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1.
Braz. j. microbiol ; 45(4): 1449-1454, Oct.-Dec. 2014. ilus, tab
Artigo em Inglês | LILACS | ID: lil-741299

RESUMO

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.


Assuntos
Humanos , Citocinas/biossíntese , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Fatores de Virulência/imunologia , Brasil , Linhagem Celular , Clonagem Molecular , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transformação Bacteriana , Fatores de Virulência/genética
2.
Electron. j. biotechnol ; 13(5): 21-22, Sept. 2010. ilus, tab
Artigo em Inglês | LILACS | ID: lil-591903

RESUMO

Electrotransformation also known as electroporation is the most reliable and efficient tool for plasmid DNA uptake. Electrotransformation efficiency is function of many factors which include (1) number of cell washes prior to electroporation, (2) electroporation cell number, (3) electroporation DNA amount, and (4) cell growth phase. Those factors have limitedly been concomitantly investigated in E. coli DH10B strain. This study is aimed to explore above key factors to define the optimal conditions for high electrotransformation efficiency. The results showed that electrotransformation efficiency of E. coli DH10B was enhanced to 1.5 x 10(9) cfu/ug by washing cells three times with 15 ml of 10 percent glycerol. This washed off extra salts from cell suspension and enhanced electrotransformation by preventing arcing and enhancing cell resistance while ensuring minimal level of conductivity. Early exponential phase at 0.15 OD600 was the best growth phase for enhancing electrotransformation of E. coli DH10B. The results also showed that higher electrotransformation efficiency was similarly achieved when 0.5 x 10(10) and 0.6 x 10(10) cell numbers were electroporated with DNA amount ranging from 10 to 40 pg. This study confirmed the optimal conditions for electro competent cell preparation and plasmid DNA electrotransformation, which can result highest transformation efficiency.


Assuntos
DNA Bacteriano/análise , Eletroporação , Escherichia coli/genética , Transformação Bacteriana , DNA Bacteriano/genética , Escherichia coli/crescimento & desenvolvimento , Transformação Genética
3.
Braz. j. microbiol ; 40(4): 923-926, Oct.-Dec. 2009. graf, tab
Artigo em Inglês | LILACS | ID: lil-528176

RESUMO

A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.


Assuntos
Bactérias Gram-Positivas/genética , Genética Microbiana , Protoplastos , Transformação Bacteriana , Vidro/análise , Métodos , Técnicas
4.
Rev. méd. Chile ; 134(4): 415-420, abr. 2006. tab
Artigo em Espanhol | LILACS | ID: lil-428539

RESUMO

Background: Klebsiella pneumoniae is an important pathogenic bacterium, frequently isolated from nosocomial samples, that exhibits wide antimicrobial resistance profiles, including third generation cephalosporins (3GC), aminoglycosides and quinolones. The resistance to 3GC is mainly due to the synthesis of extended spectrum beta lactamases (ESBL), encoded by conjugative plasmids. Aim: To investigate the potential transference of resistance to 3GC from nosocomial strains of K. pneumoniae to other clinical strains of various species of Enterobacteriaceae. Material and methods: The mating experiments were carried out in liquid media and three nosocomial strains of K. pneumoniae were used as donors. These strains were ESBL-producers and resistant to, at least, one of the 3GC assayed. One strain of Citrobacter freundii, Salmonella typhimurium, Serratia marcescens and Escherichia coli, isolated from clinical specimens, were used as recipients. The presence of bla genes was investigated by PCR. Results: The three nosocomial strains of K. pneumoniae were able to transfer the resistance to 3GC and the genes encoding the ESBL to the susceptible recipient strains of enterobacteria. The frequency of transference was as high as 3.2 x 10-2 transconjugants/recipient cell when the strain of Citrobacter freundii was used as recipient. All transconjugants exhibited high level of resistance to the 3GC assayed. Conclusions: Strains of K. pneumoniae isolated from Chilean hospitals are able to disseminate the ESBL genes to clinical strains of others species of Enterobacteriaaceae.


Assuntos
Humanos , Antibacterianos/farmacologia , Resistência às Cefalosporinas/genética , Cefalosporinas/farmacologia , Klebsiella pneumoniae/enzimologia , Transformação Bacteriana/genética , beta-Lactamases/biossíntese , Citrobacter freundii/efeitos dos fármacos , Citrobacter freundii/enzimologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologia , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/enzimologia , beta-Lactamases/genética
5.
Electron. j. biotechnol ; 8(1): 113-120, Apr. 2005. tab, graf
Artigo em Inglês | LILACS | ID: lil-448787

RESUMO

This paper describes an efficient bacterial transformation system that was established for the preparation of competent cells, plasmid preparation, and for the storage in bacterial stocks in our laboratory. Using this method, a number of different plasmids have been amplified for further experiments. Competent cells for bacterial transformation were prepared by the calcium chloride method with an optimum concentration of 75 mM. Three different strains of Escherichia coli that were tested are DH5 alpha, TG1 and XL1 blue, and the most efficient strain being XL1 blue. The optimal optical density (OD600) range for competent cell preparation varied for each of the strains investigated, and for XL1 blue it was 0.15-0.45; for TG1 it was 0.2-0.5; and for DH5 alpha it was 0.145-0.45. The storage time of competent cells and its correlation to transformation efficiency has been studied, and the result showed that competent cells can be stored at –20 ºC for 7 days and at –70 ºC for 15 days. Three critical alterations to previous methods have been made, which are the changing of the normal CaCl2 solution to TB solution, the changing of the medium from LB to S.O.C., and addition of DMSO or PEG8000 during transformation of competent cells with plasmids. Changing the medium from LB to S.O.C., resulted in much faster growth of transformants, and the transformation efficiency was increased. Addition of DMSO or PEG8000 raised transformation efficiencies by 100-300 fold. Our improved bacterial transformation system can raise the transformation efficiency about 103 times, making it becoming a highly efficient bacterial transformation system.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Plasmídeos/genética , Técnicas de Cultura de Células/métodos , Transformação Bacteriana/genética , DNA Bacteriano/genética , Tampões (Química) , Clonagem Molecular/métodos , Eletroporação , Soluções , Temperatura , Fatores de Tempo , Transformação Genética
6.
In. Santelices Cuevas, Emilio. Cuidados postoperatorios y paciente quirúrgico crítico. Santiago de Chile, Sociedad de Cirujanos de Chile, nov. 1994. p.200-4.
Monografia em Espanhol | LILACS | ID: lil-173029
7.
Braz. j. med. biol. res ; 26(3): 261-75, Mar. 1993. ilus, tab
Artigo em Inglês | LILACS | ID: lil-148691

RESUMO

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli


Assuntos
Animais , Ácidos Hidroxâmicos/metabolismo , Escherichia coli/patogenicidade , Transformação Bacteriana , Southern Blotting , Galinhas , Clonagem Molecular , Sondas de DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/análise , Virulência
8.
Iatreia ; 6(1): 46-50, mar. 1993. graf
Artigo em Espanhol | LILACS | ID: lil-434451

RESUMO

Se presenta un panorama de la resistencia bacteriana Incluyendo su fisiopatogenia y formas de presentación y se establecen algunas consideraciones generales de tipo clínico como auxiliares para racionalizar el uso de los antimicrobianos y evitar o retardar el problema de la resistencia; éste plantea la necesidad de un reordenamiento definitivo en la prescripción de antimicrobianos. No será tanto la creación o descubrimiento de nuevos antibióticos sino la racionalización del manejo de los existentes lo que permitirá alcanzar victorias sobre estos microorganismos. Es importante mantener educación continua sobre el uso adecuado de los antimicrobianos desde los puntos de vista epidemiológico, farmacocinético y fisiopatogénico.


Assuntos
Conjugação Genética , Transdução Genética , Transformação Bacteriana
9.
Rev. latinoam. microbiol ; 29(3): 263-70, jul.-sept. 1987. ilus
Artigo em Espanhol | LILACS | ID: lil-105152

RESUMO

El espectro de absorción de una solución con mertiolato y ácido desoxirribonucleico transformante de Haemophilus influenzae, fue igual al que se obtuvo al sumar sus dos espectros independientes. Después de dializar el mertiolato, el DNA mostró su espectro de absorción típico y su actividad transformantes fue igual al DNA testigo dializado que no había estado en presencia del mertiolato. Se describe un método nuevo para aislar el DNA transformante de H. influenzae que requirió: 1) lisis bacteriana con mertiolato, 2) despretinización con cloroformo: octanol, 3) preciptación del DNA con etanol frío, 4) eliminación del RNA por hidrólisis con ribonucleasa pancreática; 5) diálise y 6) conservación a - 70-C. el DNA transformante obtenido y purificado, después de la lisis con mertiolato de H. influenzae, tuvo un espectro de absorción típico con una lambda max entre 255 y 260 nm y una lambda min entre 230 y 235 nm. Los valores de E (P) a 260 y 230 nm fueron típicos de DNA nativo. La igualdad en la concentración de DNA determinada por el método espectrofotométrico o por su contenido en desorribosa, indicó la ausencia de cantidades significativas de RNA. La relación de absorbancia de 260 sobre 230 nm tuvo un valor alrededor de 2.2, sugiriendo que no había una concentración apreciable de proteínas. El DNA mostró una actividad transformante específica típica del ácido desoxirribonucleico transformante de H. influenzae


Assuntos
DNA Bacteriano/genética , Haemophilus influenzae/efeitos dos fármacos , Transformação Bacteriana , DNA Bacteriano/isolamento & purificação , Compostos de Etilmercúrio/farmacologia , Hidrólise , Timerosal/farmacologia
10.
Rev. chil. tecnol. méd ; 9(1): 395-400, 1986. ilus
Artigo em Espanhol | LILACS | ID: lil-104229

RESUMO

Se investiga la capacidad adherente y hemaglutinante de una cepa de E. COLI 36692 fimbriada y se compara con una cepa de E. COLI UCCSI no fimbriada. Mediante microscopía electrónica se demuestra la fibriación de E. COLI 36692, no observándose estas estructuras en E. COLI UCCSI. Se aísla el ADN extracromosomal de E. COLI 36692 y se obitienen 3 bandas que posiblemente corresponden a 3 diferentes plasmidios. Empleando técnicas de conjugación y transformación bacteriana se intenta transferir el ADN extracromosomal a E. COLI UCCSI sin obtenerse resultados positivos. Con el propósito de evidenciar el origen de la codificación de este tipo de fimbrias se procede a eliminar el ADN plasmidial a través de técnicas de curación. Al tratar a E. COLI 36692 con naranja de acridina se obtienen colonias carentes del ADN extracromosomal pero, no pierden su capacidad adherente ni emaglutinante. Lo anterior es confirmado mediante microscopía electrónica. De esta forma se demuestra la naturaleza cromosomal de codificación genética de las fimbrias de E. COLI 36692 pielonefritogénica


Assuntos
Ratos , Escherichia coli/genética , Escherichia coli/patogenicidade , Fímbrias Bacterianas/metabolismo , Técnicas In Vitro , Aderência Bacteriana , Conjugação Genética , DNA Bacteriano/análise , Código Genético , Pielonefrite/genética , Transformação Bacteriana
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