Molecular characterization of clinical isolates of Mycobacterium tuberculosis and their association with phenotypic virulence in human macrophages

Abstract

Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.

FIG. 1.

Distribution of IS6110 copy numbers among 125 strains of M. tuberculosis.

FIG. 2.

IS6110 RFLP patterns of M. tuberculosis isolates. Lanes M contain the digested products of the digoxigenin II molecular marker. (A) Lanes 1 to 8 contain representative isolates with IS6110 RFLP patterns belonging to the Beijing family. (B) Lanes 1 to 8 contain representative isolates with IS6110 RFLP patterns belonging to a non-Beijing family. Lanes 1, 2, and 6 contain CSF isolates (H107, H108, and H112, respectively).

FIG. 3.

Growth indexes reflecting the multiplication of M. tuberculosis organisms inside human macrophages. H107, H108, and H112 are hypervirulent strains, and H37Rv exhibited a growth index of >1. H37Ra and other strains (65 pulmonary and 57 extrapulmonary isolates) exhibited growth indexes of <1. Each strain was tested with three batches of human macrophages. The data represent means ± standard deviations.

FIG. 4.

Mean levels of TNF-α excreted by activated macrophages challenged with M. tuberculosis after 24 h (n = 2).

FIG. 5.

TNF-α gene expression (mean values) in macrophages challenged with M. tuberculosis (n = 2).