RESUMEN
A buoyant density centrifugation procedure using Percoll was developed for the isolation and purification of Mycobacterium leprae from experimentally infected armadillo liver tissue. The method separates the bacteria from host adenosine triphosphate (ATP) and tissue debris and recovers 20-25% of the bacteria within 2-2 1/2 hours under controlled conditions. The mean ATP content (585 pg/10(6] of the purified bacteria was similar to cultivable bacteria. The organisms did not leak intracellular ATP when exposed to phosphate buffer. Temperature-dependent ATP synthesis was observed within minutes and could be inhibited by 2,4-dinitrophenol. Freeze-thawing M. leprae as purified suspensions in buffer damaged the organisms, resulting in decreased ATP levels and an accelerated loss of ATP upon incubation under defined conditions. In vitro treatment with the antileprosy drug clofazimine increased the rate of ATP decay directly proportional to drug concentration.
Asunto(s)
Adenosina Trifosfato/análisis , Armadillos/microbiología , Mycobacterium leprae/análisis , Xenarthra/microbiología , 2,4-Dinitrofenol , Adenosina Trifosfato/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Dinitrofenoles/farmacología , Congelación , Hígado/microbiología , Mycobacterium leprae/crecimiento & desarrollo , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/metabolismo , Fosforilación OxidativaRESUMEN
A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/análisis , Mycobacterium/análisis , Micobacterias no Tuberculosas/análisis , Enzimas de Restricción del ADN/análisis , Electroforesis en Gel de Agar , Metilación , Mycobacterium avium/análisis , Mycobacterium lepraemurium/análisis , Mycobacterium phlei/análisis , Mycobacterium tuberculosis/análisisRESUMEN
A fluorescent staining procedure incorporating the use of fluorescein diacetate (FDA) and ethidium bromide (EB) has previously been shown to accurately measure the viability of saprophytic mycobacterial cells. Green-stained cells were shown to be viable and red-stained cells, dead. Staining Mycobacterium leprae cells with FDA/EB, however, was complicated by interfering tissue components which masked the presence of stained bacteria. A petroleum ether separation technique enables M. leprae to be segregated from armadillo liver tissue components and permitted M. leprae to be stained qualitatively equal to the saprophytic mycobacteria. An alternative and technically simpler method of staining M. leprae from human skin biopsies and mouse foot pads was developed which permitted the initiation of a clinical assessment of the staining method. Preliminary data indicate that patients who have undergone three or 24 months of chemotherapy possess a significantly lower percentage of green-stained M. leprae in their tissues than untreated patients. This would be expected if the FDA/EB staining method was providing an accurate measure of viability. M. leprae cells obtained from mouse foot pads which were harvested 5-13 months post-infection displayed more than 90% green-stained cells. There was no correlation between the FDA/EB staining method and the morphological index.
Asunto(s)
Etidio , Fluoresceínas , Mycobacterium leprae/citología , Coloración y Etiquetado/métodos , Animales , Humanos , Lepra/microbiología , Ratones , Mycobacterium leprae/aislamiento & purificación , Fenilendiaminas , Piel/microbiologíaRESUMEN
A fluorescent staining procedure has been developed which rapidly, accurately, and economically measures the viability of mycobacterial cells. M. smegmatis and M. phlei have served as prototype organisms to establish conditions which ensure optimal staining. The staining method incorporates the use of the fatty acid ester fluorescein diacetate (FDA) and ethidium bromide (EB). Non-polar, non-fluorescent FDA enters live cells where it is enzymatically hydrolyzed by acetylesterase to polar, fluorescent fluorescein which rapidly accumulates in the cytoplasm. These cells appear green when viewed under incident ultraviolet illumination. Ethidium bromide enters dead cells and intercalates between the bases of DNA molecules. These cells appear red-orange under UV illumination. Live cells are, therefore, identified on the basis of possessing acetylesterase and their ability to exclude EB; whereas dead cells are identified on the basis of lacking acetylesterase and their inability to exclude EB. The feasibility of applying the staining procedure of M. leprae has been investigated and the results are encouraging. Our findings reveal that armadillo-derived M. leprae possess acetylesterase and, therefore, stain green. M. leprae cell suspensions exposed to adverse physico-chemical conditions give rise to high proportions of red-stained cells as would be expected if the cells are being killed. An alternative means of determining the viability of M. leprae appears to be feasible.
Asunto(s)
Fluoresceínas , Mycobacterium , Coloración y Etiquetado , Medios de Cultivo , Etidio , Calor , Mycobacterium leprae , Mycobacterium phlei , Factores de TiempoRESUMEN
Two smooth and six rough strains of Salmonella typhimurium with progressively smaller amounts of sugar and protein in their outer membrane were tested for degree of virulence in normal and iron-injected mice and for ability to acquire iron in mammalian sera. The rate of mortality showed that bacterial virulence for mice was lowered with progressive decrease of outer-membrane sugar and protein. Iron injections increased the rate of mortality in mice infected either with smooth strains or with superficially rough strains but were without effect in mice infected with deep rough strains. In in vitro experiments, iron promoted with equal effectiveness the growth of all serum-exposed bacterial strains, whereas enterobactin (E) was much more effective in promoting the growth of smooth and superficial rough than in promoting that of deep rough strains. Various experiments showed that deep rough strains cannot grow in E-supplemented serum because they are not able to use the transferrin-iron-E complexes that E forms with transferrin-iron. This failure to use transferrin-iron-E complexes by deep rough strains was found to be due to the inability of these strains to absorb iron containing complexes to their outer membrane. Adsorption studies with chemically treated bacteria showed that the receptor of transferrin-iron-E or E-iron complexes is a protein of the outer membrane of bacterial cells.
Asunto(s)
Enterobactina/metabolismo , Hierro/metabolismo , Salmonella typhimurium/metabolismo , Serina/análogos & derivados , Transferrina/metabolismo , Adsorción , Animales , Sangre , Membrana Celular/metabolismo , Ratones , Receptores de Droga , Salmonella typhimurium/patogenicidadRESUMEN
Effects of iron on the growth of avirulent and virulent strains of Escherichia coli were tested in mice and in mammalian sera. Infection of the animals with iron increased mortality rates in mice infected with the avirulent strain to levels found in mice infected with the virulent strain. In vitro experiments showed that bacteria deprived of iron in bovine or human sera or milk or in chicken egg white stopped miltiplication and died in a very short time. These antibacterial effects were neutralized effectively with the addition of exogenous iron or the iron-binding bacterial product, enterochelin. In contrast to avirulent bacteria, which were effectively inhibited in mammalian serum, virulent bacteria were able to obtain iron and multiply. The ability of virulent bacteria to grow in mammalian serum is being attributed to the presence of iron-binding enterochelin and lipopolysaccharide in large amounts on the cell walls of virulent bacteria.