Phage D29 Research Articles

Use of a phage-based assay for phenotypic detection of mycobacteria directly from sputum.

The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an M. tuberculosis-spiked sputum model with (i). identification of inhibitory components of sputum and optimization of decontamination methods; (ii). simplification of the washing and development steps; (iii). reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv). optimization of virucide treatment. The following results were obtained. (i). An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii). A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii). At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv). Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable M. tuberculosis organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens.

Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow-growing mycobacteria.

Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guerin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca2+, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.

Characterization of Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed.

Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid- and sugar-containing components which form a formidable barrier that must be passed to gain access to the membrane. We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29. The phage-resistance phenotype results not from mutation but from elevated expression of a wild-type gene. It appears that the product of this multicopy phage-resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection.

Ultrastructural evidence for the accumulation of a polysaccharide-like substance during mycobacteriophage D 29 replication in Mycobacterium smegmatis

Mycobacterium smegmatis was infected with phage D 29 and five previously characterized temperature-sensitive (ts) mutants. Electron microscopy revealed an accumulation of large amounts of an electron-transparent material immediately after infection of the bacteria. This material appeared in the form of intracellular plages following lead citrate coloration. After silver proteinate staining, the bacteria were found to accumulate a polysaccharide-like substance (PLS) rich in α-1-2-glycol bonds. After bacterial lysis, this PLS disappeared, probably solubilizing in the surrounding medium. In contrast, certain ts mutants which did not cause lysis of the bacteria at the restrictive temperature resulted in bacilli which were heavily loaded with PLS. Mycobacterium smegmatis est infecté par le phage D 29 et 5 mutants thermosensibles (ts) de D 29 . L'ultrastructure des bactéries infectées, après coloration par l'acétate d'uranyl et citrate de plomb, révèle l'accumutation dans le cytoplasme d'une substance transparente aux électrons. Après coloration par le protéinate d'argent, on observe l'accumulation d'une substance polyosidique mise en évidence grâce aux liaisons α-1-2-glycols. Après la lyse bactérienne, cette substance disparaît et se dissout probablement dans le milieu. Par contre, lors de l'infection par certains mutants ts, qui ne lysent pas les bactéries à température non permissive, cette substance reste accumulée.

Inactivation of phages DS6A and D29 by acetone extracts of Mycobacterium tuberculosis and Mycobacterium bovis.

The adsorption rate constant for mycobacteriophage DS6A on clumped cells of Mycobacterium tuberculosis strain H37Rv was found to be 6.33 x 10(-13) ml per min per colony former. Acetone-extracted cells of H37Rv did not absorb phage DS6A. Acetone extracts of cells of H37Rv and of Mycobacterium bovis strain BCG inhibited plaque formation of mycobacteriophage DS6A and of phage D29. Suspensions of these extracts in heart infusion broth with 0.002 M calcium chloride and 1 per cent dioxane required 20 hours of incubation (activation) at 37 degrees C with stirring before they were capable of inactivating phage DS6A, but did not require activation for inhibition of phage D29. Sonication of water mixtures of these extracts for 45 sec yielded suspensions that were highly active against phage DS6A and independent of the 20-hour lag period. Such preparations were also active against phage D29. The extract prepared from cells of BCG was more active than the extract from cells of H37Rv. Water washing of chloroform solutions of each extract did not remove significant amounts of solid material or reduce the phage-inactivating activites of either extract.

Transduction of a streptomycin r-factor from mycobacterium smegmatis to mycobacterium tuberculosis H37Rv

Two forms of streptomycinresistanceinMycobacteriumtuberculosis H37Rv are described: (1) a genetically stable resistance that does not inactivate the antibiotic and (2) a genetically unstable form of resistance, transduced from M. smegmatis ATCC 607 by phage D29, which inactivates the antibiotic. The latter form of resistance, which is transducible at a high frequency from M. smegmatis, is irreversibly eliminated by ultraviolet light irradiation. The transduction of the streptomycin marker is not accompanied by the establishment of lysogeny by the transducing phage but is accompanied by the alteration in the phage pattern of the transduced cultures. The phage resistance is lost with the streptomycin marker after the cultures are exposed to ultraviolet light.

Further evidence against the concept of decreased phage neutralizing ability of serum of patients with sarcoidosis.

Serum from patients with sarcoidosis, tuberculosis, and normal persons matched with respect to age, sex, and race was tested for their neutralizing activity against mycobacteriophages D29 Leo, and Rl. It was found that the mean neutralizing activity of all three groups of serum was significantly similar when tested against phage Rl and likewise was similar against phage Leo. The mean neutralizing activity values of sarcoid and tuberculosis serum samples against phage D29 were significantly greater than that of normal serum. White females, less than 30 years of age, were discovered to have significantly less antibody to phage Leo than groups with other age, sex, and race combinations.

Neutralization of Mycobacteriophages by Sera of Patients with and without Sarcoidosis

SummaryOf 70 sera from different patients with sarcoidosis serum neutralization activity could be demonstrated in 91-98% against mycobacteriophages D29, Leo, or R1. Of 81 sera from nonsarcoid patients, neutralizing activity was demonstrated in 83-85% against phages D29 and Leo, and in 100% against phages R1. In test using phage R1, the magnitude of the K values of sera of patients not having sarcoidosis was much larger than those of patients with sarcoidosis.

Lysogeny in mycobacteria. I. Conversion of colony morphology, nitrate reductase activity, and tween 80 hydrolysis of Mycobacterium sp. ATCC 607 associated with lysogeny.

Colony changes from rough to smooth were observed in a rough strain of mycobacterium ATCC 607 after exposure to phages D29 and B4. The colonies surviving after exposure to these two phages were both smooth and lysogenic. Increased nitrate reductase and Tween 80 hydrolytic activities accompanied lysogenization. Loss of lysogeny was accompanied by conversion to the rough colony type and a decrease in nitrate reductase and Tween 80 hydrolytic activities. The rapid loss and gain of these multiple characteristics suggested that the genetic control lies in a plasmid of mycobacteria.