Dissertations / Theses on the topic 'Melioidosis'
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Brett, Paul James. "Immunotherapeutic strategies against melioidosis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34659.pdf.
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Cheng, Allen Cheuk-Seng, and allencheng@ozemail com au. "MELIOIDOSIS: EPIDEMIOLOGY, PATHOPHYSIOLOGY AND MANAGEMENT." Flinders University. Medicine, 2005. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20051121.141305.
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In under a century, melioidosis, the infection due to Burkholderia pseudomallei, has emerged from Whitmores series of glanders-like infections amongst the morphia addicts in Burma to a major cause of mortality in northeastern Thailand and northern Australia. Also endemic in other parts of south-east Asia, melioidosis may have varied presentations ranging from severe, overwhelming infection to chronic, low grade disease.
Observational evidence had suggested that granulocyte colony stimulating factor (G-CSF), a naturally occurring substance produced by the body in response to infection, may have been useful in reducing the high mortality associated with the more severe forms of this infection. Other observations linked the occurrence of this disease to various environmental factors, such as contamination of drinking water and the annual rainfall. This thesis explores and attempts to quantify these associations.
There are three parts to this thesis. In the first part, I reviewed the epidemiology and management of patients with melioidosis. The use of G-CSF and meropenem was associated with a fall in mortality, although other factors may have at least partially contributed to this effect.
In the second part, I progressed towards a clinical trial of G-CSF. There was no other evidence supporting the use of G-CSF in severe sepsis and ethical issues precluded a trial in Darwin. There was not evidence from laboratory models of G-CSF action in melioidosis to support the use of G-CSF in patients, although there remained some doubt regarding the applicability of such models to human disease. I examined clinical methods to identify patients at high risk of death from melioidosis. A simple scoring system based on clinical and laboratory parameters was developed and externally validated. However, clinical definitions of severe sepsis appeared to be better predictors of mortality. A clinical trial based on clinical definitions was commenced in Thailand.
In the final part, I explored the question of whether different strains or B. pseudomallei or different environmental conditions caused different patterns of infection. There was no evidence that strain types of this bacterium determine the pattern or severity of disease, but weather conditions appeared to influence the distribution of disease in northern Australia.
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Limmathurotsakul, Direk. "Title: Determinants of recurrent melioidosis." Thesis, Open University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491474.
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Recurrent melioidosis represents relapse following failure to eradicate bacteria responsible for the primary infection or re-infection with a new strain. The first results chapter (chapter 3) evaluates the proportion of recurrent melioidosis due to relapse versus re-infection. Isolates from the same patient with an identical genotype were considered as relapse and those with a different genotype as re-infection. Three quarters of recurrent cases were due to relapse and one quarter were due to re-infection. There are two ways in which this approach could be confounded. First, 're-infection' could actually represent relapse if primary infection was caused by simultaneous infection with multiple B. pseudol1lallei strains, followed by chance selection of different strains from the two episodes for genotyping. The chance of this mistake occurring is based on the rate of polyclonal B. pseudol1lallei infection. Chapter 4 describes the rate of polyclonal infection in a large group of unselected patients in northeast Thailand, which was very low (2/133 cases, 1.5%). Second, 'relapse' could actually represent reinfection in the event that re-infection was caused by a B. pseudol1lallei strain that was by chance identical to the primary strain. The probability of this happening is based on the degree of genetic diversity of B. pseudomallci in the environment. Chapter 5 demonstrates that the population of B. pscudol1lallei in even a small sampling site is extremely diverse. Thus, it is unlikely that the assessment of the causes of recurrent melioidosis contained significant errors due to polyclonal infection or low genetic diversity of the organism. Chapter 6 examines specific risk factors of relapse and reinfection. Duration and choices of antibiotics used for the primary episode were major determinants of relapse. Chapter 7 compares the clinical manifestations of relapse and re-infection and develops a simple scoring index to predict relapse or re-infection in patients presenting with recurrent melioidosis.
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Tsai, Chi-Chun Wirongrong Chierakul. "Rheumatological manifestations in melioidosis patients /." Abstract, 2006. http://mulinet3.li.mahidol.ac.th/thesis/2549/cd387/4838796.pdf.
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Gerstenmaier, Jan Frank Wirongrong Chierakul. "Pulmonary manifestations in melioidosis patients /." Abstract, 2006. http://mulinet3.li.mahidol.ac.th/thesis/2549/cd387/4838790.pdf.
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Thematic Paper (M.Sc. (Clinical Tropical Medicine))--Mahidol University, 2006.LICL has E-Thesis 0011 ; please contact computer services. LIRV has E-Thesis 0011 ; please contact circulation services.
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Leung, Beatrice Pui See. "Development of a DNA vaccine against melioidosis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0018/MQ55225.pdf.
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Kinoshita, Reimi E. "Epidemiology of melioidosis in an oceanarium: a clinical, environmental and molecular study." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B29757745.
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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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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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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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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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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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Koh, Gavin Christian Kia Wee. "The effect of glibenclamide on the pathogenesis of melioidosis." Thesis, University of Cambridge, 2012. https://www.repository.cam.ac.uk/handle/1810/242187.
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Melioidosis is an important cause of community-acquired sepsis, endemic to Southeast Asia and Northern Australia. Melioidosis is caused by the soil saprophyte, Burkholderia pseudomallei, a motile Gram-negative bacillus, and is associated with a mortality rate that approaches 50% in Northeast Thailand. The most important risk factor for melioidosis is diabetes mellitus, and two-thirds of all adult patients with melioidosis have diabetes as a risk factor. It has been noted previously, however, that patients with diabetes have lower mortality than patients without diabetes. In this dissertation, we look at a cohort of 1160 consecutive adult melioidosis patients presenting to Sappasithiprasong Hospital in Ubon Ratchathani, Thailand, 410 (35%) of whom were diagnosed with diabetes prior to admission. We confirmed previous findings that diabetes protected from mortality in melioidosis, but also found that this protective effect was confined to a smaller subset of patients (208 patients) who were treated with glibenclamide prior to admission. Patients with hyperglycaemia (but no diagnosis of diabetes prior to admission) had the same mortality rate as patients without diabetes. In vitro experiments found no inhibitory effect of glibenclamide on bacterial growth, and we therefore looked for evidence of an effect of glibenclamide on the host. We conducted a gene expression study of circulating blood leukocytes in melioidosis patients and compared them to uninfected controls. In this study, we found that glibenclamide was associated with an anti-inflammatory effect on the host response to melioidosis. To further elucidate a mechanism for the action of glibenclamide, we studied the effect of glibenclamide therapy in a mouse model of melioidosis and found that the effect of glibenclamide was specific to interleukin-1β secretion. This reduction in interleukin-1β secretion was associated with reduced cellular influx into the lungs as well as lower bacterial loads in blood, liver and spleen.
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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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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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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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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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TAYE, KISSI JIMMA. "VACCINE DEVELOPMENT AGAINST PLAGUE, GLANDERS AND MELIOIDOSIS IN THE FORMER SOVIET UNION IN COMPARSION TO THE CURRENT STATE OF GLOBAL KNOWLEDGE." Doctoral thesis, Universitätsbibliothek Leipzig, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-38336.
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The causative agents of plague (Y. pestis), glanders (B. mallei) and melioidosis (B. pseudomallei) are included in critical agents of bioterrorisim. They belong to the most intensively studied agents during cold war, specially in the former Soviet Union (FSU). Mostly what is known about these agents, particularly (Y. pestis ) is not available in English language publications. Many of the studies are written in Russian language and published in Russian scientific journals. Thus, the work is designed to evaluate, published and unpublished Russian language written data obtained, in comparisions to the current state of global knowledge on the pathogens in concern.
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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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Allwood, Elizabeth May. "Identification and Characterisation of Antigenic Proteins from Burkholderia pseudomallei and their Efficacy as Diagnostic Targets and Vaccine Candidates for Melioidosis." Thesis, Griffith University, 2009. http://hdl.handle.net/10072/366480.
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Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 hours hours after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days, which is often too late for successful management of acute disease. This delayed diagnosis is amajor contributing factor to high mortality rates, and rapid diagnosis is therefore vital for successful management of the disease. Importantly, B. pseudomallei has recently been classified as a category B bioterrorism agent by the United States Centres for Disease Control and Prevention. It is believed to have the potential to be used as a biological weapon in view of its efficient ability for aerosol dispersal, it can be readily inhaled or ingested, it can be rapidly fatal or form chronic disease and due to the fact that no vaccine exists. The issues described above highlight the need for improvement in the rapid diagnosis of melioidosis and for the production of a suitable vaccine.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Rolim, Dionne Bezerra. "Estudo epidemiolÃgico do primeiro surto de melioidose no Brasil." Universidade Federal do CearÃ, 2004. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=10691.
Full textAbstract:
O primeiro surto de melioidose no Brasil ocorreu quando quatro adolescentes
apresentaram forma grave da doenÃa, com pneumonia e sepse, numa pequena comunidade
rural do Nordeste do Brasil, em fevereiro de 2003. Burkolderia pseudomallei foi identificada,
em cultura de sangue em um caso, inicialmente por mÃtodos convencionais e confirmado por
amplificaÃÃo de Ãcido nuclÃico (PCR). O diagnÃstico foi presumÃvel nos outros trÃs casos. A
investigaÃÃo epidemiolÃgica preliminar identificou os possÃveis fatores contribuintes: inÃcio
dos sintomas durante perÃodo chuvoso,exposiÃÃo dos casos a solo e Ãgua em atividades de
lazer, presenÃa de barragem de um rio prÃximo ao domicÃlio, Ãrea ao redor do domicÃlio
exposta a grande chuva dois dias antes dos sintomas e casa com piso de terra. O fato das
crianÃas adoecerem no mesmo dia aponta para uma possÃvel exposiÃÃo comum e simultÃnea.
A barragem foi identificada como o local mais provÃvel da contaminaÃÃo e o mecanismo de
transmissÃo possivelmente pode ter ocorrido por aspiraÃÃo, inalaÃÃo ou ingestÃo de Ãgua da
barragem. O recente surto levanta o questionamento sobre a existÃncia da doenÃa no Brasil e
mostra a necessidade de estudo ambiental detalhado para compreender sua ocorrÃncia.The first melioidosis outbreak in Brazil happened when four adolescents presented a severe case of the disease, with pneumonia and sepsis, in a small rural community in the Northeast of Brazil, in February 2003. Burkolderia pseudomallei were identified through blood culture in one case, firstly using conventional methods and then confirmed by PCR. The diagnosis was presumptive in the other three cases. The preliminary epidemiological investigation identified the possible contributing factors: onset of the symptoms during the rainy season, exposure of the cases to the soil and water in leisure activities, presence of a river dam close to the residence, the area around the house had been exposed to heavy rain two days before the symptoms, and the house had dirt floor. The fact that the children got sick on the same day, points to a possible common and simultaneous exposure. The dam was identified as the most probable place of contamination, and the transmission mechanism might have happened through aspiration, inhalation or ingestion of water from the dam. The recent outbreak rises the important questioning about the existence of the disease in Brazil, and shows the need of a detailed environmental study to understand its occurrence.
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Podnecky, Nicole L., Katherine A. Rhodes, Takehiko Mima, Heather R. Drew, Sunisa Chirakul, Vanaporn Wuthiekanun, James M. Schupp, et al. "Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei: Deviation from the Norm." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/626453.
Full textAbstract:
The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus cotrimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to cotrimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Cotrimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.
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Bandeira, Tereza de Jesus Pinheiro Gomes. "CaracterizaÃÃo fenotÃpica e genotÃpica, sensibilidade a antimicrobianos e detecÃÃo de gene de virulÃncia de cepas clÃnicas e ambientais de Burkholderia pseudomallei." Universidade Federal do CearÃ, 2011. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=7112.
Full textAbstract:
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoGrupo DASA
Melioidose à uma doenÃa infecciosa grave causada por Burkholderia pseudomallei, um bacilo Gram-negativo encontrado no solo e na Ãgua. A doenÃa à endÃmica no sudeste asiÃtico e hiperendÃmica no norte da AustrÃlia, onde a letalidade permanece com uma taxa de 21%. No Brasil, à considerada uma doenÃa emergente desde marÃo de 2003. Nos Ãltimos oito anos, 12 casos ocorreram no Estado do Cearà e um notificado pelo Governo holandÃs, por se tratar de um turista que morreu de melioidose, apÃs visita ao CearÃ. Em razÃo da ocorrÃncia clÃnica de melioidose e do isolamento de B. pseudomallei no ambiente do Estado do CearÃ, este trabalho objetivou estudar as cepas clÃnicas e ambientais de B. pseudomallei isoladas no Estado no perÃodo de 2003 a 2011, visando a identificar as cepas por mÃtodos fenotÃpicos e moleculares, determinar o perfil de sensibilidade contra cinco agentes antimicrobianos (amoxicilina/clavulanato, ceftazidima, imipenem, doxicilina e sulfametoxazol/trimetoprim), realizar a genotipagem das cepas pela amplificaÃÃo aleatÃria de DNA polimÃrfico - Random Amplified Polymorphic DNA (RAPD), detectar o gene de virulÃncia Type Three Secretion System (TTSS), alÃm de avaliar os aspectos clÃnico-epidemiolÃgicos que caracterizaram a emergÃncia desta doenÃa no Brasil. Todas as 20 cepas (dez clÃnicas e dez ambientais) de B. pseudomallei foram precisamente identificadas tanto pela metodologia VITEK2 quanto pelo sequenciamento da regiÃo 16S do DNA, mostraram resultado negativo no teste de assimilaÃÃo de L-arabinose, e exibiram-se positivas para a detecÃÃo do gene de virulÃncia TTSS. As concentraÃÃes inibitÃrias mÃnimas (CIMs), obtidas por microdiluiÃÃo em caldo MÃeller-Hinton, demonstraram que todos os isolados (100%) foram sensÃveis ao imipenem, à doxicilina e ao sulfametoxazol- trimetoprim, no entanto, para amoxicilina/clavulanato e ceftazidima, a sensibilidade foi de 80 e 90%, respectivamente. A tÃcnica de RAPD evidenciou uma variabilidade genÃtica de 63% entre as cepas de B. pseudomallei oriundas do Estado do CearÃ, as quais foram agrupadas em trÃs clusters diferentes. Este trabalho decerto contribuirà para o conhecimento das caracterÃsticas fenotÃpicas e genotÃpicas das cepas de B. pseudomallei isoladas no Cearà e da atualizaÃÃo da vigilÃncia epidemiolÃgica dos casos de melioidose ocorridos no Estado, alÃm de contribuir para a conscientizaÃÃo dos ÃrgÃos de saÃde competentes para a inclusÃo do Cearà como zona endÃmica para esta enfermidade.
Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, a Gram negative rod, commonly found in soil and water. The disease is endemic in Southeastern Asia and hyperendemic in Northern Australia. Despite the initiation of empiric therapy, mortality remains at 21% in patients with melioidosis in Australia. In Brazil, it is considered an emerging disease, since April 2003, when it was first diagnosed in Ceara, Northeastern Brazil. In the last eight years, thirteen cases were reported, twelve local cases and one case reported by the Dutch government because of a tourist who died of melioidosis after a visit to Ceara. Considering the occurrence of melioidosis in CearÃ, this work aimed at studying these clinical and environmental strains of Burkholderia pseudomallei isolated from Cearà from 2003 to 2011, focusing on the bacterial and molecular identification; determining the susceptibility profile against five antimicrobial agents (amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline and trimethoprim/sulfamethoxazole); genotyping through Random Amplified Polymorphic DNA (RAPD), detecting the virulence gene Type Three Secretion System (TTSS); and analyzing epidemiological and clinical aspects that characterized the emergence of this disease in Brazil. All 20 strains (10 clinical and 10 environment) from B. pseudomallei were accurately identified by both VITEK2  and sequencing of the 16S DNA, showed to be negative for the assimilation of L-arabinose and were positive results for the detection of the virulence gene TTSS. The minimum inhibitory concentrations (MICs) obtained through microdilution in MÃeller-Hinton broth, showed that all (100%) isolates were sensitive to imipenem, doxycycline and trimethoprim-sulfamethoxazole, however, the susceptibility rate to amoxicillin/clavulanate and ceftazidime was of 80 and 90%, respectively, with no differences between clinical and environmental strains. RAPD-PCR showed a genetic relatedness of 63% among the B. pseudomallei strains from the State of CearÃ, which were grouped in two different clusters. This work will contribute to the knowledge of phenotypic and genotypic characteristics of B. pseudomallei strains isolated in Cearà and the update of epidemiological surveillance of melioidosis cases in the state, also contribute to the awareness of agencies health authority for inclusion of Cearà State as an endemic area for this disease.
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Bayliss, Marc Ashley. "Virus-like particles as a novel platform for delivery of protective Burkholderia antigens." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/25577.
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A thesis by Marc Ashley Bayliss entitled ‘Virus-like particles as a novel platform for delivery of protective Burkholderia antigens’ and submitted to the University of Exeter for the degree of Doctor of Philosophy. There is currently no licensed vaccine available for the global tropical pathogen Burkholderia pseudomallei which is the causative agent of melioidosis and a potential bio-threat agent. The capsule polysaccharide (CPS) expressed by B. pseudomallei has been shown to offer some protection against bacterial challenge. Polysaccharide immunogenicity can be enhanced by conjugation to a carrier protein and several licensed vaccines utilise this technology. Virus-like particles (VLPs) are non-infectious, non-replicating, viral proteins that self-assemble into viral structures and are in several licensed vaccines as primary antigens. VLPs are also effective delivery platforms for foreign antigens by genetic insertion or chemical conjugation. iQur, a collaborator on this project, has developed Tandem CoreTM that consists of two genetically linked hepatitis B core proteins that allow insertion of large proteins into each core whilst remaining assembly competent. The aim of this thesis was to assess the protective efficacy of Tandem CoreTM VLPs chemically conjugated to CPS and Tandem CoreTM Burkholderia protein fusion constructs. This involved three objectives; reduce the cost of CPS extraction; identify immunogenic Burkholderia proteins; and test candidate vaccine efficacy in an animal model of acute melioidosis against B. pseudomallei challenge. To reduce the cost of extraction, CPS was purified from B. thailandensis strain E555 and bacterial culture CPS concentration optimised which first required development of a quantitative ELISA. Immunogenic Burkholderia proteins were identified from the literature but Tandem CoreTM fusion constructs containing these proteins were not assembly competent. The Burkholderia proteins were added as co-antigens to the VLP CPS conjugate vaccine but did not improve efficacy. Tandem CoreTM VLPs conjugated to CPS were protective against B. pseudomallei challenge and were compared to CPS conjugated to Crm197: a commercially available carrier protein used in several licensed vaccines. At lower challenge doses, survival was greater in mice vaccinated with the VLP-CPS conjugate although at higher doses, Crm197-CPS efficacy was greater.
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Blam, Annette J. "Effects of Diabetic State and Gender on Pro-Inflammatory Cytokine Secretion by Human Macrophages Infected with Burkholderia pseudomallei." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2646.
Full textAbstract:
Burkholderia pseudomallei is a gram-negative opportunistic soil pathogen that causes the life-threatening disease melioidosis. It is endemic in Northern Australia and Southeast Asia but can be found throughout many other regions in the world. Diabetes mellitus is a predisposing risk factor for infection with this organism and it has been demonstrated that diabetic males are particularly susceptible to severe infection. Previous research suggested that monocytes isolated from the whole blood of diabetic males demonstrated a decreased ability to produce the proinflammatory cytokines IL-1β and IL-8. We hypothesized that monocyte-derived macrophages from diabetic males would also secrete lower levels of pro-inflammatory cytokines and that this difference between gender and diabetic state would be more pronounced compared to those seen previously with monocytes. Twenty volunteer with type I diabetes mellitus (ten males and ten females), along with twenty healthy age- and gender-matched controls donated blood for this study. Monocytes were collected from whole blood and allowed to differentiate into macrophages with the use of human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Macrophages were then divided into groups and infected with B. pseudomallei, B. thailandensis (a closely related by non-pathogenic bacterium that inhabits similar niches), and E. coli. An uninfected control was used as well. At six hours post-infection, mRNA was collected from all cells and qPCR was performed to determine cytokine expression levels. All mRNA values collected from cells which had been infected with bacterial agents were normalized against the corresponding concentrations of mRNA from mock-infected cells. Mean log fold increases in both IL-1β and IL-8 were computed and compared. Preliminary testing showed decreased levels of both IL-1β and IL-8 from B. pseudomallei-infected macrophages isolated from a diabetic male compared to the healthy, age-matched male control. Surprisingly, results from all forty donors demonstrated that gender and diabetic state were not significant factors in the proinflammatory responses of macrophages infected with B. pseudomallei, although further testing is needed to determine if these results were influenced by experimental parameters.
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Barker, Samuel Peter. "Early stage drug discovery screening for novel compounds active against the persister phenotype in Burkholderia thailandensis." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/26236.
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Many pathogenic microorganisms are believed to stochastically switch into low metabolic states that display resistance to supra-lethal levels of antibiotics. These so-called “persister” cells have been associated with recurrent infections and the development of antibiotic resistance. Whilst a compound that eliminates Staphylococcus aureus persister cells has been described, it is not active against Gram-negative bacteria. The aim of my PhD project was to develop a high-throughput assay for compounds that eradicate persister cells in the -proteobacterium Burkholderia thailandensis. Further to this, I aimed to develop “hit” compounds from screening into lead series through investigation of structure activity relationships and, use a chemical genetics approach to elucidate potential mechanisms of action. I developed a phenotypic assay to identify compounds that eradicate persister cells. The assay was based on the reduction of the resazurin based dye PrestoBlue. Optimization of the assay gave a Z’ prime of 0.41 when screened in high throughput at the DDU. Screening of the library of 61,250 compounds identified 2,127 compounds that gave a statistically significant reduction in persister cell numbers. Follow-up assays highlighted 29 compounds with a pIC50 greater than five. Detailed investigation allowed me to down select to six “best in class” compounds, which included the licensed drug chloroxine. A time dependent killing assay showed that chloroxine reduced levels of persister cells by three orders of magnitude over 72 hours (P = 0.01). Hit expansion around chloroxine using commercially available compounds did not identify any more potent compounds, but did highlight key features of the molecule for activity. Assay protocols were provided to collaborators at DSTL who were able to iv show that chloroxine is also active against persister cells formed by the tropical pathogen and Tier 1 biological agent Burkholderia pseudomallei. Investigations into the mechanism of action of chloroxine used Next Generation Sequencing of an over expression library, identifying two putative genes involved in inhibition of persister cells by chloroxine. My findings demonstrate a phenotypic assay against persister cells in Gram-negative bacteria, which has the power to identify potent anti-persister agents to assist in chemotherapy. Structural activity relationship and mechanism of action investigations have indicated lead series and genetic starting points for future development of this research. My PhD project has concluded with sufficient data for continuation of research following a number of leads and is at an ideal stage for instigation of a medicinal chemistry program for development of chloroxine as a clinical option for treatment of persistent melioidosis.
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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.
Full textAbstract:
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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Petit, Guillaume Axel. "Structure, Function and Inhibition of Bacterial Oxidoreductases." Thesis, Griffith University, 2021. http://hdl.handle.net/10072/407569.
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The disulfide bond forming proteins are a large and diverse family of proteins found in the periplasm of bacteria. They are involved in the maturation process, oxidation and isomerisation of disulfide bonds in their substrate periplasmic proteins, many of which are involved in pathogenicity and virulence pathways. Disulfide bond forming proteins are highly conserved yet do not appear to be critical for the survival of the bacteria under optimal conditions. Altogether these properties make them an ideal target to combat bacterial infections, with hopes and aims to reduce virulence of the bacteria while minimising selective pressure. This strategy is hypothesised to reduce the rate of resistance development compared to antibiotic and antimicrobial treatments. This thesis focuses specifically on the disulfide bond forming proteins A and B from Burkholderia pseudomallei, the bacterium causing the deadly tropical disease melioidosis.
Chapter 2 focuses on screening a small natural products library using an assay reporting on the activity of the purified B. pseudomallei disulfide bond forming proteins A and B. Three natural products including two stilbenoids, (-)-hopeaphenol and vaticanol B, and an alkaloid, spermatinamine, were found to moderately inhibit the disulfide bond forming protein A and B redox reaction. Additionally, the stilbenoids were shown to bind to the disulfide bond forming protein A of B. pseudomallei.
In chapter 3, X-ray crystallography was used to identify and characterise two druglike fragments from the Monash Institute for Pharmaceutical Sciences found to interact with B. pseudomallei disulfide bond forming protein A. These fragments were shown to bind a transient feature present only on the surface of the oxidised protein.
In chapter 4, efforts focused on crystallising the complex of B. pseudomallei disulfide bond forming protein A with its membrane protein partner - disulfide bond forming protein B. Small weakly diffracting crystals of this complex were obtained using a technique for membrane protein crystallisation called lipidic cubic phase.
Chapter 5 returns to B. pseudomallei disulfide bond forming protein A, with the objective to identify virulence-associated substrates. Having a better idea of what these substrates are, could for example, help identify antigenic epitopes for vaccine preparation or find reporters reliant on the activity of the disulfide bond forming proteins in phenotypic assays. In this research chapter, bioinformatic methods were used to filter and identify proteins suspected to be substrates of the B. pseudomallei disulfide bond forming protein A.
Disulfide bond forming proteins have been extensively studied over the past 30 years, however they are not the only proteins involved in oxidoreduction reactions within the periplasm of bacteria. Other proteins, such as the suppressor of copper sensitivity proteins, have miscellaneous functions, including copper sequestration, disulfide bond oxidation and isomerisation. These proteins share structural similarities with the disulfide bond forming proteins, especially around the active site motif, however they differ in the way they oligomerise. Since only a few members of the suppressor of copper sensitivity protein family have been characterised to date, it is not entirely clear how their functions are dependent on their oligomerisation state. The last research chapter of this thesis, chapter 6, investigated the suppressor of copper sensitivity protein C from the organism Caulobacter crescentus. Biochemical characterisation indicated that this protein has a strong affinity for copper (I) and that it was also able to isomerise incorrectly formed disulfide bonds. Finally a crystal structure of the protein was obtained indicating trimerisation via a long α-helical Nterminal domain.
In summary the work presented in this thesis investigated the structure, function and inhibition of bacterial oxidoreductases, specifically the disulfide bond forming proteins A and B from B. pseudomallei and the suppressor of copper sensitivity protein C from C. crescentus.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Mulye, Minal. "Delineating the Immune Mechanisms Required by Murine Neutrophils and Macrophages for Clearance of Burkholderia pseudomallei, the Causative Agent of Melioidosis." University of Toledo Health Science Campus / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1385032180.
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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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Dickey, Laura L. "An Infection Model for Examining the Effects of Gender and Diabetic State on Proinflammatory Cytokine Secretion by Phagocytic Cells in Response to Infection with Burkholderia pseudomallei." Diss., CLICK HERE for online access, 2007. http://contentdm.lib.byu.edu/ETD/image/etd1825.pdf.
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Schnetterle, Marine. "Résistance acquise chez les Burkholderia pseudomallei : analyse de l'expression de l'efflux et de son inhibition." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0710.
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Burkholderia pseudomallei est l’agent causal de la mélioïdose, une maladie tropicale endémique dans le Nord de l’Australie et en Asie du Sud-Est. Nous avons analysé le système d’efflux, mécanisme majoritairement impliqué la multi-résistance aux antibiotiques. Nous avons chercher à identifier des mutations dans les pompes d’efflux et des modulation de l’expression de ces dernières afin d’expliquer ces phénotypes de résistance. Les techniques de séquençage de l’ADN et de transcriptomique par RT-qPCR nous ont permis d’identifier deux mécanismes chez des souches cliniques. Un mécanisme transitoire responsable d'une résistance croisée du Cotrimoxazole, avec les quinolones, et le chloramphénicol, pour lequel nous suspectons une modulation de l’expression de l’efflux. Le second, impliqué dans la résistance au méropénème, par surexpression de l'efflux suite à une mutation dans le régulateur de la pompe AmrAB-OprA.Dans un second axe de recherche, nous avons également criblé plusieurs molécules afin d'identifier des candidats inhibiteurs de l'efflux, dérivant de la famille des phénothiazines et capables de restaurer une sensibilité aux antibiotiques. Nous avons analysé l’impact de ces molécules sur des souches modèles de multi-résistance (Burkholderia thailandensis) et sur des souches cliniques et environnementales de B. pseudomallei. Ces molécules sont capables d’impacter l’expression de l’efflux, mais nous pensons que le mécanisme majeur d’inhibition de cette famille de molécules reste l’entrée en compétition avec les antibiotiques efflués. Nous avons identifié une molécule, AST17, capable de restaurer la sensibilité au Cotrimoxazole, ainsi qu'aux quinolonesBurkholderia pseudomallei is thecausal agent of melioidosis, a tropical disease, endemic in Notrhern Australia and South-East Asia. We have analyzed efflux systeme, known to be one of the main mecanism implicated in antibiotic resistance phenotypes. We have looked for mutations in efflux pumps and for transient modulations of the efflux pumps expression, that could explain resistance phenotypes. Whole genome sequencing and a the targeted method of RT-qPCR allowed us to identified two mecanisms in clinical strains. A transient mecanism, responsible of a cross-resistance to Cotrimoxazole, quinolones and chloramphenicol, and we suspect an implication of modulation of efflux. The second one is implicated in meropenem resistance by an overexpression of the AmrAB-OprA efflux pumps, due to a mutation of its regulator. In a second time, we also have screened several compounds, all derivated from phenothiazines, in order to identify efflux pump inhibitors for a restoration of the antibiotic susceptibility. We have analyzed the impact of these molecules in multi-resistant strain models, and on several clinical and environnemental strains. These molecules are able to modulate efflux pumps expression, however, we think that the main inhibition mecanism of these derivatives is about a competition between the molecule and the antibiotics. We have identified one molecule, AST17, that is able to restore Cotrimoxazole and quinolones susceptibilities
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Telionis, Pyrros A. "Novel Applications of Geospatial Analysis in the Modeling of Infectious Diseases." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/89432.
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At the intersection of geography and public health, the field of spatial epidemiology seeks to use the tools of geospatial analysis to answer questions about disease. In this work we explore two areas: the use of geostatistical modeling as an extension of niche modeling, and the use of mobility metrics to augment modeling for epidemic responses.
Niche modeling refers to the practice of using statistical methods to relate the underlying spatially distributed environmental variables to an outcome, typically presence or absence of a species. Such work is common in disease ecology, and often focuses on exploring the range of a disease vector or pathogen. The technique also allows one to explore the importance of each underlying regressor, and the effect it has on the outcome. We demonstrate that this concept can be extended, through geostatistical modeling, to explore non-logistic phenomena such as incidence. When combined with weather forecasts, such efforts can even predict incidence of an upcoming season, allowing us to estimate the total number of expected cases, and where we would expect to find them. We demonstrate this in Chapter 2, by forecasting the incidence of melioidosis in Australia given weather forecasts a year prior. We also evaluate the efficacy of this technique and explore the impact of environmental variables such as elevation on melioidosis.
But these techniques are not limited to free-living and vector-borne pathogens. We theorize that they can also be applied to diseases that spread exclusively by person-to-person contact. Exploring this allows us to find areas of underreporting, as well as areas with unusual local forcing which might merit further investigation by the health department. We also explore this in Chapter 4, by relating the incidence of hepatitis C in rural Virginia to demographic data.
The West African Ebola Outbreak of 2014 demonstrated the need to include mobility in predictive disease modeling. One can no longer assume that neglected tropical diseases will remain contained and immobile, and the assumption of random mixing across large areas is unwise. Our efforts with modeling mobility are twofold. In Chapter 3, we demonstrate the creation of mobility metrics from open source road and river network data. We then demonstrate the usefulness of such data in a meta-population patch model meant to forecast the spread of Ebola in the Democratic Republic of Congo. In Chapter 4, we also demonstrate that mobility data can be used to strengthen outbreak detection via hotspot analysis, and to augment incidence models by factoring in the incidence rates of neighboring areas. These efforts will allow health departments to more accurately forecast incidence, and more readily identify disease hotspots of atypical size and shape.Doctor of Philosophy
The focus of this work is called “spatial epidemiology”, which combines geography with public health, to answer the where, and why, of disease. This is a growing field, and you’ve likely seen it in the news and media. Have you ever seen a map of the United States turning red in some virus disaster movie? The real thing looks a lot like that. After the Ebola outbreak of 2014, public health agencies wanted to know where the next one might hit. Now that there is another outbreak, we need to ask where and how will it spread? What areas are hardest hit, and how bad is it going to get? We can answer all these questions with spatial epidemiology. Our work adds to two aspects of spatial epidemiology: niche modeling, and mobility. We use niche modeling to determine where we could find certain diseases, usually those that are spread by insects or animals. Consider Lyme disease, you get it from the bite of a tick, and the tick gets it from a white-footed mouse. But both the mice and ticks only live in certain parts of the country. With niche modeling we can determine where those are, and we can also guess at what makes those areas attractive to the mice and ticks. Is it winter harshness, summer temperatures, rainfall, and/or elevation? Is it something else? In Chapter 2, we show that you can extend this idea. Instead of just looking at where the disease is, what if we could guess how many people will get infected? What if we could do so, a year in advance? We show that this can be done, but we need a good idea of what the weather will be like next year. In Chapter 4, we show that you can do the same thing with hepatitis C. Instead of Lyme’s ticks and mice, hepatitis C depends on drug-use, unregulated tattooing, and unsafe sex. And like with Lyme, these things are only found in certain places. Instead of temperature or rainfall, we now need to find areas with drug-problems and poverty. But we can get an idea of this from the Census Bureau, and we can make a map of hepatitis C as easily as we did for Lyme. But hepatitis C spreads person-to-person. So, we need some idea of how people move around the area. This is where mobility comes in. Mobility is important for most public health work, from detecting outbreaks to estimating where the disease will spread next. In Chapter 3, we show how one could create a mobility model for a rural area where few maps exist. We also show how to use that model to guess where the next cases of Ebola will show up. In Chapter 4, we show how you could use mobility to improve outbreak and hotspot detection. We also show how it’s used to help estimate the number of cases in an area. Because that number depends on how many cases are imported from the surrounding areas. And the only way to estimate that is with mobility.
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Lima, Leoniti Dantas Queiroz. "Avaliação do diagnóstico laboratorial de melioidose no Ceará." Universidade de Fortaleza, 2017. http://dspace.unifor.br/handle/tede/104462.
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Previous issue date: 2017-01-30Melioidosis is an emerging highly lethal infectious disease caused by Burkolderia pseudomallei, whose worldwide distribution is expanding. Its detection at Brazil is underestimated, although the country is already considered endemic. Ceará presents 94% of the cases diagnosed at Brazil. Potentially mimicking and with laboratory confirmatory diagnosis, its detection is made difficult by the lack of knowledge of health professionals and adequate laboratory structure. This study analyzed the capacity diagnostic of melioidosis at the state of Ceará. The laboratory network of the city of Fortaleza was analyzed through structured questionnaires of the capacity diagnostics: identified microorganisms; specimens analyzed; transport and storage of sample; processing and identification of bacteria; waste disposal; registration and notification of data. Another stage analyzed the diagnosis of confirmed cases of the disease in the period of 2003 to 2016: general characteristics of the cases, diagnostic criteria, diagnostic method used, time of diagnosis, clinical aspects of the cases, laboratories and hospitals in the occurrence of clinical cases. The study showed that only four of the nine laboratories evaluated have identified B. pseudomallei. The VITEK 2 automated system that has bacterial detection capability is used by seven laboratories. There is no attempt to reisolate the bacteria on suspected contamination plaques in seven laboratories, which may hinder the routine identification of B. pseudomallei. Twenty nine cases of melioidosis were diagnosed between 2003 and 2015. The microbiological diagnosis was made in 86% (25) of clinical cases and 14% (4) according to clinical-epidemiological criteria. The VITEK 2 automated method was responsible for 82% (22) of the bacterial identification. Fifty two percent of the cases diagnosed in Ceará between years 2003 to 2016 occurred after death. The State of Ceará doesn¿t have adequate diagnostic capacity to detect melioidosis in laboratories. It is necessary to improve the laboratory structure and develop strategies that facility the knowledge of the disease by health professionals, especially physicians and microbiologists. Keywords: Burkholderia pseudomallei; melioidosis; diagnosis.
Melioidose é uma doença infecciosa emergente e de elevada letalidade, causada pela Burkolderia pseudomallei cuja distribuição mundial encontra-se em expansão. Sua detecção no Brasil é subestimada, embora o país já seja considerado endêmico. O Ceará apresenta 94% dos casos diagnosticados no Brasil. Potencialmente mimetizadora e com diagnóstico confirmatório laboratorial, sua detecção é dificultada pela falta de conhecimentos dos profissionais de saúde e de estrutura laboratorial adequada. Esse estudo analisou a capacidade de diagnóstico da melioidose no Ceará. A rede laboratorial da cidade de Fortaleza foi analisada através de questionários estruturados sobre a capacidade de diagnóstico: microrganismos identificados; espécimes analisados; transporte e armazenamento de amostra; processamento e identificação de bactérias; descarte de cepas; registro e notificação de dados. Outra etapa analisou o diagnóstico dos casos confirmados da doença entre o período de 2003 a 2016: características gerais dos casos, critério diagnóstico, método diagnóstico utilizado, tempo do diagnóstico, aspectos clínicos dos casos, laboratórios e hospitais de ocorrência dos casos clínicos. O estudo evidenciou que somente quatro dos nove laboratórios avaliados já identificaram a B. pseudomallei. O sistema automatizado VITEK 2, que possui capacidade de detecção da bactéria, é utilizado por sete laboratórios. Não há tentativa de reisolamento de bactérias em placas suspeitas de contaminação em sete laboratórios, o que pode dificultar a identificação rotineira de B. pseudomallei. Vinte e nove casos de melioidose foram diagnosticados entre o período de 2003 e 2015. O diagnóstico microbiológico ocorreu em 86% (25) dos casos clínicos e 14% (4) por critério clínico-epidemiológico. O método automatizado VITEK 2 foi responsável por 82% (22) da identificação da bactéria. Cinquenta e dois por cento dos casos diagnosticados no Ceará, entre os anos de 2003 e 2016, ocorreram após o óbito. O Estado do Ceará não apresenta capacidade diagnóstica adequada para detectar melioidose em seus laboratórios. Há necessidade de melhorar a estrutura laboratorial e desenvolver estratégias que facilitem o conhecimento da doença pelos profissionais de saúde, especialmente médicos e microbiologistas. Palavras-chave: Burkholderia pseudomallei; melioidose; diagnóstico.
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Neto, José Baptista de Sant'Ana. "Perfil diferencial sérico de paciente em tratamento para melioidose : um estudo de caso." Universidade de Fortaleza, 2018. http://dspace.unifor.br/handle/tede/108159.
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Previous issue date: 2018-01-31Melioidosis is a non-contagious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei, with a greater number of cases occurring mainly in Northeast of Thailand and North of Australia, already in Brazil it permeates all the State of Ceará although its extension is not known with precision in the country. It can evolve asymptomatically, acutely with sepsis until chronification. It mimics numerous other diseases, which due to lack of clinical and laboratory knowledge are erroneously identified. In view of its high morbidity and mortality aggravated when it occurs with risk factors such as diabetes mellitus, which is especially potentiating the disease, the need for an efficient diagnosis is necessary, where time is the main obstacle, given the delay in generating - a result is obtained by the commonly used assays. Being the mass spectrometry and its other approaches, tool of improvement of the results, having great prominence that refers to the sensitivity, precision and speed of analysis, allowing the identification of thousands of proteins, through the analysis of qualitative - quantitative differential expression of protein profiles in different samples. Therefore, from this scenario, the elaboration of this study is justified when it is necessary to add another analytical method, which improves the laboratory diagnosis and consequently broadens the research options, which aim to subsidize clinical management, in the form of a characteristic protein profile of the disease, supporting better approaches to allow the correct and early diagnosis of melioidosis. The cross-sectional study led to a protein profile from a clinical serum sample, confirmed for melioidosis, obtained at different moments of the pharmacotherapeutic follow-up, elucidating a possible protein panel for this infection, through a differential analysis of the proteins expressed through the technique ESI-TOF/MS. Differentially expressed acute-chronic phase proteins were detected. It was concluded that the technique used here was useful to detect a protein profile of interest in the clinical course, aiming to reduce the response time to the therapeutic approach, however, more studies are needed as more patients to investigate other expressed proteins, as well as from the perspective of the pathogen, further expanding knowledge and improving databases that involve melioidosis. Keywords: Melioidosis. Biomarkers. Mass Spectrometry.
A melioidose é uma doença infecciosa não contagiosa causada pela bactéria Gram-negativa Burkholderia pseudomallei, com maior número de casos ocorrendo principalmente no Nordeste da Tailândia e Norte da Austrália. Já no Brasil permeia todo o estado do Ceará, embora não se saiba com precisão sua extensão no país. Pode evoluir de forma assintomática, aguda, comumente com sepse, até a cronificação. Mimetiza inúmeras outras doenças,que, por falta de conhecimento clínico-laboratorial, são erroneamente identificadas. Diante de sua alta morbidade e mortalidade, agravadas quando ocorrida com fatores de risco, como diabetes mellitus, principal potencializador da doença, a necessidade de uma diagnose eficientes e faz necessária, em que o tempo é o principal obstáculo, haja vista a demora de gerar-se um resultado através dos ensaios comumente utilizados. A espectrometria de massas e suas demais abordagens são ferramentas de aperfeiçoamento dos resultados, destacando-se o que se refere à sensibilidade, precisão e velocidade de análise. Essa técnica permite a identificação de milhares de proteínas, por meio da análise da expressão diferencial qualitativo-quantitativa dos perfis proteicos em diferentes amostras. Portanto, a partir desse cenário, a elaboração deste estudo se justifica pela necessidade de adicionar mais um método analítico, que melhore a diagnose laboratorial e, consequentemente, amplie as opções de investigação, que visam subsidiar o manejo clínico, na forma de um perfil proteico característico da doença, suportandomelhores abordagens para permitir o diagnóstico correto e precoce da melioidose. O estudo transversal suscitou em um perfil proteico, oriundo de uma amostra clínica de soro, confirmada para melioidose, obtida em momentos distintos do seguimento farmacoterapêutico, elucidando possível painel proteico para essa infecção, através de análise diferencial das proteínas expressas por meio da técnica de ESI-TOF/MS. Foram detectadasproteínas de fase agudo-crônicadiferencialmente expressas. Conclui-se, então, que a técnica aqui utilizada foi útil na detecção de perfil proteico de interesse para conduta clínica, visando reduzir o tempo de resposta à abordagem terapêutica. Entretanto, mais estudos são necessários, commaior número de pacientes, para investigar outras proteínas expressas, comotambém, pela perspectiva do patógeno, para que se possa expandir ainda mais o conhecimento e melhoraros bancos de dados que envolvem melioidose. Palavras-chaves: Melioidose. Biomarcadores. Espectrômetro de Massas.
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Rolim, Dionne Bezerra. "Estudo epidemiológico do primeiro surto de melioidose no Brasil." reponame:Repositório Institucional da UFC, 2004. http://www.repositorio.ufc.br/handle/riufc/6947.
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ROLIM, Dionne Bezerra. Estudo epidemiológico do primeiro surto de melioidose do Brasil. 2004. 82 f. Dissertação (Mestrado em Saúde Pública) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2004.Submitted by denise santos (denise.santos@ufc.br) on 2013-12-16T12:59:23Z No. of bitstreams: 1 2004_dis_dbrolim.pdf: 1239463 bytes, checksum: 739b80472e10a04a7fc8bbce9f58ba91 (MD5)
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The first melioidosis outbreak in Brazil happened when four adolescents presented a severe case of the disease, with pneumonia and sepsis, in a small rural community in the Northeast of Brazil, in February 2003. Burkolderia pseudomallei were identified through blood culture in one case, firstly using conventional methods and then confirmed by PCR. The diagnosis was presumptive in the other three cases. The preliminary epidemiological investigation identified the possible contributing factors: onset of the symptoms during the rainy season, exposure of the cases to the soil and water in leisure activities, presence of a river dam close to the residence, the area around the house had been exposed to heavy rain two days before the symptoms, and the house had dirt floor. The fact that the children got sick on the same day, points to a possible common and simultaneous exposure. The dam was identified as the most probable place of contamination, and the transmission mechanism might have happened through aspiration, inhalation or ingestion of water from the dam. The recent outbreak rises the important questioning about the existence of the disease in Brazil, and shows the need of a detailed environmental study to understand its occurrence.
O primeiro surto de melioidose no Brasil ocorreu quando quatro adolescentes apresentaram forma grave da doença, com pneumonia e sepse, numa pequena comunidade rural do Nordeste do Brasil, em fevereiro de 2003. Burkolderia pseudomallei foi identificada, em cultura de sangue em um caso, inicialmente por métodos convencionais e confirmado por amplificação de ácido nucléico (PCR). O diagnóstico foi presumível nos outros três casos. A investigação epidemiológica preliminar identificou os possíveis fatores contribuintes: início dos sintomas durante período chuvoso,exposição dos casos a solo e água em atividades de lazer, presença de barragem de um rio próximo ao domicílio, área ao redor do domicílio exposta a grande chuva dois dias antes dos sintomas e casa com piso de terra. O fato das crianças adoecerem no mesmo dia aponta para uma possível exposição comum e simultânea. A barragem foi identificada como o local mais provável da contaminação e o mecanismo de transmissão possivelmente pode ter ocorrido por aspiração, inalação ou ingestão de água da barragem. O recente surto levanta o questionamento sobre a existência da doença no Brasil e mostra a necessidade de estudo ambiental detalhado para compreender sua ocorrência.
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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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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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Vasconcelos, David Caldas. "Efeito da prometazina combinada aos antibióticos clássicos frente à forma planctônica e ao biofilme de Burkholderia pseudomallei." reponame:Repositório Institucional da UFC, 2015. http://www.repositorio.ufc.br/handle/riufc/21656.
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VASCONCELOS, D. C. Efeito da prometazina combinada aos antibióticos clássicos frente à forma planctônica e ao biofilme de Burkholderia pseudomallei. 2015. 84 f. Dissertação (Mestrado em Ciências Médicas) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015.Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-20T14:13:27Z No. of bitstreams: 1 2015_dis_dcvasconcelos.pdf: 2600778 bytes, checksum: 517035433406fc9bf380fa7e591e303e (MD5)
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Among the defense mechanisms employed by Burkholderia pseudomallei are the expression of efflux pumps such as BpeAB-OprB, AmrAB-OprA and BpeEF-OprC, which are responsible for resistance to aminoglycosides, macrolides, fluoroquinolones and sulfonamides. It is therefore necessary to find adjuvants able to minimize this resistance. In this context, the phenothiazines stand out for inhibiting those pumps. This study evaluated the in vitro inhibitory activity of promethazine alone or in combination with amoxicillin, amoxicillin/clavulanate, erythromycin, sulfamethoxazole/trimethoprim, ciprofloxacin or gentamicin against B. pseudomallei in planktonic and biofilm form. The structure of B. pseudomallei biofilm, with and without addition of promethazine, was also investigated. The sensitivity was evaluated by the broth microdilution test. The minimum inhibitory concentration (MIC) was 0.78 mg / mL and the minimum biofilm elimination concentration (MBEC) was 0.78 to 3.12 mg / mL for promethazine. Moreover, the association with promethazine significantly reduced the MIC values for erythromycin, trimethoprim / sulfamethoxazole, gentamicin and ciprofloxacin, whereas the MBEC values of all antibiotics tested significantly declined in combination with promethazine (p <0.05). Through electron and confocal microscopy, we found that promethazine was able to disrupt the biofilm matrix, possibly facilitating penetration of antibiotics. Therefore, this study demonstrates the inhibitory activity of promethazine against B. pseudomallei and its synergistic effect with traditional antibiotics against biofilms.
Dentre os mecanismos de resistência demonstrados por Burkholderia pseudomallei, há o destaque para expressão de bombas de efluxo do tipo BpeAB-OprB, AmrAB-OprA e BpeEFOprC, que são responsáveis pela resistência a aminoglicosídeos, macrolídeos, fluoroquinolonas e sulfonamidas. Torna-se assim necessária a busca por adjuvantes capazes de minimizar essa resistência. Neste contexto, destacam-se as fenotiazinas, que inibem tais bombas. O presente estudo visou avaliar a atividade inibitória in vitro da prometazina isolada e em combinação com amoxicilina, amoxicilina/clavulanato, eritromicina, sulfametoxazol/trimetoprim, ciprofloxacina e gentamicina frente à forma planctônica e ao biofilme de B. pseudomallei. Foi investigada ainda a estrutura do biofilme de B. pseudomallei, com e sem adição de prometazina. A concentração inibitória mínima (CIM) foi de 0,78 mg / mL e a concentração eliminatória mínima em biofilme (CEMB) foi de 0,78 a 3,12 mg/mL para prometazina. Ademais, a associação com prometazina reduziu significativamente os valores de CIM para eritromicina, sulfametoxazol/trimetoprim, gentamicina e ciprofloxacina, enquanto que os valores de CEMB para todos os antibióticos testados apresentaram diminuição significativa em combinação com prometazina (p<0.05). Por meio de técnicas de microscopia confocal e eletrônica, observou-se que a prometazina foi capaz de desestruturar a matriz do biofilme, possivelmente auxiliando a penetração dos antibióticos. Deste modo, o presente estudo mostrou a atividade inibitória da prometazina ante a B. pseudomallei e seu efeito sinérgico com antibióicos clássicos frente aos biofilmes.
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Couto, Manuela Soares. "Identificação molecular de cepas clínicas e ambientais de Burkholderia pseudomallei, oriundas do estado do Ceará : análise baseada nas regiões 16S e 16S-23S do DNA ribossômico nuclear." reponame:Repositório Institucional da UFC, 2009. http://www.repositorio.ufc.br/handle/riufc/6901.
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COUTO, Manuela Soares. Identificação molecular de cepas clínicas e ambientais de Burkholderia pseudomallei, oriundas do Estado do Ceará : análise baseada nas regiões 16S e 16S-23S do DNA ribossômico nuclear. 2099.0107 f. Dissertação (Mestrado em Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009.Submitted by denise santos (denise.santos@ufc.br) on 2013-12-05T14:02:29Z No. of bitstreams: 1 2009_dis_mscouto.pdf: 1672949 bytes, checksum: f161d01fb64b6c9b0e6980b0190093b1 (MD5)
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Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei, considered emerging in Brazil since the first cases were reported in 2003, on State of Ceará. This study aimed to perform the molecular identification of 31 isolates of B. pseudomallei (26 clinical and 5 environmental) maintained in the culture collection of CEMM (Specialized Center for Medical Mycology), based on sequences 16S and 16S-23S rRNA. The DNA of these samples was extracted with the kit Wizard ® Genomic DNA Purification (Promega), quantified by spectrophotometry and stored at 4°C. The amplification of a fragment of 302 bp of 16S-23S rRNA specific to B. pseudomallei was performed by PCR reaction with primers Bp1 and Bp4. The sequencing of 16S and 16S-23S rRNA was performed by using of the kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). The phylogenetic tree of 16S rRNA and the sequence identity matrix and sequence difference count matrix based on the 16S-23S rRNA were generated by the program MEGA4, version 4.1. The results confirmed the identification of 15 strains of B. pseudomallei (5 clinical and 10 environmental), which represents 48.4% of the isolates analyzed in this study. The phylogenetic tree based on 16S rRNA shows that the clinical and environmental isolates of B. pseudomallei of State of Ceará are evolutionarily clustered with the strains B. pseudomallei MSHR346 (Australia), B. pseudomallei 1106a (Thailand), B. pseudomallei K96243 (Thailand), B. pseudomallei 1710b (Thailand) and B. pseudomallei 668 (Australia). Using the same extraction kit was possible to extract DNA from B. pseudomallei directly from clinical specimen (bronchoalveolar lavage), confirming a new case of melioidosis in Ubajara/CE. In this study, the use of PCR for amplification of a fragment of 302 bp of 16S-23S rRNA identified correctly B. pseudomallei, and to confirm the discrimination between B. pseudomallei and B. mallei, the sequencing of the 16S and 16S-23S rRNA genes was performed. The technique of PCR coupled with sequencing of 16S and 16S-23S rRNA resulted in a high sensitivity and specificity of detection of B. pseudomallei in this study.
A melioidose é uma doença potencialmente fatal causada pela bactéria Burkholderia pseudomallei, sendo considerada emergente no Brasil desde que os primeiros casos foram reportados em 2003, no Estado do Ceará. Este estudo pretendeu realizar a identificação molecular de 31 isolados de B. pseudomallei (cinco clínicos e 26 ambientais) mantidos na coleção de culturas do CEMM (Centro Especializado em Micologia Médica), com base nas sequências 16S e 16S-23S DNAr. O DNA destas amostras foi extraído com o kit Wizard® Genomic DNA Purification (Promega), quantificado por espectrofotometria e armazenado a 4ºC. A amplificação de um fragmento de 302 pb da região espaçadora 16S-23S DNAr específico para B. pseudomallei foi realizada por meio de reação de PCR com os primers Bp1 e Bp4. O sequenciamento das regiões 16S e 16S-23S DNAr foi realizado pelo método da terminação da cadeia pelo didesoxinucleotídeo, usando-se o kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). A árvore filogenética da região 16S DNAr e as matrizes sequência identidade e contagem de diferenças baseadas na região 16S-23S DNAr foram geradas pelo programa MEGA4, versão 4.1. Os resultados confirmaram a identificação de 15 cepas de B. pseudomallei (cinco clínicas e dez ambientais), o que corresponde a 48.4% dos isolados em estudo. A árvore filogenética baseada na região 16S DNAr demonstra que os isolados clínicos e ambientais de B. pseudomallei do Estado do Ceará são evolutivamente agrupados com as cepas B. pseudomallei MSHR346 (Austrália), B. pseudomallei 1106a (Tailândia), B. pseudomallei K96243 (Tailândia), B. pseudomallei 1710b (Tailândia) e B. pseudomallei 668 (Austrália). Com a utilização do mesmo kit de extração também foi possível extrair DNA de B. pseudomallei diretamente de espécime clínico (lavado brônquico), confirmando um novo caso de melioidose no Município de Ubajara/CE. Em nosso estudo, o uso da PCR para a amplificação de um fragmento de 302 pb da região 16S-23S DNAr identificou corretamente B. pseudomallei, sendo que para confirmar a discriminação entre B. pseudomallei e B. mallei, o sequenciamento das regiões 16S e 16S-23S DNAr foi realizado. A técnica de PCR aliada ao sequenciamento das regiões 16S e 16S-23S do DNA ribossômico nuclear resultaram em uma elevada sensibilidade e especificidade de detecção de B. pseudomallei neste estudo.
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Baker, Anthony Lyle. "The biogeography of melioidosis." Thesis, 2012. https://researchonline.jcu.edu.au/27820/1/27820_Baker_2012_Thesis.pdf.
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Burkholderia pseudomallei causes melioidosis; an often fatal disease prevalent in tropical regions of Southeast Asia and Australasia, and emerging in other regions of the globe. Seemingly random clustering of clinical cases is linked not only to the population distribution, but also to the environmental prevalence of the organism. Previous studies based on the diversity of representative isolates have concluded that an Australian evolutionary origin of the organism is likely, with distinct populations of the organism each side of the Wallace line. However, the means of dissemination of B. pseudomallei between these regions remain uncertain. A focus of B. pseudomallei endemicity has recently been described in the Balimo region of the Western Province, Papua New Guinea; a feature of the isolates from both clinical and environmental sources is a narrow genetic diversity. This has raised questions as to the origins of the organism from this community and their relationship to those from northern Australia. Through a more thorough investigation of the reservoir and phylogeny of B. pseudomallei from northern Queensland and Papua New Guinea, this thesis aims to make a significant contribution to our understanding of the relationship between and origins of isolates in our region.
In an attempt to elucidate the origins of B. pseudomallei in the Balimo region of Papua New Guinea, multi-locus sequence typing was employed to reveal three novel sequence types (Chapter 4). Phylogenetic reconstruction using multi-locus sequence typing data and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Fimbrial gene cluster analysis however, identified a Yersinialike fimbrial gene cluster among all Balimo isolates that is found predominantly among isolates recovered from Southeast Asia. Higher resolution multi-locus variable number of tandem repeat analysis of the isolates resolved 24 genotypes with high divergence. These findings are consistent with a long term persistence of the organism in the region and a high level of environmental stability. A more thorough comparison of the Balimo isolates was made against Queensland isolates.
A field site for the collection of environmental B. pseudomallei in Queensland, Australia was sought to analyse the diversity of isolates from a region closer to Balimo (Chapter 5). Multi-locus sequence typing of 20 isolates collected from environmental sampling revealed for the first time a clinically implicated reservoir of B. pseudomallei in Townsville. Furthermore, it was discovered that naturally occurring groundwater seeps function as a vehicle for the dispersal of viable B. pseudomallei away from a primary environmental reservoir. Analysis of these isolates supported earlier findings that isolates from Queensland are distinct to those from the Northern Territory, yet no associations with B. pseudomallei from New Guinea were identified.
In an attempt to analyse B. pseudomallei from a region adjacent to Balimo, this study collected isolates from the Torres Strait region of northern Queensland, Australia (Chapter 6). The Torres Strait is recognised as an important melioidosis endemic region in northern Queensland, yet no reservoir of infection has been identified. The people of the region have a long-term history of trade and travel between the islands, New Guinea and mainland Australia. Typing of 32 clinical B. pseudomallei isolates from the Torres Strait identified statistically significant non-random distribution of sequence types, with localisation to individual islands. In addition, sequence type matches were identified with the Northern Territory, Port Moresby and Thailand, indicating potential long-distance movement of the organism, but less frequent movements between islands. These findings are consistent with the hypothesis that B. pseudomallei movements have been restricted by short stretches of ocean, and indicate a mechanism responsible for the genetic isolation of populations between Australia and Southeast Asia. The highest diversity was identified on Thursday Island, the commercial and administrative hub of the Torres Strait, indicating that human influences may have had an impact on the dispersal of melioidosis around the region. Environmental sampling on Badu Island recovered a single clone belonging to a novel sequence type, yet the isolate was noted to have closest identity to a clinical isolate from the same island. This was the first recovery of an environmental isolate from the Torres Strait and confirms it’s status as a B. pseudomallei endemic region.
Balimo B. pseudomallei were unable to be linked to others from the globe using multi-locus sequence typing and so, the analysis of whole genome sequences was used to determine their ancestry (Chapter 7). Phylogenetic reconstruction indicated that all of the Papua New Guinean and Torres Strait isolates were members of the Australian clade. Clinical isolates from Port Moresby comprised an individual branch, whilst isolates from Balimo formed a unique clade along with two isolates from the Torres Strait. These findings verify that Papua New Guinean B. pseudomallei belong to the Australian clade, and provide support for biogeographical boundaries in agreement with observed macro-flora and fauna partitioning each side of the Wallace Line. In addition, these analyses indicate that populations of B. pseudomallei have been stable in Papua New Guinea for an extended period, and are not the result of recent human importation. The results suggest that B. pseudomallei is diverse in the Port Moresby region and may represent a new foci of clinical melioidosis.
Finally, a collection of five non-arabinose assimilating B. pseudomallei-like isolates were collected during environmental sampling in the Torres Strait (Chapter 8). Sequencing of the 16S rDNA, recA genes and multi-locus sequence typing loci revealed that the isolates clustered into thee clades within the B. pseudomallei-group, with closest ancestry to B. oklahomensis. Characterisation and genome sequencing of these isolates is ongoing. The recovery of these diverse organisms from a melioidosis endemic region in Australia may have important implications for serology and biochemical based diagnostics in the region.
In conclusion, this work contained within thesis has demonstrated that isolates of B. pseudomallei from Balimo are closely related to those from Australia and the Torres Strait, with a narrow genetic diversity resulting from independent evolution following long term isolation of the Balimo population. These findings indicate that the current distribution of B. pseudomallei throughout Australasia may be linked to the geographical history of the region in a pattern congruent with the biogeography of macro flora and fauna.
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Cheng, Allen Cheuk-Seng. "Melioidosis epidemiology, pathophysiology and management /." 2005. http://catalogue.flinders.edu.au/local/adt/public/adt-SFU20051121.141305/index.html.
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Lee, Chao-Tai, and 李昭代. "The epidemiology of melioidosis in southern Taiwan." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/65084612896090074550.
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碩士中華醫事科技大學
生物科技研究所
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Melioidosis is a serious, fatal and infectious disease, caused by the bacteria of Burkholderia pseudomallei. The disease is endemic in northern Australia and Southeast Asia where the pathogen can be found in wet soil and pooled surface water. The geographical distribution is between the latitudes of 20 degrees North and 20 degrees South. Taiwan is geographically close to this endemic area, and the first case of melioidosis was reported in 1985. The sporadic cases of this disease were increased in Taiwan before 2005, and these cases were imported and indigenous. In July of 2005, owing to the flood caused by a typhoon, the first outbreak event of melioidosis was found in southern Taiwan. The cases of melioidosis which distributed over Rende township, south district of Tainan city and Qieding township. Clinical isolation of 30 B. pseudomallei were performed phenotypic identification and antimicrobial susceptibility testing, and two isolates were recovered from 2004. Isolates were analyzed by biochemical characteristics, antimicrobial susceptibility patterns, and DNA macrorestiction analysis was performed using XbaI restriction enzyme. We define the API 20NE system can provide correctly identified isolate of B. pseudomallei. The result of pulsed-field gel electrophoresis（PFGE) shows two different restriction patterns in these cases, although case-clusters of melioidosis came from the Er-Ren River Basin. The Rende area is located on upstream of the Er-Ren Rriver Basin, which shows distinct pattern from other areas. Thus, two genotypic strains were present in southern Taiwan. However, melioidosis is an endemic and emerging infectious disease in southern Taiwan. The physicians managing patients should be alert to this disease, and especially after heavy rains or typhoon.
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Barnes, Jodie Lee. "Melioidosis : an investigation of cellular immune responses /." 2004. http://eprints.jcu.edu.au/964.
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Warner, Jeffrey Mitchell. "The epidemiology of melioidosis in Papua New Guinea." Thesis, 2004. https://researchonline.jcu.edu.au/1278/1/01front.pdf.
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Melioidosis has only been sporadically reported in PNG and its contribution to the disease burden of Papua New Guineans has been questioned. The rural district of Balimo, located within the Aramia flood plain of the Western province, was chosen to test the hypothesis that melioidosis is under recognised in rural PNG due to a lack of clinical awareness and a poorly resourced laboratory sector. A prospective clinical screening program conducted at Balimo Health Centre revealed melioidosis as the cause of a previously recognised fatal febrile illness affecting children. The implementation of diagnosis and treatment protocols reduced the apparent case fatality rates from 100% to 45%. Although case numbers were small, features of melioidosis in this community include childhood predilection (average age 12-years), a lack of traditional co-morbidity and regional clustering. Simple methods of isolate identification were tested against gold standards of phenotypic and genotypic techniques and found to be sensitive and sustainable. An IHA serological study of 747 children demonstrated a correlation between sero-reactivity and clinical incidence. Furthermore, selective culture of 374 soil samples taken from the environment within this region revealed autochthonous B. pseudomallei from village communities demonstrated to be melioidosis endemic. Of the 191 samples taken from areas within these villages where children play, 3.7% were found to harbour the organism. DNA macro restriction analysis demonstrated clonality between clinical and environmental strains further substantiating the hypothesis that a driver of childhood predilection is behaviour typical of children which encourages exposure to B. pseudomallei from permanently saturated soil and/or water, most likely through preexisting abrasions or pernasal inoculation. A lack of genetic diversity of B. pseudomallei revealed by DNA macro restriction analysis is a feature. This may represent recent importation or the comfortable niche of environment - host cycling of this virulent saprophyte. This is in contrast to the diversity demonstrated in the analysis of the avirulent PNG derived B. thailandensis. In a geographical analysis of the Balimo region, the environmental attributes of low altitude (<600 m), inundation and extent of inundation and hydraquents as the predominate soil type are typical of this melioidosis implicated region. The subsequent mapping of PNG in terms of these attributes revealed only isolated regions which share these features. If the rare reports of melioidosis elsewhere in PNG is an accurate reflection of the national burden of the disease, these environmental attributes may represent important biogeographical boundaries for melioidosis in PNG. These data may serve in the remote sensing of melioidosis in PNG and throughout the Pacific-Australasian region.
To further substantiate the importance of these geographic boundaries, an indirect IgG ELISA-based sero-epidemiological assay was developed using antigen derived from PNG B. pseudomallei and used on samples taken from individuals from 16 regions throughout PNG. The assay was able to detect sero-reactivity that was dependent on region which varied according to degrees of melioidosis prevalence. The true sero-prevalence ranged from 0 - 55%, demonstrating significant spatial sero-clustering. Further, when regions were classified into risk-localities based on sero-reactivity, a correlation was revealed between regions determined high-risk by population sero-reactivity and biogeography.
A prospective study in Port Moresby where 3561 samples were selectively screened for B. pseudomallei demonstrated melioidosis to be endemic in the empirically diagnosed tuberculosis (TB) patient cohort and patients presenting with sepsis associated type 2 diabetes, although the incidence is low. In demonstrating endemic melioidosis in rural PNG for the first time, it is hoped this work will contribute to decreasing the fatality rates of pneumonia and sepsis in this rural subsistence community and may aid in the uncovering of the submerged iceberg that is melioidosis within this region.
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Fu, Tzu-Ling, and 傅子玲. "Investigation of Molecular Epidemiology of Ruminant Melioidosis in Southern Taiwan Area." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/77821195011099148937.
Full textAbstract:
碩士國立屏東科技大學
獸醫學系所
97
The contents of abstract in this thesis: The purpose of this study was to investigation of ruminant melioidosis in southern Taiwan area, and to compare the detective efficacy of melioidosis between bacterial culture and PCR test in organs on goats. We randomly collected blood samples of cattle and goats from southern Taiwan. We then established a PCR protocol for Burkholderia pseudomallei 16-23S rRNA interspacer gene and analyzed its nucleic acid sequences and phylogeneic correlations then detected melioidosis antibody by agglutination. We collected 25 suspect cases of meliodiosis in goat, isolated bacteria by culture and at the same time extracted DNA for detection of pathogenic antigen by PCR assay. The surveillance melioidosis by PCR indicated positive rates and farms positive rates of 9.3 % ( 59/634 ) and 44.7 % ( 34/76 ) in cattle；15.59 % ( 99/620 ) and 42.5 % ( 34/80 ) in goats. After comparing nucleic acid sequences of this study strain with published strains on the GeneBank, the Taiwan strain with American strain 668, 1106a, 1710b 16-23S rRNA ITS have 100 % and 99.7 % identity. The antibody titer of B. pseudomallei was negative by agglutination test. The culture method is considered as the golden standard for detection of B. pseudomallei in goat. The PCR sensitivity was 54/78 ( 69 % ), specificity was 13/13 ( 100 % ) . Results indicating that the prevalence of ruminant melioidosis was higher in southern Taiwan area. In case of confirmed results, lungs, lymph nodes, testis and breast had higher detection ratio both by bacterial culture and PCR.The bacterial culture for diagnosis of melioidosis in goat has higher examination ratio. Key words: Melioidosis, Burkholderia pseudomallei, PCR, bacteria culture.
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"Selective Enrichment Of Burkholderia Pseudomallei Outer Membrane Vesicles For Vaccination Against Melioidosis." 2016.
Find full textAbstract:
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease with a mortality rate of over 40%, and is a major public health concern in the endemic regions of Thailand and northern Australia. Bp is a resilient pathogen capable of surviving in diverse environments including soil, fresh and seawater, and plant and animal tissues for extended lengths of time. Bp is intrinsically resistant to antibiotics, which contributes to persistence and relapse in over 25% of melioidosis patients, and there is currently no vaccine. Our lab has previously shown that immunization with Bp outer membrane vesicles (OMVs) derived from Bp grown in Luria Burtani broth provides significant protection against melioidosis in mice. However, this protection was limited to the acute phase of infection and animals immunized with OMVs were unable to clear the bacteria. In this work, we show that by manipulating the growth media to simulate various bacterial niches, including the natural hypertonic soil environment (NaCl-supplemented), the limited-nutrient host macrophage intracellular environment (M9CG minimal media), and quorum sensing conditions (QS-molecule supplemented), OMV protein content can be modified to include those proteins potentially important for Bp survival and may contribute to protection against chronic or persistent infection. Here, we characterize the composition of selectively enriched Bp OMVs and demonstrate that enriched OMVs are non-toxic and well-tolerated both in vitro and in vivo. Immunization of BALB/c mice with QS OMVs elicits significantly greater OMV-, CPS-, and LPS- serum IgG along with cell-mediated immune responses compared to mice immunized with LB OMVs. LB, M9CG, and QS OMV immunization provided equal protection against aerosolized Bp through the acute phase of infection, and M9CG OMV-immunized mice demonstrated fewer signs of morbidity and less weight-loss over the course of infection, indicating potential control of the bacteria. These results suggest that immunizing with OMVs selectively enriched with intracellular proteins may elicit the necessary immune responses to protect against persistent melioidosis.Nicole L Kikendall
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Merianos, Angela. "Field placement in the Northern Territory." Thesis, 1993. http://hdl.handle.net/1885/143640.
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