Mycobacterium tuberculosis strains of the modern sublineage of the Beijing family are more likely to display increased virulence than strains of the ancient sublineage

Abstract

Strains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria.

FIG 1

Genetic polymorphisms determining phylogeny of East Asian/Beijing lineage of M. tuberculosis respective to isolates included in this study.

FIG 2

Survival of mice after infection with M. tuberculosis Beijing strains of modern and ancient sublineages. C57BL/6 mice were i.t. infected with low doses (10² CFU) (A) and high doses (2.5 × 10³ CFU) (B) of each strain and observed for 320 days and 150 days, respectively. The data were obtained in three independent experiments with 10 to 15 mice in each group. Kaplan-Meier curves and log rank test were used to evaluate statistical significance. Statistically significant differences between each group infected with the individual clinical isolate and the group infected with strain H37Rv are presented. Three different patterns of animal survival are indicated (pattern I, II, and III).

FIG 3

Bacterial growth in the lungs of mice infected with M. tuberculosis Beijing strains of modern and ancient sublineages. C57BL/6 mice were i.t. inoculated with 10² bacilli of each strain, and lungs were examined for the bacterial growth by CFU test. (A) Bacterial burdens in the lungs determined 28 days p.i. Each symbol represents the value for an individual mouse. (B) Bacterial curves within 120 days p.i. Results of at least three experiments (three mice in each group at each time point) are expressed logarithmically as the mean log₁₀ CFU ± standard deviation (SD) (error bars). Mean values that were significantly different from the mean value of the group infected by H37Rv strain are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Macropathological changes in the lungs of M. tuberculosis-infected mice. C57BL/6 mice were i.t. infected with 10² bacilli of each strain, and lungs were examined 28 days p.i. (A) Representative macroscopic lung images demonstrating gross pathology, observed as numerous giant inflammatory lesions (white nodes of different size). (B and C) Lung weights (B) and numbers of cells obtained from lungs after mechanical and enzymatic tissue disruption (C). Results of at least three experiments (three mice in each group in each experiment). Data represent means plus standard deviations (SD) (error bars). Significant differences were determined between the groups of pattern III strains (black columns), pattern II strains (gray columns), and pattern I strains (white columns). Values that are significantly different for the different groups of strains (groups with different patterns) are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Histopathological changes in the lungs of M. tuberculosis-infected mice. The lungs were obtained from infected mice as described in the legend to Fig. 3. (A) Representative hematoxylin-and-eosin-stained lung sections, demonstrating three main pathological patterns. Bars, 200 μm. (B) Representative lung sections stained by Ziehl-Neelsen method. In pattern I, induced by the strains H37Rv and M442, small and medium-sized peribronchial and perivascular granulomas, consisting of macrophages and lymphocytes, are observed. Small numbers of intracellular bacteria are seen. In pattern II, induced by strains zt264, zt272, and M467, moderate, multifocal granulomatous pneumonia is observed. Increasing numbers of intracellular bacteria are seen. In pattern III, induced by strains M299, 2172, and 1471, extensive diffuse granulomatous pneumonia with areas of necrosis and alveolitis is observed. Large numbers of intracellular and extracellular bacteria are seen. Bars, 200 μm. (C) Morphometric analysis of the inflammatory lung area. Results of two experiments (three mice in each group in each experiment). Data represent means plus SD. Significant differences were determined between the groups of pattern III strains (black columns), pattern II strains (gray columns), and pattern I strains (white columns) and indicated by asterisks as follows: **, P < 0.01; ***, P < 0.001.

FIG 6

Virulence-associated properties of the ancient and modern Beijing strains evaluated in the macrophage-based model of infection. Bone marrow-derived macrophages obtained from C57BL/6 mice were infected with different mycobacterial strains at an MOI of 1:1 and incubated for 6 days. (A) Intracellular growth of mycobacteria, measured as a fold increase in the numbers of CFU recovered from macrophages on days 0 and 6 after infection. The bacterial growth rate is presented as mean plus SD of log increases in the numbers of intracellular CFU measured in two separate experiments. (B) Induction of necrotic cell death in the macrophage cultures infected at an MOI of 10:1. LDH release from the dead cells was measured in the culture supernatants on day 5 after infection. Asterisks indicate the isolates for which the values of growth or cytotoxicity differed significantly from those of the H37Rv strain or between the indicated groups: *, P < 0.05; **, P < 0.01.