Differential growth characteristics and streptomycin susceptibility of virulent and avirulent Mycobacterium tuberculosis strains in a novel fibroblast-mycobacterium microcolony assay.
The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 microgram/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 microgram/ml. Growth of all strains was similarly inhibited by 1.0 microgram of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.
Byrd
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TF
Division of Infectious Diseases, Department of Medicine, Albuquerque Veterans Affairs Medical Center, The University of New Mexico School of Medicine, Albuquerque, New Mexico 87108, USA. tfbyrd@pol.net
Green
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GM
Fowlston
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Lyons
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D002460:Cell Line; D018920:Coculture Techniques / Q000379:methods; D015169:Colony Count, Microbial / Q000379:methods; D005110:Extracellular Space / Q000382:microbiology; D005347:Fibroblasts / Q000382:microbiology; D006801:Humans; D007424:Intracellular Fluid / Q000382:microbiology; D009169:Mycobacterium tuberculosis / Q000254:growth & development* / Q000472:pathogenicity*; D013307:Streptomycin / Q000494:pharmacology; D014774:Virulence / Q000187:drug effects
D013307:Streptomycin