Arcanobacterium haemolyticum, a Gram-positive bacterium, is an under-reportedagent of disease, causing pharyngitis, wound infections and a variety of invasive diseases.This work characterized a known A. haemolyticum toxin, phospholipase D (PLD), anddetermined its possible role in bacterial virulence. In addition, a novel toxin, arcanolysin(ALN), was identified and characterized. A draft genome sequence was determined andseveral additional virulence factors that may aid in disease pathogenesis were identified.PLD was present in all strains of A. haemolyticum tested, and was expressedmaximally during logarithmic growth. Recombinant PLD caused lipid raftrearrangement on the surface of HeLa cells in a dose-dependent manner. Thisrearrangement allowed maximal bacterial adhesion to the host, with a pld knockoutadhering only 39.7% to HeLa cells as compared to wildtype. Loss of production of PLDdid not affect bacterial invasion. However, PLD expressed by intracellular bacteria wascytotoxic to host cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate(MTS/PMS) viability assays. PLD caused host cell death via necrosis as determined bytransmission electron microscopy. PLD did not induce apoptosis, as caspases 3/7 and 9were not elevated in HeLa cells infected with wildtype A. haemolyticum.A. haemolyticum also expresses a Cholesterol-Dependent Cytolysin (CDC), ALN.Like pld, aln was present in all strains tested. ALN displays a variant undecapeptide andan unusual N-terminal extension not found in most other CDCs. Recombinant ALN11shows significantly increased activity against cultured cells and erythrocytes of humanorigin, compared with intermediate activity on rabbit and hamster cells, and low to noactivity on bovine and ovine cells as measured by hemolysis, cytotoxicity and membranebinding assays. ALN was less inhibited by free cholesterol when compared with otherCDCs, indicating the possibility of alternative receptor binding.The A. haemolyticum genome was sequenced to >20X coverage, and assembled to50 contigs covering ~95% of the genome. The genome is ~1.95Mb with a mol %G+C of53.1% and contained no plasmids. pld and aln have a reduced mol %G+C of 47.2% and46.5%, respectively, indicating the possibility of gene acquisition by horizontal transfer.Initial bioinformatics analysis identified genes encoding a protease, an extracellularDNase, two neuraminidases and three fimbrial biosynthetic operons were also identifiedwithin the genome.

No description available.

Environmental samples (air and soil) were collected from thoroughbred breeding farms with different prevalences of R. equi pneumonia to increase our understanding of the ecology of virulent R. equi on horse farms. The airborne population of virulent R. equi was a major focus of this research, as inhalation of the pathogen from the environment is considered the primary route of pulmonary infection. Air sampling was performed using an air monitoring system with selective media to facilitate the recovery of R. equi, allowing quantitative measurement of airborne virulent R. equi. Polymerase chain reaction and DNA hybridisation techniques were used to evaluate environmental samples to identify and differentiate R. equi.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/01/15). Includes bibliographical references.

Orientador: Antonio Carlos Pizzolitto / Banca: Isabel Yoko Ito / Banca: Sérgio Aparecido Torres / Banca: Maria de Lourdes Ribeiro de Souza / Banca: Clarice Queico Fujimura Leite / Resumo: Os Staphylococcus coagulase-negativa, particularmente, os Staphylococcus epidermidis são a causa mais freqüente de infecções relacionadas ao cateter por sua habilidade em aderir a uma superfície e entre si (aderência intercelular) formando biofilme em multicamadas sobre superfícies de polímeros. O objetivo do presente estudo foi avaliar cepas hospitalares de Staphylococcus coagulasenegativa isoladas de cateteres intravenosos, quanto à resistência a oxacilina, produção de slime, aderência ao poliestireno, habilidade de formar biofilme sobre superfícies abióticas (cateter esterilizado) e a presença de genes icaAD. Na presente pesquisa, a presença de icaA e icaD foi determinada pelo método PCR, em uma coleção de 27 amostras Staphylococcus coagulase-negativa (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus e 4 S. warneri). Os genes icaAD foram detectados em dez cepas S. epidermidis... (Resumo completo, clicar acesso eletrônmico abaixo) / Abstract: Coagulase-negative Staphylococcus, particularly, Staphylococcus epidermidis are frequent cause of infections associated with catheters and is attributed to the attachment ability on a surface and each other (intercellular adhesion) forming a multilayered biofilm on polymeric surfaces. The objective of the present study was to evaluate coagulase-negative Staphylococcus strains isolated from intravenous catheters by oxacillin resistance, slime production (qualitative method) and spectrophotometric assay (quantitative method), ability to form biofilm on abiotic surfaces (steriled catheter) and the presence of icaAD genes. In the present study icaA and icaD were determined by PCR method, in a collection of 27 coagulasenegative Staphylococcus (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus and 4 S. warneri). The icaA genes were detected in nine S. epidermidis and icaD in ten. The slime-producing ability was determined by culture on Congo red agar plates in which slime-producing strains formed black colonies in 10 S. epidermidis, 4 S.haemolyticus, 4 S. warneri, 2 S. xylosus and 1 S. chromogenes, while nonslimeformingones develop red colonies. The quantitative assay of coagulase-negative Staphylococcus was observed in 19 strains, including: 10 S. epidermidis, 3 S.haemolyticus, 3 S. warneri, 2 S. xylosus, 1 S. chromogenes. The ability of coagulasenegative Staphylococcus to form biofilm embedded in an amorphous substance wasobserved by scanning electronic microscope on abiotic surface in 10 S. epidermidis,3 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi,2 S. xylosus and 3 S. warneri. The oxacillin resistance was observed in 9 strains S.epidermidis, 3 S. haemolyticus, 3 S. warneri, 1 S. xylosus and 1 S. chromogenes. All strains of staphylococci were susceptible... (Complete abstract, click eletronic address below) / Doutor

MudanÃas importantes na epidemiologia das infecÃÃes fÃngicas nas Ãltimas dÃcadas tÃm resultado no isolamento frequente de leveduras do gÃnero Candida. As leveduras do complexo Candida parapsilosis, por exemplo, tÃm sido apontadas tanto como agentes de infecÃÃes como componentes da microbiota de animais. Diante disso, os objetivos deste trabalho foram realizar a identificaÃÃo molecular de cepas do complexo C. parapsilosis, isoladas de fontes veterinÃrias e mantidas no Centro Especializado em Micologia MÃdica; assim como avaliar seus atributos de virulÃncia e perfil de sensibilidade a antifÃngicos, in vitro. Para tanto, foram utilizados 28 cepas do complexo C. parapsilosis obtidos de cÃes, psitacÃdeos, rapinantes e camarÃo. Inicialmente foi realizada a fenotipagem das cepas, com base na anÃlise de suas caracterÃsticas morfolÃgicas e bioquÃmicas, que as ratificou como C. parapsilosis (lato sensu). A identificaÃÃo molecular das espÃcies foi realizada por PCR-REA. A fim de analisar o perfil de sensibilidade das cepas, empregou-se o teste de microdiluiÃÃo em caldo com anfotericina B, itraconazol, fluconazol, voriconazol e caspofungina, segundo metodologia padronizada pelo M27-A3. CLSI (2008). No tocante aos atributos de virulÃncia, a capacidade da produÃÃo de fosfolipases foi avaliada pelo mÃtodo de cultivo em Ãgar gema de ovo, a produÃÃo de proteases foi analisada atravÃs de cultivo em Ãgar albumina bovina e a formaÃÃo de biofilme foi em microplaca de poliestireno com 96 poÃos. A anÃlise genotÃpica evidenciou 13 C. parapsilosis (stricto sensu), 10 C. orthopsilosis e 05 C. metapsilosis. As concentraÃÃes inibitÃrias mÃnimas (MICs) variaram de 0,125 a 1 μg/mL para anfotericina B, de 0,5 a 16 μg/mL para fluconazol, de 0,03125 a 0,5 μg/mL para itraconazol, de 0,03125 a 0,25 μg/mL para voriconazol e de 0,0625 a 2 μg/mL para caspofungina. Foi observada resistÃncia em 03 cepas de C. parapsilosis (stricto sensu) ao fluconazol e 01 cepa apresentou MIC elevado (2 μg/mL) para caspofungina. No que tange aos isolados de C. orthopsilosis, notou-se que 05 isolados apresentaram MICs elevados (2 μg/mL) para a caspofungina. Enquanto que, 02 isolados de C. metapsilosis revelaram-se resistentes ao fluconazol. Quanto à virulÃncia, todas as cepas foram capazes de formar biofilmes, sendo, 20, 7 e 01, classificadas como produtoras moderadas, fortes e fracas respectivamente. Observou-se, ainda que 23/28 isolados apresentaram atividade proteolÃtica. Por outro lado, nenhuma foi capaz de produzir fosfolipases. Estes dados sinalizam que padrÃes de virulÃncia, patogenicidade sensibilidade antifÃngica, in vitro, podem variar entre as espÃcies do complexo C. parapsilosis. / In the past decades, important changes in the epidemiology of fungal infections have resulted in the frequent isolation of yeasts of the genus Candida. Yeasts of the Candida parapsilosis species complex, for example, have been pointed out as infectious agents and components of the microbiota of animals. Thus, the work aimed at identifying molecularly the strains of the C. parapsilosis species complex recovered from veterinary sources and maintained at the Specialized Medical Mycology Center, as well as evaluating their in vitro antifungal susceptibility profile and attributes of virulence. For such, 28 strains of the C. parapsilosis species complex, recovered from dogs, psittacines, raptors and prawn were assessed. Initially, the strains were phenotypically identified, based on their morphological and biochemical characteristics, which confirmed their identification as C. parapsilosis lato sensu. The molecular identification of the strains was then carried out through PCR-REA. In order to analyze the in vitro antifungal susceptibility, the broth microdilution assay was performed, according to the document M27-A3 of the Clinical Laboratory Standards Institute (CLSI) 2008, using amphotericin B, itraconazole, voriconazole, fluconazole and caspofungin. Concerning the virulence attributes, the ability of producing phospholipase was evaluated on egg yolk agar, while protease production was assessed on bovine serum albumin agar, and biofilm formation was tested in 96-well polystyrene microplates. The genotypical analysis identified 13 C. parapsilosis stricto sensu, ten C. orthopsilosis and five C. metapsilosis. The minimum inhibitory concentrations varied from 0.125 to 1 μg/mL foramphotericin B, from 0.03125 to 0.5 μg/mL, for itraconazole, from 0.03125 to 0,25 μg/mL for voriconazole, from 0.5 to 16 μg/mL for fluconazol and from 0.0625 to 2 μg/mL for caspofungin. Fluconazole resistance was observed in three strains of C. parapsilosis stricto sensu and two of C. metapsilosis, while one strain of C. parapsilosis stricto sensu and five C. orthopsilosis presented high MIC values for caspofungin (2 μg/mL). As for the virulence attributes, none of the tested strains produced phospholipases, while 23/28 presented proteolytic activity, and all of the strains produced biofilm, with one weak producer, 20 moderate producers and seven strong producers. These data show that the in vitro antifungal susceptibility and production of virulence attributes vary among species of the C. parapsilosis species complex, which can lead to differences in pathogenicity and therapeutic response.

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2017-2018 / Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui cause des infections pulmonaires chroniques chez les gens atteints de fibrose kystique (FK). Certaines souches, adaptées au microenvironnement des poumons FK, se distinguent, entre autres, par une résistance accrue aux antibiotiques et une formation abondante de biofilm. L'étude d'isolats provenant de patients FK est nécessaire afin de comprendre les déterminants génétiques expliquant cette adaptation et trouver de nouveaux moyens de lutter contre cette bactérie. Une librairie de mutants de LESB58, souche épidémique chez les patients FK, a été créée en 2009. Dans l’étude présentée ici, un criblage successif dans trois hôtes différents a été réalisé sur ces mutants, permettant d'identifier un mutant particulièrement peu virulent : le mutant STM PALES_11731. Ce mutant, contrairement à la souche sauvage, s'est avéré incapable de former des biofilm-like structures (BLS), une forme d'agrégation bactérienne ressemblant à du biofilm, mais flottant dans le milieu de culture. Afin d’évaluer ce phénomène chez différentes souches, la formation de biofilm adhéré et de BLS chez LESB58 et trois autres souches (la référence PAO1 et deux souches environnementales, PPF-1 et Urg-7) a été comparée à l'aide d'une nouvelle approche de quantification par analyse d'images. L’effet de cations divalents sur la formation de ces structures a également été exploré. Une diversité dans les phénotypes de formation de biofilms et de BLS et a été observée. Un manque de corrélation entre la formation de biofilms adhérés et de BLS a également été noté, suggérant que ces phénomènes pourraient ne pas être directement reliés et soulevant plusieurs questions sur les mécanismes de formation des BLS. Les résultats de ce projet de maîtrise indiquent que les BLS pourraient jouer un rôle dans la virulence de P. aeruginosa chez les patients FK et soulèvent l’importance de déterminer les mécanismes moléculaires ou physiques responsables de leur formation. / Pseudomonas aeruginosa is an opportunistic bacterial pathogen known to cause chronic lung infections in cystic fibrosis (CF) patients. Some strains, adapted to the CF lung microenvironment, show distinguishing phenotypes such as an increased resistance to antibiotic treatments and an enhanced biofilm formation. The study of P. aeruginosa isolates from CF patients is necessary to understand the genetic determinants explaining this adaptation and to allow the development of new ways to fight this bacterium. A library of LESB58 mutants has been created in 2009. LESB58 is an epidemic strain among CF patients. In the study presented here, a sequential screening in three different hosts has been performed, leading to the identification of a mutant with a strong virulence defect: the STM PALES_11731 mutant. This mutant, contrary to the wild type, was unable to form biofilm-like structures (BLSs), a type of bacterial aggregation resembling biofilm, but floating in the culture medium. To assess this phenomenon among P. aeruginosa strains, the formation of adhered biofilm and BLSs in LESB58 and three other strains (the reference strain PAO1 and two environmental strains, PPF-1 and Urg-7) was compared using a novel image analysis quantification approach. The impact of the addition of divalent cations on the formation of those structures was also assessed. The results obtained demonstrate some diversity of biofilm and BLS formation in this bacterial species. They also reveal a lack of correlation between the BLS and adhered biofilm formation response to the ion treatment, suggesting that these two phenomena might not be directly related and raising questions about the mechanisms of BLS formation. The results of this project indicate that BLSs could play a role in the virulence of P. aeruginosa in CF patients and highlight the importance, in a future study, of studying the molecular and physical mechanisms responsible for their formation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les parasites protozoaires du genre Leishmania sont les agents microbiens responsables d’un groupe de maladies connues sous le nom de leishmanioses. L’infection productive dépend de la capacité de survie du parasite suite au premier contact initial du système immunitaire de l’hôte. Par conséquent, la prolifération du parasite à l’intérieur des phagolysosomes sera responsable de la pathologie. Les promastigotes stationnaires, récupérés en culture axénique de laboratoire sont semblables aux promastigotes métacycliques. Ces derniers sont fortement immunomodulateurs et sont considérés, traditionnellement comme la forme la plus infectieuse du parasite. Les protéines sécrétées de différents organismes ont été directement impliquées dans plusieurs pathologies. Donc, il est possible que les protéines sécrétées par Leishmania, soient également impliquées dans la capacité du parasite à subvertir le système immunitaire. Les avancées récentes dans l’étude du sécrétome de plusieurs types de Leishmania ont permis d’affirmer que le sécrétome est fort complexe. Nous avons déterminé qu’environ 300 protéines sont sécrétées par le parasite, la plupart d’entre elles ayant aucun signal canonique de sécrétion. Le sécrétome de Leishmania est donc composé surtout de protéines qui sont libérées par sécrétion non conventionnelle, . Afin d’étudier le sécrétome associé à la virulence, nous avons développé et validé une approche qui a permis l’étude des composants de l’exoprotéome des parasites stationnaires et logarithmiques. Cette approche était basée sur la culture continue des parasites dans un milieu de culture sans supplément de sérum de bovin fœtal, cRPMI, dans lequel la virulence des parasites est maintenue. Grâce à cette approche nous avons mis en évidence un exoproteome distinct de ceux jusqu’à date répertorié. La méthode de production et de la récupération de l’exoproteome sont donc très importants. Notre exoprotéome est dominé par la GP63, une glycoprotéine dont l’importance centrale dans l’infection a été déjà validée. La culture continue des parasites est donc essentiel pour avoir un exoprotéome représentatif. Nous avons également déterminé que la cultutre en continu pouvait amener à une diminution de la virulence et quarante divisions sont nécessaires pour une perte de virulence significative. Par conséquent toutes nos études se sont fait chez des parasites comptant moins de 20 divisions. Le principal mécanisme associé à la perte de virulence a été identifié comme une incapacité de se différencier en amastigotes. Le cRPMI a donc permis la culture des parasites pour l’étude de l’exoprotéome tout en maintenant la virulence des parasites. La présence de vésicules, décrite déjà comme un composant de l’ exoprotéome, a été confirmée aussi par notre approche continue et a été confirmé, d’ailleurs, dans l’exoproteome des parasites en phase de croissance logarithmique. Les vésicules récupérées des parasites logarithmiques diffèrent de celles recueillies de parasites en phase stationnaire de croissance. En effet des protéines potentiellement impliquées dans la rénovation et le recyclage du contenu protéique, tels certains composants du ribosome étaient enrichies dans les parasites en phase logarithmique tandis que les vésicules des parasites stationnaires ont un contenu protéomique ayant des caractéristiques similaires aux corps apoptotiques des cellules de mammifères. En dehors de la GP63, plusieurs autres protéines décrites comme immunomodulatrices ont été retrouvées dans l’exoprotéome des parasites stationnaires, ce qui indique que celui-ci contient un ensemble de protéines avec un potentiel d’interaction directe avec les cellules du système immunitaire. Immunologiquement, l’exoprotéome récupéré des parasites stationnaires a été capable d’activer les cellules dendritiques, laissant supposer une fonction importante dans la création d’un environnement inflammatoire précoce lors des premières étapes de l’infection. En conclusion, la recherche développée a contribué à l’avancement des connaissances actuelles sur la biologie du Leishmania, grâce au développement et à la validation d’une nouvelle approche afin d’étudier son exoprotéome. L’exoprotéome récupéré était dynamique, il avait une composition spécifique, dépendant du stade du parasite. Cet exoprotéome avait des effets spécifiques sur les cellules dendritiques et il jouait un rôle important dans les étapes précoces de l’infection. Cette étude a ouvert des nouvelles perspectives sur l’exoprotéome de Leishmania spp. permettant la découverte de nouvelles protéines immunomodulatrices et, en corrolaire, de nouvelles cibles pour le contrôle de la maladie associée au parasite. / Protozoa parasites of the genus Leishmania are the responsible for a group of diseases known as leishmaniasis. The infection is associated with the capacity of these parasites to survive in the phagolysosomes of infected macrophages. Successful infections with pathogenic Leishmania spp. are linked to the capacity of the parasite to survive the initial impact of the host immune system and to interfere with the infected cells rendering them incapable of eliminating the parasites. The secreted proteins from the parasite are expected to be in the front line for interactions with the host. Recent advances in the study of the secretome of Leishmania spp. depicted it as highly complex with the majority of proteins without any predictable secretion signal. The secretome is composed of proteins that are released by different mechanisms like conventional and unconventional secretion. Several proteins secreted by Leishmania spp. are known to interact and influence the outcome of the disease by directly interfering with the host immune cells. The proteomic studies on the Leishmania spp. secretome identified more than three hundred proteins released into the exterior. The stationary promastigotes recovered in axenic culture were enriched in the most virulent promastigote form, the metacyclic parasites. Therefore we aimed at evaluating the exoproteome associated with the stationary parasites. To achieve this we developed and validated an approach that would enable the study of the exoproteome components of stationary and logarithmic parasites. This approach was based on the continuous cultivation of the parasites in a medium without any protein supplementation that maintained the basic virulence of the parasites. The continuous approach produced a GP63-rich exoproteome that was distinct from the traditional approaches indicating that the process of recovery induced a significant bias in the study. Furthermore as the continuous approach was chosen, we determined the mechanisms associated with loss of virulence assuring that fully virulent parasites were used. At least forty parasite divisions were required for a short-term loss of virulence. The main mechanism associated with loss of virulence was identified as a growing incapacity to differentiate into amastigotes. The defined time interval of forty divisions enabled us to evaluate the exoproteome without loss of virulence related to the subculture. The protein-free medium developed, cRPMI, retained parasite virulence and morphology similar to that of parasites grown in standard media. The exoproteomes recovered using cRPMI were dominated by proteins without any recognizable secretion sequence, in concordance with reports on other Leishmania spp. The presence of vesicles, already reported as a component of the exoproteome, was also confirmed using our continuous approach. Furthermore, the presence of vesicles in the logarithmic parasites exoproteome was confirmed. The protein content of these vesicles presented a dynamic profile that was dependent on the parasite stage. The vesicles recovered from logarithmic parasites seemed to be related to protein turnover, being significantly enriched in ribosomal components. The vesicles from stationary parasites are of different composition, presenting some characteristics similar to apoptotic bodies. Immunologically the exoproteome recovered from stationary parasites was able to activate dendritic cells suggesting that the exoproteome might have a function in the creation of an early inflammatory environment leading to the recruitment of neutrophils and monocytes that might function as safe heavens for the parasites. In conclusion, our research has contributed to the advance of the current knowledge of Leishmania biology, through the development and validation of a novel approach to study the Leishmania secretome. The exoproteome recovered from stationary parasites had specific immune-modulating effects on bone marrow derived dendritic cells, indicating that it can play an important role in the precocious steps of infection. This study opened new perspectives into the Leishmania spp. exoproteome that will enable the search of new immunomodulatory proteins that might become the future targets to leishmaniasis control.

No description available.