The immunogenicity and protective efficacy of a DNA vaccine encoding a truncated groEL heat shock gene (pcDNA3/groEL) from Burkholderia pseudomallei was evaluated in vaccinated BALB/c mice infected with B. pseudomallei or B. cenocepacia. After vaccination, the levels of anti-GroEL total IgG and IgG2a were increased in mouse sera. The clonal expansion of the spleen cells increased, and the GroEL protein induced IFN-£^ production by spleen cells. The anti-GroEL antibody-mediated opsonic killing effect was not able to eliminate the growth of B. pseudomallei but was able to eliminate the growth of B. cenocepacia. After intravenous challenge of the vaccinated Balb/c mice with B. pseudomallei, the number of bacteria colonizing the in liver and/or spleen was not reduced. Over 50% of vaccinated mice infected with B. pseudomallei died within 7 days post-infection. By contrast, the bacterial loads in organs were significantly reduced if the vaccinated mice were infected with B. cenocepacia. All of vaccinated mice were alive 7 days post-infection. Liver damage, as assessed by histological observation, and abnormalities in the levels of liver enzymes rapidly resolved in vaccinated mice. We suggest that B. pseudomallei groEL plasmid DNA immunization of Balb/c mice induces a Th1-type immune response and provides cross-protection against B. cenocepacia but not against B. pseudomallei infection.

Persister cells are able to survive in the presence of high concentrations of antibiotic, and re-grow once the antibiotic has been removed. Unlike conventional antibiotic resistance, the antibiotic tolerance of persister cells is due to phenotypic switching, and is non-inherited. There is growing evidence for a role of persisters in various persistent bacterial diseases. Burkholderia pseudomallei is a pathogen which causes melioidosis, which often persists in the host despite antibiotic treatment. As persister cells may contribute to persistent melioidosis, this study investigated persisters in B. thailandensis, as a model for B. pseudomallei. Treatment of B. thailandensis with ceftazidime, ciprofloxacin, imipenem or trimethoprim demonstrated persister cells which survived antibiotic treatment. Persister frequencies were increased in the absence of oxygen, and higher in stationary phase cultures compared with growing cultures. Drug concentration did not affect persister frequencies, and inherited antibiotic resistance was not detected. Different persister fractions were detected using treatment with multiple antibiotics, indicating heterogeneous susceptibility to antibiotics. In order to increase understanding of the molecular basis of B. thailandensis persister cells, a transposon mutagenesis-based sequencing approach was used on persister cultures. This indicated some issues with genome coverage and mutant diversity. Genes were identified from mutants present before and/or after ciprofloxacin treatment. In order to try to eradicate persister cells from a culture, two anti-persister strategies were tested. Itaconate appeared to stimulate growth of B. thailandensis, increasing susceptibility to the antibiotic ceftazidime. However, the overall effect of the combination was no greater than ceftazidime alone in the conditions tested. Metronidazole was effective against a persister culture under anaerobic conditions, suggesting it may be useful in treating anaerobic persisters. Treatment of B. pseudomallei infected mice with metronidazole and ceftazidime did not improve survival over ceftazidime treatment alone.

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

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

No description available.

Soybean cyst nematode (H. glycines), reniform nematode (R. reniformis), and Root-Knot nematode (M. incognita) are three damaging plant-parasitic nematodes on soybean. Syntaxin proteins are involved in the process of membrane fusion. T wo G. max syntaxin genes (Gm-SYP22-1, and Gm-SYP22-2) that were similar in amino acid composition have been found to contribute to the ability of Glycine max to defend itself from infection by the plant parasitic nematode Heterodera glycines. Syntaxin genes SYP22-1 and SYP22-2 were identified to be expressed specifically in syncytia undergoing a resistant reaction to H. glycines parasitism. The Gm-SYP22-1 and Gm-SYP22-2 genes were isolated by molecular means and genetically engineered in G. max [Williams 82/PI 518671], a genotype typically susceptible to H. glycines parasitism. Genetically engineered control plants in G. max [Williams 82/PI 518671] that lack the overexpression of Gm-SYP22-1 or Gm-SYP22-2 genes were produced to serve as a comparison. The transgenic Gm-SYP22-1 or Gm-SYP22-2 overexpression lines with their pRAP15 control have then been infected with H. glycines. In another study, tests include three separate tests in 2015 and one test in 2016 that evaluated different biological products, application rates and product combinations as seed treatments on soybeans. Results collected from soybean plants that were infested with either H. glycines, M. incognita or R. reniformis indicated that many of these biological products significantly reduced the nematode reproduction compared to control. The number of cyst, juveniles, and eggs recovered were significantly reduced compared with the non-treated control. Other findings identified Burkholderia renojensis variant 2 (BioST Nematicide) as being a more consistent nematicide candidate when referencing data from all nematodes and rate ranges. Combinations of B. renojensis variant 2 with selected SAR (systemic acquired resistant) products numerically improved the efficacy and consistency of the biological nematicide. Another study focused about investigated of biological seed treatments on H. glycines, and F. virguliforme indicated that many of these biological products significantly reduced the nematode reproduction over the fungicide only check. Foliar disease severity happened more in the treatments that infested with H. glycines + F. virguliforme combination than F. virguliforme alone.

Burkholderia pseudomallei, the etiological agent of melioidosis, is a saprophytic bacterium existing endemically in the water and soil of SE Asia and Northern Australia. This organism has shown the ability to remain dormant in its host for decades. B. thailandensis is a closely related non-pathogenic near neighbor that is also found in these soils. It has been suggested that free-living amoeba could be natural reservoirs for these organisms. The interactions of Burkholderia species and Acanthamoeba castellanii, a species of free-living amoeba, were studied to better understand the natural ecology of these organisms and to determine the effects amoeba interactions might have on pathogenesis. In this study, the adherence and persistence of several B. pseudomallei clinical isolates were compared to that of B. thailandensis within both amoeba and a human monocyte cell line. Results showed that B. pseudomallei isolates can enter amoeba and survive therein at varying levels of efficiency. Some isolates were able to persist inside the amoeba for up to three weeks. Optimal entry time into an amoeba trophozoite was found to be about three hours for all ten B. pseudomallei isolates. Interestingly, it was found that after internalization by amoeba, B. pseudomallei have a significantly increased ability to both attach to, and grow within human monocytes, suggesting that such interactions increase the virulence capabilities of soil isolates.

Fungal infections have a significant impact on the world population, with estimates of over 1.6 million deaths a year. One contributing factor is the increasing number of fungi resistant to the current clinical treatments, including the last approved family of antifungal compounds introduced into the market over a decade ago. This is driving the search for new antifungals with different biological targets. A new potential antifungal occidiofungin, is a cyclic glycolipopeptide isolated from the soil bacterium Burkholderia contaminans MS14 with a broad spectrum of activity against both human and plant pathogens. Kill kinetics demonstrated that this compound is fungicidal and activates the cell wall integrity pathway at suboptimal dosing as determined by Mkc1 MAPK phosphorylation studies. As three of the four classes of currently available antifungals target ergosterol or ergosterol biosynthesis, the bioactivity of occidiofungin was assayed in the presence of ergosterol containing DOPC vesicles and was shown to retain antifungal properties. Occidiofungin was also found to have a similar activity profile against the S. cerevisiae -1,3-glucan synthesis mutant, indicating that it does not share a target with the fourth class of antifungals. Stability testing showed occidiofungin retained in vitro potency in the presence of human serum, across a broad range of pH and temperature conditions, and was resistant to gastric proteases. Based on cell morphology, occidiofungin did not target a specific stage of the yeast cell cycle, however cells were smaller in size and acquired ‘dancing bodies’, both properties of apoptosis. This was confirmed with data showing concentration dependent increases in DNA fragmentation, reactive oxygen species, and extracellular localization of phosphatidylserine. In addition to these findings, cells deleted for the yeast caspase gene exhibit a 2old resistance to occidiofungin. While SEM showed no morphological differences between treated and untreated cells, TEM did identify a thinning of the cell wall and inclusion bodies in cells treated with occidiofungin. As a stable fungicidal compound that induces apoptosis in yeast, occidiofungin has a great potential to become a new candidate drug for clinical use in treating fungal infections, including those resistant to current antifungals.

No / Burkholderia cepacia is known to invade and survive within respiratory epithelial cells. Previous studies have employed transformed cell lines and it is not known whether the bacterium is capable of manifesting the same phenomena in primary cell culture. Two strains of B. cepacia of environmental (NCTC 10661) and clinical origin (C1359) were examined for their ability to invade and survive (over a 24 h period) within type II pneumocytes in primary culture using a gentamicin¿ceftazidime antibiotic protection assay. Both strains of B. cepacia were capable of invasion of type II pneumocytes in primary culture. Strain C1359 was capable of multiplying intracellularly as indicated by a seven-fold increase in the numbers of bacteria from 4¿24 h, whereas strain 10661, although unable to replicate intracellularly, was found to survive in the pneumocytes for at least 24 h. Future studies on the invasiveness of B. cepacia can employ A549 cells as a valid surrogate for primary cell culture assays which are time-consuming, labour-intensive and expensive to perform.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Northern Australia and Southeast Asia. Melioidosis can present with acute, chronic and latent infections and can relapse several months or years after initial presentation. Currently not much is known about the ways in which B. pseudomallei can persist within the host, although it has been speculated that the ability to survive within an anaerobic environment will play some role. B. pseudomallei is able to survive anaerobically for extended periods of time but little is known about the molecular basis of anaerobic respiration in this pathogenic species. Bioinformatic analysis was used to determine the respiratory flexibility of both B. pseudomallei and B. thailandensis, identifying multiple genes required for aerobic and anaerobic respiration, and molybdopterin biosynthesis. Using B. thailandensis as a model organism a transposon mutant library was created in order to identify genes required for anaerobic respiration. From this library one transposon mutant was identified to have disrupted moeA, a gene required for the molybdopterin biosynthetic pathway. This B. thailandensis transposon mutant (CA01) was unable to respire anaerobically on nitrate, exhibiting a significant reduction in nitrate reductase activity, altered motility and biofilm formation, but did not affect virulence in Galleria mellonella. It was hypothesised that the reduction in nitrate reductase activity was contributing to the phenotypes exhibited by the B. thailandensis moeA transposon mutant. To determine whether this was true an in-frame narG deletion mutant was created in B. pseudomallei. Deletion of B. pseudomallei narG (ΔnarG) resulted in a significant reduction in nitrate reductase activity, anaerobic growth, motility and altered persister cell formation, and but did not affect virulence in G. mellonella or intracellular survival within J774A.1 murine macrophages. This study has highlighted the importance of anaerobic respiration in the survival of B. thailandensis and B. pseudomallei.