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1
Jones, Amanda L. "Burkholderia pseudomallei, host interactions." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq20744.pdf.
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Lowe, Carolyn Ann. "Iron regulation in Burkholderia cepacia and Burkholderia pseudomallei." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246990.
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Ellis, Jill Frances. "Antibody detection of Burkholderia pseudomallei and Burkholderia mallei." Thesis, Aston University, 2000. http://publications.aston.ac.uk/10974/.
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Monoclonal and polyclonaI antibodies have been produced for use in immunological assays for the detection of Burkholderia pseudomallei and Burkholderia mallei. Monoclonal antibodies recognising a high molecular weight polysaccharide material found in some strains of both species have been shown to be effective in recognising B. pseudomallei and B. mallei and distinguishing them from other organisms. The high molecular weight polysaccharide material is thought to be the capsule of B. pseudomallei and B. mallei and may have important links with virulence. B. pseudomallei and B. mallei are known to be closely related, sharing many epitopes, but antigenic variation has been demonstrated within both the species. The lipopolysaccharide from strains of B. pseudomal/ei and B. mallei has been isolated and the silver stain profiles found to be visually very similar. A monoclonal antibody raised to B. mallei LPS has been found to recognise both B. mallei and B. pseudomallei strains. However, in a small number of B. pseudomallei strains a visually atypical LPS profile has been demonstrated. A monoclonal ant ibody rai sed against this atypical LPS showed no recognition of the typical LPS profile of either B. mallei or B. pseudomallei. This atypical LPS structure has not been reported and may be immunologically distinct from the typical LPS. Molecular biology and antibody engineering techniques have been used in an attempt to produce single-chain antibody fragments reactive to B. pseudomallei. Sequencing of one of the single-chain antibody fragments produced showed high homology with murine immunoglobulin genes, but none of the single-chain antibody fragments were found to be specific to B. pselldomallei.
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Senkiw, Michelle D. "Intracellular existence of Burkholderia pseudomallei." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ49655.pdf.
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Atkins, Timothy Philip. "Virulence determinants of Burkholderia pseudomallei." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325608.
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張健文 and Kin-man Cheung. "B-lactamases in Burkholderia pseudomallei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31225792.
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Davies, Clare. "Molecular characterisation of Burkholderia pseudomallei." Thesis, University of Plymouth, 2001. http://hdl.handle.net/10026.1/2281.
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A programme of research was carried out to attempt the molecular characterisation of the human and animal pathogen, Burkholderia pseudomallei, the causative agent of melioidosis and the newly described avirulent species, B.thailandensis for comparative purposes. Melioidosis is still little understood, and so the clinical approach to the prevention and control of melioidosis must ultimately rest upon the basic understanding of the causative organism, particularly the pathogenic properties of B.pseudomallei. A range of B.pseudomallei and B.thailandensis isolates were cultured and the extracellular products were isolated and concentrated and an initial study conducted to identify potential target molecules for cloning. Those isolates tested were shown to have somewhat differing ECP profiles when analysed with SDS-PAGE and antigenic profiles when subject to irnmunoblotting using convalescent human serum although isolates within and between species shared a number of common bands. The ECPs were also tested for a range of activities and it was established that both species had proteolytic and phospholipase activities neither had a haemolytic activity and only isolates of B.pseudomallei had a hexosaminidase activity a putative pathogenicity determinant. Genomic DNA of B.pseudomallei was used to construct genomic libraries in a range of E. coli host vector systems. A λGTII genomic library was screened with antisera for the presence of B.pseudomallei antigens and a number of natural and synthetic substrates for the presence of haemolytic and proteolytic components. Screening yielded one stable immunopositive clone with a novel positive reaction in the form of a "halo" of reaction around the plaque. The 5 kbp cloned fragment was subcloned into a plasmid vector, and the resulting recombinant molecule, pBPGT2 was DNA sequenced and found to contain a putative pilin gene. Attempts were made to determine the size of the recombinant antigen and to further express the pilin gene product all of which were unsuccessful. A southern blot procedure confirmed the fidelity of the cloning procedure proving that the fragment was from the host organism, B.pseudomallei. A further southern blot procedure tested for the presence of the pilin sequence in a range of B.pseudomallei and B.thailandensis isolates proving the presence of the gene in only isolates of B.pseudomallei. PCR primers were designed to amplify the DNA encoding the active site of the ADP-ribosylating toxin (ET A) of Pseudomonas aeruginosa and a PCR reaction was carried out on a number of B.pseudomallei and B.thailandensis isolates. The reaction yielded a 500 bp product in only B.pseudomallei isolates and DNA sequencing of the product revealed no obvious homology to ETA of P.aeruginosa but was used as a probe to isolate a larger fragment of DNA which was found to encode a number of interesting putative genes. These included one with homology to a porin similar to that of the pathogen Neisseria gonorrhoea, with a role in virulence. During attempts to digest the genomic DNA of B.pseudomallei isolate 4845 with the restriction enzyme Sau3A two 12 kbp bands of DNA were resistant to the endonuclease activity. Attempts were made to clone these bands into a range of plasmid vectors with two clones containing deleted products. DNA sequencing proved inadequate with only a small amount of sequence information obtained. However, towards the final stages of the research project sequence information from the B.pseudomallei genome sequencing project facilitated the recognition of a 38 kbp fragment containing the sequence information from one of the clones, which encodes an alkaline protease and a putative haemagglutinin and is postulated to be a Pathogenicity Island encoding secreted virulence factors. The sequencing project also facilitated the isolation of two putative hexosaminidase genes postulated to be responsible for the activities observed when testing the B.pseudomallei isolates concentrated ECPs. Future studies for the putative genes identified and other components of B.pseudomallei are discussed.
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Cheung, Kin-man. "B-lactamases in Burkholderia pseudomallei /." Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25205158.
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梁嘉玲 and Ka-ling Leung. "Novel molecular targets of Burkholderia pseudomallei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31224726.
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Leung, Ka-ling. "Novel molecular targets of Burkholderia pseudomallei /." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23440144.
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Flegg, Cameron. "Burkholderia Pseudomallei: Interaction with Host Cells." Thesis, Griffith University, 2012. http://hdl.handle.net/10072/366738.
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Melioidosis is the serious and often fatal systemic disease of humans and animals resulting from infection from Burkholderia pseudomallei. B. pseudomallei is a Gram-negative bacillus that inhabits the soil and stagnant water of tropical and sub-tropical countries. Infection usually occurs through inhalation or inoculation though ingestion is a common route for animal infections. The disease can occur in almost any organ and symptoms are non-specific leading to common misdiagnosis. B. pseudomallei has increased prevalence for infection in immunocompromised and diabetic patients.
Due to the soil dwelling nature of B. pseudomallei this bacteria has a high level of antibiotic resistance. Treatment is often difficult and prolonged, though modern medical treatment has reduced mortality to around 40%. Recent studies have revealed that B. pseudomallei contains numerous virulence mechanisms including bacterial protein secretion systems, lipopolysaccharides, and quorum sensing mechanisms. These systems allow the bacteria to survive and perpetuate especially in host macrophages. It is this interaction and the responses of the host that remain to be elucidated.
Many studies have described activation of host signalling pathways or proteins during infection. Often B. pseudomallei mutants are produced to establish the affect these altered bacteria have on the host pathways. This study attempted to examine intact virulence mechanisms by utilising drug and small peptide inhibitors of the host pathways to examine the effect that these have on the invading bacteria. Furthermore, it set out to investigate the processes involved in B. pseudomallei-host interactions. It examined the MAPK, NFAT and NF-B signalling pathways activated in infected macrophages. Further, the resultant chemokine expression and receptor activation on these infected cells was established.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Keith, Karen Elizabeth. "Biochemical studies and proteomics of Burkholderia pseudomallei and Burkholderia cenocepacia." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409526.
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Duangsonk, Kwanjit. "Genomic and genetic analysis of Burkholderia pseudomallei." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437528.
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Jenkins, Christopher. "Investigating the lytic transglycosylases of Burkholderia pseudomallei." Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42166.
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Peptidoglycan is a mesh like structure that is an integral part of the bacterial cell wall. Comprised of glycan chains interconnected with short peptide chains, it is responsible for protecting the bacteria from hydrostatic pressures as well as providing a scaffold for many cell wall spanning structures. Tight regulation of the synthesis, maintenance and turnover of peptidoglycan is essential to maintaining integrity of bacteria. One family of enzymes involved in the regulated cleavage of peptidoglycan are lytic transglycosylases (Ltgs). These proteins are highly conserved in bacteria and function in the restructuring of peptidoglycan during growth and division but also in the insertion of large macromolecular structures including secretion systems and flagella. One class of Ltgs, called resuscitation-promoting factors (Rpf) have also been implicated in resuscitation of dormant Actinobacteria. Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease prevalent particularly in South East Asia and Northern Australia which claims the lives of an estimated 89,000 people per year. Based on sequence homology to Ltgs of E. coli, we have identified 5 putative Ltgs (LtgA-E) encoded by genes on chromosome 1 of Burkholderia pseudomallei strain K96243. This study aimed to understand the role of these proteins in B. pseudomallei biology and investigate their capacity as drug targets. Using a range of approaches I have shown the muralytic activity of four of these Ltgs. The X-ray crystal structure of LtgE was solved and the catalytic site identified using site directed mutagenesis. In complementary studies single and multiple deletion mutants of B. pseudomallei have been generated. Excision of single or multiple ltg genes from B. pseudomallei resulted in mutants with severely altered cellular morphology (increased cell length and defects in cell division), motility and reduced biofilm formation. ΔltgEBDC had a small increase in susceptibility to carbenicillin, doxycycline and ceftazidime. ΔltgB, ΔltgD, ΔltgE were all attenuated in the BALB/c mice model of melioidosis. In addition, I began to develop a model for the generation of non culturable Burkholderia. Incubation in NaCl concentrations >2.5% (w/v) for 7 days resulted in a complete loss of culturability on solid media. Live/dead staining however, revealed a substantial population of seemingly viable bacteria. Attempts to resuscitate these into actively growing cells however were unsuccessful. Given the role in virulence, the assays available and optimised for use with B. pseudomallei Ltgs and the X-ray crystal structure of LtgE, it would appear that Ltgs are good candidates for novel drug targets and that the tools are now in place for high throughput screening of Ltg inhibitors.
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Norville, Isobel Harriet. "The identification and characterisation of PPIases from Burkholderia pseudomallei and Burkholderia thailandensis." Thesis, University of Exeter, 2011. http://hdl.handle.net/10036/3152.
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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.
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Lim, J. "The characterization of the lipoprotein VacJ in Burkholderia pseudomallei and Burkholderia thailandensis." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2014. http://researchonline.lshtm.ac.uk/2124339/.
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Burkholderia pseudomallei, the causative agent of melioidosis, has evolved multiple strategies to facilitate survival in the environment and can cause serious disease in the human host. The lipoprotein BPSL3147 (VacJ) was previously shown to be important in the growth and survival of B. pseudomallei in an in vivo mouse model and a VacJ transposon mutant was highly attenuated. This work has focused on elucidating the role of VacJ as a virulence determinant in B. pseudomallei. The gene was characterized using bioinformatic and genetic techniques, utilizing comparisons with B. thailandensis to study the in vivo and in vitro roles. In this study a rationally defined B. pseudomallei VacJ deletion mutant was constructed, verified and evaluated. The VacJ mutant was able to colonize mice organs during the initial infection phase, but was unable to sustain the infection. In in vitro assays the VacJ mutant did not display any defect in early steps of the intracellular lifecycle. However, VacJ appears to play a contributory role to human serum resistance, as evidenced by the serum susceptibility of an acapsular B. pseudomallei ΔBPSL3147 mutant and B. thailandensis VacJ mutants. Taken together, VacJ contributes to virulence by affecting the outer membrane of B. pseudomallei and B. thailandensis affecting serum resistance sensitivity. The B. pseudomallei VacJ mutant was also investigated for potential as a live attenuated vaccine and displayed partial protection against a lethal challenge in an acute intranasal mice infection model.
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Fehlhaber, Beate. "Identifizierung und Charakterisierung von Virulenzfaktoren bei Burkholderia pseudomallei." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=96804042X.
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Lertmemongkolchai, Ganjana. "Mechanisms of cellular immune response against Burkholderia pseudomallei." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271142.
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Devarajoo, Shashikala Devi. "Purification of the exotoxin(s) of Burkholderia pseudomallei." Thesis, University of Salford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360452.
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Shalom, Gil. "Identification of macrophage-induced genes in Burkholderia pseudomallei." Thesis, University of Sheffield, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251370.
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Donaldson, Matthew. "Antimicrobial carbohydrate vaccines : development of Burkholderia pseudomallei immunogens." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/48103/.
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The potential bio-terror threat posed by Burkholderia pseudomallei highlights the need for an effective vaccine. Immunisation and challenge experiments in mice have demonstrated that the capsular polysaccharide (CPS-1) of B. pseudomallei, which is composed of β-1,3-linked 6-deoxy-D-manno-heptopyranose residues, is a promising candidate for vaccine development. This thesis set out to explore routes to potential vaccine candidates for Burkholderia pseudomallei infection based on both bottom-up synthetic monosaccharide constructs and top-down CPS-1 conjugates. Initially this thesis focused on the development of a monosaccharide antigen based on the 6-deoxy-D-manno-heptose building block of CPS-1. The antigens were conjugated to TetHc carrier protein via an 8-(methoxycarbonyl)octyl linker using traditional acyl hydrazide conjugation chemistry. The TetHc monosaccharide conjugates were analysed by SDS-PAGE and MALDI-ToF mass spectrometry before undergoing immunisation trials in sheep. Immunological assessment of the resulting polyclonal antisera was conducted by ELISA, slot-blot and the Octet biosensor system. These studies demonstrated the generation of antibodies that were specific for the cognate monosaccharide. Preliminary investigations showed that the 6-deoxy-D-mannoheptose- derived antibodies reacted positively with the natural CPS-1. The linker was shown to be important in the specificity of the polyclonal sera, leading to cross reactivity with non-parent monosaccharide antigens. A second generation of antigens was developed using a short linker, 3-(methyl mercaptopropionate). The expression and purification of the natural B. pseudomallei CPS-1 was achieved in an avirulent strain of Burkholderia, B. thailandensis E555. Characterisation of the capsular polysaccharide highlighted the co-expression of an α-1,3-mannan polysaccharide. Mild acid hydrolysis of the CPS-1 preparation, combined with capillary electrophoresis was trialled with a view to the generation of oligosaccharide fragments of CPS-1 for conjugation and immunisation studies. While initial carbohydrate conjugates were prepared with a previously described tetanus toxoid Hc fragment, the development and in planta expression of a chemically conjugatable Hepatitis B core virus-like particle system was also achieved.
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Pankhania, Depesh. "The serine/threonine protein kinases of Burkholderia pseudomallei." Thesis, University of Leicester, 2015. http://hdl.handle.net/2381/32255.
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Burkholderia pseudomallei is a Gram-negative bacterium that is endemic in tropical and sub-tropical regions. B. pseudomallei is an opportunistic pathogen and is the causative agent of the human disease, melioidosis, which accounts for 20% of all septicaemic deaths in Thailand. The phosphorylation via serine/threonine protein kinases (STKs) coordinates a vast array of signal pathways in both eukaryotic and prokaryotic organisms. In bacteria these pathways include: biofilm formation, cell wall biosynthesis, metabolism, sporulation, stress response and virulence. In this study, I identified six genes in the B. pseudomallei K96243 genome, bpsl0220, bpsl0571, bpsl0597, bpsl1828, bpss1584 and bpss2102 encoding putative STKs. The importance of these enzymes in B. pseudomallei was not determined to date, and thus the aim of this study is to Understand the roles of these proteins in B. pseudomallei virulence and regulations of Cellular processes. Four of the genes, bpsl0220, bpsl0597, bpsl1828, and, bpss2102, were successfully cloned and over-expressed the putative STKs in Escherichia coli and the recombinant proteins were purified. Each of the recombinant proteins exhibited autophosphorylation as well as phosphorylation of a general kinase substrate in vitro, thus demonstrating that each of the four proteins has protein kinase activity. Furthermore, I constructed specific mutants in each of the four STK-encoding genes in B. pseudomallei. These mutants were assessed in a variety of in vivo and in vitro, assays. The inactivation of the genes in B. pseudomallei, identified a switch in colony morphotype may be associated with a change in lipopolysaccharide structure and the motility of the bacteria, which warrants further study. One of the mutants, Δbpsl1828 displayed a conditional temperature sensitivity in distilled water, and thus the protein encoded by this gene could potentially contribute to the ability of B. pseudomallei to persist in the tropical and sub-tropical environments. However, future work is required to confirm a role of BPSL1828 in the regulation of environmental stress. A second mutant, Δbpsl0220, was avirulent in the Galleria mellonella model of disease, and introducing the full-length gene into the mutant complemented the phenotype. This result suggests that BPSL0220 may play a role in virulence. However, future work should assess the Δbpsl0220 deletion mutant in the mouse model of melioidosis. If these results confirm the role of BPSL0220 in pathogenicity, subsequent work could investigate if BPSL0220 maybe used as a target for therapeutic treatment of B. pseudomallei.
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Pramanpol, Nuttawan. "Structural studies on immunogenic proteins of Burkholderia pseudomallei." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/3807/.
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Patel, Natasha. "Experimental models of pulmonary vaccination against burkholderia pseudomallei." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549760.
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Virginio, Camila Gomes. "Aspectos fenotÃÂpicos de amostras de Burkhoderia pseudomallei isoladas de uma microepidemia do municÃÂpio de TejuÃÂuoca-Ce." Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=768.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorA Burkholderia pseudomallei à um bacilo Gram-negativo nÃo-fermentador, saprÃfita ambiental capaz de causar melioidose em homens e animais. A doenÃa à considerada endÃmica em diversos paÃses, entre os quais destacam-se TailÃndia e AustrÃlia. Em fevereiro de 2003, ocorreu no Brasil, o primeiro isolamento e identificaÃÃo da bactÃria em quatro crianÃas da localidade de TejuÃuoca, CearÃ. Este trabalho consiste na caracterizaÃÃo fenotÃpica de 3 amostras de B. pseudomallei originÃrias dos pacientes do municÃpio de TejuÃuoca, com o propÃsito de comparar os dados obtidos de tais amostras com os dados da literatura. Foram avaliados: morfologia das colÃnias em diferentes meios de cultivo, assimilaÃÃo de L-arabinose, testes bioquÃmicos manuais e em sistema semi-automatizado API 20NE, teste de sensibilidade antibacteriano e diagnÃstico por PCR, a partir de cultivos bacterianos. Nos resultados obtidos, foi observado o padrÃo morfolÃgico caracterÃstico de B. pseudomallei em Ãgar sangue, chocolate, Ashdown, Mac Conkey, CLED e tripticase soja. As amostras 1 e 3 foram classificadas como mucÃides e a amostra 2 como rugosa. As trÃs amostras apresentaram os padrÃes fenotÃpicos caracterÃsticos de B. pseudomallei tanto nos testes bioquÃmicos manuais: motilidade, crescimento à 42ÂC, oxidase positiva e resistÃncia à polimixina B, como no Kit DiagnÃstico API 20NE. Neste Ãltimo, houve diferenÃa na esculina entre as amostras, o que nÃo interferiu no resultado final de identificaÃÃo, quando a leitura foi realizada com 48h. Todas as trÃs amostras foram incapazes de assimilar o carboidrato L-arabinose, quando testadas em meio sais mÃnimo e API 20NE, caracterÃstica de amostras virulentas de B. pseudomallei e utilizada tambÃm para diferenciar esta espÃcie da B. thailandensis, que à capaz de assimilar este carboidrato. O padrÃo de sensibilidade resultante do TSA em disco difusÃo apresentado pelas trÃs amostras foi o caracterÃstico da espÃcie B. pseudomallei. Os isolados foram resistentes à gentamicina, cefalotina, ciprofloxacina (1 amostra apresentou resistÃncia intermediÃria) e sulfa-trimetoprim; 2 amostras apresentaram sensibilidade intermediÃria à ceftriaxona. Todas as trÃs amostras foram sensÃveis à piperacilina-tazobactam, ticarcilina-Ãcido clavulÃnico, ceftazidima, imipenem, tetraciclina e cloranfenicol. Com o protocolo fenol-clorofÃrmio modificado de extraÃÃo de DNA, a PCR apresentou banda de 718 pb, o que confirmou o diagnÃstico da bactÃria tambÃm por mÃtodo molecular. O estudo confirma a presenÃa da B. pseudomallei em territÃrio brasileiro, com fenotipagem semelhante à descrita na literatura internacional.
Burkholderia pseudomallei is a Gram-negative non-fermentative bacilli, environmental saprophyte able to cause melioidosis on men and animals. The disease is considered endemic in several countries, especially in Thailand and Australia. The bacteria was isolated and identified for the first time in Brazil, february 2003, from four children that lived in a place called TejuÃuoca, CearÃ. This work consists of the phenotypic characterization of 3 strains of B. pseudomallei isolated from the patients from TejuÃuoca. The main aim of this study is to compare the data from these samples with the ones from the literature. It was assessed: the colonies morphology in different culture mediums, assimilation of L-arabinose, manual and semi-automatized biochemical tests in API 20NE, antibacterial sensitivity test and diagnosis by PCR, from bacterial cultures. In the obtained results, it was observed the morphological pattern of B. pseudomallei in blood agar, chocolate, Ashdown, Mac Conkey, CLED and trypticase soy agar. The strains 1 and 3 were classified as mucoid and the strain 2 as wrinkled. The three strains had shown the usual phenotypic patterns of B. pseudomallei as much in biochemical manual tests: motility, growth at 42ÂC, positive oxidase and resistance to polimixin B, as in the API 20NE Diagnosis Kit. In this last one, there was difference in the esculin test among the strains, when the reading was carried out with 48 hours, which did not change on the final identification. All of the three strains were unable to metabolize the carbohydrate L-arabinose, when tested in minimum medium salts and API 20NE, which is a characteristic of virulent strains of B. pseudomallei and is also used to differ this species from B. thailandensis, that is able to use the carbohydrate. The three isolates had shown a poor sensitivity pattern from disk diffusion on TSA, which were resistant to gentamicin, cefalotin, ciprofloxacin (one strain presented intermediate resistance) and sulfa-trimethoprim; two strains presented intermediate sensitivity to ceftriaxone. All of them were sensitive to piperacilin-tazobactam, ticarcilin-clavulanate, ceftazidime, imipenem, tetracycline and chloramphenicol. A modified extraction protocol based on phenol-chloroform was used to obtain DNA and later to test it by PCR, which had shown a 718 bp product, what also confirmed the diagnosis of the organisms by molecular method. The study confirms the presence of B. pseudomallei in Brazil with similar phenotype to that described in the international literature.
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Virginio, Camila Gomes. "Aspectos fenotípicos de amostras de Burkhoderia pseudomallei isoladas de uma microepidemia do município de Tejuçuoca-Ce." reponame:Repositório Institucional da UFC, 2005. http://www.repositorio.ufc.br/handle/riufc/1818.
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VIRGINIO, Camila Gomes. Aspectos fenotípicos de amostras de burkholderia pseudomallei isoladas de uma microepidemia no municípios de Tejuçuoca-CE. 2005. 136 f. Dissertação (Mestrado em Microbiologia Médica) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2005.Submitted by denise santos (denise.santos@ufc.br) on 2012-01-04T13:45:00Z No. of bitstreams: 1 2005_dis_cgvirgínio.pdf: 7687643 bytes, checksum: d6c0e4ff2f09282d3738c27cd4b0bc8f (MD5)
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Burkholderia pseudomallei is a Gram-negative non-fermentative bacilli, environmental saprophyte able to cause melioidosis on men and animals. The disease is considered endemic in several countries, especially in Thailand and Australia. The bacteria was isolated and identified for the first time in Brazil, february 2003, from four children that lived in a place called Tejuçuoca, Ceará. This work consists of the phenotypic characterization of 3 strains of B. pseudomallei isolated from the patients from Tejuçuoca. The main aim of this study is to compare the data from these samples with the ones from the literature. It was assessed: the colonies morphology in different culture mediums, assimilation of L-arabinose, manual and semi-automatized biochemical tests in API 20NE, antibacterial sensitivity test and diagnosis by PCR, from bacterial cultures. In the obtained results, it was observed the morphological pattern of B. pseudomallei in blood agar, chocolate, Ashdown, Mac Conkey, CLED and trypticase soy agar. The strains 1 and 3 were classified as mucoid and the strain 2 as wrinkled. The three strains had shown the usual phenotypic patterns of B. pseudomallei as much in biochemical manual tests: motility, growth at 42°C, positive oxidase and resistance to polimixin B, as in the API 20NE Diagnosis Kit. In this last one, there was difference in the esculin test among the strains, when the reading was carried out with 48 hours, which did not change on the final identification. All of the three strains were unable to metabolize the carbohydrate L-arabinose, when tested in minimum medium salts and API 20NE, which is a characteristic of virulent strains of B. pseudomallei and is also used to differ this species from B. thailandensis, that is able to use the carbohydrate. The three isolates had shown a poor sensitivity pattern from disk diffusion on TSA, which were resistant to gentamicin, cefalotin, ciprofloxacin (one strain presented intermediate resistance) and sulfa-trimethoprim; two strains presented intermediate sensitivity to ceftriaxone. All of them were sensitive to piperacilin-tazobactam, ticarcilin-clavulanate, ceftazidime, imipenem, tetracycline and chloramphenicol. A modified extraction protocol based on phenol-chloroform was used to obtain DNA and later to test it by PCR, which had shown a 718 bp product, what also confirmed the diagnosis of the organisms by molecular method. The study confirms the presence of B. pseudomallei in Brazil with similar phenotype to that described in the international literature.
A Burkholderia pseudomallei é um bacilo Gram-negativo não-fermentador, saprófita ambiental capaz de causar melioidose em homens e animais. A doença é considerada endêmica em diversos países, entre os quais destacam-se Tailândia e Austrália. Em fevereiro de 2003, ocorreu no Brasil, o primeiro isolamento e identificação da bactéria em quatro crianças da localidade de Tejuçuoca, Ceará. Este trabalho consiste na caracterização fenotípica de 3 amostras de B. pseudomallei originárias dos pacientes do município de Tejuçuoca, com o propósito de comparar os dados obtidos de tais amostras com os dados da literatura. Foram avaliados: morfologia das colônias em diferentes meios de cultivo, assimilação de L-arabinose, testes bioquímicos manuais e em sistema semi-automatizado API 20NE, teste de sensibilidade antibacteriano e diagnóstico por PCR, a partir de cultivos bacterianos. Nos resultados obtidos, foi observado o padrão morfológico característico de B. pseudomallei em ágar sangue, chocolate, Ashdown, Mac Conkey, CLED e tripticase soja. As amostras 1 e 3 foram classificadas como mucóides e a amostra 2 como rugosa. As três amostras apresentaram os padrões fenotípicos característicos de B. pseudomallei tanto nos testes bioquímicos manuais: motilidade, crescimento à 42ºC, oxidase positiva e resistência à polimixina B, como no Kit Diagnóstico API 20NE. Neste último, houve diferença na esculina entre as amostras, o que não interferiu no resultado final de identificação, quando a leitura foi realizada com 48h. Todas as três amostras foram incapazes de assimilar o carboidrato L-arabinose, quando testadas em meio sais mínimo e API 20NE, característica de amostras virulentas de B. pseudomallei e utilizada também para diferenciar esta espécie da B. thailandensis, que é capaz de assimilar este carboidrato. O padrão de sensibilidade resultante do TSA em disco difusão apresentado pelas três amostras foi o característico da espécie B. pseudomallei. Os isolados foram resistentes à gentamicina, cefalotina, ciprofloxacina (1 amostra apresentou resistência intermediária) e sulfa-trimetoprim; 2 amostras apresentaram sensibilidade intermediária à ceftriaxona. Todas as três amostras foram sensíveis à piperacilina-tazobactam, ticarcilina-ácido clavulânico, ceftazidima, imipenem, tetraciclina e cloranfenicol. Com o protocolo fenol-clorofórmio modificado de extração de DNA, a PCR apresentou banda de 718 pb, o que confirmou o diagnóstico da bactéria também por método molecular. O estudo confirma a presença da B. pseudomallei em território brasileiro, com fenotipagem semelhante à descrita na literatura internacional.
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Bandeira, Tereza de Jesus Pinheiro Gomes. "Caracterização fenotípica e genotípica, sensibilidade a antimicrobianos e detecção de genes de virulência de cepas clínicas e ambientais de Burkholderia pseudomallei." reponame:Repositório Institucional da UFC, 2011. http://www.repositorio.ufc.br/handle/riufc/5313.
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BANDEIRA, Tereza de Jesus Pinheiro Gomes. Caracterização fenotípica e genotípica, sensibilidade a antimicrobianos e detecção de genes de virulência de cepas clínicas e ambientais de Burkholderia pseudomallei. 2011. 151 f. Tese (Doutorado em Microbiologia Médica) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2011.Submitted by denise santos (denise.santos@ufc.br) on 2013-07-08T16:20:33Z No. of bitstreams: 1 2011_tese_tjpgbandeira.pdf: 2968543 bytes, checksum: 7ca158d99b2739cf86254cd483a4ca24 (MD5)
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Melioidose E UMA Doença infecciosa causada túmulo POR Burkholderia pseudomallei, um bacilo Gram-Negativo ENCONTRADO nenhum sozinho e na Água. A Doença endêmica e No Sudeste Asiático e hiperendêmica nenhum norte da Austrália, Onde a letalidade permanece com UMA taxa de 21%. No Brasil, è considerada UMA Doença emergente desde Marco de 2003. N.os ULTIMOS Oito Anos, 12 Casos ocorreram no Estado do Ceará e hum notificado Pelo Governo Holandês, POR SE TRATAR de hum turista Que Morreu de melioidose, APOS Visita ao Ceará. Em Razão da Ocorrência Clínica de melioidose e faça Isolamento de B. pseudomallei nenhum Ambiente do Estado do Ceará, Este Trabalho objetivou Estudar como cepas Clínicas e Ambientais de B. pseudomallei isoladas sem Estado não PERÍODO de 2003 a 2011, visando a identificar como cepas POR MÉTODOS fenotípicos e moleculares, determinar o Perfil de sensibilidade contra cinco Agentes antimicrobianos (amoxicilina / clavulanato, Ceftazidima, imipenem, doxicilina e sulfametoxazol / trimetoprim), Realizar uma genotipagem das cepas Pela amplificação aleatória de DNA polimórfico - Random Amplified Polymorphic DNA (RAPD), detectar o tipo de virulência Três Sistema de Secreção gene (TTSS), ALÉM de avaliar OS Aspectos clínico-epidemiológicos Que caracterizaram uma Emergência Desta Doença no Brasil. TODAS como 20 cepas (dez Clínicas e dez Ambientais) de B. pseudomallei FORAM precisamente identificadas Tanto Pela Metodologia VITEK2 ® Quanto Pelô sequenciamento da Região 16S fazer DNA, mostraram Resultado Negativo nenhum teste de assimilação de L-arabinose, e exibiram-se Positivas parágrafo fazer a detecção de genes de virulência TTSS. Como Concentrações inibitórias Mínimas (CIM), obtidas POR microdiluição los caldo Müeller-Hinton, demonstraram Opaco de Todos os Isolados (100%) sensíveis FORAM AO imipenem, à doxicilina e AO sulfametoxazol-trimetoprim, não entanto, parágrafo amoxicilina / clavulanato e Ceftazidima, um FOI sensibilidade de 80 e 90%, respectively. A Técnica de RAPD evidenciou UMA Variabilidade genética de 63% empreendedorismo como cepas de B. pseudomallei oriundas do Estado do Ceará, como cais Quais d'Orsay FORAM Agrupadas in Tres aglomerados Diferentes. Este Trabalho decerto contribuirá par o Conhecimento das Características fenotípicas e genotípicas das cepas de B. pseudomallei isoladas nenhuma Ceará e da atualização da Vigilância epidemiológica dos Casos de melioidose ocorridos no Estado, ALÉM de contribuir para à Conscientização dos Órgãos de Saúde competentes Pará a Inclusão fazer Ceará Como zona endêmica parágrafo ESTA enfermidade.
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Alla, Ravi Chandran Reddy. "Structural Analyses of Lipid A from Burkholderia pseudomallei and Burkholderia thailandensis by Mass Spectrometry." University of Toledo / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1440078701.
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Rolim, Dionne Bezerra. "Burkholderia pseudomallei no Estado do CearÃ: caracterizaÃÃo de reservÃrias." Universidade Federal do CearÃ, 2009. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=14171.
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nÃo hÃA melioidose, uma enfermidade causada pelo bacilo Gram-negativo, Burkholderia pseudomallei, à endÃmica no sudeste da Ãsia e na AustrÃlia e tem distribuiÃÃo esporÃdica em outras partes do mundo. A doenÃa à descrita nas AmÃricas, sendo emergente no Brasil desde que casos em humanos sÃo bem documentados no Estado do CearÃ. Esta pesquisa pretendeu compreender melhor a ecologia da bactÃria por meios da caracterizaÃÃo de suas reservÃrias. O estudo foi realizado em uma pesquisa ambiental de B. pseudomallei no solo dos MunicÃpios de TejuÃuoca e Banabuià e na realizaÃÃo de inquÃrito soro-epidemiolÃgico para a populaÃÃo rural residente nesses locais. Para o estudo ambiental, foram coletadas amostras mensais de solo da superfÃcie atà 40 cm de profundidade durante o perÃodo de janeiro a dezembro do ano de 2007. Cinco sÃtios de coleta em cada municÃpio, delimitados na residÃncia de pessoas que tiveram melioidose. Para a realizaÃÃo do estudo sorolÃgico, foram coletadas amostras de soro de 321 residentes nessas Ãreas e efetivado inquÃrito, que incluiu informaÃÃes sobre dados demogrÃficos, histÃria de doenÃas prÃvias e atividades com exposiÃÃo relativas a solo e Ãgua. A determinaÃÃo dos tÃtulos sorolÃgicos de anticorpos foi realizada mediante teste imunoenzimÃtico, utilizando microplacas adsorvidas com antÃgeno filtrado de B. pseudomallei. A bactÃria foi encontrada no solo dos MunicÃpios de TejuÃuoca e Banabuià em 4,3 % (26/600) das amostras investigadas. As duas regiÃes apresentaram aspectos geoclimÃticos e componentes ambientais, como tipo de solo e vegetaÃÃo, Ãndice pluviomÃtrico, temperatura similares entre si. A detecÃÃo de B. pseudomallei ocorreu em clima tropical semi-Ãrido, com Ãndice pluviomÃtrico anual baixo e vegetaÃÃo de caatinga arbustiva, demonstrando influÃncia de fatores locais que facilitam a sobrevivÃncia e multiplicaÃÃo do microorganismo. O inquÃrito sorolÃgico evidenciou 51, 27% (161/327) para oisotipo IgM e 58,49 % (186/317) para o isotipo IgG. A freqÃÃncia dos tÃtulos de IgM foi maior em crianÃas do que em adultos, enquanto a freqÃÃncia de IgG aumentou com a idade. Houve associaÃÃo significativa entre a ocupaÃÃo em atividades de agricultura e os tÃtulos de IgM (44.15%, p<0.005) e de IgG (44.15%, p<0.005) e entre trabalhadores de construÃÃo civil e os tÃtulos de IgG (84.6%, , p=0.005). A maioria das amostras com tÃtulos elevados mostrou reatividade com as banda na posiÃÃo 33 a 45 kD que correspondem ao polissacarÃdeo O da cadeia do lipossacarÃdeo da bactÃria.
Melioidosis, a disease caused by the Gram-negative bacteria Burkholderia pseudomallei, is endemic in southeast Asia and in Australia and shows a sporadic distribution in other parts of the world. The disease has been described in the Americas, it is an emergent illness in Brazil, as human cases are well documented in Cearà state. This research aims to better understand the ecology of the bacteria. The study was carried out by means of an environmental search for B. Pseudomallei in the ground of the cities TejuÃuoca and Banabuià and by a epidemiological surveillance on the local rural population. For the environmental study, soil samples were collected monthly from the surface down to 40 cm depth from January to December 2007. Five sampling sites in each municipality were chosen near to the homes of people who had melioidosis. For the serological study, serum samples of 321 inhabitants of those areas were collected, and an inquiry was taken that included data about demography, previous illnesses and activities related to exposition to water and soil. Serological titers were determined by means of an immunoenzymatic test, using microplates adsorbed with filtered antigen of B. Pseudomallei. The bacteria was found in the soil of TejuÃuoca and Banabuià municipalities in 4.3% (26/600) of the samples. Both regions show similar geoclimatic and environmental aspects, such as soil and vegetation, rainfall index, and temperatures. Detection of B. Pseudomallei occurred in tropical semi-arid climate, with low annual pluviometric index and a shrubby âcaatingaâ vegetation, showing an influence of local factors enabling the survival and multiplication of the microorganism. Epidemiological surveillance showed 51.27% (161/327) for the IgM isotype and 58.49% (186/317) for IgG isotype. Frequency of IgM titers was higher among children than adults, while IgG frequency raised with age. There was a significant association between agricultural occupations and IgM (44.15%, p<0.005) and IgG titers (44.15%, p<0.005) and between construction workers and IgG titers (84.6%, p=0.005). Most samples with high titers showed reactivity to the 33 and 45 kD bands, which correspond to the polysaccharide O of the liposaccharide chain of the bacteria.
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Southern, Stephanie. "Evaluation of two novel antimicrobial targets in Burkholderia pseudomallei." Thesis, University of Southampton, 2014. https://eprints.soton.ac.uk/367092/.
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Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of the disease melioidosis. Melioidosis is endemic in regions of Southeast Asia and northern Australia, with human infection associated with a high mortality rate. The disease can manifest in several forms, including pneumonia, septicaemia or a chronic infection which can affect multiple organs and persist for months or years. This makes treatment of B. pseudomallei infection extremely problematic, complicated further by its inherent antibiotic resistance. For this reason new antimicrobials are required that target novel pathways within the cell. The aim of this study was to evaluate two proteins in B. pseudomallei as future targets for antimicrobial drugs. These proteins included an essential target, inhibition of which would result in cell death, and a second that was predicted to be crucial for virulence. The Min system is responsible for the correct placement of the cell division apparatus. It is made up of three proteins; MinC, MinD and MinE, where MinE is predicted to be essential in B. pseudomallei. The virulence target chosen was PspA, the main effector of the Phage-shock protein (Psp) response. The Psp response is an extracytoplasmic response system that is vital for maintenance of the inner membrane when the cell encounters stressful conditions. In order to validate MinE as an essential target in B. pseudomallei, a number of conditional mutagenesis techniques were used to inactivate the gene. This study found that the min operon was not essential when all three genes were inactivated, but an imbalance any of the min genes did have a detrimental effect on the survival of the bacteria, indicating that this would provide an ideal target for inhibitors. The Psp response was fully characterised by creating a knockout mutant in the pspA gene. Deletion of pspA caused a growth defect during prolonged growth in a liquid culture, also displaying reduced survival in a macrophage infection during this stage of its lifecycle. However, the ΔpspA mutant did not show attenuation when tested in multiple infection models and so was not thought to play a major role in the virulence of B. pseudomallei. The results from this study indicate the PspA would not make an effective candidate for an antimicrobial target.
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Rolim, Dionne Bezerra. "Burkholderia pseudomallei no estado do Ceará : caracterização de reservárias." reponame:Repositório Institucional da UFC, 2009. http://www.repositorio.ufc.br/handle/riufc/12249.
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ROLIM, Dionne Bezerra. Burkholderia pseudomallei no estado do Ceará : caracterização de reservárias. 2009. 156 f. Tese (Doutorado em Ciências Médicas) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2009.Submitted by denise santos (denise.santos@ufc.br) on 2015-05-18T13:23:34Z No. of bitstreams: 1 2009_ tese_dbrolim.pdf: 3141910 bytes, checksum: 3afbdab5e7f63e729d72f0de548bd1f4 (MD5)
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Melioidosis, a disease caused by the Gram-negative bacteria Burkholderia pseudomallei, is endemic in southeast Asia and in Australia and shows a sporadic distribution in other parts of the world. The disease has been described in the Americas, it is an emergent illness in Brazil, as human cases are well documented in Ceará state. This research aims to better understand the ecology of the bacteria. The study was carried out by means of an environmental search for B. Pseudomallei in the ground of the cities Tejuçuoca and Banabuiú and by a epidemiological surveillance on the local rural population. For the environmental study, soil samples were collected monthly from the surface down to 40 cm depth from January to December 2007. Five sampling sites in each municipality were chosen near to the homes of people who had melioidosis. For the serological study, serum samples of 321 inhabitants of those areas were collected, and an inquiry was taken that included data about demography, previous illnesses and activities related to exposition to water and soil. Serological titers were determined by means of an immunoenzymatic test, using microplates adsorbed with filtered antigen of B. Pseudomallei. The bacteria was found in the soil of Tejuçuoca and Banabuiú municipalities in 4.3% (26/600) of the samples. Both regions show similar geoclimatic and environmental aspects, such as soil and vegetation, rainfall index, and temperatures. Detection of B. Pseudomallei occurred in tropical semi-arid climate, with low annual pluviometric index and a shrubby “caatinga” vegetation, showing an influence of local factors enabling the survival and multiplication of the microorganism. Epidemiological surveillance showed 51.27% (161/327) for the IgM isotype and 58.49% (186/317) for IgG isotype. Frequency of IgM titers was higher among children than adults, while IgG frequency raised with age. There was a significant association between agricultural occupations and IgM (44.15%, p<0.005) and IgG titers (44.15%, p<0.005) and between construction workers and IgG titers (84.6%, p=0.005). Most samples with high titers showed reactivity to the 33 and 45 kD bands, which correspond to the polysaccharide O of the liposaccharide chain of the bacteria.
A melioidose, uma enfermidade causada pelo bacilo Gram-negativo, Burkholderia pseudomallei, é endêmica no sudeste da Ásia e na Austrália e tem distribuição esporádica em outras partes do mundo. A doença é descrita nas Américas, sendo emergente no Brasil desde que casos em humanos são bem documentados no Estado do Ceará. Esta pesquisa pretendeu compreender melhor a ecologia da bactéria por meios da caracterização de suas reservárias. O estudo foi realizado em uma pesquisa ambiental de B. pseudomallei no solo dos Municípios de Tejuçuoca e Banabuiú e na realização de inquérito soro-epidemiológico para a população rural residente nesses locais. Para o estudo ambiental, foram coletadas amostras mensais de solo da superfície até 40 cm de profundidade durante o período de janeiro a dezembro do ano de 2007. Cinco sítios de coleta em cada município, delimitados na residência de pessoas que tiveram melioidose. Para a realização do estudo sorológico, foram coletadas amostras de soro de 321 residentes nessas áreas e efetivado inquérito, que incluiu informações sobre dados demográficos, história de doenças prévias e atividades com exposição relativas a solo e água. A determinação dos títulos sorológicos de anticorpos foi realizada mediante teste imunoenzimático, utilizando microplacas adsorvidas com antígeno filtrado de B. pseudomallei. A bactéria foi encontrada no solo dos Municípios de Tejuçuoca e Banabuiú em 4,3 % (26/600) das amostras investigadas. As duas regiões apresentaram aspectos geoclimáticos e componentes ambientais, como tipo de solo e vegetação, índice pluviométrico, temperatura similares entre si. A detecção de B. pseudomallei ocorreu em clima tropical semi-árido, com índice pluviométrico anual baixo e vegetação de caatinga arbustiva, demonstrando influência de fatores locais que facilitam a sobrevivência e multiplicação do microorganismo. O inquérito sorológico evidenciou 51, 27% (161/327) para oisotipo IgM e 58,49 % (186/317) para o isotipo IgG. A freqüência dos títulos de IgM foi maior em crianças do que em adultos, enquanto a freqüência de IgG aumentou com a idade. Houve associação significativa entre a ocupação em atividades de agricultura e os títulos de IgM (44.15%, p<0.005) e de IgG (44.15%, p<0.005) e entre trabalhadores de construção civil e os títulos de IgG (84.6%, , p=0.005). A maioria das amostras com títulos elevados mostrou reatividade com as banda na posição 33 a 45 kD que correspondem ao polissacarídeo O da cadeia do lipossacarídeo da bactéria.
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Zainal, Abidin Nurhamimah. "Studies on the intracellular life of the melioidosis pathogen Burkholderia pseudomallei." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31336.
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Melioidosis, caused by the environmental Gram negative bacillus Burkholderia pseudomallei, is an emerging infectious disease affecting both animals and humans. B. pseudomallei has the ability to enter the host cell and escape from the phagosome. Once in the cytoplasm, the pathogen proliferates and expresses a virulence-associated protein known as BimA which polymerises cellular actin at the pole of the bacterium to promote its movement inter- and intracellularly, a process known as actin-based motility. This actin-based motility is also used as a strategy to evade host immune responses and survive intracellularly. In the first part of the thesis, we demonstrate that a B. pseudomallei ΔbimA mutant displays impaired intracellular survival compared to the isogenic parent strain in BALB/C bone-marrow derived macrophages (BMDMs), notably at later time points post-infection. Macrophages are the key innate immune cells that control B. pseudomallei in vivo and in vitro, and BALB/C mice provide an excellent model of acute human melioidosis. We also have determined that in BMDMs, the ΔbimA mutant is able to escape from the phagosome and enters the cytosol where it is unable to form actin tails. We used targeted, hypothesis-driven experiments to identify potential cell-autonomous innate mechanism/s of killing the mutant. First, we speculated that BimA mediates escape from autophagy. However our studies, including LC3-conversion assays, and bacterial co-localisation studies, failed to demonstrate a role for autophagy in clearance of the ΔbimA mutant from infected BMDMs. In the second part of this thesis, we investigated the role of Toll-like Receptors (TLR) in recognition and elimination of B. pseudomallei. MyD88 (Myeloid differentiation primary-response gene 88) and TRIF (TIR-domain-containing adaptor protein inducing IFNβ) are the main adaptor proteins involved in TLR signalling. We utilised the gene silencing technique using short interfering RNAs (siRNAs) to knockdown MyD88 transcript, and in a separate experiment used MyD88- or TRIF-blocking peptides. In addition, we investigated the involvement of canonical and non-canonical inflammasome pathways in cell-autonomous immunity of the BMDMs. However, none of these pathways were shown to be involved in clearance of the ΔbimA mutant from infected BMDMs. Finally we took an unbiased approach by microarray to characterise the global host transcriptome in BALB/C BMDMs upon B. pseudomallei infection, and to identify specific responses to the ΔbimA mutant. Analyses performed at the gene level revealed that several interferon signalling-related pathways are activated in cells infected with either the WT or ΔbimA mutant strains. A number of other pro-inflammatory mediators that are commonly seen in general inflammatory infections, such as IL-1α, IL-1β, IL-12β, and IL-6, were also upregulated. Interestingly, the cytoplasmic RNA sensors RIG-1 and MDA-5, thought primarily to be involved in the detection of RNA viruses, were also induced upon B. pseudomallei infection. Very few pathways were associated with a specific macrophage response to the ΔbimA mutant, indicating that an as yet undescribed pathway may play a role in sensing and eliminating the ΔbimA mutant. We conclude that actin-based motility mediates escape of B. pseudomallei from macrophage intracellular killing through a novel pathway which has yet to be unravelled.
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Jitprasutwit, Niramol. "Investigating the role of IQGAP1 in intracellular life of Burkholderia pseudomallei." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31142.
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Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes melioidosis, a serious disease of humans and animals in tropical countries. This pathogen can subvert the host cell actin machinery by a process known as actibased motility, for promoting its movement both within and between cells. The bacterial factor required for this process is known as BimA (Burkholderia intracellular motility A). Intracytoplasmic bacterial pathogens use distinct mechanisms for actin-based motility, hijacking host cytoskeletal proteins for their benefit. However, the molecular mechanism by which BimA subverts the cellular actin machinery is ill-defined. From an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerisation, a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner was identified. A subset of these proteins was independently validated with specific antisera including IQ motif containing GTPase activating protein 1 (IQGAP1). IQGAP1 is a ubiquitous scaffold protein that integrates several key cellular signalling pathways including those involved in actin dynamics. Previous studies demonstrated IQGAP1 was targeted by pathogens to regulate the actin cytoskeleton, for example promoting Salmonella invasion into epithelial cells or supporting cell attachment and pedestal formation of Enteropathogenic Escherichia coli. The aim of this study is to explore the roles of IQGAP1 in the intracellular life of B. pseudomallei. This present study revealed that IQGAP1 was recruited to B. pseudomallei actin tails in infected HeLa cells. This protein has not previously been associated with actin-based motility of other intracellular pathogens. To examine the effect on actibased motility of B. pseudomallei, siRNA was utilised to knockdown IQGAP1 in HeLa cells. After optimisation of siRNA transfection, IQGAP1 expression in HeLa cells was suppressed by approximately 70% as assessed by IQGAP1 immunoblotting. The siIQGAP1 knockdown cells were infected with B. pseudomallei. The bacteria could still form actin tails in the knockdown cells, however, the data showed a statistically significant increase in overall tail length with a concomitant decrease in actin density, compared with the tails formed by B. pseudomallei in control cells. Actin-based motility is essential in the life cycle of several cytoplasmic bacterial pathogens, particularly in cell-to- cell spread. After entry into the host cell cytosol, B. pseudomallei polymerises actin in a BimA-dependent manner and propels itself within and between cells. This is accompanied by cell fusion which generates multi-nucleated giant cells (MNGCs), a process mediated by a Type 6 Secretion System that is co-regulated with BimA. To gain an understanding of the impact of IQGAP1 on the intracellular life of B. pseudomallei, IQGAP1 was successfully knocked-out from HeLa cells using CRISPR-Cas9 technique. Interestingly, Burkholderia invasion was not affected in HeLa cells lacking IQGAP1. However, the bacteria showed a defect in intracellular survival in IQGAP1 knockout cells that was revealed after 6 hours post-infection. Moreover, there was no difference in the proportion of bacteria associated with actin in the control and knockout cells at 16 hours post-infection, although the bacteria formed longer actin tails in control cells with similar actin density. Consequently, the number of MNGCs decreased dramatically in the cells lacking IQGAP1, which was indicated by the absence of plaque formation. Another element of this study was to determine whether BimA and IQGAP1 are direct interacting partners. Using either an in vitro pulldown assay or in vivo yeast two-hybrid system, a direct interaction between these proteins could not be detected. It is, therefore, likely that IQGAP1 is recruited to B. pseudomallei actin tails through its intrinsic ability to interact with F-actin. Despite the lack of a direct interaction between these two proteins, an N-terminal IQGAP1 fragment significantly augmented BimA-mediated actin polymerisation in vitro. Taken together, this study provides the first evidence of the presence of IQGAP1 in B. pseudomallei actin tails and presents the importance of IQGAP1 in actin-based motility and intracellular life of this bacterium. Understanding the mechanism of B. pseudomallei actin-based motility is useful to gain insights into host cell actin dynamics and its role in pathogenesis. Targeting host cellular proteins that are required for the intracellular life of pathogens are a topical area of research, with the potential to be useful alternatives to classic antibiotic therapy. Indeed, IQGAP1 could be a potential novel therapeutic target to develop drugs for treating B. pseudomallei infection.
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Valente, Lívia Gurgel do Amaral. "Identificação de melanina em cepas clínicas e ambientais de Burkholderia pseudomallei e atividade in vitro do farnesol como inibidor de β-lactamases." reponame:Repositório Institucional da UFC, 2012. http://www.repositorio.ufc.br/handle/riufc/8692.
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VALENTE, Livia Gurgel do Amaral. Identificação de melanina em cepas clínicas e ambientes de Burkholderia pseudomallei e atividade in vitro do farnesol como inibidor de ß-lactamases. 2012. 81 f. Dissertação (Mestrado em Microbiologia Médica) - Universidade Federal do Ceará, Fortaleza, 2012.Submitted by denise santos (denise.santos@ufc.br) on 2014-08-12T16:03:59Z No. of bitstreams: 1 2012_dis_lgavalente.pdf: 2099115 bytes, checksum: 202d0f130cefb452d49f8b0f3f7f0594 (MD5)
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Burkholderia pseudomallei é um bacilo Gram-negativo causador da melioidose uma doença infecciosa severa e geralmente fatal, a qual pode ser adquirida através da inoculação, inalação e ingestão do microrganismo que se encontra distribuído no ambiente. No Brasil a melioidose é considerada uma doença emergente descrita pela primeira vez em 2003 no Nordeste brasileiro O presente estudo consistiu em identificar o gene hppD que codifica a produção de um precursor importante na síntese de melanina e avaliar fenotipicamente a produção do pigmento em cepas de B pseudomallei através de meios contendo substratos fenólicos. Aliado ao estudo foi realizada a comparação do perfil de sensibilidade e curva de morte entre cepas melanizadas e não melanizada ante ao imipenem além da avaliação da possível ação do sesquiterpeno farnesol como um inibidor de β-lactamases Os isolados utilizados no estudo pertencem ao Laboratório de Patógenos Reemergentes e Emergentes-LAPERE UFC Para o cumprimento da metodologia as cepas foram recuperadas do estoque realizado a extração de DNA e a identificação do gene hppD através de reação de PCR A expressão fenotípica de melanina foi avaliada através de subcultivos em meio Brain Heart infusion (BHI) acrescido de ácido caféico. O teste de sensibilidade com cepas melanizadas e não melanizadas foi realizado utilizando-se o teste de microdiluição em caldo padronizado pelo CLSI segundo documento M07-A8 A curva de morte foi realizada a partir da incubação do inóculo com imipenem no intervalo de 0,125 µg/ mL a 0,5 µg/ mL por 1 e 2 horas, seguida da contagem das colônias A avaliação do farnesol como um possível inibidor de β-lactamases foi realizada através da combinação in vitro do farnesol com antibióticos β-lactâmicos (amoxicilina ampicilina oxacilina imipenem amoxicilina-ácido clavulânico utilizando-se o teste de microdiluição em caldo. O gene hppD foi detectado em todas as cepas de B pseudomallei testadas além da produção de pigmento em meios contendo substratos fenólicos Em relação ao perfil de sensibilidade as cepas não melanizadas e melanizadas apresentaram o mesmo valor de CIM porém as cepas melanizadas demonstraram maior capacidade de sobrevivência quando em contato com a droga do que as cepas não melanizadas. Em relação ao farnesol todas as cepas testadas foram inibidas por este composto com CIM variando de 75 a 150µM Dentre as combinações dos β-lactâmicos testadas com farnesol, todas as drogas apresentaram redução estatisticamente significativa: amoxicilina (p=0,0001) amoxicilina-ácido clavulânico (p=0,0005), ampicilina (p=0,0026), oxacilina (p=0,0001) e imipenem (p=0,0105). Por fim as combinações com amicacina e gentamicina não apresentaram redução estatisticamente significativa pois os aminoglicosídeos praticamente repetiram seus valores quando combinados com farnesol Esse estudo abre perspectivas acerca de um novo fator de virulência melanina ainda não descrita para B pseudomallei, além do sesquiterpeno farnesol como um possível inibidor de β-latamases nessa espécie.
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Correia, Giovanna Riello Barbosa. "Inhibitory effect in vitro of farnesol against Burkholderia pseudomallei biofilms." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=16814.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorA intrÃnseca resistÃncia apresentada pela bactÃria Burkholderia pseudomallei à um grave empecilho para o tratamento da melioidose. Muitas pesquisas focam na busca de adjuvantes que aumentem a sensibilidade desta bactÃria aos antimicrobianos. Nesse contexto, a aÃÃo inibitÃria do farnesol frente Ãs cepas de B. pseudomallei, na forma planctÃnica, jà foi relatada em estudo prÃvio. Diante disso, esse estudo objetivou analisar a atividade in vitro do farnesol contra cepas de B. pseudomallei na forma de biofilme. Aliado à anÃlise da aÃÃo do farnesol isoladamente, foi investigada a combinaÃÃo desse composto com os antimicrobianos amoxicilina, ceftazidima, doxiciclina, imipenem e sulfametoxazol/trimetoprim frente ao biofilme. A sensibilidade foi avaliada por meio do teste de microdiluiÃÃo em caldo e a leitura da viabilidade celular feita com a resazurina. A concentraÃÃo inibitÃria mÃnima em biofilme (CEMB) para o farnesol foi de 75 a 2400 mM. Ademais, o farnesol reduziu em atà 256, 16, 4 e 4 vezes os valores de CEMB para ceftazidima, amoxicilina, doxiciclina e sulfametoxazol/trimetoprim, respectivamente (P<0.05). Por meio de tÃcnicas de microscopia, tais como Ãptica, confocal e eletrÃnica, observou-se que o farnesol foi capaz de causar danos na matriz do biofilme, facilitando assim, a penetraÃÃo dos antibiÃticos. Deste modo, o presente estudo mostrou a eficÃcia do farnesol contra biofilmes de B. pseudomallei e seu efeito potenciador, em especial com ceftazidima, amoxicilina, doxiciclina e sulfametoxazol/trimetoprim
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
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Pongmala, Khemngeun. "Influence des propriétés physiques et chimiques du sol et de leur variation saisonnière sur l'occurrence et la distribution de Burkholderia pseudomallei dans une rizière au centre du Laos." Electronic Thesis or Diss., Toulouse 3, 2022. http://thesesups.ups-tlse.fr/5342/.
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Burkholderia pseudomallei (BP) bactérie commune des sols tropicaux, provoque une maladie grave, la mélioïdose. Cette thèse, basée sur un travail de terrain dans le centre de la RDP Lao où BP est endémique, vise à clarifier les déterminants de la distribution de BP dans le sol en fonction des conditions biogéochimiques du sol et climatiques, à des échelles emboîtées. PB a été identifiée dans le sol jusqu'à 300 cm de profondeur, à des concentrations variables (maximales en dessous de 100 cm), à la fois à l'échelle du millimètre, en lien avec l'état d'oxydation du fer, et à l'échelle du profil du sol. La variabilité saisonnière des concentrations de BP, plus marquée près de la surface qu'en profondeur, est cohérente avec sa persistance accrue dans les couches saturées d'eau tout au long de l'année. Cette thèse démontre l'importance de considérer la complexité du sol pour mieux comprendre l'écologie de BPBurkholderia pseudomallei (BP) a pathogenic bacterium found in tropical soils, causes a severe disease, melioidosis. This thesis, based on field work in the main region of BP endemicity in Lao PDR, aims to clarify the determinants of BP distribution in soil. We applied a multi-scale approach to characterize the distribution of BP in relation with soil physico-chemical variability and highlight seasonal variations in BP distribution. BP occurred at all soil depths down to 300 cm but its concentrations varied drastically, both at the millimetre scale, concomitantly with the oxidation state of iron, and at the soil profile scale, unexpectedly reaching peak values below 100 cm. Seasonal variability of BP concentrations was higher in shallow than deep soil horizons, consistent with increased BP persistence in layers saturated with water year round. This thesis demonstrate the importance of considering the complexity of soil to better understand the ecology of BP
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Gasqué, Mégane. "Étude de l'occurence de Burkholderia pseudomallei dans la région Antilles-Guyane." Electronic Thesis or Diss., Sorbonne université, 2024. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2024SORUS169.pdf.
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Burkholderia pseudomallei est l'agent pathogène responsable de la mélioïdose, une infection opportuniste. Elle est endémique en Asie du Sud, en Asie du Sud-Est et dans le Nord de l'Australie. En 2015, le nombre de cas annuels de mélioïdose était estimé à 165 000 dans le monde. Cette bactérie a la capacité d'infecter l'homme et les animaux par inhalation, ingestion ou contact cutané. Depuis plusieurs années, des cas humains de mélioïdose sont signalés en dehors des zones endémiques connues, notamment dans la région Antilles-Guyane. Depuis 1993, vingt cas humains de mélioïdose ont été recensés en Martinique et cinq en Guadeloupe. Des enquêtes épidémiologiques ont révélé que de nombreux patients n'avaient jamais voyagé dans des zones endémiques, suggérant une contamination locale. L'objectif de cette thèse était de confirmer l'implantation locale de B. pseudomallei dans la région Antilles-Guyane, en utilisant une approche « Une seule santé » qui étudie l'homme, l'animal et l'environnement. À cette fin, une collecte exhaustive des cas humains a été réalisée, accompagnée d'enquêtes sérologiques chez les animaux et d'enquêtes environnementales. En parallèle, un outil de typage moléculaire a été développé pour prédire la provenance géographique des souches isolées.Une revue de la littérature et des entretiens avec les cliniciens ont été menés pour recenser les cas humains. Les analyses sérologiques ont été effectuées à l'aide d'un kit ELISA-GLANDA. Deux types d'enquêtes environnementales ont été menées : des enquêtes ciblées dans les élevages présentant des animaux séropositifs et des enquêtes aléatoires en Guadeloupe et aux Saintes.Le recensement des cas humains a permis d'identifier vingt cas publiés dans la littérature, auxquels s'ajoutent cinq cas supplémentaires suite à des entretiens avec les cliniciens. L'enquête sérologique a révélé la présence d'animaux séropositifs sur tous les territoires, suggérant une présence locale de la bactérie, avec des taux de séroprévalence plus élevés chez les équins (24%) et les bovins (16%) en Guyane, chez les équins (9%) en Martinique et chez les caprins (39%) aux Saintes. Les enquêtes environnementales ciblées dans les élevages aux Saintes ont permis d'isoler une souche de B. pseudomallei, tandis que les enquêtes aléatoires en Guadeloupe et aux Saintes ont révélé des sols positifs en PCR, confirmant ainsi la présence locale de la bactérie.Cette thèse représente la première étude de ce type réalisée dans la région Antilles-Guyane. La mise en place d'une approche « Une seule santé » a permis d'obtenir des données préliminaires sur la présence de B. pseudomallei dans cette région. Ce projet constitue une première étape vers d'autres initiatives. Pour confirmer la présence de B. pseudomallei en Martinique et en Guyane, il est nécessaire de mener des enquêtes environnementales dans les élevages où des animaux séropositifs ont été identifiés. De plus, de nouvelles recherches pourraient être entreprises en Guadeloupe pour caractériser les types de sols où la présence de B. pseudomallei a été détectée, enrichissant ainsi la littérature scientifique. Cette étude souligne la possibilité d'appliquer une approche interdisciplinaire basé sur concept « Une seule santé » pour mieux comprendre la répartition d'autres bactéries environnementales pathogènesBurkholderia pseudomallei is the pathogen responsible for melioidosis, an opportunistic infection. It is endemic in South Asia, Southeast Asia and northern Australia. This bacterium is capable of infecting humans and animals through inhalation, ingestion or skin contact and in 2015, the number of annual global burden of melioidosis was estimated at 165,000. Human cases of melioidosis have been reported outside known endemic areas, notably in the Antilles-Guyane region. Since 1993, twenty human cases of melioidosis have been recorded in Martinique and five in Guadeloupe. Epidemiological investigations revealed that many of these patients had no history of travel to endemic areas, suggesting local contamination. The aim of this thesis was to confirm the local establishment of B. pseudomallei in the Antilles-Guyane region, using a "One Health" approach that studies humans, animals and the environment. To this end, an exhaustive search for human cases was implemented, accompanied by serological investigations in animals and environmental surveys. At the same time, a molecular typing tool was developed to predict the geographical origin of isolated strains.A literature review and interviews with clinicians were conducted to identify human cases. Animal serological analyses were carried out using an ELISA-GLANDA kit. Two types of environmental surveys were carried out: targeted surveys in farms with seropositive animals, and random surveys in Guadeloupe and Les Saintes.The survey of human cases identified twenty cases published in the literature, plus five additional cases based on interviews with clinicians. The serological survey revealed the presence of seropositive animals in all territories, suggesting a local presence of the bacterium, with higher seroprevalence rates among equines (24%) and cattle (16%) in French Guiana, equines (9%) in Martinique and goats (39%) in Les Saintes. Targeted environmental surveys on two goat farms in Les Saintes resulted in the isolation of one strain of B. pseudomallei, while random surveys in Guadeloupe and Les Saintes revealed PCR-positive soil, confirming the local presence of the bacterium.This thesis represents the first study of this type to be carried out in the Antilles-Guyane region. The implementation of an interdisciplinary "One Health" based approach has enabled us to obtain preliminary data on the presence of B. pseudomallei in this region and represents a first step towards other initiatives. To confirm the presence of B. pseudomallei in Martinique and French Guiana, it is necessary to carry out environmental surveys on farms where seropositive animals have been identified. In addition, new research could be undertaken in Guadeloupe to more finely characterize the types of soil where the presence of B. pseudomallei has been detected, thus enriching the scientific literature. This study highlights the interest of applying the "One Health" approach to better understanding the distribution of other pathogenic environmental bacteria
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Day, Matthew. "Structural studies on protein targets from the pathogenic bacterium Burkholderia pseudomallei." Thesis, University of Sheffield, 2012. http://etheses.whiterose.ac.uk/3396/.
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The main body of this thesis deals with a small-scale structural genomics project for proteins from the bacterium, Burkholderia pseudomallei. Potential pathogenicity determinants were selected based on their possession of at least one of two criteria. The first was their expression in the pathogenic Burkholderia pseudomallei but not the closely related, nonpathogenic Burkholderia thailandensis. The second required their expression be controlled as part of a stress response, which controls other known virulence factors. Of the nine selected targets, five were successfully overexpressed, purified and crystallised and the structure of one was determined. The selected targets were all of unknown function and initial sequence analysis highlighted a number of interesting characteristics for certain targets. BPSL1958 contains a highly repetitive sequence conserved at both the protein and DNA level, suggesting the gene was created in a recent duplication event and there has not been enough time for the sequence of the individual repeats to drift. Four of the selected targets, BPSS0211 - BPSS0214, formed an operon, of these BPSS0212 and BPSS0213 were homologous, with BPSS0211 also representing the C-terminal domain of the two proteins. The structure of BPSS0211 was determined to 2.17 Å using multiplewavelength anomalous dispersion experimental phasing. BPSS0211 is a small protein annotated as containing a conserved domain of unknown function, DUF1843. The tertiary structure of BPSS0211 entails two alpha helices stacked together which assemble to form a tetramer comprising a dimer of dimers. The pattern of conservations between residues in DUF1843 within BPSS0211, BPSS0212 and BPSS0213 is such that interactions between the three proteins would be possible. The proposed function of DUF1843 is as an oligomerisation domain, allowing the formation of homo and hetero, dimer and tetramer, complexes of three proteins encoded within the BPSS0211-BPSS0214 operon. Other work included involves the determination of the structure of thioredoxin from Burkholderia pseudomallei as part of an on-going project into this system. The structure was solved to 1.07 Å using molecular replacement and agreed well with previously solved thioredoxin structures from other organisms.
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Sangiambut, Sutha. "Cloning, expression and characterisation of potential therapeutic targets of Burkholderia pseudomallei." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271682.
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Marshall, Laura Emma. "The identification and characterisation of novel antimicrobial targets in Burkholderia pseudomallei." Thesis, University of Exeter, 2012. http://hdl.handle.net/10036/4074.
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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.
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Mahfouz, Magdy Elsayed. "Molecular characterisation of a two-component regulatory system from Burkholderia pseudomallei." Thesis, University of Plymouth, 2001. http://hdl.handle.net/10026.1/2538.
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Studies were undertaken to clone and characterise a two-component regulatory system from a clinical isolate (204) of the human and animal pathogen Burkholderia pseudomallei. A number of genomic libraries were constructed in E. coli host-vector systems and screened for the presence of a two-component system using oligonucleotide probes based on nucleotide sequence homology. Fragments of genomic DNA were cloned and sequenced and found to possess two open reading frames (ORFs) that overlap with a single nucleotide and are believed to encode a novel two-component regulatory system. A possible promoter region was identified upstream of the two ORFs, mrgR and mrgS, which read in the same direction and may represent an operon. The deduced translation of mrgR reveals a protein, MrgR, which possesses conserved motifs that are consistent with the phosphorylation domains and DNA-binding helix-turn-helix structure of a family of response regulatory proteins. The deduced translation of mrgS reveals that the MrgS protein possesses all the invariant amino acids that characterise other sensor regulatory proteins. Southern hybridisation studies showed that the mrgRS locus was present in 19 isolates of B. pseudomallei from a wide geographical derivation, but not in any closely related bacterial species, including Burkholderia thailandensis. The expression of the two genes was verified using antibodies developed to synthetic peptides based on sequences from the C- and N-terminal regions of MrgR and MrgS, respectively. The specificity of the antibodies was confirmed in Western blotting studies in which almost all of mrgR and the proximal quarter of mrgS were translationally fused with malE (MBP-MrgR and MBP-MrgS) and expressed in E. coli K12. The antibodies were used to probe Western blots of cellular and extracellular extracts of different isolates of B. pseudomallei and identified multiple bands in whole-cell lysates. The sizes of two of these bands were 24 kDa and 115 kDa, which may represent the unprocessed forms of MrgR and MrgS, respectively. It was proposed that the other bands represented either isoforms or degradation products of the full-length proteins. The recognition of all bands was abolished following pre-incubation of the antibodies with the immunising peptide but remained unaffected if an irrelevant peptide, was used for this purpose. Western blot analysis demonstrated that serum antibodies from a patient with acute melioidosis recognised MBP-MrgR but not MBP-MrgS suggesting a possible role for MrgR in the disease process. The expression of mrgR and mrgS was found to be constitutive in B. pseudomallei that had been cultured using different combinations of temperature, pH and NaCl suggesting that the genes perform a number of biological functions. There is some evidence that at 42°C the processing of MrgR and MrgS may be altered and the possible mechanisms for this are discussed. B. pseudomallei grew better at 42°C and pH 5 and less well at 25°C and pH 8 and this was influenced by NaCl concentration partly reflecting the environmental distribution and intracellular nature of the pathogen. Environmental and clinical isolates of B. pseudomallei differed in the pH optimum for growth at 42°C. The DNA flanking the mrgRS locus in isolate 204 was cloned, sequenced, and seven ORFs were identified including a transcriptional regulatory gene similar to bvgR of Bordetella pertussis. Southern blot analysis using three different DNA probes revealed restriction fragment length polymorphisms (RFLPs) in the region downstream of mrgRS. Two distinct RFLP patterns were identified among 16 different isolates of B. pseudomallei. The potential effects of this variation on gene expression and protein function await further investigation.
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Baldwin, Victoria Mae. "Next generation approaches to polysaccharide preparation for Burkholderia pseudomallei vaccine development." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/30158.
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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.
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Vander, Broek Charles William. "Subversion of host cellular processes by the melioidosis pathogen, Burkholderia pseudomallei." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25402.
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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.
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Butt, Aaron Trevor. "Identification and characterisation of toxin-antitoxin systems (TA) in Burkholderia pseudomallei." Thesis, University of Exeter, 2013. http://hdl.handle.net/10871/9303.
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The aim of this study was to identify and characterise type II toxin-antitoxin (TA) systems in Burkholderia pseudomallei, the causative agent of the human disease melioidosis. 8 putative TA systems were identified within the genome of B. pseudomallei K96243. 5 of these were located witihn genome islands. Of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ΔhipBA background) caused a reduction in the number of culturable bacteria. HicA also caused growth arrest in B. pseudomallei K96243 ΔhicAB. These toxin induced phenotypes were enhanced by an <3kDa extracellular factor that accumulated in the spent medium during growth. Expression of the cognate antitoxins could restore growth and culturability of cells. Expression of hicA in E. coli gave an increased number of persister cells in response to ciprofloxacin or ceftazidime. Site directed mutagenesis studies identified two key residues within the HicA toxin that were essential for both the reduced culturability and increased persistence phenotypes. Deletion of hicAB from B. pseudomallei K96243 did not affect persister cell or survival frequencies compared to the wild type following treatment with a variety of stress conditions. Deletion of the ΔhipBA locus from B. pseudomallei K96243 also had no affect on bacterial persistence or survival under the conditions tested.
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Schmidt, Imke [Verfasser]. "Rolle von antioxidativen Enzymen bei Infektionen mit Burkholderia pseudomallei / Imke Schmidt." Greifswald : Universitätsbibliothek Greifswald, 2015. http://d-nb.info/1073726088/34.
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Legutki, Joseph Barten. "Analysis of the immunogenicity of peptide mimotopes of burkholderia pseudomallei exopolysaccharide." The Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1163549512.
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Santanirand, Pitak. "The role of cytokines in the immune response to Burkholderia pseudomallei." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391948.
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Boddey, Justin Andrew. "Molecular and cellular characterisation of Burkholderia pseudomallei interactions with host cells." Thesis, Griffith University, 2006. http://hdl.handle.net/10072/365664.
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Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of serious and oftenfatal diseases, collectively termed melioidosis. B. pseudomallei is capable of interacting specifically with host cells; it can adhere, invade, and survive and replicate intracellularly and induce the formation of multinucleated giant cells (MNGCs). While recent work has begun characterising these interactions at the molecular level, much is still unknown. In this work, B. pseudomallei interactions with eukaryotic cells were investigated at the cellular and molecular level. Many putative loci were studied by, firstly, generating isogenic mutant strains lacking a gene or locus of interest; and secondly, by characterising each mutant strain using a number of in vitro and in vivo models. These included models for adherence to eukaryotic cells, microcolony development, biofilm formation, twitching motility, bacterial cell aggregation, invasion of eukaryotic cells, actinbased motility, intracellular proliferation, multinucleated giant cell (MNGC) development and characterisation, gene expression, and virulence in vivo. Three putative type IV pilus (TFP) systems were identified in nine loci in the B. pseudomallei K96243 genome; type IVA, IVB, and IVB Flp pilus systems. Studies revealed that TFP are produced on the surface of B. pseudomallei K96243 but that TFP play only a minor role in adherence to eukaryotic cells using the assays described. However, collaborators showed that B. pseudomallei K96243 uses the type IVA pilin, PilA, to adhere to eukaryotic cells using a different adherence assay. Interestingly, two strains of B. pseudomallei adhered to eukaryotic cells to different degrees and used TFP (pilA) differently. B. pseudomallei K96243 adhered significantly less to four cell lines than B. pseudomallei 08. Furthermore, adherent microcolony formation was shown to be a temperaturedependent phenotype that enhanced bacterial association with eukaryotic cells; however, while B. pseudomallei 08 required pilA to form microcolonies, strain K96243 did not form microcolonies, and cell association was markedly reduced for K96243 relative to strain 08. TFP were not required for the formation of biofilms on PVC; however, type IVA and IVB Flp pili were required for optimal virulence in BALB/c mice using the intranasal route of infection, indicating the importance of TFP for B. pseudomallei survival in vivo.
This work also identified that growth temperature, growth medium, and association with eukaryotic cells were important regulatory signals for adherence/cell association, microcolony formation, biofilm development, and TFP (pilA) expression. A homologue of eukaryotic 'senescence marker protein30' (SMP30) was identified in B. pseudomallei 08 and K96243 and was termed Lfp1 (for lactonase family protein 1). lfp1 is located within a genomic island and is conserved in both prokaryotes and eukaryotes. A homologue of lfp1 (lfp2) was also present in B. pseudomallei K96243. Though sharing considerable homology, prokaryotic and eukaryotic homologues of lfp1 were shown to be phylogenetically distinct. Expression of lfp1 mRNA by B. pseudomallei 08 was significantly increased in association with eukaryotic cells, relative to maintenance media alone; however, lfp1 was not required for adherence, invasion, intracellular proliferation, actinbased motility, or cell fusion by B. pseudomallei. Importantly though,
B. pseudomallei 08infected macrophagelike cells rapidly fused into MNGCs, assuming an lfp1specific osteoclastlike pattern of gene expression that was distinct from B. thailandensisinfected MNGCs, and the process may be different to the LPSdependent mechanisms of osteoclastlike cell induction described previously for other bacteria. B. pseudomalleiinduced MNGC formation correlated with potent increases in mRNA levels for the cell fusion and multinucleation factors MCP1, CCL9/MIP1?, RANTES, and NFATc1 in B. pseudomalleiinfected cells, implicating these molecules in cell fusion. B. pseudomalleiinduced osteoclastlike MNGCs could not authentically resorb dentine; however, regions of apparently demineralised dentine were observed, suggesting a defect in the excavation of the organic phase of bone, analogous to that observed in CTSK knockout mice. Finally, an lfp1 null mutant was significantly attenuated for virulence in both the Syrian hamster model and the BALB/c inhalation model, indicating a role for lfp1 in virulence.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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Alharbi, Mona. "Structural investigation of the GAF domain protein BPSL2418 from Burkholderia pseudomallei." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/8314/.
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A new family of methionine-sulfoxide reductase (Msr) was recently discovered, and was named free methionine sulfoxide reductase (fRMsr). This family includes enzymes with a reductase activity toward the free R isomer of a methionine sulfoxide substrate. The fRMsrs have a GAF domain topology, a domain, which was previously identified as having in some cases a cyclic nucleotide phosphodiesterase activity. The classification of fRMsrs as GAF domains revealed a new function can be added to the GAF domain family. Interestingly the four members identified in the fRMsr family share the GAF domain structure and the presence of three conserved cysteines in the active site with free R methionine sulfoxide substrate specificity. This thesis presents the crystal structures of reduced, free Met-SO substrate-bound and MES-bound forms of a new fRMsr from Burkholderia pseudomallei (BPSL2418). BPSL2418 was cloned, overexpressed and purified to enable protein crystallization. The crystallization trials for reduced, Met-SO-bound and MES-bound forms of BPSL2418 were prepared and reasonable crystals of each form were produced. The crystal structures of BPSL2418MES, BPSL2418Met-SO and BPSL2418Reduced were solved at 1.18, 1.4 and 2.0Å, respectively by molecular replacement. The BPSL2418MES crystal belongs to space group P 21 21 21 while BPSL2418Met-SO and BPSL2418Reduced crystals belong to space group P 1 21 1. All three forms share the GAF domain structure of six antiparallel β-strands and four a-helices with connecting loops. The antiparallel β- strands (β1, β2, β5 and β6) are located in the center of the BPSL2418 structure flanked on one side by a three a-helices (a1, a2 and a4) and on the other side by a (loop1, β3, loop2, a3, β4 loop4) unit where loop4 forms a capping flap and covers the active site. The structural comparison of the three forms of BPSL2418 indicates that the catalytically important cysteine is CYS109, where the resolving cysteine is CYS75, which forms a disulfide bond with CYS109. They also suggest that the third conserved cysteine in the active site, CYS85, which is located in a3, is a nonessential cysteine for the catalytic function but it may play a role in the binding of the substrate. The structural comparison of the three forms reveals that conformational changes appear in the active site particularly involving loop4 and CYS109 during catalysis. The 3D structure of BPSL2418 shows strong structure similarity to fRMsrs enzymes, which further suggests that BPSL2418 acts as a free Met-R-SO reductase and shares the catalytic mechanism of fRMsr family.
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Correia, Giovanna Riello Barbosa. "Efeito inibitório in vitro do composto farnesol frente ao biofilme de Burkholderia pseudomallei." reponame:Repositório Institucional da UFC, 2015. http://www.repositorio.ufc.br/handle/riufc/16552.
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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)
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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.