Neste trabalho foi estuda a interação da E. coli patogênica aviária (APEC), agente etiológico da colibacilose aviária, e células não fagocitárias. A APEC é uma ExPEC (E. coli extra-intestinais), grupo que também inclui a UPEC (E. coli uropatogênica) e a NMEC (E. coli de meningite neonatal). Foi analisado o comportamento de 8 cepas APEC - MT78, IMT2470, A2363, UEL31, UEL13, UEL17, IMT5155, UEL29 - frente a duas linhagens de células não-fagocitárias, fibroblastos aviários CEC-32 e células endoteliais humanas EAhy926. Foi realizada a genotipagem de 33 genes associados à virulência, verificou-se capacidade de associação (adesão e invasão), de invasão e de multiplicação intracelular, de citotoxicidade e de ativação das caspases 3/7 das cepas após infecção de fibroblastos aviários. Foi observado que enquanto todas as cepas foram capazes de aderir aos fibroblastos aviários, somente a cepa MT78 foi capaz de invadí-los em níveis comparáveis à bactéria invasiva Salmonella Typhymurium SL1344. As cepas APEC não induziram ativação de capases 3/7, nem foram citotóxicas aos fibroblastos. Uma vez que a cepa invasiva, MT78, e a não invasiva, IMT2470, apresentam genótipos de virulência muito similares, foi realizado o estudo da expressão por RT-PCR dos genes de virulência da bactéria crescida com e sem fibroblastos aviários, por 3 h. Os resultados mostraram a expressão de adesinas, sideróforos e protectinas/estruturas de resistência ao soro para ambas as cepas na ausência e presença de fibroblastos. A análise da expressão dos genes fimH, ompA, ibeA e gimB pela técnica RT-qPCR revelou a repressão do gene fimH na MT78, mas indução do mesmo na IMT2470. Já as invasinas, gimB e ibeA, estavam induzidas em MT78 e reprimidas em IMT2470, enquanto o gene ompA estava sendo expresso em ambas as cepas. A expressão das invasinas gimB e ibeA explica em parte o fenótipo invasivo da MT78. No presente estudo também foi analisado o comportamento das 8 cepas APEC em relação ao perfil de associação, invasão e multiplicação intracelular ao infectarem células EAhy926. As cepas não mostraram capacidade de invadir as células mas apresentaram um nível de associação muito superior ao dos fibroblastos aviários (até 14 vezes superior para algumas cepas). Este trabalho mostrou que o ensaio in vitro com CEC-32 é um modelo adequado para o estudo da interação celular entre APEC e células eucarióticas e agregou mais conhecimento sobre o patotipo. / In this work the interaction between avian pathogenic E. coli (APEC), the etiological agent of avian colibacillosis, and non-phagocytic cells was studied. APEC is an ExPEC (extraintestinal E. coli), a group that also includes UPEC (uropathogenic E. coli) and NMEC (E. coli neonatal meningitis). We analysed the behavior of 8 APEC strains - MT78, IMT2470, A2363, UEL31, UEL13, UEL17, IMT5155, UEL29 - against two non-phagocytic cell lines, avian fibroblasts (CEC-32) and human endothelial cells (Eahy926). Strains were genotyped for 33 virulence associated genes, and investigated the association capacity (adhesion and invasion), the invasion ability, intracellular multiplication, cytotoxicity and activation of caspases 3/7 of the strains after infecting avian fibroblasts. While all strains were able to adhere to avian fibroblasts, only the strain MT78 was able to invade them at levels comparable to the invasive bacterium Salmonella Typhymurium SL1344. APEC strains could not induce activation of caspases 3/7, nor were cytotoxic to fibroblasts. Since the invasive MT78 and the non-invasive IMT2470 strains presented very similar virulence genotypes, the expression of virulence genes of the bacteria grown in the absence and presence of avian fibroblasts by 3 h was analysed by RT-PCR. Results showed the expression of adhesins, siderophores and protectins/serum resistance structures for both strains in the two culture conditions, with and without fibroblasts. Analysis of the expression of fimH, ompA, ibeA and gimB by RT-qPCR revealed the repression of fimH in MT78, but the induction in IMT2470. In relation to ibeA and gimB invasins, they were induced in MT78 but repressed in IMT2470, while the ompA gene was expressed in both strains. The expression of invasins partly explains the invasive phenotype of MT78. It was also analysed the behaviour of the strains in relation to the association profile, invasion and intracellular multiplication when infecting EAhy926 human endothelial cells. The strains showed no ability to invade endothelial cells but showed a high level of association (up to 14 times higher than for fibroblasts cells for some strains). This study showed that the CEC-32 in vitro model is suitable for the study of cellular interaction between APEC and eukaryotic cells, and added more knowledge about the pathotype.

Neste trabalho foi estuda a interação da E. coli patogênica aviária (APEC), agente etiológico da colibacilose aviária, e células não fagocitárias. A APEC é uma ExPEC (E. coli extra-intestinais), grupo que também inclui a UPEC (E. coli uropatogênica) e a NMEC (E. coli de meningite neonatal). Foi analisado o comportamento de 8 cepas APEC - MT78, IMT2470, A2363, UEL31, UEL13, UEL17, IMT5155, UEL29 - frente a duas linhagens de células não-fagocitárias, fibroblastos aviários CEC-32 e células endoteliais humanas EAhy926. Foi realizada a genotipagem de 33 genes associados à virulência, verificou-se capacidade de associação (adesão e invasão), de invasão e de multiplicação intracelular, de citotoxicidade e de ativação das caspases 3/7 das cepas após infecção de fibroblastos aviários. Foi observado que enquanto todas as cepas foram capazes de aderir aos fibroblastos aviários, somente a cepa MT78 foi capaz de invadí-los em níveis comparáveis à bactéria invasiva Salmonella Typhymurium SL1344. As cepas APEC não induziram ativação de capases 3/7, nem foram citotóxicas aos fibroblastos. Uma vez que a cepa invasiva, MT78, e a não invasiva, IMT2470, apresentam genótipos de virulência muito similares, foi realizado o estudo da expressão por RT-PCR dos genes de virulência da bactéria crescida com e sem fibroblastos aviários, por 3 h. Os resultados mostraram a expressão de adesinas, sideróforos e protectinas/estruturas de resistência ao soro para ambas as cepas na ausência e presença de fibroblastos. A análise da expressão dos genes fimH, ompA, ibeA e gimB pela técnica RT-qPCR revelou a repressão do gene fimH na MT78, mas indução do mesmo na IMT2470. Já as invasinas, gimB e ibeA, estavam induzidas em MT78 e reprimidas em IMT2470, enquanto o gene ompA estava sendo expresso em ambas as cepas. A expressão das invasinas gimB e ibeA explica em parte o fenótipo invasivo da MT78. No presente estudo também foi analisado o comportamento das 8 cepas APEC em relação ao perfil de associação, invasão e multiplicação intracelular ao infectarem células EAhy926. As cepas não mostraram capacidade de invadir as células mas apresentaram um nível de associação muito superior ao dos fibroblastos aviários (até 14 vezes superior para algumas cepas). Este trabalho mostrou que o ensaio in vitro com CEC-32 é um modelo adequado para o estudo da interação celular entre APEC e células eucarióticas e agregou mais conhecimento sobre o patotipo. / In this work the interaction between avian pathogenic E. coli (APEC), the etiological agent of avian colibacillosis, and non-phagocytic cells was studied. APEC is an ExPEC (extraintestinal E. coli), a group that also includes UPEC (uropathogenic E. coli) and NMEC (E. coli neonatal meningitis). We analysed the behavior of 8 APEC strains - MT78, IMT2470, A2363, UEL31, UEL13, UEL17, IMT5155, UEL29 - against two non-phagocytic cell lines, avian fibroblasts (CEC-32) and human endothelial cells (Eahy926). Strains were genotyped for 33 virulence associated genes, and investigated the association capacity (adhesion and invasion), the invasion ability, intracellular multiplication, cytotoxicity and activation of caspases 3/7 of the strains after infecting avian fibroblasts. While all strains were able to adhere to avian fibroblasts, only the strain MT78 was able to invade them at levels comparable to the invasive bacterium Salmonella Typhymurium SL1344. APEC strains could not induce activation of caspases 3/7, nor were cytotoxic to fibroblasts. Since the invasive MT78 and the non-invasive IMT2470 strains presented very similar virulence genotypes, the expression of virulence genes of the bacteria grown in the absence and presence of avian fibroblasts by 3 h was analysed by RT-PCR. Results showed the expression of adhesins, siderophores and protectins/serum resistance structures for both strains in the two culture conditions, with and without fibroblasts. Analysis of the expression of fimH, ompA, ibeA and gimB by RT-qPCR revealed the repression of fimH in MT78, but the induction in IMT2470. In relation to ibeA and gimB invasins, they were induced in MT78 but repressed in IMT2470, while the ompA gene was expressed in both strains. The expression of invasins partly explains the invasive phenotype of MT78. It was also analysed the behaviour of the strains in relation to the association profile, invasion and intracellular multiplication when infecting EAhy926 human endothelial cells. The strains showed no ability to invade endothelial cells but showed a high level of association (up to 14 times higher than for fibroblasts cells for some strains). This study showed that the CEC-32 in vitro model is suitable for the study of cellular interaction between APEC and eukaryotic cells, and added more knowledge about the pathotype.

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T06:09:15Z (GMT). No. of bitstreams: 1 Cordeiro_MelinaAparecida_M.pdf: 3889960 bytes, checksum: 3e92b071b7e108ebb8bff09fe751b180 (MD5) Previous issue date: 2015 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

Leptospirose é uma doença infecciosa emergente cujos agentes etiológicos são espécies patogênicas do gênero Leptospira. Leptospiras patogênicas possuem inúmeros genes específicos codificando proteínas com funções desconhecidas, sugerindo que as leptospiras apresentam fatores de virulência únicos. Adesinas bacterianas são importantes fatores de virulência e, assim, a identificação de adesinas conservadas em espécies patogênicas de Leptospira pela construção de bibliotecas genômicas expostas na superfície de bacteriófagos (shotgun phage display), seguida por seleção em células e/ou componentes da matriz extracelular (biopanning), pode revelar novos antígenos e alvos para o tratamento e prevenção da leptospirose. Bibliotecas foram construídas com o DNA genômico de L. interrogans fragmentado e o fagomídeo pG8SAET, sendo testadas algumas abordagens para clonagem como a ligação entre extremidades cegas (blunt-end) e técnicas baseadas em ligação entre extremidades coesivas, incluindo a obtenção de ORESTES e a utilização de adaptadores em grampo. Apesar de serem encontradas algumas limitações, a clonagem por ligação blunt-end se mostrou a mais eficiente para a construção de bibliotecas, sendo adotada para a construção de três bibliotecas em maior escala. A seleção de novas possíveis adesinas a partir das bibliotecas construídas foi realizada em células eucarióticas através da metodologia BRASIL. A primeira biblioteca (BBT1) exibiu 106 clones totais, a partir da qual foram selecionados quatro proteínas em fase apenas com a proteína VIII do fago (pVIII). No entanto, nenhuma delas seria exposta por programas de predição na bactéria. Outras duas bibliotecas foram construídas (BBT2 e BBT3), as quais obtiveram um número ideal de clones para uma ampla cobertura do genoma (>2x107 clones). Por apresentar maior proporção de clones válidos, a BBT2 foi utilizada para a seleção de adesinas, resultando em onze clones em fase com pVIII e/ou sequência sinal do fago. Análises por programas de predição revelaram três proteínas hipotéticas, denominadas LepA962, LepA069 e LepA388, as quais poderiam estar expostas ou ser secretadas pela bactéria, sugerindo uma possível função de adesina. O estudo da proteína LepA388 levou ao reconhecimento de outras doze proteínas semelhantes e pertencentes a uma família paráloga contendo um domínio denominado DUF_61, motivo de função desconhecida presente em proteínas compartilhadas somente entre as espécies patogênicas mais virulentas de Leptospira. Por esta razão, a proteína LepA388 foi a mais estudada. A clonagem de três porções da proteína (LepA388P, LepA388NR e LepA388F) para expressão heteróloga resultou em proteínas recombinantes insolúveis e, considerando a riqueza em resíduos de cisteína presente em sua estrutura, não foi possível renaturá-las adequadamente. Diante dos obstáculos encontrados, apenas a porção contendo a sequência apresentada pelo fago (LepA388P) foi utilizada para obtenção de antissoros em camundongos, os quais apresentaram altos títulos, demonstrando a alta imunogenicidade da proteína LepA388P. O reconhecimento de proteínas nativas da família paráloga DUF_61 em extratos de diferentes sorovares de Leptospira não foi observado, assim como sua expressão in vitro a partir de bactérias em diferentes condições de cultivo. Estudos adicionais sobre a expressão in vivo e funções dos membros desta família são necessários para uma compreensão mais ampla de seu papel na biologia de leptospiras e, possivelmente, na patogênese da leptospirose. / Leptospirosis is an emerging infectious disease whose etiologic agents are pathogenic species of the genus Leptospira. Pathogenic leptospires have countless specific genes encoding proteins with unknown functions, suggesting that leptospires have unique virulence factors. Bacterial adhesins are important virulence factors and so the identification of conserved adhesins in pathogenic Leptospira species from shotgun phage display libraries, followed by selection (biopanning) in cells and/or extracellular matrix components, can reveal new antigens and strategies for leptospirosis treatment and prevention. Libraries were constructed using fragmented genomic DNA from L. interrogans and pG8SAET phagemid vector. Cloning approaches included blunt-end ligation and techniques based in cohesive-end ligation, such as ORESTES strategy and hairpin linkers. Despite some limitations, cloning by blunt-end ligation was the most efficient for library construction, being adopted for the construction of three libraries on a larger scale. Selection of new possible adhesins was performed by biopanning of the libraries in eukaryotic cells through BRASIL methodology. The first library called BBT1 exhibited approximately 106 total clones, and its biopanning resulted in four proteins fused to phage protein VIII, but none of them were expected to be exposed by the bacteria. Other libraries were built (BBT2 and BBT3) which reached the expected number of clones to obtain a larger genome representation (> 2x107 clones). Since it showed the highest proportion of positive clones, BBT2 was selected to perform a second biopanning, resulting in eleven proteins fused to phage protein VIII and/or signal peptide. In silico analysis revealed three hypothetical proteins, named LepA962, LepA069 and LepA388, that would be exposed or secreted by the bacteria, suggesting a possible adhesin function. The study of LepA388 protein led to the recognition of twelve other similar proteins belonging to a paralogous family that contains a domain called DUF_61, domain of unknown function that is present in proteins shared only among the most virulent pathogenic species of Leptospira. For this reason, the LepA388 protein was the most studied. The cloning of three portions of the protein (LepA388P, LepA388NR and LepA388F) for heterologous expression resulted in insoluble recombinant proteins, and given the presence of many cysteine residues in its structure, it was not possible to renature them appropriately. In face of the imposed obstacles, only the portion containing the sequence presented by the bacteriophage (LepA388P) was used to obtain antisera in mice, which showed high titers, demonstrating the high immunogenicity of the protein LepA388P. Recognition of native DUF_61 paralogous family proteins in extracts from distinct Leptospira serovars was not observed, as well as its in vitro expression from bacteria cultured in different conditions. Additional studies on the in vivo expression and functions of members of this family are needed for a broader understanding of their role in leptospiral biology and possibly in the pathogenesis of leptospirosis.

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa. / Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce\'s disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce\'s disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence.

Made available in DSpace on 2014-07-29T15:26:19Z (GMT). No. of bitstreams: 1 Tese Sarah Veloso Nogueira.pdf: 4968384 bytes, checksum: 1a3070a4c2260fba10919cd4f6ccd4f4 (MD5) Previous issue date: 2010-03-11 / Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis, prevalent in Latin America. Extracellular matrix (ECM) is a complex net where collagens, laminin and fibronectin can be found and, when exposed, is the first site for the fungus adhesion. Our aim was to study genes involved in the adhesion process using Representational Difference Analysis (RDA). RDA is a PCR-coupled subtractive method that allows the isolation of genes differentially expressed in two different cDNA populations. Hence, cDNAs were synthesized from RNAs extracted from P. brasiliensis yeast cells adhered to collagen and fibronectin to identify overexpressed genes. Genes involved in a wide range of cellular process were found and PbCtr3 (cooper transporter) and enolase (PbEno) were chosen to further studies. A synthetic peptide (PbCTR3) and the recombinant enolase (rPbEno) were utilized together with the anti-rPbEno polyclonal antibody in functional analysis with ECM components and plasminogen. The studies suggest that P. brasiliensis enolase, in the surface, is able to generate plasmin from plasminogen by plasminogen activator. Therefore, it was also demonstrated that this protein is secreted and able to promote fungus adhesion and invasion to cells. These findings clearly establish the role of enolase in the patogenicity of P. brasiliensis. / Paraccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM), uma micose sistêmica, prevalente na América Latina. A matriz extracelular (MEC) é uma rede complexa formada por colágeno, laminina, fibronectina, entre outros componentes, que, quando exposta, é o local inicial de adesão do fungo. Nosso objetivo foi estudar genes envolvidos nesse processo de adesão utilizando Análise Diferencial Representacional (RDA). RDA é um método de subtração acoplado a PCR que permite o isolamento de genes diferencialmente expressos entre duas populações de cDNAs diferentes. Assim, cDNAs foram sintetizados a partir de RNAs extraídos de células leveduriformes de P. brasiliensis aderidos à colágeno e fibronectina para identificar genes super-expressos nestas condições. Genes envolvidos com vários processos celulares foram observados e PbCtr3 (transportador de cobre) e enolase (PbEno) foram escolhidos para análises adicionais. Um peptídeo sintético (PbCTR3) e a proteína recombinante (rPbEno) foram utilizados, juntamente com o anticorpo policlonal antirPbEno em análises funcionais com componentes da MEC e plasminogênio. Os estudos sugerem que a enolase de P. brasiliensis, localizada na parede celular, é capaz de gerar plasmina a partir do plasminogênio mediada pelo ativador de plasminôgenio. Além disso, foi também demonstrado que esta proteína é secretada sendo capaz de promover a adesão e invasão do fungo a células. Esses estudos claramente estabelecem o papel da enolase na patogenicidade de P. brasiliensis.

Pseudomonas aeruginosa and Candida albicans have shown antagonistic relationships, both in vivo and in vitro. The interaction between P. aeruginosa and C. albicans is one of the many prokaryotic-eukaryotic interactions existing in nature and needs more research due to their complex pathogenicity in humans. In this work, we have studied growth dynamics of Candida in a mixed population of Pseudomonas and Candida, and their receptor and ligand specificities, both from an ecological and a molecular point of view. Initially, growth, viability, and morphogenesis of Candida were studied in the presence of Pseudomonas and the conditioned medium of Pseudomonas using two Candida strains, namely CAF2 and tup1 mutant. The killing effect of Pseudomonas was more pronounced on the hyphal form of Candida. However, growth of Candida was inhibited by Pseudomonas irrespective of its morphological form. The conditioned medium had no effect on the growth rate of Candida. Nevertheless, it completely inhibited the germination of Candida. The attachment of Pseudomonas to Candida was studied using different strains of both, and with Pseudomonas cells harvested at different stages of its growth. The percent attachment varied with the age of the textit Pseudomonas culture. A lecB mutant of Pseudomonas showed a two-fold reduction in attachment compared to the wild type PAK strain. Carbohydrate inhibition studies confirmed that LecB is not directly involved in the attachment mechanism, but indirectly involves through regulating the expression of other proteins required for attachment. A genomic DNA library of Pseudomonas PAO1 was screened for clones that had acquired the ability to attach to C. albicans. A clone was isolated with a small increase in attachment and was subjected to genetic analysis. It contained nucleotides 458565 to 475917 of the Pseudomonas genome including some genes of the Pil-Chp gene cluster. Seven transposon mutants that represent mutations in ChpA,B,C,D,E operon and three other ORFs were selected, and their attachment ability was tested. All seven mutants showed a reduction in attachment indicating that this was a non specific effect, which could be attributed to the in vitro manipulation of the bacteria. We conclude that the attachment of Pseudomonas to Candida is multi-factorial.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική μελέτη των λοιμώξεων από πηκτάση-αρνητικούς σταφυλοκόκκους (CNS) σε ασθενείς με προσθετικά υλικά, όπως ενδαγγειακούς καθετήρες και η σύγκριση με στελέχη που προκαλούν βακτηριαιμία. Συνολικά μελετήθηκαν 168 Staphylococcus epidermidis και 58 S. haemolyticus από βακτηριαιμίες (BSIs, 100 στελέχη) και εντοπισμένες λοιμώξεις σχετιζόμενες με την εφαρμογή προσθετικών υλικών (PDAIs, 126 στελέχη) από ασθενείς του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών (ΠΓΝΠ) και του Νοσοκομείου Παίδων Πεντέλης (ΝΠΠ). Η πλειοψηφία των στελεχών (89.8%) ήταν ανθεκτικά στην methicillin (MR-CNS) και πολυανθεκτικά (90.7%). Βιομεμβράνη συνέθεταν τα 106/226 στελέχη, ενώ 208 παρήγαγαν β-λακταμάση. Τα γονίδια σύνθεσης προσκολλητινών aap, fnbA και bap βρέθηκαν σε συχνότητα 40.3%, 35.8% και 20.4% αντίστοιχα. Οι S. epidermidis έφεραν τα γονίδια atlE και fbe σε ποσοστά 88.1% και 81%, αντίστοιχα. Από τα γονίδια σύνθεσης τοξινών, συχνότερο ήταν το γονίδιο της τοξίνης τοξικής καταπληξίας tst (8.4%) ενώ τα γονίδια που κωδικοποιούν τις εντεροτοξίνες sea, sec βρέθηκαν μόνο σε μικρό ποσοστό στελεχών S. epidermidis και S. haemolyticus (5.3% και 3.1% του συνολικού πληθυσμού αντίστοιχα). Κανένα στέλεχος δεν έφερε τα γονίδια σύνθεσης των εντεροτοξινών seb και sed. Ο πληθυσμός των στελεχών S. epidermidis έδειξε μεγάλη γενετική ποικιλομορφία, με 67 PFGE τύπους, μεταξύ των οποίων δύο κύριοι τύποι (a, b) με 50 και 36 στελέχη αντίστοιχα. Έλεγχος με MLST ανέδειξε τρεις κύριους κλώνους (ST2, ST5 και ST16) που ανήκαν στο ίδιο κλωνικό σύμπλεγμα (Clonal Complex 2). Τα στελέχη του PFGE τύπου a παρουσίασαν υψηλότερα ποσοστά αντοχής στα αντιμικροβιακά clindamycin, ciprofloxacin, fusidic acid, SXT και στις αμινογλυκοσίδες, ενώ τα στελέχη του τύπου b έφεραν συχνότερα το γονίδιο aap (p=0.049). Τα στελέχη S. haemolyticus παρουσίασαν μικρότερη γενετική ποικιλομορφία, με έναν κύριο PFGE τύπο (h), που περιελάμβανε 44/58 στελέχη (75.9% του συνολικού πληθυσμού). Τα στελέχη CNS από BSIs ήταν συχνότερα ανθεκτικά στην methicillin (p<0.001) και στα υπόλοιπα αντιμικροβιακά (p<0.05), ενώ υπερείχαν και στην παραγωγή biofilm (p=0.003). Αντιθέτως, οι CNS από PDAIs έφεραν συχνότερα τα γονίδια των προσκολλητινών aap (p=0.006) και bap (p=0.045). Σε ένα δεύτερο σκέλος της παρούσας ερευνητικής εργασίας μελετήθηκε ένας πληθυσμός S. lugdunensis από το ΠΓΝΠ (37 στελέχη) και το ΝΠΠ (1 στέλεχος). Ο S. lugdunensis κατέχει ιδιαίτερη θέση μεταξύ των CNS, καθώς μπορεί να μιμηθεί την παθογόνο δράση του S. aureus και να προκαλέσει σοβαρές λοιμώξεις. Είκοσι δύο S. lugdunensis απομονώθηκαν από ασθενείς με λοιμώξεις δέρματος και μαλακών μορίων (Skin and Soft Tissue Infections: SSTIs), εννέα από εν τω βάθει λοιμώξεις (Deep Sited Infections: DSIs), συμπεριλαμβανομένων τριών στελεχών από ασθενείς με βακτηριαιμία, και επτά στελέχη από λοιμώξεις σχετιζόμενες με προσθετικά υλικά, κυρίως ενδαγγειακούς καθετήρες, (PDAIs). Όλα τα στελέχη ήταν ευαίσθητα στην methicillin (MS-CNS), στις αμινογλυκοσίδες (kanamycin, gentamicin), καθώς και στα: ciprofloxacin, rifampicin, teicoplanin, vancomycin, linezolid και daptomycin, ενώ μόνο τέσσερα στελέχη ήταν πολυανθεκτικά. Οι S. lugdunensis της συλλογής μας έδειξαν μικρή γενετική ποικιλομορφία. Τα 38 στελέχη ταξινομήθηκαν σε επτά κλώνους, με δύο κύριους PFGE τύπους (C και D), οι οποίοι περιελάμβαναν 22 και εννέα στελέχη αντίστοιχα. Τα 26 από τα 38 στελέχη έφεραν το οπερόνιο ica, ενώ συνολικά 14 ήταν biofilm-θετικά. Δεν παρατηρήθηκε συσχέτιση της παρουσίας του ica με κάποιο κλώνο, αλλά ούτε και με την παραγωγή βιομεμβράνης. Οι S. lugdunensis από PDAIs ήταν συχνότερα biofilm-θετικοί σε σχέση με τα στελέχη από SSTIs και DSIs, ενώ ο κύριος κλώνος C παρήγαγε biofilm σε μεγαλύτερο ποσοστό από τον D, δεύτερο σε συχνότητα κλώνο. Το γονίδιο fbl ανιχνεύθηκε σε όλα τα στελέχη S. lugdunensis που εξετάστηκαν, επιβεβαιώνοντας την φαινοτυπική ταυτοποίηση σε επίπεδο είδους. Ο επόμενος κατά σειρά συχνότητας παράγοντας παθογένειας που ανιχνεύθηκε ήταν το γονίδιο atlL, το οποίο βρέθηκε σε 36 από τα 38 στελέχη (94.7%). Ακολουθούν οι παράγοντες vwbl και slush, που βρέθηκαν σε 31 (81.6%) και 15 (39.5%) S. lugdunensis, αντίστοιχα. Τα στελέχη από εν τω βάθει λοιμώξεις (DSIs) έφεραν σε μεγαλύτερο ποσοστό τα γονίδια vwbl και slush σε σχέση με αυτά από PDAIs και SSTIs . Ο κλώνος C υπερείχε στην παρουσία του ermC, ενώ τα στελέχη που ανήκαν στον κλώνο D έφεραν σε μεγαλύτερο ποσοστό τα γονίδια vwbl και slush. / Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and S. haemolyticus, have emerged as opportunistic pathogens in patients with low immune response or indwelling medical devices. In the present study, bloodstream (BSIs) and prosthetic-device associated infections (PDAIs) CNS isolates were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, adhesin and toxin genes carriage. A collection of 226 CNS (168 S. epidermidis and 58 S. haemolyticus) recovered from BSIs (100) and PDAIs (126) of different patients in the Patras tertiary-care University General Hospital (UGHP) and Pentelis Paediatric Hospital in Athens (PPHA), was tested for biofilm formation, antimicrobial susceptibility, mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, seb, sec, sed) genes carriage. All CNS were classified into pulsotypes by PFGE, whereas S. epidermidis strains were assigned to sequence types by MLST. In total, 106 isolates (46.9%) produced biofilm, whereas 150 (66.4%) carried ica operon. Most isolates carried mecA and were multidrug resistant (90.7%). The adhesin encoding genes aap, fnbA and bap were identified in 40.3%, 35.8% and 20.4% of the total population, respectively. Genes encoding AtlE and Fbe were found in 88.1% and 81% of S. epidermidis isolates, respectively. CNS recovered from BSIs prevailed in biofilm formation (P=0.003), resistance to antimicrobials and mecA carriage (P<0.001) as compared to isolates derived from PDAIs. CNS from PDAIs carried more frequently aap and bap genes (P=0.006 and P=0.045, respectively). No statistically significant difference in toxin genes carriage was identified (P>0.05). Even though PFGE showed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by MLST, revealed three major clones (ST2, ST5, ST16). A clonal relationship was found concerning antimicrobial susceptibility, ica and aap gene carriage, reinforcing the aspect of clonal expansion in hospital settings. Pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capability of CNS. In the second part of this study we analyzed a collection of S. lugdunensis isolates recovered from different inpatients hospitalized in UGHP (37 isolates) and PPHA (one isolate) during a six-year period (2008-2013). S. lugdunensis has emerged as a significant human pathogen with distinct clinical and microbiological characteristics. A collection of 38 S. lugdunensis was tested for biofilm formation, antimicrobial susceptibility, clonal distribution, virulence factors (ica operon, fbl, atlL, vwbl, slush) and antibiotic resistance genes (mecA, ermC) carriage. The majority (22) was isolated from skin and soft tissue infections (SSTIs), nine from deep-sited infections (DSIs), including three bacteraemias and seven from PDAIs. All isolates were oxacillin-susceptible, mecA-negative and fbl-positive. The higher resistance rate was detected for ampicillin (50%), followed by erythromycin and clindamycin (18.4%). Fourteen isolates (36.8%) produced biofilm, 26 carried ica operon, but no relation between ica carriage and biofilm formation was identified. Biofilm formation was more frequent in isolates recovered from PDAIs. Thirty six strains (94.7%) carried atlL, 31 (81.6%) vwbl, whereas, slush was detected in fifteen (39.5%). PFGE revealed low level of genetic diversity: strains were classified into seven pulsotypes, with two major clones C and D including 22 and nine strains, respectively. Type C strains, recovered from all infection sites, prevailed in biofilm formation and ermC carriage, whereas, type D strains, associated with SSTIs and DSIs, carried more frequently vwbl, slush or both genes. Despite susceptibility to antimicrobials, clonal expansion and carriage of virulence factors combined with biofilm-producing ability render this species an important pathogen that should not be ignored.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.