<p dir="ltr">Tuberculosis remains a global health challenge, ranking as the second leading cause of mortality worldwide in 2022. The resilience of <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, is enhanced by the high expression of efflux pumps that confer antibiotic tolerance and the formation of biofilms that confer resistance to antibiotics. Carbon nanomaterials (CNMs) exhibit a broad-spectrum of antibacterial efficacy, making them promising candidates for combating drug-resistant bacterial strains. The effects of the novel carbon allotropes called fullertubes (C₉₀) on any living cell have not been studied. In our study, we employed <i>Mycobacterium smegmatis</i> as a model organism for <i>M. tuberculosis</i> and exposed it to fullertubes and fullerenes. We explored the impact of these CNMs on efflux activity and biofilm formation through biochemical assays like ethidium bromide transport assay and crystal violet assay. We also investigated their impact on lipid biosynthesis associated with log-phase growth and biofilm formation using metabolic radiolabeling studies. We also investigated the effects of the efflux pump inhibitors (EPIs) piperine, berberine, 1-(1-naphthylmethyl)-piperazine and thioridazine on efflux activity, biofilm formation, and lipid biosynthesis associated with log-phase growth and biofilm formation in <i>M. smegmatis.</i> We utilized metabolic radiolabeling methods using ¹⁴C-palmitic acid and ¹⁴C-acetic acid which are precursors of lipid biosynthesis and analyzed the lipids by silica-thin layer chromatography and autoradiography. Our studies revealed that CNMs do not influence efflux activity. However, efflux pump inhibitors effectively block efflux activity in <i>M. smegmatis</i>. Biofilm formation was decreased by CNMs and EPIs. In biofilm cells, fullertubes increased the incorporation of radiolabeled ¹⁴C-palmitic acid into glycopeptidolipids on the cell surface as well as inside the cell. Piperine and berberine affected the incorporation of the radiolabels into lipids such as trehalose monomycolate, phosphatidylethanolamine and cardiolipin in planktonic and biofilm cells. Our study provides insights into the diverse effects of CNMs and efflux pump inhibitors on <i>M. smegmatis</i>.</p>

The overall hypothesis of this work is that the physiological microbial stress response could serve as a rapid, sensitive, and mechanistically-based indicator of process upset in biological wastewater treatment systems that receive sporadic shock loads of toxic chemicals. The microbial stress response is a set of conserved and unique biochemical mechanisms that an organism activates or induces under adverse conditions, specifically for the protection of cellular components or the repair of damaged macromolecules. Using traditional immunochemical analysis techniques, the heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals at all concentrations tested (cadmium, pentachlorophenol, and acetone) or heat stress. As total cadmium concentrations increased above 5 mg/L, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls, but there was no additional increase in GroEL levels. Stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but GroEL may not be a good candidate protein for this purpose due to the lack of a dose/response relationship. Additionally, production of stress proteins did not explain the significant deflocculation upsets that were characteristic of many of the industrially-relevant chemicals tested, including pentachlorophenol and cadmium. Although the purpose of stress response mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. The goal of the second research phase was to determine whether the bacterial glutathione-gated, electrophile-induced potassium efflux system is responsible for deflocculation observed due to shock loads of toxic electrophilic (thiol reactive) chemicals. The results indicate significant K+ efflux from the activated sludge floc structure to the bulk liquid in response to shock loads of 1-chloro-2,4-dinitrobenzene (CDNB), N-ethylmaleimide (NEM), 2,4-dinitrotoluene (DNT), 1,4-benzoquinone (BQ), and Cd2+ to a bench-scale sequencing batch reactor (SBR) system. In most cases, the stressor chemicals caused significant deflocculation, as measured by an increase in effluent volatile suspended solids (VSS), at concentrations much less than that required to reduce the maximum specific oxygen uptake rate by 50% (IC50). This suggests that electrophile-induced activated sludge deflocculation is caused by a protective bacterial stress mechanism (as hypothesized) and that the upset event may not be detectable by aerobic respirometry. More importantly, the amount of K+ efflux appeared to correlate well with the degree of deflocculation. The transport of other cations including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared to K+ efflux. In bench-scale SBRs, it was also determined that the K+ efflux occurred immediately (within minutes) after toxin addition and then was followed by an increase in effluent turbidity. K+ efflux and deflocculation responses were similar for bench-scale SBRs and continuous-flow reactor systems, indicating that the periods of elevated exogenous substrate levels typical in SBR systems are not required to activate electrophile-induced K+ efflux or deflocculation. This also suggests that the initial and rapid efflux of K+ immediately following electrophile addition is the factor that leads to deflocculation, not the increase in bulk liquid K+. Sphingomonas capsulata, a bacterium consistent with that found in biological wastewater treatment systems, Escherichia coli K-12, and activated sludge cultures exhibited very similar dynamic efflux/uptake/efflux responses due to the electrophilic stressors, NEM and CDNB, and the thiol reducing agent, dithiothreitol (DTT). The polyether ionophore antibiotic, nigericin, was used to artificially stimulate K+ efflux from S. capsulata and activated sludge cultures. Thus, glutathione-gated K+ efflux (GGKE) activity may cause K+ release from the cytoplasm of activated sludge bacteria into the floc structure and extracellular polymeric substances (EPS) and then diffusion-limited transport into the bulk liquid. It was not possible to resolve the effect of the GGKE system on changes in bulk liquid or floc-associated pH. However, calculations indicate that the localized K+ concentration within the floc structure immediately after chemical stress is consistent with that known to induce floc disruption as a result of KCl addition. Using alkaline phosphatase as a periplasmic marker as well as fluorescent membrane-permeable and impermeable nucleic acid stains, it was determined that a negligible amount of the K+ efflux response was due to lysis of activated sludge microorganisms. The current results are very promising and are the first to suggest that activated sludge upset (i.e. deflocculation) may be caused by a specific protective stress response in bacteria. / Ph. D.

Fertilization is a common, cost effective treatment for increasing forest productivity within managed forests of the southeastern United States. However, little is known about how fertilization affects the below-ground processes that drive soil CO2 efflux in loblolly pine (Pinus taeda L.). A thorough understanding of below-ground carbon dynamics is necessary for the estimation of net ecosystem productivity and the carbon storage potential of these managed systems. In April 2004, we began monitoring total soil CO2 efflux (EC), heterotrophic (RH), and root respiration (RR) in response to fertilization with diammonium phosphate (DAP). Respiratory components were measured prior to fertilization, weekly following fertilization, and bi-weekly after respiratory components stabilized using a dynamic closed chamber and an infrared gas analyzer. We found that EC differed significantly (P<0.0001) between fertilized and unfertilized plots, but the direction was dependent on date. In the early period of the study, fertilized plot values were lower than control plots. However, by the latter periods fertilized plot values returned to control levels except for one sampling date in March 2005 when fertilized plot values were greater then control plots. Heterotrophic respiration was consistently and significantly (P=0.0002) lower in fertilized plots. Root respiration was significantly (P=0.0597) increased in fertilized plots when analyzed over the study and showed a 20% increase due to fertilization. We concluded that an increase in RR and possibly root biomass was enough to balance the decrease in RH leading to no difference in EC later in the growing season. We performed a pair of greenhouse studies to observe the effects of fertilization in the form of diammonium phosphate (DAP) on RR. The objectives were to determine how nutrient additions initially affect RR in one-year-old loblolly pine seedlings. Secondly, we wanted to determine if Captan [N-(trichloromethylthio) cyclohex-4-ene-1, 2-dicarboximide], a mild fungicide, could be used to reduce or eliminate ecto-mycorrhizae upon visual inspection. Both studies showed that initially, at a high rate (100 ppm N and 49 ppm P) of fertilization, RR was significantly (P<0.10) increased relative to seedlings that did not receive fertilization. This increase was only temporary with rates returning to, or decreasing below, control levels by the end of the study. No consistent trend was found between low (25 ppm N and 13 ppm P) and moderate (50 ppm N and 25 ppm P) rates of fertilization. Captan was shown to generally have no affect on RR. Captan and fertilization both showed (visual inspection) a decrease in fine-roots and mycorrhizae, which could explain the reduction in respiration rates observed in these treatments by the end of the studies. / Master of Science

Soil carbon dioxide efflux resulting from microbial and root respiration is a major component of the forest carbon cycle. We undertook this investigation to better understand the nature of soil carbon dioxide efflux of plantation loblolly pine, an important ecological and economical resource in the southeastern United States. Specifically, we hoped to learn how soil carbon dioxide efflux differs both spatially and temporally for four age classes of plantation loblolly pine on the Virginia piedmont. During a 12-month period, soil carbon dioxide efflux was repeatedly measured for four age classes of plantation loblolly pine using a dynamic, closed-chamber infrared gas analyzer. The age classes examined were 1- to 2-year-old, 4- to 6-year-old, 8- to 12-year-old, and 20- to 25-year-old stands. Mean soil carbon dioxide efflux rates measured during the 12-month study were 1.72, 2.58, 2.84, and 2.90 micromole/sq m/s for 1- to 2-year-old, 4- to 6-year-old, 8- to 12-year-old, and 20- to 25-year-old stands, respectively. Stand age had a significant effect on efflux rate during 10 of the 12 monthly sampling sessions. Additionally, mean efflux rates were consistently higher near the tree and a significant positional difference was detected during 8 of the 12 monthly sampling sessions. Mean soil carbon dioxide efflux rates, by position, for the 12-month study were 2.72 and 2.28 micromole/sq m/s for the near and away measurement positions, respectively. Based on monthly mean soil carbon dioxide efflux rates, annual carbon losses were estimated at 651, 976, 1074, and 1082 g C/sq m/yr for 1- to 2-year-old, 4- to 6-year-old, 8- to 12-year-old, and 20- to 25-year-old stands, respectively. Regression analysis was used to examine the influence of soil and climatic factors on seasonal changes in soil carbon dioxide efflux. The most influential factors affecting soil carbon dioxide efflux during the 12-month study were soil temperature, soil moisture, stand age, and measurement position. We believe respiring roots significantly influence soil carbon dioxide efflux of plantation loblolly pine and account for differences observed between stands of different ages as well as spatial differences observed within a given stand. / Master of Science

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution. We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons. We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed. Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

Pseudomonas aeruginosa est une bactérie Gram-négative qui présente une grande résistance aux antibiotiques, lui permettant de sévir dans le milieu hospitalier en infectant plus particulièrement les patients immunodéprimés. Cette résistance est principalement due au système d’efflux membranaire MexAB-OprM, capable d’exporter les antibiotiques en dehors de la cellule. Cette pompe à efflux est composée de trois protéines, MexA, MexB et OprM, incorporées dans les membranes internes et externes de la paroi bactérienne. Les structures de MexA, OprM et AcrB -une protéine présente chez E. coli, homologue de MexB- ont été déterminées individuellement par cristallographie des rayons X. Cependant, la structure du complexe entier, regroupant les trois protéines en interaction, ainsi que le mécanisme de cette pompe font toujours défaut. Le renforcement de nos connaissances structurales et fonctionnelles est donc capital pour lutter plus efficacement contre ces bactéries, par de nouvelles stratégies médicamenteuses. Ce travail porte sur l’étude de la structure et de la stœchiométrie de l’assemblage des protéines OprM et MexA au sein d’une membrane lipidique. La caractérisation du complexe OprM/MexA a été réalisée à l’aide de nouvelles techniques de caractérisation physico-chimique des surfaces, telle que la Microbalance à Cristal de Quartz avec Mesure de Dissipation (QCM-D), et par des méthodes d’imagerie, telles que la Cryo-Microscopie Electronique en Transmission (CryoMET) et la Cryo-Tomographie Electronique (CryoTE). En QCM-D, les mesures d’interaction entre OprM et MexA ont été réalisées sur support solide en contrôlant l’orientation d’OprM placée dans un environnement lipidique. Après ajout de la protéine MexA, la formation de complexes OprM/MexA a été mise évidence. Pour comprendre l’organisation de ce complexe, nous avons procédé à une étude comparative de l’organisation des protéines OprM, MexA et du complexe OprM/MexA incorporés dans une membrane lipidique, par CryoMET. Trois types d’organisation, respectivement spécifiques d’OprM, de MexA et du complexe OprM/MexA, ont été mis en évidence. Une analyse structurale de ces trois différents assemblages, pris en sandwich entre deux membranes lipidiques, a été menée par CryoTE. La reconstitution de la protéine OprM conduit à la formation de protéoliposomes, dû à des interactions intervenant entre les protéines OprM au niveau de leurs hélices périplasmiques. La protéine MexA s’organise sous forme d’une structure annulaire de 13 nm de hauteur au sein des membranes lipidiques, et d’une structure plus complexe de 26 nm de hauteur, résultant de l’empilement tête-bêche de deux structures annulaires de 13 nm. Ce travail révèle les dimensions exactes de l’assemblage formé par MexA, et permet de localiser à proximité des membranes les domaines non résolus dans la structure cristallographique. La reconstitution du complexe OprM/MexA révèle une disposition régulière des deux protéines dans les membranes lipidiques. Au sein des complexes, les protéines OprM sont présentes sous forme de trimères. Dans la membrane opposée, à l’aplomb d’une molécule d’OprM, MexA ne forme pas une structure annulaire similaire à celle décrite précédemment, indiquant un état d’oligomérisation différent de celui observé dans les assemblages MexA. Les densités de MexA sont compatibles avec la présence de quelques molécules de MexA. Cependant des structures annulaires de MexA, positionnées à l’aplomb de trois trimères d’OprM sont visibles. Notre étude montre que MexA adopte des structures oligomériques spécifiques en fonction de ses interactions avec les membranes lipidiques ou avec son partenaire OprM. / The structure determination of membrane protein in lipid environment can be carried out using cryo electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of membrane protein reconstitued into a lipid membrane using both cryo electron tomography and single particle analysis which is an alternative approach to electron crystallography for solving 3D structure. We show the organization of the successive layers of OprM molecules revealing the protein-protein interactions between OprM molecules of two successive lipid bilayers.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Sýkorová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters Tyrosine kinases are an important class of enzymes controlling cell proliferation, carcinogenesis, apoptosis and cell differentiation. Deregulation of these enzymes can transform normal cell into a cancerous one. Blocking their function by tyrosine kinase inhibitors (TKi) is considered a promising treatment for various types of cancer. ATP-binding cassette (ABC) transporters form a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes via ATP-dependent drug efflux pumps. They modulate drug pharmacokinetics, but on the other hand, lead to therapy failure due to overexpression in cancer cells. In our previous study, we evaluated inhibition properties of two selected TKi (alectinib, brivanib) in MDCKII cell lines (parent one and those transduced with human ABCB1, ABCC1 and ABCG2). Alectinib significantly inhibited ABCB1, ABCG2 but not ABCC1 transporter. Brivanib showed triple inhibition of all studied transporters. In the present work, we...

La résistance aux médicaments antiépileptiques (MAEs) est un des problèmes majeurs des épilepsies infantiles, comme par exemple le syndrome de Dravet. La pharmacoresistance de l’épilepsie pourrait s’expliquer par une diminution du passage des MAEs dans le cerveau, à travers la Barrière Hémato-Encéphalique (BHE). La BHE comporte des transporteurs des familles « ATP-binding cassette » (ABC) et « SoLute Carrier » (SLC) localisés au niveau de la membrane des cellules endothéliales qui contrôlent leur passage entre le sang et le cerveau. La pharmacoresistance des épilepsies a été associée à ces transporteurs car des MAEs ont été identifiés comme substrats de transporteurs comme la glycoprotéine-P (P-gP) et la « Breast Cancer Resistance Protein » (BCRP). L’hypothèse de cette relation est confortée par l’observation de l’augmentation de l’expression de ces transporteurs d’efflux dans le foyer épileptogène et par l’identification des polymorphismes dans les gènes des transporteurs chez des patients pharmacorésistants. L’interaction au cours du développement cérébral entre les cellules endothéliales et les neurones et astrocytes pourrait modifier le profil des transporteurs de la BHE. Les MAEs sont aussi connus pour être soit des inducteurs, soit des inhibiteurs des enzymes du métabolisme des médicaments et des transporteurs membranaires. Ces données nous permettent de faire les hypothèses suivantes: 1) La BHE en développement présente un profil de transporteurs différent de la BHE mature qui pourrait modifier le passage des MAEs vers le cerveau ; et 2) le traitement chronique administré au cours du syndrome de Dravet pourrait changer le phénotype des transporteurs de la BHE en développement. Nous résultats ont montré que la P-gP et la BCRP augment leur expression au cours du développement. La maturation de la BHE a aussi un impact sur le passage des MAEs étudiés. Nous avons constaté une augmentation de l’expression des différents transporteurs ABC et SLC étudiés pendant le développement de la BHE, suite au traitement chronique avec la thérapie du Syndrome de Dravet. L’acide valproïque, un des MAEs utilisé dans ce traitement, diminue l’activité d’efflux de la P-gP chez les rats en développement et adultes, ce qui a été confirmé dans un modèle in-vitro de BHE immature. Ces résultats mettent en évidence l’interaction entre la BHE en développement et le traitement chronique par les MAEs peut modifier leur distribution au niveau du cerveau et la réponse aux MAEs. / Resistance to Antiepileptic Drugs (AEDs) has been a major concern in infantile epilepsies such as for example the Dravet Syndrome. One hypothesis concerning the pharmacoresistance in epilepsy is that a decreased delivery of these drugs to the brain may occur in relation to changes in the Blood-Brain Barrier (BBB) function. BBB exhibits ATP-binding cassette (ABC) and SoLute Carrier (SLC) transporters at the surface of endothelial cells that control the blood-brain transport. Pharmacoresistance in epilepsy may be linked to changes in the functions of these transporters since some AEDs are substrates of the P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP) transporters. The increased expression of efflux transporters in epileptogenic tissue and the identification of polymorphisms in the efflux transporters genes of resistant patients further support this potential relationship. The interaction of endothelial cells with astrocytes and neurons during brain development could change the pattern of transporters in the BBB. AEDs are also known as either inducers or inhibitors of drug metabolic enzymes and membrane transporters. Taken together, these facts led us to test the following hypothesis: 1) the developing BBB in immature animals presents a different pattern of transporters that could change AEDs disposition in the brain of immature subjects; and 2) the chronic pharmacotherapy used in infantile epilepsies such as the Dravet Syndrome may change the transporters phenotype of the BBB. Our work showed that the expression of P-gP and BCRP increases during development as a function of age. We also showed the maturation of the BBB has an impact on brain disposition of the studied AEDs. We finally observed an increase in the expression of various ABC and SLC transporters induced by the pharmacotherapy of the Dravet Syndrome in immature animals. One of the drugs used, valproic acid, appeared nonetheless to reduce the efflux activity of P-gP in developing and adult animals, which was confirmed in an in-vitro model of the immature BBB. Taken together, these results demonstrated that the interaction between the developing BBB and the AEDs chronic treatment may lead to differences in brain disposition of the AEDs that may impact on the response to AEDs.

A resistência aos antibióticos é um problema de saúde pública global com sérias consequências para o tratamento de várias infecções bacterianas. Os fatores de transcrição da família MarR têm sido descritos controlando resistência a antibióticos e vários outros processos em bactérias. Neste trabalho, estudamos mecanismos de resistência a antibióticos em Chromobacterium violaceum, uma bactéria Gram-negativa ambiental que pode atuar como um patógeno oportunista em humanos. A estratégia envolveu a varredura de um painel de treze linhagens mutantes de fatores de transcrição da família MarR de C. violaceum disponíveis, por testes de susceptibilidade a 24 antibióticos. Estes ensaios revelaram que apenas o mutante ?emrR apresentou resistência aumentada ao antibiótico ácido nalidíxico em relação à linhagem selvagem. Esta resistência aumentada do mutante ?emrR ao ácido nalidíxico foi revertida em uma linhagem complementada deste mutante, conforme verificado por ensaios de viabilidade, ensaios de difusão em disco e concentração inibitória mínima (MIC). O fenótipo de diminuída produção de violaceína deste mutante, observado em meio líquido, também foi complementado. Além disso, foi realizado o isolamento de mutantes espontâneos de C. violaceum resistentes a ácido nalidíxico com mutação pontual em emrR. Os ensaios de microarranjo de DNA mostraram que EmrR reprime algumas dezenas de genes, incluindo o operon emrCAB, o qual codifica a bomba de efluxo EmrCAB. Os ensaios de Northern blot confirmaram que o EmrR reprime o operon emrCAB, e que a expressão desta bomba é induzida por salicilato, mas não outros compostos, como ácido nalidíxico ou brometo de etídeo. Os ensaios de alteração de mobilidade eletroforética (EMSA) mostraram que a proteína EmrR purificada se liga diretamente às 8 regiões promotoras de emrR, emrCAB e vários outros genes do regulon EmrR, para exercer uma regulação negativa direta sobre esses genes. Um mutante nulo ?emrCAB foi obtido, mas a ausência desta bomba de efluxo não tornou C. violaceum mais susceptível ao ácido nalidíxico, sugerindo que ela é importante somente em condições nas quais é induzida. Estas condições indutoras talvez incluam estresse oxidativo, uma vez que enzimas antioxidantes são parte do regulon de EmrR e a proteína EmrR formou dímeros covalentes na presença de agentes oxidantes in vitro. Portanto, nossos dados revelam que mutações pontuais ou moléculas como salicilato abolem a atividade repressora do fator de transcrição EmrR sobre o operon emrCAB, levando a superexpressão da bomba de efluxo EmrCAB e aumentando a resistência ao ácido nalidíxico em C. violaceum. / Antibiotic resistance is a global public health problem with serious consequences for the treatment of various bacterial infections. MarR family transcription factors have been described controlling antibiotic resistance and several other processes in bacteria. In this work, we studied mechanisms of antibiotic resistance in Chromobacterium violaceum, an environmental Gramnegative bacterium that can act as a human opportunistic pathogen. The strategy involved a screening of an available collection of thirteen C. violaceum mutant strains of MarR family transcription factors, by susceptibility testing for 24 antibiotics. These assays revealed that only the ?emrR mutant showed increased resistance to the antibiotic nalidixic acid in relation to the wild-type strain. This increased resistance of the ?emrR mutant to nalidixic acid was reversed in a complemented strain of this mutant, as verified by viability, disk diffusion, and minimal inhibitory concentration (MIC) assays. The phenotype of decreased violacein production of this mutant, observed in a liquid medium, was also complemented. In addition, it was performed the isolation of spontaneous mutants of C. violaceum resistant to nalidixic acid with a point mutation in emrR. DNA microarray assays showed that EmrR represses a few dozen of genes, including the emrCAB operon, which encodes the EmrCAB efflux pump. Northern blot assays confirmed that EmrR represses the emrCAB operon and that the expression of this pump is induced by salicylate, but not other compounds, such as nalidixic acid or ethidium bromide. Electrophoretic mobility shift assays (EMSA) showed that the purified EmrR protein binds directly to the promoter regions of emrR, emrCAB and several other genes of the EmrR regulon, to exert a direct negative regulation of these genes. A ?emrCAB null mutant strain was obtained, but the absence of this efflux pump did not make C. violaceum more susceptible to nalidixic acid, suggesting that it is important only under conditions in which it is induced. These inducing conditions may include 10 oxidative stress since antioxidant enzymes are part of the EmrR regulon and the EmrR protein has formed covalent dimers in the presence of oxidizing agents in vitro. Therefore, our data reveal that point mutations or molecules such as salicylate abolish the repressive activity of the EmrR transcription factor on the emrCAB operon, causing overexpression of the EmrCAB efflux pump and increasing the resistance to nalidixic acid in C. violaceum.

Introdução: Isolados clínicos de Pseudomonas aeruginosa multirresistentes estão associados a elevadas taxas de mortalidade. A resistência ao imipenem é uma urgência global, uma vez que é considerado o tratamento de escolha para infecções associadas a bactérias Gram negativas multirresistentes. Assim, elucidar os mecanismos de resistência é de vital importância para realizar um controle epidemiológico efetivo da disseminação deste tipo de isolado. Objetivos: Caracterizar os principais mecanismos de resistência ao imipenem em 76 isolados clínicos brasileiros de Pseudomonas aeruginosa, recuperados em 2004/2007, de 4 centros hospitalares do Estado de São Paulo. Material e métodos: Foram investigados: i) o perfil de resistência com determinação da CIM do imipenem; ii) a detecção de metalo-betalactamases (MBL) através de métodos fenotípicos e genotípicos; iii) a sensibilidade e especificidade do método de dupla difusão do disco na detecção de MBL; iv) a presença de genes codificadores de metilases 16S RNAr e sua associação com fenótipos aminoglicosídeo resistentes; v) alterações da permeabilidade por perda da porina OprD; vi) a presença ou ausência do gene oprD por PCR; vii) triagem fenotípica para expressão de bombas de efluxo através da determinação da CIM de quinolonas, cefalosporinas e carbapenêmicos na presença/ausência de inibidores específicos, realizando uma análise comparativa com o método de disco combinado; viii) os genes associados às bombas de efluxo mexA e mexE, através de PCR; ix) caracterizar a expressão das bombas de efluxo MexAB-OprM e MexEF-OprN, x) a clonalidade dos isolados por tipagem genotípica, através de ERIC-PCR, avaliando a relação genética (dendrograma) e sua associação com o predomínio de um determinado mecanismo de resistência. Resultados: Dentre os isolados de P. aeruginosa resistentes ao imipenem estudados (n=76, CIM50 e CIM90 = 32 &#181;g/mL e > 512 &#181;g/mL, respectivamente) 82% apresentaram um fenótipo de multirresistência. O principal mecanismo de resistência ao imipenem foi a produção de MBL, detectada em 74% dos isolados, e destes, 62% carregavam o gene blaSPM-1 e 12% carregavam o gene blaVIM-like. O método de dupla difusão do disco identificou a produção de MBL em 61% dos isolados. A combinação CAZ/MAA apresentou maior sensibilidade na detecção de MBL associada à SPM-1 (89%), mostrando uma especificidade de 86%. A presença do gene rmtD foi confirmada em 66% das amostras resistentes aos aminoglicosídeos, sendo que a presença concomitante do gene rmtD e do gene blaSPM-1 foi confirmada em 61% dos isolados. A deleção da porina OprD foi observada em 71% dos isolados. Dentre os isolados MBL positivos, 66% apresentaram ausência desta porina e, dentre as amostras MBL negativas, 85% não apresentaram OprD. Assim, para a resistência ao imipenem foi confirmada a contribuição de dois mecanismos, mediados pela presença de MBL e ausência de porina OprD. Em 13% (10/76) isolados, a deleção da porina OprD esteve associada à presença de seqüências de inserção (SI) em uma região anterior ao gene oprD. Por outro lado, a ausência de amplificação da região 736/1394 do gene oprD, em 11% (9/76) dos isolados, sugeriu a presença de polimorfismos. O gene mexA esteve presente em 92% dos isolados, enquanto que o gene mexE esteve presente em 82% dos isolados. A triagem de bombas de efluxo por disco combinado e análise da CIM na presença de reserpina, CCCP e PA&#946;N, utilizando levofloxacina, meropenem, aztreonam, imipenem ou levofloxacina, não teve correlação com a superexpressão dos sistemas MexAB-OprM e MexEF-OprN. Ambos os métodos careceram de especificidade e sensibilidade quando comparados ao PCR em tempo real. A superexpressão dos sistemas mexA e mexE foi confirmada em 35% (7/20) isolados MBL negativos, enquanto que 11% (6/56) isolados MBL positivos apresentaram superexpressão do gene mexA ou mexE, sendo que 7% (4/56) isolados MBL positivos superexpressaram ambos os genes. A superexpressão dos sistemas MexAB-OprM e MexEFOprN como único mecanismo de resistência ao meropenem e imipenem foi confirmada em 10% (2/20) dos isolados MBL negativos. Nos 76 isolados, a tipagem genotípica por ERIC-PCR, identificou a presença de 24 clusters (considerando 90% de similaridade na análise do dendrograma). Conclusão: A convergência de múltiplos mecanismos de resistência em P. aeruginosa parece ser um evento favorável para a seleção de clones endêmicos multirresistentes disseminados na região Sudeste do Brasil. / Introduction: Clinical isolates of Pseudomonas aeruginosa are associated with high mortality rates. Resistance to imipenem is a global concern, since it is a drug of choice for the treatment of infections produced by multidrug-resistant Gram-negative bacteria. Thus, research on resistance mechanisms is crucial to carry out an effective program for infection control and epidemiology of imipenem-resistant strains. Objective: to characterize the major mechanisms of imipenem resistance in 76 clinical isolates of P. aeruginosa recovered from clinical samples collected, from 2004 to 2007, in four hospitals in the State of São Paulo, Brazil. Material and methods: Isolates were screened for: i) resistance profile to antibacterial agents, determining the MIC of imipenem; ii) the detection of metallo-beta-lactamase (MBL) by phenotypic and genotypic methods, iii) MBL detection by using a double-disk diffusion test (D-test), determining the sensitivity and specificity of the assay; iv) the presence of genes encoding 16S rRNA methylases and their association with aminoglycoside-resistant phenotypes, v) changes in the bacterial permeability due to porin (OprD) loss; vi) the presence or absence of the oprD gene by using PCR; vii) phenotypic expression of efflux pumps by determining the MIC of quinolones, cephalosporins and carbapenems in the presence/absence of specific inhibitors, performing a comparative analysis with a combined-disk method, viii) genes encoding efflux pumps proteins (mexC and mexX) by PCR; ix) MexAB-OprM and MexEF efflux pumps expression; x) clonal relatedness, by ERIC-PCR genotyping, regarding the predominance of major resistance genotypes. Results: Among imipenem-resistant P. aeruginosa strains (n=76, MIC50 e MIC90 = 32 &#181;g/mL e > 512 &#181;g/mL, respectively) 82% showed a multidrug-resistant phenotype. The main mechanism of imipenem resistance was the MBL production detected in 74% strains, of which 62% harbored the blaSPM-1 gene, and 12% harbored the blaVIM-like gene. The D-test identified MBL production in 61% strains. In this regard, CAZ/MAA was the most sensitive combination for MBL detection associated to SPM-1 enzyme (89%), exhibiting 86% specificity. The presence of the rmtD 16S rRNA methylase gene was confirmed in 66% aminoglycoside-resistant strains. Moreover, presence of both rmtD and blaSPM-1 genes was identified in 61% strains. Loss of OprD porins was observed in 71% strains. In this regard, 66% MBL positive strains and 85% MBL negative strains showed OprD loss. Thus, MBL production and OprD loss contributed to imipenem resistance in P. aeruginosa. Most likely, in 13% (10/76) strains the porin loss was associated to insertion sequences (SI) inserted upstream of the oprD gene. On the other hand, in 11% (9/76) strains the absence of a PCR product targeting the 736/1394 region of the oprD gene, suggested the presence of polymorphisms. The mexA gene was identified in 92% strains, whereas the mexE gene was identified in 82% strains. Results obtained from efflux pump screening by using a combined-disk assay and MIC determination in the presence of reserpine, CCCP e PABN (using levofloxacin, meropenem, aztreonam or imipenem) was not correlated with results obtained from MexAB-OprM and MexEF-OprN overexpression analysis by RT-PCR. In this regard, both combined-disk and MIC assay showed lack of specificity and sensitivity in comparison to RT-PCR. Overexpression of mexA and mexE genes was confirmed in 35% (7/20) MBL-negative and 11% (6/56) MBL-positive strains, respectively, being 7% (4/56) MBL-positive strains overexpressed both genes. The overexpression of MexAB-OprM and MexEF-OprN efflux pumps, as only mechanism of resistance to meropenem and imipenem was observed in 10% (2/20) MBL-negative strains. ERICPCR typing revealed the presence of 24 clusters among 76 imipenem-resistant P. aeruginosa strains (&#8805; 90% similarity). Conclusion: The convergence of multiple mechanisms of resistance in Pseudomonas aeruginosa seems to be a favorable event for the selection of multiresistant clones endemic in the southeastern region of Brazil.