DDI - Teses de doutoramento
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- Decrypting the diversity of microbiome in aquaculturePublication . Salgueiro, Vanessa; Caniça, Manuela; Manageiro, Vera; Nogueira, IsabelAquaculture can play an important role in reducing the overexploitation of natural re- sources and feeding the world’s growing population. However, the use of e.g., antibiotics in aquaculture can favor the development of resistant bacteria and jeopardize the safety of its products. Thus, this Ph.D. thesis aimed to contribute to the deciphering of aquaculture’s mi- crobiome and resistome, as well as to the understanding of the role of mobile genetic elements (MGE) in the dissemination of resistance genes in these environments. Several approaches were used, to obtain the results that most reflect the microbiome and resistome of seabream and bivalve mollusks from aquaculture. All microbiomes studied were very diverse, encompassing commensal and pathogenic bacteria from seabream and bivalve mollusks (e.g., Aeromonas,Kocuria, Pseudomonas and Vibrio genera), as well as bacteria important in human medicine (e.g., Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae). Twenty-one new se- quence types were described in Aeromonas spp., Citrobacter sp., Enterobacter spp., Shewanella spp., Staphylococcus sp. and Vibrio spp. Decreased susceptibilities to phenicols, oxytetracy- cline, β-lactams (namely carbapenems), quinolones, glycopeptides, mupirocin, erythromycin, and colistin were found. The resistome also revealed a great diversity of genes in all samples studied associated with antibiotics (e.g., blaTEM-1B, mecA, sul2, mcr-9.1), disinfectants (e.g.,formA-type), and heavy metals (e.g., sil) resistance. Twenty-five different genes related with increased virulence were also detected. Thirteen new β-lactams resistance genes were identi- fied (e.g., blaCTX-M-246, blaFOX-18, and blaOXA-958) and 35 other resistance genes, namely for antibi- otics (e.g., mcr-9 and qnrD2), heavy metals (e.g., emrA and mdtE) and disinfectants ( sitABCD- type), and virulence factors (e.g., astA and hlyF) were here described for the first time associated with aquaculture. Our results suggest that some of these resistance genes (e.g., erm(T)-type,qnrB19, catA1-type, tet(A), dfrA-type, aph(6)-Id, qacE∆ 1 and merA) are being disseminated by MGE such as plasmids, class 1 integrons, and Tn As1. These findings not only expand our knowledge about aquaculture’s microbiome and resistome, but also provide the necessary xiv information to implement the most suitable measures to control antibiotic resistance in aqua- culture environments.
- Aspergillus no contexto “One Health”: Epidemiologia molecular, resistência a azóis e novos compostos com atividade antifúngica frente a isolados clínicos de humanos e de aves aquáticasPublication . Melo, Aryse Martins; Xavier, Melissa Orzechowski; Sabino, RaquelO contexto "One health" considera a saúde em três pilares: saúde animal, saúde humana e saúde do ecossistema. Nesta abordagem, evidencia-se o papel das mudanças ambientais na emergência de doenças crônicas e infecciosas, e, como forte aliada no desenvolvimento de estratégias de prevenção de doenças. Fungos do gênero Aspergillus se enquadram substancialmente nesta abordagem, tendo em vista sua ubiquidade, bem como sua importância como potenciais patógenos animais e humanos. Além da resistência intrínseca apresentada por algumas espécies, a emergência da resistência a azóis adquirida por isolados de Aspergillus fumigatus sensu stricto é uma preocupação da comunidade científica a nível global. Este trabalho teve como objetivo a avaliação da epidemiologia molecular de isolados clínicos de Aspergillus de aves e humanos do sul do Rio Grande do Sul, Brasil, e avaliar perfil de suscetibilidade e mecanismos de resistência aos azóis. Como resultados, realizamos uma ampla revisão de literatura no contexto One Health, com enfoque no papel da aves como indicador ambiental e na dispersão de cepas de Aspergillus; descrevemos pela primeira vez a aspergilose em albatrozes durante a reabilitação; e descrevemos casos de aspergilose em aves aquáticas de vida livre. Também obtivemos importantes resultados in vitro sobre a atividade antifúngica do composto disseleneto de difenila sozinho e em combinação com antifúngicos clássicos. Em relação a pesquisa de resistências aos azóis em A. fumigatus, não foram detectadas mutações relacionadas ao contexto ambiental da emergência da resistência nessa espécie fúngica na região estudada no Brasil. Além disso, a pesquisa de diversidade genética demonstrou elevada diversidade nos isolados estudados, pelo que se conclui que a técnica utilizada é de grande valia para o acompanhamento clínico dos pacientes, além de permitir confirmar que o ambiente de recuperação de animais é uma potencial fonte de infecção para aves (no caso estudado, pinguins) durante reabilitação. A detecção de um mesmo genótipo em estirpe de A. fumigatus isolado em um paciente humano e um pinguim de vida livre demonstraram a importância de uma abordagem One Health do Aspergillus e da aspergilose, uma vez que uma mesma estirpe possui potencial de colonização/infecção tanto em animais como em humanos. Por fim, como resultado da parceria internacional estabelecida durante o desenvolvimento desse trabalho, tivemos o primeiro reporte da mutação de resistência TR34/L98H em ambiente indoor em Portugal, e o resultado da vigilância em Aspergillus realizada por um laboratório de referência em Portugal, no qual encontrou-se grande prevalência de espécies crípticas de Aspergillus em isolados clínicos e ambientais, além de resistência panazol em estirpes de A. fumigatus, confirmando-se os mecanismos moleculares envolvidos com a resistência antifúngica no gene cyp51A. Os resultados obtidos neste trabalho mostram a importância da abordagem multidisciplinar do Aspergillus e da aspergilose, e a necessidade de ampliar a vigilância de Aspergillus no contexto One Health, com objetivo de entender melhor os mecanismos envolvidos na dispersão de estirpes, e na adoção de medidas mais efetivas de monitoramento da emergência da resistência, bem como para adoção de estratégias de controle do crescimento desse problema global.
- Phenotypic and genotypic characterization of Salmonella spp. isolates in PortugalPublication . Silveira, Leonor; Gomes, João; Machado, Jorge; Mota, LuísSalmonellosis remains one of the most common causes of gastrointestinal infection in the world, and represents a considerable burden in developing and developed countries. As such, Salmonella surveillance is essential to control infection and decrease the economic burden of the disease, and the Portuguese National Institute of Health has been working in this field roughly since 1950. Considering the importance of the surveillance of Salmonella enterica, the main goal of this work was to contribute to a better knowledge of relevant serovars circulating in Portugal, either the most common, or serovars exhibiting important fitness traits that confer ecological advantages over other strains. In order to achieve this objective, first we analysed decades of data and established the trends of Salmonella enterica serovars circulating in Portugal. Additionally, whole-genome sequencing (WGS) was implemented at the National Reference Laboratory to the surveillance of Salmonella enterica, and was evaluated as a substitute of traditional typing methods, such as serotyping and multiple-locus variable number tandem repeat analysis. WGS proved to be a highly discriminatory method for the detection of suspected outbreaks and even previously undetected clusters. We identified several isolates carrying multiple resistance markers and mobile genetic elements that confer the potential of persistence and spread of these bacteria. This PhD dissertation highlights the need for the adjustment of the existing regulations for foodstuffs monitoring and the investigation of safe control measures in animal production, as a means to control the spread of resistant and persistent clones that may contribute to the increase of severe infections and to the spread of those markers to other bacteria. This work stands as an important contribution for the insight on Salmonella enterica isolates circulating in Portugal, as well as a contribution for the reinforcement of the capacitation of the Portuguese National Reference Laboratory.
- The role of wild leporids as reservoirs of infectious agentsPublication . Carvalho, Carina Luísa da Costa; Duarte, Elsa Leclerc; Duarte, Margarida; Zé-Zé, LíbiaThe European wild rabbit (Oryctolagus cuniculus) and the Iberian hare (Lepus granatensis), are keystones species in various ecosystems of the Iberian Peninsula and on the local game-based economy. This thesis aimed to investigate the rabbit haemorrhagic disease virus 2 (RHDV2), detected in Portugal in 2012 and currently disseminated in the continent and autonomous regions (Azores, Madeira, Berlengas). Given its alarming impact in the wild rabbit populations, the Portuguese Government has recently activated a plan aiming the control of the disease (Despatch 4757/2017 of May 31st). A second objective of this thesis was the epidemiological surveillance of Francisella tularensis zoonotic vector-borne pathogenic bacterium, with potential impact on Human Health. The Iberian hare is considered reservoir and potential sentinel species for this pathogen. Several methodologies were used to carry out the studies presented in this work, including basic pathology, microbiology and molecular methods. Data analysis involved the resource to phylogenetic inference, statistical programs and bioformatics (e.g. R software). This work enabled the development and validation of the first molecular diagnostic method for RHDV2, currently figuring in the OIE manual, and allowed insights into to the virus dynamic evolution in different epidemiologic and geographic contexts, widening the comprehension of RHDV2 phylogenetic relations among the strains that circulate in Portugal from 2012 until 2017. This study also made possible to ascertain the tularaemia epidemiologic situation in Portugal, confirming the role of wild leporids as reservoirs for the agent and enabling preliminary conclusions on Public Health risk in the country. Moreover, this work allowed confirming ticks as the main vectors for Francisella tularensis in Portugal. Globally, this work contributed to the state-of-the-art of both infections and produced relevant information that can be used to adjust the medical and sanitary prophylactic measures of both diseases to the present reality.
- Borrelia lusitaniae and Lyme borreliosis in Portugal: a contribution to the study of an emerging bacterial zoonosis with an impact on public healthPublication . Lopes de Carvalho, Isabel; Núncio, Maria Sofia; Carolino, M. ManuelaLyme borreliosis, is a tick-borne disease caused by Borrelia burgdorferi sensu lato, that is considered an emerging disease in some regions of the world, namely in Portugal. The B. burgdorferi s. l. complex includes 16 different species that are transmitted to the vertebrate host by the tick vector. The principal vector in Europe is Ixodes ricinus. LB is a multisystem disease involving many organs such as the skin, the nervous system, the joints, and the heart. The main goal of this project is to examine specific biological and molecular aspects of B. lusitaniae. The aims of this dissertation are divided into two main topics: Human infection and the eco-epidemiological study of B. burgdorferi s.l. in Portugal. Although LB is a mandatorily notifiable disease in Portugal, the evaluation of CEVDI/INSA data concerning human cases and the number of notified cases shows that this disease is clearly underreported in Portugal. In the period of 1999-2004, the estimated incidence was 0.4 per 100 000 inhabitants, which is relatively low when compared with some endemic countries. In this work it was reported an isolation of B. lusitaniae from a human patient presenting with a vasculitis-like syndrome that confirm the patogenicity of this genospecies. This clinical report supports also the hypothesis that the clinical manifestations associated with infection by B. lusitaniae include some clinical symptoms until this moment considered unspecific for LB. In a prospective study, twelve patients with clinical diagnostic of LB were analysed and enabled the first detection of B. afzelii DNA in a patient with neurological symptoms. A survey of ixodid ticks collected in Madeira Island and southern region revealed that both are hotspot regions for the presence of B. lusitaniae, however other Borrelia genospecies were detected in Madeira Island. Moreover, Ixodes ricinus species was confirmed as the principal vector of this zoonosis in Portugal. Other species such as Dermacentor marginatus were found to contain Borrelia DNA, but the ability of this tick to transmit B. burgdorferi is unknown. The sequences analysis of the isolates obtained from ticks from the southern region reveal that some recombinants strains are present which could indicate that an intersection in the allelic profiles of the B. lusitaniae populations exists. In this dissertation is also described the first report of dual infections of Rickettsia helvetica, R. monacensis and R. slovaca, each of which infected with B. lusitaniae, highlighting the increased likelihood of infection by multiple agents to humans. Concerning the study on migratory shorebirds the detection of B. garinii DNA was observed in three different genera (black-tailed godwits Limosa l. limosa, Icelandic black-tailed godwit Limosa l. islandica and little stints Calidris minuta) and also Francisella tularensis subsp. holarctica DNA in a little stint. The studies conducted in animal species prove the involvement of the small mammals, Apodemus sylvaticus, as a potential reservoir of B. lusitaniae with the first isolate from this animal species. Seropositive samples were obtained from M. spretus and R. rattus species. Phylogenetic analyses grouped this new isolate near other B. lusitaniae strains isolated within this dissertation and most closely aligned to the North African clade. Furthermore the importance of lizards (Teira dugessi) in the maintenance of B. burgdorferi s.l. was confirmed. Potential B. valaisiana isolation from lizard tissues and detection on parasitizing ticks was reported for the first time, but this observation needs conformation. B. lusitaniae DNA was also detected in lizard tissues and in ticks that are collected from them. However, the sequence data from lizards’ isolation suggest the co-existence of B. lusitaniae. This data indicates that there may exist a sylvatic cycle specific to the B. lusitaniae, maintained by a variety of hosts and reservoirs. Many aspects introduced and explored in this work will require more detailed investigations. However, this dissertation contributes to clarify some important epidemiological aspects of the clinical and laboratorial diagnosis of LB, a disease with impact in public health in Portugal.
- Assessing antibiotic resistance in Gram negative bacteria from animals and the wider environmentPublication . Jones-Dias, Daniela; Caniça, Manuela; Nogueira, IsabelThe alarming increase in the levels of antibiotic resistant bacteria in clinical practice launched the call for a broader understanding of this event. This Ph.D. thesis aimed to unravel the main mobile antibiotic resistance determinants circulating in Gram negative bacteria from non-human sources, showing the contribution of mobile genetic elements for the overall process. The susceptibility and molecular epidemiological studies performed on different collections of bacterial isolates showed the predominance of multidrug resistant Escherichia coli in animals of different origins, soil and vegetables. E. coli and other species of Gram negative bacteria were related with carriage of diverse antibiotic resistance genes [e.g. blaCTX-M-1, blaCTX-M-15, blaCMY-2, blaGES-11, qnrS1, aac(6’)-Ib-cr, strAB, tet, drfA, aadA, mcr-1] that were associated with a transferable genetic support. Indeed, an assortment of mobile genetic elements (e.g. IncI1 and IncF plasmids, ISEcp1 insertion sequences, Tn402 and Tn7 transposons and class 1, 2 and 3 integrons) was detected in the genetic proximity of those antibiotic resistance genes, suggesting their profound involvement, not only in interspecies dispersion, but also in the movement of the genes within the cell. Specific genomic investigation of two Enterobactericeae isolates and the proteomic study of a third, underscored the potential of massive omic approaches to study antibiotic resistance as a global process. One of the key findings released from these studies is that antibiotic resistance is not only linked to virulence and pathogenicity, but can also be connected to core bacterial metabolic processes. The results obtained throughout this thesis extend our knowledge on the distribution of mobile antibiotic resistance genes in animals, environment and, ultimately, in the food chain. If the gathered results increased our concerns towards the current distribution of antibiotic resistant bacteria, they can also be an encouragement to address this problem at a global scale.
- Deteção, transmissão e diversidade genotípica de Giardia duodenalisPublication . Júlio, Cláudia; Tenreiro, Rogério Paulo de Andrade; Ângelo, Helena FernandesGiardia duodenalis é um microrganismo eucariota unicelular flagelado, sendo um dos protozoários parasitas mais comuns no intestino do homem e o agente etiológico da giardiose. Em Portugal é reduzido o número de trabalhos publicados sobre este parasita pelo que, os objetivos deste estudo foram avaliar a frequência de infeção e fatores de risco associados, principais vias de transmissão e caraterização genotípica dos diferentes isolados encontrados. Foram estudadas 844 crianças selecionadas aleatoriamente em 25 centros de saúde, utentes do programa nacional de vacinação, do distrito de Lisboa. Foi recolhida uma amostra de fezes e um questionário de cada criança. A frequência de infeção por Giardia foi diagnosticada por exame direto e pelo método ELISA. Para estudo das fontes de infeção recolheram-se 74 amostras de água, processadas pelo Método USEPA 1623 para detetar Giardia spp. e Cryptosporidium spp. Os indicadores fecais e os parâmetros físico-químicos foram analisados de acordo com a Diretiva (2006/7/EC). Foram igualmente recolhidos 13 líquidos duodenais, 304 biopsias e 217 fezes de individuos sintomaticos. Na caraterização genotípica foram amplificados os genes gdh, bg, tpi e o gene de rRNA 18S. A frequência de infeção nas crianças assintomáticas foi 6,9%. As variáveis analisadas que constituíram fator de risco associado à infeção foram o nível de escolaridade dos pais, coinfecção com H. pylori, ausência de sistema de saneamento básico e contato com animal de estimação. Os resultados obtidos nas amostras ambientais apontam para uma ampla distribuição do protozoário nas praias fluviais portuguesas embora o risco para a saúde pública seja baixo. Foi encontrada uma alta frequência do genótipo A comparativamente ao B, tal como acontece nos restantes países do sul da Europa, e confirmada a existência de transmissão antroponótica em crianças e adultos em Portugal. Foi detetado, pela primeira vez, um surto de giardiose em Portugal continental.
- Sistema dinâmico da doença meningocócica em Portugal:caracterização e modelo de transmissãoPublication . Simões, Maria João; Pereira, J. Moniz; Mendes, Pedro; Brum, Laura[PT] Neisseria meningitidis e um comensal frequente na nasofaringe humana que ocasionalmente causa doença meningocócica invasiva (DM). A DM constitui um problema de Saúde Pública devido a incidência alta em crianças, com elevadas taxas de letalidade e sequelas. A vacinação constitui uma medida de controlo. Receia-se que a introdução da vacina conjugada que apenas protege para meningococos C (MenC) origine a substituição dos serogrupos de meningococos, cenário já conhecido após vacinação de crianças com vacina conjugada para pneumococos, que e igualmente comensal da nasofaringe humana com elevada variabilidade genética. E questão desta tese saber se, depois da introdução da vacina MenC em Portugal, o nicho deixado por estirpes C vira a ser ocupado por outros serogrupos, aumentando a incidência da DM causada por serogrupos não incluídos na vacina. Respondeu-se e esta questão com a descrição da epidemiologia da DM, caracterização da doença em Portugal e desenvolvimento de um modelo, baseado na metodologia de Dinâmica de Sistemas, que traduz o comportamento dinâmico da DM. O modelo permite conhecer a importância relativa das variáveis que afectam a epidemiologia e simular cenários de evolução da doença. As características epidemiológicas (incidência e serogrupos/grupo etário, genótipos, proporção de complexos clonais hiper virulentos em estirpes invasivas, efeito da vacina MenC) revelaram-se semelhantes em Portugal e noutros países europeus. O total anual de doentes devolvido pelo modelo é sobreponível ao número de doentes na base de dados nacional. A idade, número de fumadores e capacidade de infecção/invasão das estirpes são as variáveis que mais afectam o modelo. Meningococos de serogrupos não C não ocuparam o nicho deixado pelas estirpes C depois da introdução da vacina. Contudo, a recombinação genética, base da clonalidade da população bacteriana associada a doença invasiva, ocorre continuamente, pelo que e espectável a emergência de estirpes não C, endémicas se bem adaptadas, ou epidémicas se com elevada capacidade de infecção/invasão.
- Dynamics of β-lactamases in Gram-negative bacteriaPublication . Manageiro, Vera; Caniça, Manuela; Caeiro, Maria Filomenaβ-Lactamase production is the most important resistance mechanism among Gram-negative bacteria. The overall aim of this PhD thesis was to contribute to the knowledge of molecular epidemiology of β-lactamases and to the understanding of their diversity in a structural-functional level. To accomplish this aim, several studies with different approaches were performed. The emergence of β-lactamase-producing isolates, as well as the appearance of new epidemic clones, is of great concern. The studies presented in the first chapter of results, have clearly shown that specific extended-spectrum β-lactamase (ESBL)-, plasmid-mediated AmpC β-lactamase (PMAβ)- and carbapenem-hydrolyzing class D β-lactamase (CHDL)-producing clones are able to persist in clinical settings for long periods, resulting in a complex β-lactamase endemic situation. A high diversity of β-lactamases was encountered, specifically: CTX-M family which is the most prevalent ESBL, and PMAβ (e.g., DHA-1, CMY-2, CMY-39, MIR-1, MIR-3, FOX-5 and the novel CMY-46 and CMY-50), both in Enterobacteriaceae, as well as CHDLs OXA-23 and OXA-24/40 in Acinetobacter baumannii. The results obtained in this thesis also highlight different strategies for bacterial spread of resistance that can occur through either clonal spread or horizontal gene transfer of mobile genetic elements. In the second chapter of results, structure/function correlation of five novel clinical important β-lactamases, namely three inhibitor-resistant SHV (SHV-72, SHV-84 and SHV-107), one ESBL (SHV-55) and one parental SHV (SHV-99), are presented. One of the key findings we can infer from results is that the conserved motif Lys234-Thr/Ser235-Gly236, present in class A β-lactamases, is a hot-spot for β-lactamase inhibition, meaning that new compounds can be designed to address this structural feature. In summary, the work performed in this thesis allows the elucidation on the dynamics of β-lactamases in Gram-negative bacteria, in Portugal. Molecular characterization together with biochemical data is essential for understanding the emergence of new resistance mechanisms and their spread.