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

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

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

Non-antimicrobial drugs: Etodolac as a possible antimicrobial or adjuvant agent against ESKAPE pathogens (The Open Microbiology Journal (2018), 12 (288-296), 10.2174/1874285801812010288)

The correct numbering of author ‘s affiliation is mentioned below: Sónia G. Pereira1, *, Vanessa S. Domingues2, João Theriága1, Maria de Jesus Chasqueira1, Paulo Paixão1 The original numbering of author ‘s affiliation provided was: Sónia G. Pereira1, *, Vanessa S. Domingues2, João Theriága2, Maria de Jesus Chasqueira2, Paulo Paixão2.publishersversionpublishe

Pooling Saliva Sample as an Effective Strategy for the Systematic CMV Screening of Newborns-A Multicentric Prospective Study

BACKGROUND: Cytomegalovirus is the most common cause of congenital infections worldwide. Screening all newborns in the first 2 weeks of life is the only way to detect all cases of congenital infection, allowing the monitoring of children with asymptomatic infection at birth and early intervention. AIM: In this multicenter study, we aimed to evaluate the feasibility of using a saliva pool strategy for mass screening in 7 Portuguese hospitals, and to estimate the current prevalence of this congenital infection in these hospitals. METHODS: A total of 7033 newborns were screened between June 2020 and June 2022, and 704 pools of 10 saliva samples were analyzed by polymerase chain reaction (PCR). RESULTS: Of the 704 pools analyzed, 685 were negative and 19 had positive PCR results for cytomegalovirus. After individual PCR testing, 26 newborns had positive saliva results, of which 15 were confirmed by urine testing. Thus, this study's prevalence of congenital infection was 0.21% (95% confidence interval: 0.12%-0.35%). CONCLUSIONS: In this study, the pooling strategy proved to be effective for the systematic screening of newborns, although this low prevalence raises questions regarding the cost-effectiveness of implementing universal screening. However, this prevalence is probably the result of the control measures taken during the pandemic; therefore, the rates are expected to return to prepandemic values, but only a new study after the pandemic will be able to confirm this.publishersversionepub_ahead_of_prin