of Integrated Epidemiological Surveillance of Legionnaires' Disease and 48 were recovered in a hospital in the Lisbon area, with several cases of hospital infection for 21 years, and with systematic isolation over the years in the water of the distribution system. For typing of these isolates, two methodologies recommended by the European Group, the monoclonal antibodies (MAbs) of the Dresden Panel and the sequence-based typing (SBT) were used. In the group of isolates from cases of hospital infection, two other methods were applied, amplified fragment length polymorphisms (AFLP) and whole genome sequencing (WGS). The typing results showed that all isolates belong to the species Legionella pneumophila and mainly to serogroup 1, and all but one reacts with the monoclonal MAb3/1. In sequence-based typing (SBT), 39 different profiles were identified, 16 of which were new profiles, therefore never previously identified. In the whole genome sequencing, of the 48 isolates from hospital infection, it was possible to group them in the same clone, with a microevolution marked essentially by the fixation of point mutations. Among the isolates, it was possible to identify three sub-lineages, based on the number of nucleotide differences. The direct characterization on clinical samples by a nested PCR technique allowed the identification of some alleles, and it was verified that only in samples from supernatants of amoeba cultures the complete allelic profile was detected. It was also carried out the study of the phylogenetic relationship between the allelic profiles identified in Portugal and those reported to the European Database by other countries. The population of Legionella responsible for cases of disease in Portugal consists of a mixture of specific profiles (exclusive of Portugal) plus profiles common to other countries, and it was verified in this evaluation that 34 of the profiles have relation with at least one profile of the European Database. In the second part of this thesis, the study of the interaction Legionella-natural host was developed, using the species Acanthamoeba castellanii. The rates of internalization and multiplication were evaluated at 4, 14 and 22h, sensitivity to sodium, osmotic shock with potassium, and bacterial transcriptome, 22 hours after the start of co-culture. The results showed specificity in relation to the two strains used, and although they presented a transmissible phenotype, it was verified that the pattern of gene expression is similar to that evidenced by strains in the replicative phase, suggesting that Legionella at the final phase of its intracellular multiplication cycle is already preparing the next replicative phase

Cytomegalovirus Reactivation in Patients with Sepsis in an Intensive Care Unit in Portugal

INTRODUCTION: In the last few years, cytomegalovirus reactivation has been considered an aggravating factor for septic patients in Intensive Care units. The main objectives of this study were to determine cytomegalovirus reactivation in patients with a diagnosis of sepsis admitted to an intensive care unit, and whether this reactivation was related to the evolution of the patient's clinical condition. MATERIAL AND METHODS: The detection of cytomegalovirus DNA was performed by real-time polymerase chain reaction and the concentration of nine cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL- TNF-α, and INFγ) were determined by a Multiplex ELISA technique. RESULTS: Eight of 22 septic patients (36.3%) from the Intensive Care Unit of the Hospital da Luz had cytomegalovirus reactivation. No association was found between cytomegalovirus reactivation and gender, age, length of Intensive Care unit stay, duration of mechanical ventilation, and patient death. No significant differences were found in cytokine concentrations in patients with and without reactivation. However, patients with cytomegalovirus reactivation had a longer hospital stay from Intensive Care unit entry to hospital discharge or patient death (p = 0.025). DISCUSSION: Despite the low sampling rate, the present study suggests that reactivation is a frequent event in patients diagnosed with sepsis and may be related to prolonged hospital stay in these patients. CONCLUSION: The overall analysis of the results obtained and the literature review do not support the concept that cytomegalovirus monitoring should be implemented in routine practice, but it seems prudent to wait for further randomized trials using antiviral prophylaxis, before assuming a definitive attitude towards the role of cytomegalovirus in sepsis.publishersversionpublishe

Antibiotic susceptibility pattern of Portuguese environmental Legionella isolates

Publisher Copyright: Copyright © 2023 Cruz, Rodrigues, Fernandes, Santos, Paixão and Chasqueira.[This corrects the article DOI: 10.3389/fcimb.2023.1141115.].publishersversionpublishe

Antibiotic susceptibility pattern of Portuguese environmental Legionella isolates

IntroductionLegionnaires’ Disease is a pneumonia caused by Legionella spp., currently treated empirically with fluoroquinolones and macrolides. In this study, we aim to describe the antibiotic susceptibility pattern of environmental Legionella recovered in the south of Portugal.MethodsMinimal inhibitory concentration (MIC) determination of 57 Legionella isolates (10 Lp sg 1, 32, Lp sg 2-14 15 L. spp) was achieved by broth microdilution, as described by EUCAST, for azithromycin, clarithromycin, ciprofloxacin, levofloxacin, and doxycycline. ResultsFluoroquinolones were the most active antibiotic, displaying the lowest MIC values in contrast to doxycycline which had the highest. MIC90 and epidemiological cut-off (ECOFF) values were, respectively, 0.5/1 mg/L for azithromycin, 0.125/0.25 mg/L for clarithromycin, 0.064/0.125 mg/L for ciprofloxacin, 0.125/0.125 mg/L for levofloxacin and 16/32 mg/L for doxycycline.DiscussionMIC distributions were higher than reported by EUCAST for all antibiotics. Interestingly, two phenotypically resistant isolates with high-level quinolone resistance were identified. This is the first time that MIC distributions, lpeAB and tet56 genes have been investigated in Portuguese environmental isolates of Legionella

Lower prevalence of congenital cytomegalovirus infection in Portugal: possible impact of COVID-19 lockdown?

info:eu-repo/semantics/publishedVersio

possible impact of COVID-19 lockdown?

59301]. Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.Cytomegalovirus (CMV) is the most frequent cause of congenital infection all over the world. Its prevalence ranges from 0.2 to 2.2%. Transmission from children to their pregnant mothers is a well-known risk factor, particularly if they attend a childcare centre. This study aims to compare the prevalence of CMV congenital infection (CMV_CI) in Portugal (Lisbon) between two studies, performed respectively in 2019 and 2020. In the 2019 study, performed in two hospitals, we found a 0.67% CMV_CI prevalence, using a pool strategy previously tested with saliva samples. In the 2020 study, using the same pool approach in four hospitals (the previous and two additional), and based on 1277 samples, the prevalence was 0.078%. Conclusion: The close temporal coincidence with COVID-19 lockdown suggests that these measures may have had a significant impact on this reduction, although other explanations cannot be ruled-out.What is Known:• Cytomegalovirus is the leading cause of congenital infection.• Behavioural measures decrease cytomegalovirus seroconversion in pregnant women.What is New:• From 2019 to 2020 there was a significant reduction in the prevalence of congenital CMV infection.publishersversionpublishe

Estratégias de ensino das ciências promotoras de criatividade e pensamento crítico

Tese de mestrado, Educação (Didáctica das Ciências), Universidade de Lisboa, Faculdade de Ciências, 2009O estudo pretende dar resposta ao seguinte problema central: “Será que se verificam melhorias dos níveis de criatividade e de pensamento crítico quando os alunos realizam actividades de ensino-aprendizagem que apelam para o desenvolvimento da criatividade e do pensamento crítico?”. Trata-se de um estudo de design quase-experimental / grupo controlo-grupo experimental / pré-teste-pós-teste / grupos não aleatórios. As observações foram realizadas num colégio particular da região do concelho de Lisboa e nele participaram alunos de duas turmas de Ciências Físico-Químicas do 8º ano de escolaridade. Com este trabalho não se pretendeu apenas fazer uma medição dos níveis de criatividade e de pensamento crítico dos alunos. Um dos grandes objectivos deste trabalho consistia na elaboração e implementação de materiais didácticos passíveis de ser utilizados em sala de aula como promotores de criatividade e pensamento crítico. Foram seleccionadas sete actividades que integraram um programa de intervenção que foi implementado durante um período lectivo no grupo experimental. Antes e depois do programa de intervenção foram administrados testes de medição de pensamento criativo (Teste de Pensamento Criativo de Torrance) e testes de medição de pensamento crítico (Teste de Pensamento Crítico de Cornell - Nível X). A análise dos resultados obtidos no pré-teste permitiu constatar que os dois grupos eram equivalentes no que diz respeito aos níveis de Criatividade e de Pensamento Crítico. Após a conclusão do estudo verificou-se que não se registaram melhorias estatisticamente significativas nos níveis de pensamento criativo e crítico dos alunos envolvidos no estudo. Verificou-se contudo que o nível de pensamento crítico dos alunos do grupo experimental no aspecto assumpções aumentou significativamente. O estudo permitiu a reflexão sobre a importância da promoção da Criatividade e do Pensamento Crítico através das práticas lectivas dos professores e concluir sobre a necessidade de formação de professores no que diz respeito à concepção e implementação de materiais didácticos.The present study seeks to answer the following central problem: “Are there improvements of the levels of creativity and critical thinking when pupils perform teaching-learning activities that call for the development of creativity and critical thinking?”. It is a study of quasi-experimental design/ control group-experimental group/ pre-test-post-test/ non-random groups. The data were collected in a private school localized in central Lisbon and involved pupils of two classes of Physics and Chemistry of the 8th grade. The purpose of this work consisted in the development and implementation of didactic materials to be used in the classroom as promoters of creativity and critical thinking and in the measurement of the levels of creativity and critical thinking of the pupils involved. Seven activities were selected and incorporated in an intervention programme that was implemented during a school term in the experimental group. At the beginning and at the end, tests measuring creativity (Torrance Test of Creative Thinking) and critical thinking (Cornell Critical Thinking Test, Level X) were administered. Analysis of the results obtained in the pre-test revealed that the two groups were equivalent in respect of levels of creativity and critical thinking as well as critical thinking aspects. After the end of the study it was found that there were no statistically significant improvements in the levels of creative and critical thinking of pupils involved in the study. It was noted, however, that the level of the critical thinking aspect, assumptions, increased significantly in the experimental group. This study allowed to reflect on the importance of promoting creativity and critical thinking through the teachers’ practices and conclude on the need for training of teachers in relation to the design and implementation of didactic materials

Etodolac as a possible antimicrobial or adjuvant agent against ESKAPE pathogens

Introduction: Multiple-drug resistant bacteria are emerging exponentially in healthcare units, threatening public health and requiring novel therapeutic approaches. In 2017, World Health Organization published a list that frames antimicrobial resistant bacteria into priority levels for research of novel drugs to fight them. Methods & Materials: Antimicrobial resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter sp.) and Enterococcus faecalis and Escherichia coli pathogens are present in this list. Representative isolates of each species were used to test the Antibacterial and anti-biofilm formation activities of Etodolac (a Non-Steroidal Anti-Inflammatory Drug, NSAID) at 10 and 1 mM using a broth microdilution technique. Results & Discussion: Statistically significant (p< 0,05) results were observed against all tested gram-positives, particularly anti-biofilm activity against E. faecium. Etodolac had an almost null influence on tested gram-negatives, with the exception of one A. baumannii clinical isolate regarding biofilm formation inhibition. Observed differences deserve further analysis and prospection of the involved mechanisms, to unravel possible novel bacterial targets for drug development. Similar work with other NSAID’s may also be worth exploring to ascertain novel therapeutic applications for these drugs, particularly regarding biofilm formation inhibition, per si or as adjuvants of current antibiotherapy, mainly against gram-positives, as suggested by present work. Conclusion: Already approved drugs in terms of pharmacokinetics and safety may deploy faster solutions for antimicrobial therapy against priority pathogens. Current work intends to bring attention to that possibility, particularly regarding NSAIDs, anti-biofilm formation and top priority pathogens.publishersversionpublishe