Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

The opportunistic pathogen Pseudomonas aeruginosa ranks among the leading causes of nosocomial infections.  The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins (effectors) into host cells to suppress the host's innate immune response.  Expression of all T3SS-related genes is strictly dependent upon the transcription factor ExsA.  Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest.  The ExsA-ExsC-ExsD-ExsE signaling cascade ties host cell contact to the up-regulation of T3SS gene expression.  Prior to T3SS induction, the antiactivator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions.  Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC, resulting in the release of ExsA and an up-regulation of the T3SS. ExsA is an AraC/XylS-type transcriptional regulator and belongs to a subfamily of activators that regulate the T3SS in a variety of Gram-negative pathogens.  These regulators are characteristically difficult to purify due to the low solubility of their C-terminal DNA binding domains.  A new method for purifying ExsA was developed and produced ExsA with improved solubility.  The interaction of ExsA and its PexsD promoter was examined using fluorescence anisotropy.  An in vitro transcription assay was developed and it was determined that ExsA is sufficient to activate T3SS transcription. Next, the ExsD--ExsA inhibitory mechanism was examined.  It was demonstrated for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in  vitro without the aid of any other cellular factors.  More significantly and contrary to previously published results, it was discovered that independently folded ExsD and ExsA are capable of interacting, but only at 37 degrees C and not at 30 degrees C.  Guided by the crystal structure of ExsD, a monomeric variant of the protein was designed to demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30 degrees C. To further elucidate the ExsD-ExsA inhibitory mechanism, the ExsD-ExsA interface was examined.  ExsD variants were generated and used to determine which region of ExsD interacts with ExsA.  Interestingly, ExsD was also found to bind DNA, although it is unclear whether or not this plays a role in ExsA inhibition.  Fully understanding the mechanism by which ExsD inhibits ExsA may enable the development of drugs that target ExsA in order to shut down the T3SS, thereby eliminating P. aeruginosa infection. / Ph. D.

Bacterial biofilms are communities of micro-organisms encased a self-produced extracellular matrix. While they form readily in a nature, biofilm formation in man-made systems have economic and health consequences. Prior research demonstrated that topographical features comprised of uniform, micro-meter sized particles hindered the biofilm formation of Pseudomonas aeruginosa (P. aeruginosa), an opportunistic human pathogen. The goal of the present work is to 1) further develop a potential anti-biofilm coating by improving its robustness and 2) study the mechanism(s) by which surface topography hinders biofilm formation. The robustness of a topographical coating comprised of an array of silica particles is improved by the introduction of silica bridges through a sol-gel reaction. To study the mechanism(s), specifically, we hypothesized that the motion, or surface motility, of P. aeruginosa is hindered by the presence of micro-meter scale obstacles via physical obstruction. To test this, we analyzed the behavior of single P. aeruginosa cells at micron-scale spatial resolutions using time-lapse fluorescence microscopy, image analysis, and particle tracking techniques. We fabricated various types of micron-scale topography with curvature (particle arrays) and recti-linear features (vertical steps) and varied the critical dimension within the range of 0.5 – 10 µm which spans the dimensions of a typical P. aeruginosa cell. We found that there was a threshold feature size of 1-2 µm at which bacterial surface motility is drastically impacted. On positively curved topography (particle arrays), we found that the frequent obstacles reduced the average speed of a bacterium from 6.2  0.3 µm per 5 min on a flat surface to 2.1  0.3 µm per 5 min on an array of 2 µm particles. Furthermore, we observed that bacteria often move in-between particles, suggesting that bacteria have difficulty climbing over tall obstacles. To further investigate P. aeruginosa's ability to cope with topography, we examined the effect of recti-linear features (vertical steps) on surface motility. We found that step heights > 0.9 µm drastically reduced the probability of crossing and that the average speed when approaching the step is reduced by a factor of 2. Interestingly, we find that bacteria have a slight preference to traverse down which is against the direction of gravity in our system. In summary, these results offer insights into how a surface motile bacterium copes with a topographical surface. Our data indicate that the topography of a surface can impede the surface motility of bacterium and thus, may be an important mechanism by which topography prevents biofilm formation. / Doctor of Philosophy / Bacteria and other micro-organisms can grow on surfaces such as medical devices and cause infections. Other examples of where bacteria can grow are on drains and pipes causing clogging, and on the hulls of ships, thus increasing drag. The goal of the current work is to investigate material coatings that resist the attachment and growth of bacteria on surfaces. We demonstrate that changing the roughness of the surface can reduce the number of bacteria found on the surface. More specifically, we have made surfaces covered with spheres that are approximately the same size as a bacterium, about 1 micrometer (10x smaller than the diameter of hair). We find that the spheres act as physical obstacles that block bacteria from moving on a surface. These results suggest that changing the micro-scale geometry of a surface may reduce the rate of infections on medical devices or hinder the growth of bacteria in other systems

Microbes in nature live collaboratively in adherent communities, known as biofilms. Biofilms can be contextually beneficial or detrimental. In medical implants, biofilms cause infections leading to additional healthcare costs of billions of dollars. Studies have found that micro/nanoscale surface topography can significantly alter (i.e., promote or hinder) the process of biofilm formation. The formation of biofilm starts with planktonic microbes attach to the surface. To further understand the biophysical underpinning of this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. To this end, aligned nanofiber coating with controlled fiber diameter and edge-to-edge spacing were manufactured using the Spinneret-based Tunable Engineered Parameters (STEP) techniques. The effect of surface topography on bacterial near-surface motility was studied. The experimental results showed that the bacterial attachment and near-surface motion can be greatly impacted by surface topography. Furthermore, the finding was applied to ureteral stents. The results showed that the aligned nanofiber can significantly reduce the biofilm formation process on ureteral stents. / Master of Science / Many microbes in nature live in adherent communities called biofilm. Biofilms contain individual microbes inside polymeric matrix which protect them from environmental stressors such as antibiotics. Biofilms are a significant contributor to the infection of implantable medical devices, which leads to additional healthcare costs of billions of dollars annually in the U.S. alone. Studies have found that sub-micron scale surface topography can significantly promote or hinder biofilm formation; however, the exact mechanism remains poorly understood. To further understand this process, the effect of aligned nanoscale surface structures on microbial adhesion was studied. The formation of microbial biofilm starts with swimming bacteria sensing the liquid-solid interface and attaching to the surface. Microbes are more likely to settle on a surface if a surface is favorable to attach. However, the decision-making process has not been fully understood. Our experimental results showed that the bacterial attachment and near-surface motion can be greatly influenced by surface topography. Furthermore, the finding was applied to ureteral stents, which is a type of medical implants used to maintain the flow of urine in the urinary tract. Ureteral stents serve great for medical purposes, but as foreign bodies, they also lead to urinary tract infection. The results showed that some types of aligned fiber coating increased microbial attachment density, while other types of aligned fiber coating reduced the bacterial surface coverage by up to 80%, which provides directions for future studies.

Yes / Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 C) and an arbitrarily-selected temperature of 46 C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. / University of Malaya High Impact Research (HIR) UM-MOHE HIR Grants (UM.C/625/1/HIR/MOHE/CHAN/14/1, No. H-50001-A000027; UM.C/625/1/HIR/MOHE/CHAN/01, No. A000001-50001); PPP Grant (PG081-2015B)

Pseudomonas aeruginosa are medically important bacteria that are notorious for causing nosocomial infections. To gain more knowledge into understanding how this organism operates, it was decided to explore the pyrimidine biosynthetic pathway. Pyrimidine synthesis, being one half of the DNA structure, makes it a very important pathway to the organism’s survivability. With previous studies being done on various genes in the pathway, pyrE has not been studied to the fullest extent. To study the function of pyrE, a site directed mutagenesis was done to completely knock out pyrE, which encodes the protein orotate phosphoribosyl transferase that is responsible for converting orotate into orotate monophosphate (OMP). A mutation in this step leads to accumulation and secretion of orotate into the medium. Analyzing virulence factors produced by the mutant and comparing to the wild type, some intriguing features of the mutant were discovered. One of the findings was the over expression of virulence factors pyoverdin and pyocyanin. Pyocyanin over expression, based on the results of this study, is due to the accumulation of orotate while over production of pyoverdin is due to the accumulation of dihydroorotate. The other virulence factors studied were motility assays, exoproducts, and growth analysis. All virulence factor production was reduced significantly in the mutant compared to the wild type. The casein protease assay showed absolutely no production of proteases in the mutant. The conclusion is that orotate accumulation leads to a significant reduction in virulence factor production in Pseudomonas aeruginosa. In addition to that, it was found that excess orotate in the wild type led to a decrease in quorum sensing regulated products.

Une coagulopathie aigue endogène (EAC) est présente chez 25% des patients de traumatologie dès leur arrivée. Des résultats d’études récentes montrent que cette EAC est liée à l’activation de la voie de la protéine C (aPC). Quelques heures après, se développe un état pro-coagulant associant un niveau abaissé d’aPC et un taux plasmatique élevé de l’inhibiteur de l’activateur du plasminogene (PAI-1). Nous trouvons que l’incidence des pneumopathies associées à la ventilation est significativement augmentée chez ces patients sans toutefois connaître le rôle exact de ces anomalies de coagulation. Basé sur cette hypothèse central de susceptibilité augmentée a l’infection et plus particulièrement aux pneumopathie a P.aeruginosa (PA) le but de ce travail est d’identifier les mécanismes par lesquels PAI-1 et aPC peuvent moduler la perméabilité de la barrière alveolo capillaire et ceci a travers 3 objectifs spécifiques1 – Objectif 1 : déterminer les mécanismes par lequel PA augmente la perméabilité endothéliale. 2 – Objectif 2 : déterminer le rôle d’aPC dans la modulation des effets de PA sur l’œdème pulmonaire lésionnel.3 – Objectif 3 : déterminer le rôle de PAI-1 dans la modulation des effets de PA sur l’œdème pulmonaire lésionnel.En utilisant un inhibiteur spécifique des petites GTPases nous démontrons le rôle centrale joué par RhoA dans le développement de l’œdème pulmonaire induit par PA. PAI-1 et aPC sont impliquées dans le mécanisme lésionnel pulmonaire. aPC et l’inhibition de la voie du RhoA attenue le développement de l’œdème pulmonaire et diminue la dissémination systémique bactérienne. Cependant le blocage invivo de la voie de PAI-1 est associé à une surmortalité et à une augmentation de la charge bactérienne suggérant un rôle de PAI-1 dans l’activation de la réponse inflammatoire nécessaire a l’éradication de PA / A clinically significant acute endogenous coagulopathy (EAC) is present in 25% of major trauma patients upon arrival in the emergency department, before any fluid resuscitation. Results from recent clinical studies indicate that EAC is primarily caused by the activation of the anticoagulant protein C pathway. Several hours later, there is the development of a systemic procoagulant activity associated with low plasma levels of activated protein C (aPC) and an inhibition of the fibrinolysis caused by elevated plasma levels of plasminogen activator inhibitor 1 (PAI-1). We have found that the incidence of ventilator-associated pneumonia (VAP) is significantly increased in trauma patients with these coagulation abnormalities [6, 9]. However, whether these coagulation abnormalities play a mechanistic role in the increased susceptibility to nosocomial lung infection observed after severe posttraumatic hemorrhage is unknown. Thus, the central hypothesis is that the increased susceptibility to P. aeruginosa (PA) pneumonia following severe trauma with tissue hypoperfusion is mediated in part by these posttraumatic coagulation abnormalities within the airspaces of the lung. Specifically, in this work, we will identify through 3 specific aims the mechanisms by which PAI-1 and aPC modulate PA–mediated increase in alveolar-capillary barrier permeability.1 - Specific Aim 1: To determine the mechanisms by which PA increases lung endothelial permeability.2 - Specific Aim 2 : To determine the Role of aPC in modulating the effect of PA on the lung endothelial barrier function3 - Specific Aim 3 : To determine the Role of PAI-1 in modulating the effect of PA on the lung endothelial barrier functionIn the present work, we demonstrated the central role small GTPases RhoA plays in the increase of permeability induced by pseudomonas infection. PAI-1 and aPC are deeply involved in the control of early lung inflammation. aPC and inhibition of the RhoA pathway attenuates the development of pulmonary edema and decrease in the systemic dissemination of P. aeruginosa. However, in vivo disruption of PAI-1 signalling is associated with higher mortality at 24 h and significant increase in the bacterial burden suggesting that PAI-1 is required for the activation of the innate immune response necessary for the eradication of PA from the distal airspaces of the lung

Dans un contexte mondial d’émergence de bactéries résistantes aux antibiotiques, il est nécessaire d’explorer de nouvelles voies de recherche pour trouver de nouveaux traitements. Cet état de fait est particulièrement marqué dans le cadre d’infections chroniques associées à une colonisation des tissus par des biofilms bactériens. L’endocrinologie microbienne est un champ de recherche axé sur l’étude des mécanismes de communication inter-règnes qui peut s’établir entre des bactéries et leurs hôtes. Les molécules humaines qui permettent ce dialogue constituent de potentiels outils capables de moduler la physiologie des bactéries pour empêcher leur développement. Dans cette optique, l’objectif de ma thèse était d’approfondir nosconnaissances sur l’effet des peptides natriurétiques, une famille d’hormones humaines, sur la physiologie du pathogène opportuniste P. aeruginosa. Les travaux que j’ai menés ont permis de préciser les mécanismes de l’action anti-biofilm du peptide natriurétique de type C ou CNP. J’ai également montré qu’un autre peptide, le peptide natriurétique atrial (ANP), est capable de disperser un biofilm établis de P. aeruginosa. Dans un second temps, j’ai pu identifier que l’opéron ami, dans son intégralité, est indispensable aux effets des peptides natriurétiques et qu’en plus les protéines codées par les gènes de l’opéron ami ont un rôle important dans la régulation de la virulence bactérienne et dans la formation des biofilms. Ainsi, j’ai pu mettre en évidence que les protéines AmiE et AmiR, en plus de leur rôle dans le métabolisme secondaire, sont impliquées dans la régulation de la virulence et de la formation de biofilm de P. aeruginosa. / In a global context of emergence of antibiotic-resistant bacteria, it is necessary to explore new paths of research to find new treatments. This state of affairs is particularly marked in the context of chronic infections associated with tissue colonization by bacterial biofilms. Microbial endocrinology is a field of research focused on the study of inter-kingdom communication that can be established between bacteria and their hosts. The human molecules that allow this dialogue are potential tools capable of modulating bacterial physiology to prevent their development. In this perspective, the aim of my thesis was to deepen our knowledge about the effect of natriuretic peptides, a family of human hormones, on the physiology of the opportunistic pathogen P. aeruginosa. The work that I carried out allowed us to characterize the mechanisms of the anti-biofilm action of the natriuretic peptide type C or CNP. I have also shown that another peptide, the atrial natriuretic peptide (ANP), is able to disperse an established biofilm of P. aeruginosa. In a second step, I was able to identify that the entire ami operon is essential for the effects of the natriuretic peptides and that the proteins encoded by the genes of the ami operon have an important role in bacterial virulence regulation and in the formation of biofilms. Thus, I was able to demonstrate that the AmiE and AmiR proteins, in addition to their role in secondary metabolism, are involved in the regulation of virulence and biofilm formation of P. aeruginosa.

A contaminação de corpos d\'água por fármacos é um tema de extrema relevância, tendo em vista problemas como a escassez de água, florações de cianobactérias tóxicas e lançamentos clandestinos de efluentes domésticos. Sendo assim, este trabalho teve como objetivo determinar a presença de cafeína (CAF), fluoxetina (FLX), levotiroxina (LVX) e bezafibrato (BZF) em mananciais do estado de São Paulo, bem como avaliar a toxicidade desses compostos à cianobactéria Microcystis aeruginosa LTPNA 08. Um método por LC-MS/MS foi desenvolvido e validado, de acordo com a RDC nº 166 da ANVISA, para a detecção de CAF, FLX, LVX e BZF em amostras ambientais. As represas Guarapiranga e Billings, bem como os rios Taiçupeba, Sorocaba, Baixo Cotia, Grande e Paraíba foram monitorados de abril a setembro de 2017. A toxicidade dos fármacos foi avaliada por meio do monitoramento do crescimento, produção de microcistinas e viabilidade celular da cianobactéria M. aeruginosa LTPNA 08. CAF foi detectada em todas as amostras analisadas, com concentrações que variaram de 6,6 ng.L-1 a 16,47 µg.L-1. No Rio Cotia foram verificadas as maiores concentrações de CAF, FLX e BZF (16,47 µg.L-1; 3,5 ng.L-1 e 322 ng.L-1, respectivamente). A LVX, cujos produtos de biotransformação não foram monitorados, não foi detectada em nenhuma amostra analisada. A concentração de 50 µg.L-1 de FLX inibiu o crescimento da cianobactéria em 82,3% (CE50: 31,4 µg.L-1). Em relação à produção de microcistinas totais, os fármacos inibiram a liberação da fração extracelular para a maior concentração testada ao longo do tempo de monitoramento, embora não tenham demonstrado efeito sobre a viabilidade celular. Sendo assim, considerando-se que fármacos estão presentes nos mananciais monitorados no estado de São Paulo e que a FLX pode causar efeito sobre a M. aeruginosa, os efeitos decorrentes da exposição a concentrações ambientais contínuas e cumulativas de fármacos em corpos d\'água devem ser estudados. Além disso, uma vez que a ocorrência destas substâncias e outros contaminantes antropogênicos no ambiente aquático natural é uma questão emergente devido aos efeitos adversos potenciais que estes compostos representam para a vida aquática e os seres humanos, os tipos e níveis destes compostos, que têm um impacto maior na qualidade da água, deve ser constantemente monitorada. Práticas de gestão que investem em saneamento e na redução da descarga de efluentes não tratados, e um plano de proteção de recursos hídricos com o objetivo de garantir a segurança da água seriam medidas essenciais para reduzir o aporte de contaminantes nos corpos d\'água do estado de São Paulo. / Contamination of water bodies by drugs is a subject of extreme relevance considering related problems such as water scarcity, harmful cyanobacterial blooms and discharge of untreated domestic effluents. Therefore, the aim of this work was to determine the presence of caffeine (CAF), fluoxetine (FLX), levothyroxine (LVX) and bezafibrate (BZF) in springs in the State of São Paulo, and to evaluate the toxicity of these compounds in cyanobacteria Microcystis aeruginosa LTPNA 08. A LC-MS/MS method was developed and validated according to RDC nº 166 of ANVISA to assess the concentration of CAF, FLX, LVX and BZF in environmental samples. Guarapiranga and Billings reservoirs, as well as the Taiçupeba, Sorocaba, Baixo Cotia, Grande and Paraíba rivers were monitored from April to September 2017.The drugs toxicity in M. aeruginosa LTPNA 08 was assessed by monitoring their effects on cyanobacterial growth, microcystins production and cell viabilityby flow cytometry. CAF was detected in all analyzed samples at concentrations ranging from 6.6 ng to 16.47 µg.L-1.Among studied sites, Cotia river showed the highest concentrations of CAF, FLX and BZF (16.47 µg.L-1, 3.5 ng.L-1 and 322 ng.L-1, respectively). LVX, which biotransformation products were not monitored, was not detected in any of the analyzed samples. Regarding the drugs toxicity, 50 µg.L-1 of FLX inhibited the cyanobacterial grow thin 82.3% (EC50 of 31.4 µg.L-1). Although no effect on cell viability was seen by flow cytometry, the highest concentrations of all compounds tested were able to inhibit the release of microcystins. Therefore, considering that some of the drugs monitored showed to be present in water sources in São Paulo State and that FLX affects cyanobacteria M. aeruginosa growth, the effects of continuous and cumulative exposure at environmental drug concentrations of in water bodies should be evaluated. Also, since the occurrence of these substances and other anthropogenic contaminants in the natural aquatic environment is an emerging issue due to the potential adverse effects these compounds pose to aquatic life and humans, thet ypes and levels of these compounds, which have a greater impact on water quality, should be constantly monitored. Management practices investing in sanitation and in reducing discharge of untreated effluents, as well as a plan for water resources protection with the goal of ensuring water security would be essential measures in reducing drugs loading into water bodies situated in São Paulo State.

A infecção de corrente sanguínea (ICS) causada por bactérias multirresistentes tem alta mortalidade em pacientes receptores de transplante de células-tronco hematopoiéticas (TCTH). A Pseudomonas aeruginosa é um dos agentes mais frequentes e de difícil tratamento nessa população de pacientes. Objetivos: Avaliar características clínicas, microbiológicas e moleculares de 30 isolados de P. aeruginosa resistente à carbapenêmicos (PARC) em ICS de pacientes submetidos a TCTH e a presença de sinergismo antimicrobiano in vitro. Métodos: Os dados clínicos foram obtidos retrospectivamente de prontuários médicos e registrados em banco de dados. Análises bivariadas e multivariadas foram realizadas para avaliar determinantes de desfechos clínicos e uma curva de sobrevida foi construída. Determinou-se a concentração inibitória mínima (CIM) dos antimicrobianos por meio de microdiluição, foram realizados ensaios de sinergismo por método de checkerboard e time-kill, avaliação da clonalidade por eletroforese em campo pulsado e detecção de genes codificadores de mecanismos de resistência e virulência por reação em cadeia de polimerase. O sequenciamento do genoma completo (WGS) dos principais clones foi realizado por Nextera XT, utilizando a tecnologia Illumina MiSeq. Resultados: A maioria dos pacientes era do gênero feminino, com mediana de idade de 48 anos. Neutropenia foi presente em 93% dos pacientes e colonização prévia por PARC em 32%. A mortalidade em 14 dias foi 68%; a maioria dos pacientes que morreram foram transplantados alogênicos (79% vs. 17% entre receptores de transplante autólogo; p=0,012). Pacientes tratados com duas ou três drogas não apresentaram diferença estatisticamente significante na mortalidade até 14 dias após a ICS. Foram avaliados 30 isolados bacterianos. Todos apresentaram alto nível de resistência ao meropenem (MERO): CIM90 > 512 ug/mL; dois terços eram resistentes à amicacina (AMK) (CIM 2-512 ug/mL) e todos mantinham sensibilidade à colistina (COL). Muitos isolados (17/30) alcançaram efeito sinérgico in vitro pelo método time-kill com a combinação MERO mais COL, mas não com AMK. Nenhum antagonismo foi observado. Houve menor mortalidade em pacientes cujo isolado apresentou sinergismo entre COL e MERO quando comparados a pacientes portadores de isolados sem sinergismo, sem significância estatística. O gene de carbapenamase mais identificado foi blaSPM e 6 isolados apresentaram blaSPM e blaKPC. Os isolados apresentaram genes relacionados com virulência, tais como toxA, exoS e lasB; pacientes com ICS causada por P. aeruginosa que abrigava o gene lasB apresentaram maior risco de evoluir para o óbito. O WGS mostrou que os clones abrigavam SPM-1, Tn4371, mutações em porinas, em partes das bombas de efluxo, nas proteínas ligadores de penicilina (PBP) e pertenciam a ST277. Conclusão: As ICS por PARC cursaram com alta mortalidade em pacientes submetidos à TCTH. Houve uma grande proporção de resultados positivos para sinergismo entre os antimicrobianos in vitro, mas não foi possível demonstrar benefício estatisticamente significante no uso da terapia combinada com três drogas. Os clones carreavam SPM-1, Tn4371 e pertenciam a ST277 / Bloodstream infection (BSI) has high mortality in hematopoietic stem cell transplant (HSCT) recipients and Pseudomonas aeruginosa is an important and challenging organism. Objectives: To evaluate clinical, microbiological and molecular features of carbapenem-resistant P. aeruginosa (CRPA) isolates from BSI identified among HSCT patients and address in vitro synergy of antibiotic combination. Methods: Patient medical records were retrospectively reviewed and registered in a database. We used bivariate and multivariate analyzes to investigate determinants of clinical outcomes, and demonstrated overall mortality using a survival curve. We determined minimal inhibitory concentrations (MIC) for antimicrobials and in vitro synergies using checkerboard and time-kill assays, pulsed-field electrophoresis (PFGE) for clonality assessment and polymerase chain reaction (PCR) to detect carbapenamases and virulence genes were performed for all isolates. Whole genome sequence (WGS) of main clones was performed by Nextera XT, using Illumina MiSeq technology. Results: Most patients were female, median age was 48 years old. Main baseline disease was acute leukemia and 68% received allogeneic HSCT. 93% of patients had neutropenia and 32% had prior CRPA gut colonization.14-day mortality was 68%; mortality was higher among allogeneic HSCT recipients compared to autologous HSCT recipients (79% vs. 17% p = 0,012). Patients treated with two or three drugs did not present a statistically significant difference in 14-day mortality after BSI. In total, 30 bacterial isolates were analyzed; all presented a high resistance level to meropenem (MERO): MIC90 > 512ug/mL; two thirds were also resistant to amikacin (AMK) (MIC 2-512 ug/mL) and all were susceptible to colistin (COL). Many (17/30) isolates achieved in vitro synergistic effect in time-kill assay with the association of MERO and COL, but synergistic effect was not observed with AMK, by time-kill. No antagonistic effect was observed. There was a tendency towards better survival in patients whose CRPA isolate had in vitro synergy between COL and MERO without statistical significance. The most frequent carbapenamase gene identified was blaSPM, and six co-harboured both blaKPC and blaSPM. Isolates presented genes related to virulence factors such as toxA, exoS and more patients with BSI caused by P. aeruginosa harbouring gene lasB evolved to death. WGS analysis showed that clones harboured SPM-1, Tn4371 and belonged to ST277. They also presented mutations in genes related with porins and efflux pumps, as well in penicillin binding proteins (PBPs). Conclusion: CRPA BSI as associated with high mortality in HSCT recipients. A large proportion of isolates had in vitro synergy; however, we could not demonstrate statistically significant benefit in the use of combination therapy. Clones carried SPM-1, Tn4371 and belonged to ST277