Serratia marcescens bacteremia associated with schistosomiasis mansoni

The case of a 21 -year-old man coming from rural Paraíba, northeastern Brazil, with schistosomiasis mansoni associated with Serratia marcescens bacteremia, is reported. His main complaints on admission were fever, diaphoresis and chills for ten days, and diarrhoea that lasted for four days. On physical examination he hadjaundice and hepatosplenomegaly. Diagnosis of S. marcescens bacteremia was made by isolation of the bacterium in blood culture, and schistosomiasis was diagnosed by rectal and liver biopsies. This is the first time that the association of S. marcescens bacteremia and schistosomiasis mansoni is recognized. Although our case does not fit into the classic definition of prolonged bacteremia associated with schistosomiasis, it can be considered as a mild form of this association. With the improvement of medical assistance and laboratory facilities, early diagnosis of this association will be made more frequently, cases with short duration will be diagnosed few days after the start of the symptoms, and classic prolonged cases will become rarer.

Protein stability in a proteomic perspective

Dissertation presented to obtain the Ph.D degree in Biochemistry

Exopolysaccharide production by Enterobacter A47: optimization of cultivation conditions and study of polymer functional properties

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Enterococcus faecalis V583 prophages: Dynamic interactions and contribution to bacterial pathogenic traits

Dissertation presented to obtain the Ph.D degree in Biology.

Desulfovibrio vulgaris defenses against oxidative and nitrosative stresses

Dissertation presented to obtain the Ph.D degree in Biochemistry, Microbial Biochemistry

Estudo da expressão génica da Legionella pneumophila estirpe paris após co-cultura em Acanthamoeba castellanii

RESUMO: A Legionella é um bacilo Gram-negativo que replica dentro de protozoários como Acanthamoeba castellanii (A. castellanii) e no interior de macrófagos alveolares humanos, podendo resultar numa pneumonia grave. A Legionella em meio líquido tem um ciclo de vida bifásico, apresentando traços replicativos na fase exponencial e expressando factores transmissíveis na fase estacionária. Estudos recentes demonstraram que a Legionella precisa de assegurar um tempo preciso no seu ciclo de vida para efectuar com êxito a infecção das células hospedeiras. Muitos modelos de estudo foram desenvolvidos a fim de aumentar o conhecimento sobre o ciclo de vida intracelular e identificar os genes necessários para a modulação da célula hospedeira. Embora o conhecimento sobre a interacção bactéria-hospedeiro ainda seja limitado, parece que esta interacção gera um conjunto de características de virulência permitindo que a bactéria infecte células fagocíticas humanas e cause doença. O objectivo do presente projecto de investigação foi investigar e seleccionar genes críticos para a infecciosidade da Legionella pneumophila estirpe Paris (Lp Paris), desenhar e optimizar uma técnica de PCR em tempo real para o estudo da expressão génica e comparar o perfil de expressão da Lp Paris antes e depois da co-cultura em A. castellanii. Os resultados mostraram que oito dos 12 genes em estudo alteraram a sua expressão relativa após co-cultura em A. castellanii quando os ensaios foram realizados com culturas de Lp Paris na fase estacionária precoce (cinco foram induzidos e três reprimidos) Quando os ensaios foram realizados com culturas de Lp Paris na fase estacionária tardia 11 genes apresentaram repressão na sua expressão relativa. Analisando os resultados, concluímos que o perfil de expressão de Lp Paris foi modificado pela interacção com A. castellanii, no entanto essa mudança foi dependente da fase do seu ciclo de vida.-------ABSTRACT: Legionella is a pathogenic Gram-negative bacterium that replicates not only within aquatic protozoa like Acanthamoeba castellanii (A. castellanii), but also within human alveolar macrophages, which can result in a severe pneumonia. Legionella has a biphasic life cycle in broth, where exponential phase cultures display replicative traits and stationary bacteria express transmissive factors. Recent studies demonstrated that for successful infection of host cells, Legionella needs to ensure a precise timing of its life cycle. Many models of study were developed in order to learn about the intracellular life cycle and to identify the genes necessary for the host cell modulation. Although knowledge about the bacteria-host interaction is still limited, it appears that this interaction generate a pool of virulence traits, allowing the bacterium to infect human phagocytic cells and cause disease. The purpose of the present study was to investigate and select de critical genes for the infectivity of Legionella pneumophila strain Paris (Lp Paris), design and optimize a real time PCR technique for gene expression study and compare the expression profile of Lp Paris before and after co- culture of A. castellanii. The results show that eight of 12 genes in study changed its relative expression after coculture in A. castellanii when we performed the intracellular assays with early stationary phase Lp Paris cultures (five were induced and tree were repressed). When we performed the intracellular assays with late stationary phase Lp Paris cultures 11 genes showed a repressed relative expression. Analysing the results, we conclude that the expression profile of Lp Paris was modified by interaction with A. castellanii but this change was dependent of the timing of its life cycle.

Surto de listeria monocytogenes na Região de Lisboa e Vale do Tejo : 2009-2011

RESUMO - O género Listeria contém oito espécies (L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, e a L. rocoutiae), das quais duas são patogénicas. L. monocytogenes é patogénica para humanos e animais; L. ivanovii primeiramente infecta animais e raramente causa doenças em humanos. A Listeria monocytogenes é uma bactéria patogénica Gram-positiva facultativa intracelular, ubíqua na natureza. Nos últimos anos o número de casos de listeriose tem vindo a aumentar. Pode causar uma doença rara e grave chamada listeriose, especialmente nas mulheres grávidas, nos idosos ou em indivíduos com o sistema imunitário debilitado, de maneira esporádica ou em forma surtos. Realizou-se um estudo observacional, descritivo com o objectivo de se fazer a descrição e a caracterização do surto de listeriose ocorrido na região de Lisboa e Vale do Tejo entre 2009-2011. O período de maior número de casos diagnosticados de listeriose ocorreu entre o mês de Abril e Agosto de 2010. Mas a janela temporal em que ocorreu o surto estendeu-se de Março de 2009 a Janeiro de 2012. Ocorreram 51 casos de internamento com diagnóstico de listeriose, dos quais 25 casos foram confirmados, pela técnica de PFGE, pertencer à mesma estirpe I, sorotipo 4b e pulsotipo 070 e 0101. Na maioria dos casos eram do sexo feminino, com uma média de 57,14 anos de idade e com residência na região de Lisboa e Vale do Tejo. Em 96,08% dos doentes internados com listeriose apresentavam factores de predisposição, comorbilidade e /ou imunossupressão. A bacteriemia foi a manifestação clínica mais frequente, seguida da meningite. A letalidade da listeriose foi de 15,69%.

Studies of the assembly of the Streptococcus pneumoniae capsular polysaccharide

Dissertation presented to obtain the Ph.D degree in Biology.

Dissecting the function of the SpoIIIJ and YqjG membrane protein insertases during bacterial spore development

Dissertation presented to obtain the Ph.D degree in Biology, Microbial Biology

Recommendations for the detection of Leptospira in urine by PCR

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.

Deciphering the genome of Desulfovibrio gigas: the role of the hydrogenases

The work presented in this thesis describes the functional characterization of hydrogenases in the overall energy metabolism of the sulfate reducing bacterium Desulfovibrio gigas. With the complete annotation of the D. gigas genome, we were able to verify that only the two previously described hydrogenases are present in this organism, the periplasmic [NiFe] HynAB and the cytoplasmic membrane-bound [NiFe] Ech.(...)

Spore differentiation in relation to the infectious cycle of the enteric pathogen Clostridium difficile

Clostridium difficile is a gram positive, spore former, anaerobic bacterium that is able to cause infection and disease, with symptoms ranging from mild diarrhea to pseudomembranous colitis, toxic megacolon, sepsis and death. In the last decade new strains have emerged that caused outbreaks of increased disease severity and higher recurrence, morbidity and mortality rates, and C. difficile is now considered both a main nosocomial pathogen associated with antibiotic therapy as well as a major concern in the community.(...)

Detection of viable and viable nonculturable Vibrio cholerae O1 through cultures and immunofluorescence in the Tucumán rivers, Argentina

Vibrio cholerae has been sporadically isolated from rivers in Tucumán, Argentina, since the outbreak in 1991. The aim of this study was to determine the environmental reservoir of the bacterium in these rivers, assessing the presence of Vibrio cholerae non-O1 and O1 (the latter both in its viable culturable and non culturable state) and its relationship to environmental physicochemical variables. 18 water samplings were collected in the Salí River (in Canal Norte and Banda) and the Lules River between 2003 and 2005. Physical-chemical measurements (pH, water temperature, electrical conductivity and dissolved oxygen) were examined. Vibrio cholerae was investigated with conventional culture methods and with Direct Immunofluorescence (DFA-VNC) in order to detect viable non culturable organisms. All isolated microorganisms corresponded to Vibrio cholerae non-O1 and non-O139 (Lules 26%, Canal Norte 33% and Banda 41%). The majority was found during spring and summer and correlated with temperature and pH. Non culturable Vibrio cholerae O1 was detected year round in 38 of the 54 water samples analyzed. Application of the Pearson correlation coefficient revealed that there was no relationship between positive immunofluorescence results and environmental physicochemical parameters. Genes coding for somatic antigen O1 were confirmed in all DFA-VNC-positive samples, whereas the virulence-associated ctxA and tcpA genes were confirmed in 24 samples.

Follow the red road of triheme cytochromes in Geobacter sulfurreducens

The bacterium Geobacter sulfurreducens (Gs) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membrane in a process designated as extracellular electron transfer. The Gs genome was fully sequenced and a family composed by five periplasmic triheme cytochromes c7 (designated PpcA-E) was identified. These cytochromes play an important role in the reduction of extracellular acceptors. They contain approximately 70 amino acids, three heme groups with bis-histidinyl axial coordination, and share between 57 and 77% sequence identity. The triheme cytochrome PpcA is highly abundant in Gs and is most likely the reservoir of electrons destined for outer surface. In addition to its role in electron transfer pathways this protein can perform e-/H+ energy transduction, a process that is disrupted when the strictly conserved aromatic residue phenylalanine 15 is replaced by a leucine (PpcAF15L). This Thesis focuses on the expression, purification and characterization of these proteins using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The orientations of the heme axial histidine ring planes and the orientation of the heme magnetic axis were determined for each Gs triheme cytochrome. The comparison of the orientations obtained in solution with the crystal structures available showed significant differences. The results obtained provide the paramagnetic constraints to include in the future refinement of the solution structure in the oxidized state. In this work was also determined the solution structure and the pH-dependent conformational changes of the PpcAF15L allowing infer the structural origin for e-/H+ energy transduction mechanism as shown by PpcA. Finally, the backbone and side chain NH signals of PpcA were used to map interactions between this protein and the putative redox partner 9,10-anthraquinone-2,6-disulfonate (AQDS). In this work a molecular interaction was identified for the first time between PpcA and AQDS, constituting the first step toward the rationalization of the Gs respiratory chain.

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.