CORREIA, G. R. B. Efeito inibitório in vitro do composto farnesol frente ao biofilme de Burkholderia pseudomallei. 2015. 88 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-05-02T15:58:57Z No. of bitstreams: 1 2015_dis_grbcorreia.pdf: 1260823 bytes, checksum: 8ebde2fdd525176303b72981cc66cfa0 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-05-02T15:59:07Z (GMT) No. of bitstreams: 1 2015_dis_grbcorreia.pdf: 1260823 bytes, checksum: 8ebde2fdd525176303b72981cc66cfa0 (MD5) / Made available in DSpace on 2016-05-02T15:59:07Z (GMT). No. of bitstreams: 1 2015_dis_grbcorreia.pdf: 1260823 bytes, checksum: 8ebde2fdd525176303b72981cc66cfa0 (MD5) Previous issue date: 2015-07-14 / The intrinsic antimicrobial resistance of Burkholderia pseudomallei is a serious challenge to the treatment of melioidosis. Many studies have searched for adjuvants that increase susceptibility bacteria to antimicrobials. In this context, the antimicrobial activity of farnesol against B. pseudomallei in planktonic growth has been reported. Thus, the aim of this study was to analyze the in vitro activity of farnesol alone against Burkholderia pseudomallei biofilms, as well as its combination with the antibacterials amoxicillin, doxycycline, ceftazidime and sulfamethoxazole-trimethoprim. Susceptibility was assessed by the broth microdilution test and cell viability was read with the oxidation-reduction indicator dye resazurin. The interaction between farnesol and antibacterial drugs against B. pseudomallei biofilms was evaluated through the calculation of the fractional inhibitory concentration index. The minimum biofilm erradication concentration (MBEC) for farnesol was 75 to 2400 mM. In addition, farnesol significantly reduced the MBEC values for ceftazidime,amoxicillin, doxycycline and sulfamethoxazole-trimethoprim by 256, 16, 4 and 4 times respectively (P<0.05). Optical, confocal and electronic microscopic analyses of farnesol treated B. pseudomallei biofilms demonstrated that this compound damages biofilm matrix,facilitating antimicrobial penetration in the biofilm structure. This study demonstrated the effectiveness of farnesol against B. pseudomallei biofilms and its potentiating effect on the activity of antibacterial drugs, in particular ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim. / A intrínseca resistência apresentada pela bactéria Burkholderia pseudomallei é um grave empecilho para o tratamento da melioidose. Muitas pesquisas focam na busca de adjuvantes que aumentem a sensibilidade desta bactéria aos antimicrobianos. Nesse contexto, a ação inibitória do farnesol frente às cepas de B. pseudomallei, na forma planctônica, já foi relatada em estudo prévio. Diante disso, esse estudo objetivou analisar a atividade in vitro do farnesol contra cepas de B. pseudomallei na forma de biofilme. Aliado à análise da ação do farnesol isoladamente, foi investigada a combinação desse composto com os antimicrobianos amoxicilina, ceftazidima, doxiciclina, imipenem e sulfametoxazol/trimetoprim frente ao biofilme. A sensibilidade foi avaliada por meio do teste de microdiluição em caldo e a leitura da viabilidade celular feita com a resazurina. A concentração inibitória mínima em biofilme (CEMB) para o farnesol foi de 75 a 2400 mM. Ademais, o farnesol reduziu em até 256, 16, 4 e 4 vezes os valores de CEMB para ceftazidima, amoxicilina, doxiciclina e sulfametoxazol/trimetoprim, respectivamente (P<0.05). Por meio de técnicas de microscopia, tais como óptica, confocal e eletrônica, observou-se que o farnesol foi capaz de causar danos na matriz do biofilme, facilitando assim, a penetração dos antibióticos. Deste modo, o presente estudo mostrou a eficácia do farnesol contra biofilmes de B. pseudomallei e seu efeito potenciador, em especial com ceftazidima, amoxicilina, doxiciclina e sulfametoxazol/trimetoprim.

The aim of this study was to identify and characterise peptidyl-prolyl cis-trans isomerases (PPIases) from the bacterium Burkholderia pseudomallei, the causative agent of the disease melioidosis. The longer term goal was to assess their potential as vaccine candidates or antimicrobial targets. Using bioinformatic approaches, six putative FK506-binding proteins (FKBPs) proteins and three putative parvulin proteins were identified in B. pseudomallei. Of these, six were expressed and purified as recombinant proteins. The purified proteins were used to immunise BALB/c mice, with some providing protection against a subsequent B. pseudomallei infection. These proteins could therefore be proposed as potential vaccine candidates. Homologues of Mip or SurA, which are associated with virulence in other bacterial species, were identified in B. pseudomallei and closely related B. thailandensis. Recombinant Mip or SurA homologues from B. pseudomallei were shown to have characteristic PPIase enzyme activity. To evaluate the role of the Mip homologue from B. pseudomallei in virulence, an unmarked deletion mutant was constructed. The mutant had reduced intracellular survival; defects in putative virulence mechanisms and attenuated virulence in mice. To assess the role of a SurA homologue, closely related B. thailandensis was used as a model organism, with deletion of the gene resulting in defects in intracellular infection, outer membrane integrity and virulence. This indicates that PPIases from B. pseudomallei and B. thailandensis represent novel virulence determinants and potential antimicrobial targets for therapeutics against melioidosis.

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for host cell invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. The aims of this study are twofold. The first is to expand the repertoire of known effector proteins using modern proteomics techniques. The second is to explore the function of a subset of effector proteins to better understand their interaction with host cells. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hyper-secreting mutants of B. pseudomallei with the isogenic parent strain as well as a mutant incapable of effector protein secretion. This study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. To determine the possible function of two effector proteins, BipC and BapA, a yeast two-hybrid system was used to identify host cell proteins the effectors interact with. The proteins were screened against a library of human proteins for interactions. BapA interacted with 2 proteins while BipC interacted with 14. Both BapA and BipC were shown to interact with human C1QBP, a mitochondrial protein involved in inflammation, immunity and autophagy. Finally, the Bsa T3SS protein BipC was characterised in its ability to interact with actin. This study is the first evidence that BipC has the ability to bind to filamentous actin, but not monomeric actin. This binding is direct and no intermediate proteins are required for the interaction. Ectopic expression of BipC in eukaryotic cells caused cytoskeletal rearrangements consistent with an actin-binding protein. The core secretome represents a substantial resource of targets that will be mined for improved diagnostic assays and vaccines. Diagnostics that will detect early stages of disease to allow for more effective antimicrobial intervention are currently lacking. Furthermore, there is scope to design diagnostic assays with dual use such as to detect both melioidosis and infection of cystic fibrosis patients with the closely related opportunistic pathogen B. cepacia. The description of novel T3SS effector proteins is also of considerable value since T3SS proteins are often potent B- and T- cell antigens representing promising components of sub-unit vaccines. Such effector proteins commonly modulate cellular processes such as phagocytosis, inflammasome activation and cell cycle progression, hence the function of the predicted T3SS effectors will provide a series of future research opportunities.

MOREIRA, C. A. Estudo de sensibilidade do biofilme de Burkholderia pseudomallei a antibióticos de uso clínico e farnesol. 2013. 115 f. Tese (Doutorado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-10-03T15:38:15Z No. of bitstreams: 1 2013_tese_camoreira.pdf: 2691368 bytes, checksum: 33ff8ee13ee8b744ebce1f67d391647c (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-10-03T15:38:53Z (GMT) No. of bitstreams: 1 2013_tese_camoreira.pdf: 2691368 bytes, checksum: 33ff8ee13ee8b744ebce1f67d391647c (MD5) / Made available in DSpace on 2016-10-03T15:38:53Z (GMT). No. of bitstreams: 1 2013_tese_camoreira.pdf: 2691368 bytes, checksum: 33ff8ee13ee8b744ebce1f67d391647c (MD5) Previous issue date: 2013-09-27 / The melioidosis is an emerging infectious disease, especially in northeastern Brazil, with serious international implications, as well as a public health problem. Caused by the bacillus Burkholderia pseudomallei, this infection appears from tables asymptomatic to severe and often fatal frames. The high mortality rate of the disease (20-50%) make it a priority for global health agencies. In Brazil, cases of melioidosis were first reported in 2003 in Tejuçuoca municipality in Ceará. Since then, new cases of melioidosis were diagnosed in six other municipalities in Ceará. B. pseudomallei is intrinsically resistant to many antibiotics and recent studies have described cases of antimicrobial resistance used in the treatment of melioidosis. The investigation of the biofilm forming capacity of B. pseudomallei seems to be essential, since the biofilm allows the development of microcolonies within a protected environment, which relates to the environmental protection, adhesion, colonization, immune evasion and bonding the environmental cell. Therefore, the development of therapeutic alternatives antibiofilm, including new drugs is needed. Indeed, this study aimed to characterize the strains of B. pseudomallei of LAPERE collection as the biofilm production capacity and sensitivity in planktonic growth and biofilm. Selected drugs were ceftazidime (CAZ), doxycycline (DOX), imipenem (IPM), amoxicillin / clavulanate (AMC) and trimethoprim / sulfamethoxazole (SXT); and farnesol (FNS). All strains were classified as biofilm producers, being divided into: hard (5 strains), moderate (3 strains), and low (1 strain). Mean values ​​of minimum inhibitory concentration (MIC), minimum inhibitory concentration of biofilm (MBIC) and concentration minimum biofilm eradication in (MBEC) for these strains were determined for AMC (MIC 10.2 / 5.1 mg / L, 21 MBIC 3 / 10.6 mg / L and MBEC 27.6 / 13.8 mg / L) to CAZ (MIC 5.6 mg / L, MBIC 120.4 mg / L and MBEC 419.6 mg / L) to doxorubicin (MIC 0.28 mg / L, MBIC 1.3 mg / L and MBEC 3.8 mg / L) to IPM (MIC 0.597 mg / L, MBIC ≥ 256 mg / L and MBEC> 256 mg / G) to SXT (MIC 1.25 / 23.75 mg / L, MBIC ≤ 0.5 / 9.5 mg / L and MBEC <0.72 / 13.72 mg / L), and NSF (MIC and MBIC equal to 150 uM / L). The data obtained show especially for the inhibitory potential of three antibiotics studied - AMC, DOX and SXT and farnesol against strains of B. pseudomallei associated with biofilm. However, further studies are needed to investigate the mechanisms of action of these drugs on the biofilm, as well as the design of in vivo experiments to confirm the significance of these findings clinically. Moreover, the growth of these strains melanin formation conditions and biofilm showed that this combination makes them more resistant strains to the action of imipenem and farnesol. / A melioidose é uma doença infeciosa emergente, notadamente no nordeste do Brasil, com sérias implicações internacionais, assim como um problema de saúde pública. Causada pelo bacilo Burkholderia pseudomallei, esta infecção se apresenta desde quadros assintomáticos a quadros graves e frequentemente fatais. As altas taxas de mortalidade da doença (20 a 50%) a tornam uma prioridade para orgãos globais de saúde. No Brasil, casos de melioidose foram relatados pela primeira vez em 2003, no município de Tejuçuoca no Ceará. Desde então, novos casos de melioidose foram diagnosticados em outros seis municípios cearenses. B. pseudomallei é intrinsecamente resistente a muitos antibióticos e estudos recentes já descrevem casos resistência aos antimicrobianos utilizados no tratamento da melioidose. A investigação da capacidade de formação de biofilme por B. pseudomallei parece ser fundamental, já que o biofilme permite o desenvolvimento de microcolônias dentro de um ambiente protegido, o qual se relaciona com a proteção ambiental, adesão, colonização, evasão do sistema imune e ligação a células ambientais. Portanto, o desenvolvimento de alternativas terapêuticas antibiofilme, incluindo novas drogas, é necessário. Com efeito, este estudo teve como objetivo caracterizar as cepas de B. pseudomallei da coleção do LAPERE quanto à capacidade de produção de biofilme e sensibilidade em crescimento planctônico e em biofilme. As drogas selecionadas foram: ceftazidima (CAZ), doxiciclina (DOX), imipenem (IPM), amoxicilina/clavulanato (AMC) e sulfametoxazol/trimetoprim (SXT); e farnesol (FNS). Todas as cepas foram classificadas como produtoras de biofilme, sendo divididas em: forte (5 cepas), moderada (3 cepas), e fraca (1 cepa). Valores médios de concentração inibitória mínima (MIC), concentração inibitória mínima em biofilme (MBIC) e concentração de erradicação mínima em biofilme (MBEC) para estas cepas foram determinados para AMC (MIC 10,2/5,1 mg/L, MBIC 21,3/10,6 mg/L e MBEC 27,6/13,8 mg/L), para CAZ (MIC 5,6 mg/L, MBIC 120,4 mg/L e MBEC 419,6 mg/L), para DOX (MIC 0,28 mg/L, MBIC 1,3 mg/L e MBEC 3,8 mg/L), para IPM (MIC 0,597 mg/L, MBIC ≥ 256 mg/L e MBEC > 256 mg/L), para SXT (MIC 1,25/23,75 mg/L, MBIC ≤ 0,5/9,5 mg/L e MBEC < 0,72/13,72 mg/L), e para FNS (MIC e MBIC iguais a 150 µM/L). Os dados obtidos apontam especialmente para o potencial inibitório de três dos antibióticos estudados - AMC, DOX e SXT, e farnesol contra cepas de B. pseudomallei associadas a biofilme. Todavia, são necessários novos estudos para investigar os mecanismos de ação dessas drogas sobre o biofilme, bem como o delineamento de experimentos in vivo para confirmar a significância desses achados clinicamente. Ademais, o crescimento dessas cepas em condições de formação de melanina e biofilme evidenciou que esta associação torna as cepas mais resistentes à ação de imipenem e farnesol.

COUTO, Manuela Soares. Identificação molecular de cepas clínicas e ambientais de Burkholderia pseudomallei, oriundas do Estado do Ceará : análise baseada nas regiões 16S e 16S-23S do DNA ribossômico nuclear. 2099.0107 f. Dissertação (Mestrado em Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009. / Submitted by denise santos (denise.santos@ufc.br) on 2013-12-05T14:02:29Z No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-12-05T14:03:42Z (GMT) No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5) / Made available in DSpace on 2013-12-05T14:03:43Z (GMT). No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5) Previous issue date: 2009 / Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei, considered emerging in Brazil since the first cases were reported in 2003, on State of Ceará. This study aimed to perform the molecular identification of 31 isolates of B. pseudomallei (26 clinical and 5 environmental) maintained in the culture collection of CEMM (Specialized Center for Medical Mycology), based on sequences 16S and 16S-23S rRNA. The DNA of these samples was extracted with the kit Wizard ® Genomic DNA Purification (Promega), quantified by spectrophotometry and stored at 4°C. The amplification of a fragment of 302 bp of 16S-23S rRNA specific to B. pseudomallei was performed by PCR reaction with primers Bp1 and Bp4. The sequencing of 16S and 16S-23S rRNA was performed by using of the kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). The phylogenetic tree of 16S rRNA and the sequence identity matrix and sequence difference count matrix based on the 16S-23S rRNA were generated by the program MEGA4, version 4.1. The results confirmed the identification of 15 strains of B. pseudomallei (5 clinical and 10 environmental), which represents 48.4% of the isolates analyzed in this study. The phylogenetic tree based on 16S rRNA shows that the clinical and environmental isolates of B. pseudomallei of State of Ceará are evolutionarily clustered with the strains B. pseudomallei MSHR346 (Australia), B. pseudomallei 1106a (Thailand), B. pseudomallei K96243 (Thailand), B. pseudomallei 1710b (Thailand) and B. pseudomallei 668 (Australia). Using the same extraction kit was possible to extract DNA from B. pseudomallei directly from clinical specimen (bronchoalveolar lavage), confirming a new case of melioidosis in Ubajara/CE. In this study, the use of PCR for amplification of a fragment of 302 bp of 16S-23S rRNA identified correctly B. pseudomallei, and to confirm the discrimination between B. pseudomallei and B. mallei, the sequencing of the 16S and 16S-23S rRNA genes was performed. The technique of PCR coupled with sequencing of 16S and 16S-23S rRNA resulted in a high sensitivity and specificity of detection of B. pseudomallei in this study. / A melioidose é uma doença potencialmente fatal causada pela bactéria Burkholderia pseudomallei, sendo considerada emergente no Brasil desde que os primeiros casos foram reportados em 2003, no Estado do Ceará. Este estudo pretendeu realizar a identificação molecular de 31 isolados de B. pseudomallei (cinco clínicos e 26 ambientais) mantidos na coleção de culturas do CEMM (Centro Especializado em Micologia Médica), com base nas sequências 16S e 16S-23S DNAr. O DNA destas amostras foi extraído com o kit Wizard® Genomic DNA Purification (Promega), quantificado por espectrofotometria e armazenado a 4ºC. A amplificação de um fragmento de 302 pb da região espaçadora 16S-23S DNAr específico para B. pseudomallei foi realizada por meio de reação de PCR com os primers Bp1 e Bp4. O sequenciamento das regiões 16S e 16S-23S DNAr foi realizado pelo método da terminação da cadeia pelo didesoxinucleotídeo, usando-se o kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). A árvore filogenética da região 16S DNAr e as matrizes sequência identidade e contagem de diferenças baseadas na região 16S-23S DNAr foram geradas pelo programa MEGA4, versão 4.1. Os resultados confirmaram a identificação de 15 cepas de B. pseudomallei (cinco clínicas e dez ambientais), o que corresponde a 48.4% dos isolados em estudo. A árvore filogenética baseada na região 16S DNAr demonstra que os isolados clínicos e ambientais de B. pseudomallei do Estado do Ceará são evolutivamente agrupados com as cepas B. pseudomallei MSHR346 (Austrália), B. pseudomallei 1106a (Tailândia), B. pseudomallei K96243 (Tailândia), B. pseudomallei 1710b (Tailândia) e B. pseudomallei 668 (Austrália). Com a utilização do mesmo kit de extração também foi possível extrair DNA de B. pseudomallei diretamente de espécime clínico (lavado brônquico), confirmando um novo caso de melioidose no Município de Ubajara/CE. Em nosso estudo, o uso da PCR para a amplificação de um fragmento de 302 pb da região 16S-23S DNAr identificou corretamente B. pseudomallei, sendo que para confirmar a discriminação entre B. pseudomallei e B. mallei, o sequenciamento das regiões 16S e 16S-23S DNAr foi realizado. A técnica de PCR aliada ao sequenciamento das regiões 16S e 16S-23S do DNA ribossômico nuclear resultaram em uma elevada sensibilidade e especificidade de detecção de B. pseudomallei neste estudo.

No description available.

The bacterium Burkholderia pseudomallei causes the disease melioidosis, a significant public health threat in endemic regions and is a potential biowarfare agent. Treatment of melioidosis is intensive and prolonged and there is no licensed vaccine to protect against it. The aim of this study was to characterise novel targets for antimicrobials to improve treatment of melioidosis. A holistic down selection process was undertaken in order to identify a range of possible novel and exploitable antimicrobial targets in Burkholderia pseudomallei. Four targets: FtsA, FtsZ, MraW and TonB were selected for characterisation by mutagenesis study. FtsA and FtsZ are early effectors of cell division and are considered potential antimicrobial drug targets in other pathogenic bacteria. Genes for both were shown likely to be essential for viability in Burkholderia pseudomallei, following attempted deletion of the genes, thus confirming their potential for drug targeting for treatment of melioidosis. MraW, a highly conserved methyltransferase, and TonB, the energiser for high affinity iron uptake in Gram negative bacteria, were also selected for characterisation as antimicrobial targets. In-frame deletions of the genes encoding these targets were constructed in B. pseudomallei K96243. In order to determine the roles played by MraW and TonB during infection, these mutants were characterised in several models of Burkholderia pseudomallei infection. Deletion of mraW rendered the bacteria non-motile and led to attenuation during infection of Balb/C mice. A small growth defect was seen early during infection of macrophages by this mutant, whilst no attenuation was seen on deletion of mraW in Galleria mellonella. Burkholderia pseudomallei ΔtonB required free iron supplementation for growth. This mutant had an improved ability to invade murine macrophages, though the mutant was attenuated in both Galleria mellonella and Balb/C mice. Attenuation of both mutants in a mammalian model of infection, support the strategy to target either of these proteins as novel targets for inhibition with small molecules during Burkholderia pseudomallei infection. However, an improved ability to infect macrophages by Burkholderia pseudomallei ΔtonB and non-complementation of this mutant by iron supplementation to Galleria mellonella suggests additional roles to iron uptake alone for TonB in Burkholderia pseudomallei, such as bacterial iron sensing and signalling.

Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.

Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. These diseases are endemic mainly in southeastern Asia and northern Australia, but they also pose a bioterrorism threat in the developed world. These diseases have high mortality, partially due to the lack of vaccines and rapid, accurate diagnostic assays. The work discussed here represents a part of a larger project to develop a dependable diagnostic assay for use in both developing endemic areas and the developed world, as well as a subunit vaccine to protect against disease. In this study, several proteins from B. pseudomallei, B. mallei, and the closely related but less virulent B. thailandensis have been cloned, expressed and purified in order to develop highly sensitive and specific diagnostic reagents for the detection of B. pseudomallei and B. mallei in infected patient samples. Protein targets expressed in this study were also used in subunit vaccine development for melioidosis and glanders. / text

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Grupo DASA / Melioidose à uma doenÃa infecciosa grave causada por Burkholderia pseudomallei, um bacilo Gram-negativo encontrado no solo e na Ãgua. A doenÃa à endÃmica no sudeste asiÃtico e hiperendÃmica no norte da AustrÃlia, onde a letalidade permanece com uma taxa de 21%. No Brasil, à considerada uma doenÃa emergente desde marÃo de 2003. Nos Ãltimos oito anos, 12 casos ocorreram no Estado do Cearà e um notificado pelo Governo holandÃs, por se tratar de um turista que morreu de melioidose, apÃs visita ao CearÃ. Em razÃo da ocorrÃncia clÃnica de melioidose e do isolamento de B. pseudomallei no ambiente do Estado do CearÃ, este trabalho objetivou estudar as cepas clÃnicas e ambientais de B. pseudomallei isoladas no Estado no perÃodo de 2003 a 2011, visando a identificar as cepas por mÃtodos fenotÃpicos e moleculares, determinar o perfil de sensibilidade contra cinco agentes antimicrobianos (amoxicilina/clavulanato, ceftazidima, imipenem, doxicilina e sulfametoxazol/trimetoprim), realizar a genotipagem das cepas pela amplificaÃÃo aleatÃria de DNA polimÃrfico - Random Amplified Polymorphic DNA (RAPD), detectar o gene de virulÃncia Type Three Secretion System (TTSS), alÃm de avaliar os aspectos clÃnico-epidemiolÃgicos que caracterizaram a emergÃncia desta doenÃa no Brasil. Todas as 20 cepas (dez clÃnicas e dez ambientais) de B. pseudomallei foram precisamente identificadas tanto pela metodologia VITEK2 quanto pelo sequenciamento da regiÃo 16S do DNA, mostraram resultado negativo no teste de assimilaÃÃo de L-arabinose, e exibiram-se positivas para a detecÃÃo do gene de virulÃncia TTSS. As concentraÃÃes inibitÃrias mÃnimas (CIMs), obtidas por microdiluiÃÃo em caldo MÃeller-Hinton, demonstraram que todos os isolados (100%) foram sensÃveis ao imipenem, à doxicilina e ao sulfametoxazol- trimetoprim, no entanto, para amoxicilina/clavulanato e ceftazidima, a sensibilidade foi de 80 e 90%, respectivamente. A tÃcnica de RAPD evidenciou uma variabilidade genÃtica de 63% entre as cepas de B. pseudomallei oriundas do Estado do CearÃ, as quais foram agrupadas em trÃs clusters diferentes. Este trabalho decerto contribuirà para o conhecimento das caracterÃsticas fenotÃpicas e genotÃpicas das cepas de B. pseudomallei isoladas no Cearà e da atualizaÃÃo da vigilÃncia epidemiolÃgica dos casos de melioidose ocorridos no Estado, alÃm de contribuir para a conscientizaÃÃo dos ÃrgÃos de saÃde competentes para a inclusÃo do Cearà como zona endÃmica para esta enfermidade. / Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, a Gram negative rod, commonly found in soil and water. The disease is endemic in Southeastern Asia and hyperendemic in Northern Australia. Despite the initiation of empiric therapy, mortality remains at 21% in patients with melioidosis in Australia. In Brazil, it is considered an emerging disease, since April 2003, when it was first diagnosed in Ceara, Northeastern Brazil. In the last eight years, thirteen cases were reported, twelve local cases and one case reported by the Dutch government because of a tourist who died of melioidosis after a visit to Ceara. Considering the occurrence of melioidosis in CearÃ, this work aimed at studying these clinical and environmental strains of Burkholderia pseudomallei isolated from Cearà from 2003 to 2011, focusing on the bacterial and molecular identification; determining the susceptibility profile against five antimicrobial agents (amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline and trimethoprim/sulfamethoxazole); genotyping through Random Amplified Polymorphic DNA (RAPD), detecting the virulence gene Type Three Secretion System (TTSS); and analyzing epidemiological and clinical aspects that characterized the emergence of this disease in Brazil. All 20 strains (10 clinical and 10 environment) from B. pseudomallei were accurately identified by both VITEK2  and sequencing of the 16S DNA, showed to be negative for the assimilation of L-arabinose and were positive results for the detection of the virulence gene TTSS. The minimum inhibitory concentrations (MICs) obtained through microdilution in MÃeller-Hinton broth, showed that all (100%) isolates were sensitive to imipenem, doxycycline and trimethoprim-sulfamethoxazole, however, the susceptibility rate to amoxicillin/clavulanate and ceftazidime was of 80 and 90%, respectively, with no differences between clinical and environmental strains. RAPD-PCR showed a genetic relatedness of 63% among the B. pseudomallei strains from the State of CearÃ, which were grouped in two different clusters. This work will contribute to the knowledge of phenotypic and genotypic characteristics of B. pseudomallei strains isolated in Cearà and the update of epidemiological surveillance of melioidosis cases in the state, also contribute to the awareness of agencies health authority for inclusion of Cearà State as an endemic area for this disease.