La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l'étiologie est multifactorielle. Les lésions iléales des patients atteints de MC sont anormalement colonisées par des souches pathogènes de Escherichia coli appartenant au pathovar AIEC pour " Adherent-Invasive E. coli ". Ces souches sont capables d'adhérer et d'envahir les cellules épithéliales intestinales, et ont la capacité de survivre et de se multiplier fortement en macrophages en induisant une synthèse intense de TNF-α. L'objectif de ce travail s'inscrit dans la compréhension des mécanismes permettant aux bactéries AIEC de coloniser la muqueuse intestinale et d'induire les stades précoces de la pathologie. Une précédente étude menée au laboratoire avait permis de mettre en évidence l'importance de l'activation de la voie de régulation dépendante du facteur bactérien sigma alternatif RpoE (ou σE) dans le processus d'adhésion et d'invasion des cellules épithéliales intestinales par la souche AIEC de référence LF82 via l'expression des pili de type 1 et des flagelles. En continuité de ces travaux, nous montrons que l'activation de la voie de signalisation dépendante du facteur σE est également primordiale pour la capacité des souches AIEC à former des biofilms, et une analyse bioinformatique ayant pour but d'identifier les gènes régulés par σE a montré que l'opéron waaWVL, impliqué dans la biosynthèse du lipopolysaccharide, est primordial pour la formation de biofilm par les souches AIEC. De plus, nous avons mis en évidence que les long polar fimbriae (LPF) sont impliqués dans le ciblage de l'épithélium associé aux plaques de Peyer par les bactéries AIEC, et ceci en leur permettant de cibler spécifiquement les cellules M. L'inactivation du gène Nod2, gène de susceptibilité à la MC, conduit à une augmentation du nombre de plaques de Peyer ainsi que des cellules M à leur surface, indiquant que les bactéries AIEC pourraient tirer avantage d'une susceptibilité génétique pour cibler les plaques de Peyer.

Listeria monocytogenes est une bactérie pathogène impliquée dans la toxi-infection alimentaire à l'origine de la listeriose, une maladie peu fréquente mais avec un taux de mortalité de 25 % chez l'homme. Cette bactérie est capable de former un biofilm lui permettant de mieux résister aux stress environnementaux ainsi qu'aux traitements de décontamination. Une nouvelle stratégie d'analyse génomique a été développée et a permis de cibler des systèmes de sécrétion et des protéines potentiellement impliqués dans la formation de biofilm. L'inactivation de la voie SecA2 entraîne la formation d'un biofilm aérien et par conséquent fragile. Ce morphotype est capable de croître de façon sessile à 20°C sur du polystyrène alors que ce n'est pas le cas pour la souche sauvage. De nouvelles protéines sécrétées de façon SecA2 dépendante ont été identifiées par l'étude de l'exoprotéome du mutant ΔsecA2 en comparaison avec celui de la souche sauvage. Le rôle des lipoprotéines dans la formation de biofilm ainsi que leur maturation par les peptidases signal de type II, LspA et LspB, a également été abordé. La combinaison d'une analyse de l'expression des gènes codant les lipoprotéines au cours de la formation de biofilm avec l'analyse génomique basé sur le sécrétome a permis de cibler trois lipoprotéines, dont LpeA qui serait impliquée dans les phases tardives de formation de biofilm. Enfin, l'importance majeure de LspA dans la maturation des lipoprotéines, a été mise en évidence par l'étude de l'exoprotéome des doubles mutant ΔlgtΔlspA et ΔlgtΔlspB en comparaison avec celui de Δlgt.

Staphylococcus aureus, pathogène opportuniste de l'homme, est responsable de 30% des infections nosocomiales. L'apparition de souches multi résistantes aux antibiotiques en font un problème majeur de santé publique. La pathogénie de la bactérie résulte de l'expression d'une pléthore de facteurs de virulence, mais quels sont les mécanismes de régulation contrôlant l'expression de ces gènes ? Aujourd'hui, il est clairement établi que les ARN non-codant sont des molécules clés dans la régulation de l'expression des gènes. Plus de 50 ARN ont été identifiés chez S. aureus. Néanmoins la fonction de peu d'entre eux est connue. Durant ce travail de thèse, l'étude de la fonction et du mécanisme de régulation des ARN RsaA et RsaE a été entreprise. RsaA est un ARN sous le contrôle du facteur de stress sigmaB. Les résultats obtenus montrent que ce dernier régule la traduction de l'ARNm mgrA qui code pour un facteur de transcription important dans l'expression des gènes de virulence et la régulation de l'autolyse. Par appariement de base, RsaA cible l'ARNm en utilisant deux sites distants et coopératifs, permettant un interaction forte qui empêche la traduction de l'ARNm. In vivo, la délétion du gène rsaA perturbe la synthèse de biofilm de capsule. En régulant la traduction de sa cible, RsaA permet de relier l'adaptation au stress à l'expression des gènes de virulence. De manière plus générale, les réseaux de régulation des ARN se connectent les uns aux autres pour permettre à la bactérie d'intégrer une multitude de signaux provenant du milieu extracellulaire afin de moduler finement l'expression des gènes.

Dans les environnements naturels, industriels ou médicaux, les microorganismes sont majoritairement présents en étant associés aux surfaces dans des communautés hautement organisées appelées biofilms. Ces édifices biologiques constituent une stratégie de survie étonnement efficace témoignant d'une grande capacité de résistance à différent stress environnementaux tels que les traitements de nettoyage et de désinfection. L'impact des biofilms d'un point de vue sanitaire est donc considérable du fait qu'ils permettent la persistance et la transmission de germes pathogènes dans l'environnement. Dans ce contexte, ce travail de thèse avait pour objectif une meilleure compréhension des phénomènes limitant l'efficacité de désinfectants au sein des biofilms en s'appuyant notamment sur des techniques innovantes d'imagerie de fluorescence non-invasive. Le but final étant d'apporter des éléments utiles à l'optimisation des traitements de désinfection. Dans une première partie, une méthode d'investigation structurale à haut-débit par microscopie confocale a été développée et utilisée pour étudier la diversité architecturale des biofilms bactériens formés par un large panel de souches. Cette étude nous a permis d'identifier des souches d'intérêt en termes de structures de biofilms formés pour la suite du travail. Nous avons notamment pu mettre en évidence la capacité de B. subtilis à former des structures importantes et avec une architecture spécifique dans un système immergé. Dans une deuxième partie, les dynamiques d'action spatiotemporelles de désinfectants ont été visualisées dans les biofilms de souches de P. aeruginosa ou B. subtilis par des approches de microscopie confocale de fluorescence en temps réel. L'utilisation de cette technique nous a permis de mettre en évidence les difficultés de pénétration du chlorure de benzalkonium au sein des structures formées par différentes souches de P. aeruginosa. La corrélation des paramètres cinétiques d'inactivation et des données obtenues par la caractérisation biochimique de la matrice suggère un rôle majeur des substances extracellulaires dans la limitation de pénétraton du désinfectant. Nous avons également pu montrer une résistance marquée du biofilm formé par une souche de B. subtilis isolée d'un dispositif médical à l'acide péracétique, à la concentration et au temps d'utilisation du biocide dans le milieu médical. De plus, les structures tridimensionnelles formées par cette souche étaient capables de protéger le pathogène Staphylococcus aureus dans un biofilm mixte vis-à-vis du même traitement soulignant l'importance des interactions multi-espèces dans la résistance des bactéries aux désinfectants et la persistance de pathogènes dans nos environnements.

Fully-dynamic, continuous fugacity-based fate and transport models have been developed to examine all natural processes and interactions in the aquatic water systems. Within a body of surface water such as a lake or a river, a dynamic interaction among different media takes place. Chemical compounds are continuously dissolving, adsorbing into solid particles, attaching to suspended particles, resuspending, reacting, diffusing, and advecting. As the inclusion of all these interactions into a model is complex, the use of fugacity concept instead of concentration, renders the modeling task relatively easy. Fugacity, which is described as the escaping tendency of a chemical from a medium, is continuous among different phases, thus easier to follow the movement of the chemical. The first model has been developed to be used as an emergency response model by decision makers, which models the fate and transport of any contaminant in a lake. Due to uncertainties involved in the analysis, Monte Carlo simulations are performed. The fate of three representative contaminants; polychlorinated biphenyls (PCBs), atrazine, and benzene in air, water, and sediment compartments are examined. The second model developed is a continuous, dynamic river fugacity-based water quality model. In order to develop a continuous model, the hydrodynamics of the river system is solved first. Water depth and velocity at each point along the river are used in the advection-dispersion equation to determine the fate and transport of a contaminant. Interactions between different phases are also incorporated into the advection-dispersion equation which is solved numerically and coupled with a mass balance equation derived for the same contaminant in the sediments. The third model is a multispecies contaminant fate and transport model which can be used for the fate of a single contaminant and its daughter products. Trichloroethylene (TCE) and its daughter products, dichloroethylene (DCE) and vinyl chloride (VC), are used as representative of multispecies contaminants. The fate and transport of TCE and its daughter products has been analyzed first in a lake environment, and then in a river environment with the addition of a biofilm compartment where all biotransformations take place.

Background There is a transition towards minimally invasive restorative techniques in restorative dentistry based upon reducing bacterial viability and encouraging remineralization of caries infected tissue. To improve the predictability of the antibacterial and remineralization potential of carious dentine by either the application of medicaments or placement of restorative materials that encourage remineralization would be a significant benefit in disease management. Materials and Methods An experimental model was developed using a chemostat for in vitro analysis of the effects of silver fluoride followed by potassium iodide (AgF/KI) and ozone treatment on non demineralized and demineralized dentine. Electron Probe Micro Analysis (EPMA) and Scanning Electron Microscopy (SEM) on the treated dentine were conducted to investigate ion transfer, and biofilm formation. Bacteria growth was measured by optical density. An in vitro caries model using a chemostat was developed to determine the ability of glass ionomer cement and composite resin to inhibit dentinal degradation in adjacent dentine and to measure ion exchange at the restorative interface. Tests were made to determine the bond strength between dentine and glass ionomer cement after application of silver fluoride to the surface of the dentine. Results S. mutans migrated through all dentine samples. Samples treated with AgF/KI had significantly lower optical densities than the corresponding controls. Optical density readings were significantly lower in demineralized dentine treated with AgF/KI than non demineralized dentine. There were lower but not significant differences in the optical density readings between ozonated and non ozonated dentine. An S. mutans biofilm covered all control discs. No biofilm was detected on discs treated with AgF/KI and these discs were significantly more resistant to further demineralization than the control discs. Detectable amounts of silver and fluoride were found up to 450 μm in the AgF and AgF/KI sections. Ozone infusion prevented S. Mutans and L. acidophilus biofilm formation on all the treated dentine samples, biofilm was present on all control specimens. There was calcium and phosphorus present in all auto cure glass ionomer cements to a depth beyond 50 microns. Aluminium and strontium ions were also present in dentine except strontium subjacent to Ketac Molar restorations. Fluoride uptake was significantly higher under glass ionomer cement restorations where the dentine was pretreated with AgF/KI compared to non treated specimens. Silver and iodine deposits were present in demineralized dentine treated with AgF/KI. Calcium and phosphorus levels up to 130 microns from the restorative interface were similar to non demineralized dentine adjacent to auto cure glass ionomer cements and half that adjacent to composite resin. There was significant surface degradation in auto cure glass ionomer cements compared to composite resin. Washing away the AgF/KI precipitate produced higher bond strengths to dentine than samples where the precipitate remained. Conclusions Under the conditions of these in vitro studies, the application of AgF/KI and ozone pharmacologically reduces the initiation and rate of dentine caries. Glass ionomer cements were shown to protect dentine from experimental carious degradation and assist with remineralization. AgF/KI application enhances remineralization beneath glass ionomers and does not interfere with bond strengths. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1344616 / Thesis (Ph.D.) - University of Adelaide, Dental School, 2008

The aim of this study was to observe biofilm formation by Enterobacter sakazakii (E. Sakazakii) from different clinical, dairy and environmental origins on three infant feeding tubes made of different materials. Infant formula milk was selected as the medium for E. sakazakii growth. Seventeen isolates from different origins were retrieved and tested for purity, using a plating method and biochemical tests to eliminate the non E. sakazakii strains from this study. A method to rapidly and accurately detect viable cells of E. sakazakii on infant feeding tube surfaces using of the BacTrac® 4000 microbiological growth analyser was developed. The sources of errors such as from cleaning, operation and handling procedures were assessed prior to experimental runs. The strength of biofilm formation by different isolates of E. sakazakii on plastic surfaces was scrutinised using a microtiter plate assay. The results from the microtitre plate assay were based on the absorbance at 550 nm of crystal violet stained films and showed that all the clinical isolates were able to attach and form strong biofilms on the plate. Some environmental isolates formed strong or weak biofilms and some did not produce biofilm at all. However, dairy isolates formed both strong and weak biofilms in the microtitre plate when incubated in 10% reconstituted infant formula milk. The further studies were to quantify biofilm formation by three isolates of different origin on three different materials of infant feeding tubes using a batch system. Tubing pieces were incubated with infant formula milk inoculated with E. sakazakii cells at approximately 8 log CFU mL-1 and the biofilm formation was assessed at three time intervals: 4, 12 and 24 hours. Biofilm formation on the tubing by clinical isolates was also observed using epifluorescence microscopy and the scanning electron microscope. E. sakazakii from clinical, dairy and environmental isolates were able to form biofilm on three different materials of infant feeding tubes. The results showed that the initial attachment at 4 h on silicone tubing was low compared with the other two tubes. The scanning electron micrographs showed the surface characteristics of each tubing and the biofilm formation by E. sakazakii clinical isolates after 4, 12 and 24 hours. Silicone tubing appeared to be the best choice for premature babies that need feeding using feeding tubes, as it was slow to become colonised compared with the PVC and polyurethane tubing.

A detailed study has been carried out to demonstrate the importance of external and internal mass transfer on the nitrification rates in three distinct treatment processes: flocculent and granular activated sludge, and suspended carrier reactor (SCR) systems. The major emphasis was on external mass transfer, and the impact of system hydrodynamics on this mechanism. Laboratory-scale flocculent and granular sequencing batch reactors were operated for the nitrification of a synthetic wastewater. A two-stage, continuous, nitrifying SCR was operated using the same wastewater feed. Within each stage, biofilm was grown on two types of commercial carriers- the Natrix C10/10 from ANOX AB (Sweden); and the K1 carrier from Kaldnes Miljøteknologi (Norway). Biofilm carriers obtained from each of these reactors was utilised for the mass transfer investigations. The major findings, and contributions of the work to the field of biological wastewater treatment, are described in the following paragraphs. In order to complete the work, a novel experimental tool, the TOGA (Titrimetric and Off-Gas Analysis) sensor was created, which utilises off-gas mass balancing, coupled with pH titration to provide detailed measurement of biological reaction rates. An original method for off-gas mass balancing was developed, within a reactor that allowed modification of the hydrodynamic conditions using gas phase mixing independent of dissolved oxygen control within the liquid phase. This sensor has already proven to be a highly effective tool not only for the measurement of oxygen but also for carbon dioxide and various nitrogen species, and has application for numerous other compounds present in the gas phase of biological reactors (e.g. hydrogen, methane). The application of the TOGA sensor signals to the nitrification process was demonstrated, which enabled the online measurement of oxygen, ammonia, and nitrite reaction rates. The TOGA sensor development underpinned the majority of the subsequent experimental work within this thesis. Dissolved oxygen microelectrodes were also used, enabling microscale measurements to be made in conjunction with the macroscale TOGA sensor analyses. Combined with size and microbiological analyses a detailed study of mass transfer and reaction was able to be carried out on the various systems. For suspended aggregate systems (flocs and granules): A spherical particle model was developed and used to predict the potential for external mass transfer limitation in flocs and granules. The significance of this limitation was confirmed experimentally, by observing changes in reaction rate or concentration boundary layer (in the TOGA sensor or microelectrode study, respectively) upon modification of the system’s flow conditions. Despite this flow effect being small, and only observable under low bulk liquid substrate concentrations, the external mass transfer limitation was concluded to be significant for biological flocs and granules even at higher substrate concentrations. As particle size and the maximum volumetric reaction rate of the biomass increases, external mass transfer effects become increasingly significant. The work highlights the impact of mass transfer limitation on the measurement of Monod half saturation coefficients (KS) in flocs and granules. Without accounting for external or internal mass transfer limitation, KS is seriously overestimated and becomes a lumped parameter, reflecting not only the microbial response but also the mass transfer limitations observed within the system under study. To avoid confusion or generation of erroneous results, care should be taken in defining, measuring and utilising the half saturation coefficient in biological systems where the biomass is not present as individual cells or extremely small flocs. For Suspended Carrier Reactor systems: External and internal mass transfer are both concluded to be important rate limiting steps within suspended carrier reactors. The demonstration of a significant impact of fluid flow conditions on the nitrification rates highlights the impact of external mass transfer limitation within these systems. Application of a one-dimensional biofilm model to the experimental results led to the conclusion that there is little difference between the external mass transfer limitation of the two different carrier types, for carriers grown under the same environmental conditions. However, there was a significantly higher areal nitrification rate observed on the Natrix carriers compared to the Kaldnes carriers. It is the biofilm structure that is critically important in characterising the mass transfer steps. Systems operated under high nitrogen loads, producing filamentous biofilms on the carrier surface, were found to have larger external mass transfer coefficients and responses to changes in fluid flow than those carriers which were operated under nitrogen-limited conditions (producing a flatter, more gel-like biofilm). The structure of the biofilm colonising the carrier surface was far more important in defining the mass transfer coefficient than the actual carrier type used. In a remarkably similar trend to that of the external mass transfer coefficient, the biofilm morphology was again significantly more important than carrier type in determining both the magnitude and response to fluid flow of the gas-liquid mass transfer coefficient for oxygen (kLa) calculated within the laboratory TOGA sensor. These findings led to the postulation that direct gas-biofilm interfacial mass transfer mechanism is occurring within the SCR systems. This hypothesis is an alternative to the standard mechanism of gas transfer from the bubble into the liquid phase, and then into the biofilm. Understanding of interfacial transfer is likely to be important for developing the knowledge of SCR processes. Overall, both external and internal mass transfer phenomena have been demonstrated to create important rate limitations to suspended aggregate systems (flocs and granules) and biofilms grown in suspended carrier reactors. This significantly advances the conceptual understanding of these biological treatment processes.

A detailed study has been carried out to demonstrate the importance of external and internal mass transfer on the nitrification rates in three distinct treatment processes: flocculent and granular activated sludge, and suspended carrier reactor (SCR) systems. The major emphasis was on external mass transfer, and the impact of system hydrodynamics on this mechanism. Laboratory-scale flocculent and granular sequencing batch reactors were operated for the nitrification of a synthetic wastewater. A two-stage, continuous, nitrifying SCR was operated using the same wastewater feed. Within each stage, biofilm was grown on two types of commercial carriers- the Natrix C10/10 from ANOX AB (Sweden); and the K1 carrier from Kaldnes Miljøteknologi (Norway). Biofilm carriers obtained from each of these reactors was utilised for the mass transfer investigations. The major findings, and contributions of the work to the field of biological wastewater treatment, are described in the following paragraphs. In order to complete the work, a novel experimental tool, the TOGA (Titrimetric and Off-Gas Analysis) sensor was created, which utilises off-gas mass balancing, coupled with pH titration to provide detailed measurement of biological reaction rates. An original method for off-gas mass balancing was developed, within a reactor that allowed modification of the hydrodynamic conditions using gas phase mixing independent of dissolved oxygen control within the liquid phase. This sensor has already proven to be a highly effective tool not only for the measurement of oxygen but also for carbon dioxide and various nitrogen species, and has application for numerous other compounds present in the gas phase of biological reactors (e.g. hydrogen, methane). The application of the TOGA sensor signals to the nitrification process was demonstrated, which enabled the online measurement of oxygen, ammonia, and nitrite reaction rates. The TOGA sensor development underpinned the majority of the subsequent experimental work within this thesis. Dissolved oxygen microelectrodes were also used, enabling microscale measurements to be made in conjunction with the macroscale TOGA sensor analyses. Combined with size and microbiological analyses a detailed study of mass transfer and reaction was able to be carried out on the various systems. For suspended aggregate systems (flocs and granules): A spherical particle model was developed and used to predict the potential for external mass transfer limitation in flocs and granules. The significance of this limitation was confirmed experimentally, by observing changes in reaction rate or concentration boundary layer (in the TOGA sensor or microelectrode study, respectively) upon modification of the system’s flow conditions. Despite this flow effect being small, and only observable under low bulk liquid substrate concentrations, the external mass transfer limitation was concluded to be significant for biological flocs and granules even at higher substrate concentrations. As particle size and the maximum volumetric reaction rate of the biomass increases, external mass transfer effects become increasingly significant. The work highlights the impact of mass transfer limitation on the measurement of Monod half saturation coefficients (KS) in flocs and granules. Without accounting for external or internal mass transfer limitation, KS is seriously overestimated and becomes a lumped parameter, reflecting not only the microbial response but also the mass transfer limitations observed within the system under study. To avoid confusion or generation of erroneous results, care should be taken in defining, measuring and utilising the half saturation coefficient in biological systems where the biomass is not present as individual cells or extremely small flocs. For Suspended Carrier Reactor systems: External and internal mass transfer are both concluded to be important rate limiting steps within suspended carrier reactors. The demonstration of a significant impact of fluid flow conditions on the nitrification rates highlights the impact of external mass transfer limitation within these systems. Application of a one-dimensional biofilm model to the experimental results led to the conclusion that there is little difference between the external mass transfer limitation of the two different carrier types, for carriers grown under the same environmental conditions. However, there was a significantly higher areal nitrification rate observed on the Natrix carriers compared to the Kaldnes carriers. It is the biofilm structure that is critically important in characterising the mass transfer steps. Systems operated under high nitrogen loads, producing filamentous biofilms on the carrier surface, were found to have larger external mass transfer coefficients and responses to changes in fluid flow than those carriers which were operated under nitrogen-limited conditions (producing a flatter, more gel-like biofilm). The structure of the biofilm colonising the carrier surface was far more important in defining the mass transfer coefficient than the actual carrier type used. In a remarkably similar trend to that of the external mass transfer coefficient, the biofilm morphology was again significantly more important than carrier type in determining both the magnitude and response to fluid flow of the gas-liquid mass transfer coefficient for oxygen (kLa) calculated within the laboratory TOGA sensor. These findings led to the postulation that direct gas-biofilm interfacial mass transfer mechanism is occurring within the SCR systems. This hypothesis is an alternative to the standard mechanism of gas transfer from the bubble into the liquid phase, and then into the biofilm. Understanding of interfacial transfer is likely to be important for developing the knowledge of SCR processes. Overall, both external and internal mass transfer phenomena have been demonstrated to create important rate limitations to suspended aggregate systems (flocs and granules) and biofilms grown in suspended carrier reactors. This significantly advances the conceptual understanding of these biological treatment processes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A ocorrência de fenótipos multirresistentes de Corynebacterium pseudodiphtheriticum e sua associação a infecções graves, com elevada mortalidade em pacientes imunocomprometidos, aliados ao escasso conhecimento da virulência e patogenia destas infecções, motivou esta pesquisa, que teve como objetivo investigar mecanismos de virulência e resistência microbiana deste agente entre pacientes de um hospital universitário brasileiro. Um total de 113 amostras de C. pseudodiphtheriticum identificadas por métodos bioquímicos convencionais e sistema API-Coryne isoladas de pacientes de diferentes grupos etários. Os micro-organismos eram, em sua maioria, relacionados a infecções no trato respiratório (27,45%), urinário (29,20%) e sitios intravenosos (18,60%) e cerca de 32,70% das amostras foram provenientes de pacientes com pelo menos uma das condições predisponentes: insuficiência renal; transplante renal, tuberculose em paciente HIV+, câncer, cirrose hepática, hemodiálise e uso de cateter. As amostras testadas revelaram-se multirresistentes sendo a maioria resistente à oxacilina, eritromicina e clindamicina. A adesão das cepas ao poliestireno e ao poliuretano indicou o envolvimento de hidrofobicidade da superfície celular na fase inicial da formação de biofilmes. O crescimento subsequente conduziu à formação de microcolônias, agregados bacterianos densos incorporados na matriz exopolimérica rodeada por espaços vazios, típica de biofilmes maduros. Adicionalmente, a interação do micro-organismo com fibrinogênio e fibronectina humana indica o envolvimento destes componentes séricos na formação de biofilme, sugerindo a participação de diferentes adesinas neste processo e a capacidade deste agente formar biofilme in vivo. A afinidade por esses componentes e a formação de biofilme podem contribuir para o estabelecimento e disseminação da infecção no hospedeiro. Adicionalmente, as cepas de C. pseudodiphtheriticum isoladas de pacientes com infecções localizadas (ATCC10700/Pharyngitis) e sistêmicas (HHC1507/Bacteremia) exibiram um padrão de aderência agregativa-like a células HEp-2, caracterizado por aglomerados de bactérias com aparência de um "empilhado de tijolos". Através do teste FAS e ensaios de interação na presença de inibidores de citoesqueleto, demonstramos o envolvimento da polimerização de actina na internalização das cepas testadas. A internalização bacteriana e rearranjo do citoesqueleto pareceu ser parcialmente desencadeado pela ativação da tirosina-quinase. Finalmente, C. pseudodiphtheriticum foi capaz de sobreviver no ambiente intracelular e embora não tenha demonstrado capacidade de replicar intracelularmente, células HEp-2 foram incapazes de eliminar o patógeno completamente no ambiente extracelular no período de 24 horas. Todas as cepas estudadas foram capazes de induzir apoptose em células epiteliais 24 horas pós-infecção evidenciada pelo aumento significativo no número de células mortas e pela ocorrência de alterações nucleares reveladas através dos métodos de coloração pelo azul Trypan, pelo DAPI e microscopia electrônica de transmissão. Alterações morfológicas incluindo a vacuolização, a fragmentação nuclear e a formação de corpos apoptóticos foram observadas neste período. A citometria de fluxo demonstrou ainda uma diminuição significativa no tamanho das células infectadas e a utilização de dupla marcação (iodeto de propídio / anexina V) permitiu a detecção da ocorrência de necrose e apoptose tardia. Em conclusão, o conhecimento de tais características contribuiu para a compreensão de mecanismos envolvidos no aumento da frequência de infecções graves com elevada mortalidade em pacientes no ambiente hospitalar, por C. pseudodiphtheriticum, um patógeno rotineiramente subestimado em países em desenvolvimento. / The occurrence of multiresistant phenotypes and associated with severe infections, with high mortality in immunocompromised hosts due to Corynebacterium pseudodiphtheriticum, allied to little known about virulence and pathogenesis these infections, led to present investigation. The investigation aims to examine the virulence mechanisms and resistance to antimicrobial agents of C. pseudodiphtheriticum among patients with bacterial infections at a Brazilian teaching hospital. A total of 113 C. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups. Micro-organisms were mostly related to infections in the respiratory tracts (27.45%), urinary (29.20%) and intravenous sites (18.60%) and approximately 32.70% samples were obtained of patients presenting at least one of the pre-disposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains were resistant to oxacillin, erythromycin and clindamycin. Adherence to polystyrene and polyurethane indicated the involvement of cell surface hydrophobicity in the initial stage of biofilm formation. Further growth led to the formation of dense bacterial aggregates embedded in the exopolymeric matrix surrounded by voids, typical of mature biofilms. Data also showed C. pseudodiphtheriticum recognizing human fibrinogen (Fbg) and fibronectin (Fn) and involvement of these sera components in biofilm formation in conditioning films. These findings suggest that biofilm formation may be associated with the expression of different adhesins. C. pseudodiphtheriticum may form biofilm in vivo possibly by an adherent biofilm mode of growth in vitro currently demonstrated on hydrophilic and hydrophobic abiotic surfaces. The affinity to Fbg and Fn and the biofilm-forming ability may contribute to the establishment and dissemination of infection caused by C. pseudodiphtheriticum. Additionally, C. pseudodiphtheriticum strains isolated from patients with localized (ATCC10700/Pharyngitis) and systemic (HHC1507/Bacteremia) infections exhibited an aggregative adherence-like pattern to HEp-2 cells characterized by clumps of bacteria with a stacked-brick appearance. The fluorescent actin staining test demonstrated that actin polymerization is involved in the internalization of the C. pseudodiphtheriticum strains. Bacterial internalization and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 hours. All samples were able to induce apoptosis in HEp-2 cells 24 h post-infection, evidenced by significant increase in the number of dead cells and nuclear alterations were observed by the Trypan blue assay, DAPI and transmission electron microscopy. Morphological changes in HEp-2 cells observed 24 h post-infection included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an significant decrease in cell size of infected HEp-2 cells. Furthermore, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. In conclusion, these characteristics may contribute to understanding of mechanisms involved on increase of severe infection, with high mortality in nosocomial enviroment patients by C. pseudodiphtheriticum, a pathogen usually overlooked in emerging countries.