RESUMEN
Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.
Asunto(s)
Vectores Genéticos/genética , Micobacteriófagos/genética , Mycobacterium/aislamiento & purificación , Clonación Molecular/métodos , Cósmidos/genética , Expresión Génica , Genes Reporteros/genética , Cinética , Luciferasas/biosíntesis , Luciferasas/genética , Lisogenia , Pruebas de Sensibilidad Microbiana/métodos , Micobacteriófagos/crecimiento & desarrollo , Mycobacterium/virología , Regiones Promotoras Genéticas/genética , Mapeo Restrictivo , SobreinfecciónRESUMEN
The interactions of mycobacteriophage D29 and Mycobacterium leprae were examined. It was demonstrated that after adsorption D29 injected its DNA in M. leprae. While the synthesis of host proteins and lipids were inhibited in M. tuberculosis and in M. smegmatis during infection by D29, the results were inconclusive in the case of M. leprae because these bacteria did not incorporate the appropriate substrates.
Asunto(s)
Micobacteriófagos/crecimiento & desarrollo , Mycobacterium leprae/fisiología , ADN Bacteriano/fisiología , Metabolismo de los Lípidos , Mycobacterium leprae/metabolismoRESUMEN
The minimal inhibitory concentrations of antituberculosis and antileprosy drugs were determined for Mycobacterium aurum. The concentrations that reduced the final yield of bacteriophage D29R1 by 50% and the time during the replication cycle at which the drugs completely inhibited phage production were estimated. THe 50% inhibitory concentration/minimal inhibitory concentration ratios were close to 1.0 for clofazimine, colistin, rifampin, and streptomycin; these ratios were high for dapsone (diaminodiphenylsulfone) and isoniazid. Ethambutol (minimal inhibitory concentration, 1.0 micrograms/ml) was without effect on viral growth.