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Microbiology

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Browsing Microbiology by Author "Ashiru, Olubisi T."

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    Interaction between Mycobacterium tuberculosis and pulmonary epithelium.
    (2013) Ashiru, Olubisi T.; Sturm, Adriaan Willem.
    Background Mycobacterium tuberculosis isolates such as the Beijing and F15/LAM4/KZN families dominate in patients. The emergence of extensively drug resistant (XDR) M. tuberculosis isolates raises concern. The need to better understand the pathogenesis of M. tuberculosis isolates resulted in this work. Methods M. tuberculosis clinical isolates that belonged to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and laboratory strains were used. Isolates were grown in the presence of oxygen and then exposed to A549 alveolar and BBM bronchial epithelial cells. The number of bacilli that adhered to the epithelial cells were viewed and counted using light microscopy. Isolates grown in the presence of oxygen and under oxygen deprivation were used for subsequent assays. Invasion of A549 and BBM cells by isolates grown under these different circumstances was investigated. Based on the results, the remaining assays were performed with A549 cells only. Cytotoxicity was quantified using the Cyto Tox96 Non-Radioactive Cytotoxicity Assay kit. Morphological changes in A549 cells after exposure to the isolates were observed using the scanning electron microscopy (SEM). Real-time quantitative PCR was performed to assess the relative expression levels of four genes potentially associated with virulence (hbhA; mdp1; fdxA; hspX). Results were normalized against 16S rRNA and ftsZ gene transcription and reported as fold difference as compared to H37Rv. Results All isolates adhered to and invaded A549 cells in significantly higher numbers than BBM cells (P<0.0029). Isolates grown under oxygen deprivation displayed higher levels of virulence than their aerobic phenotype. Grouped together, the isolates belonging to the Beijing and F15/LAM4/KZN families of strains showed greater adhesion capacity (28%) than isolates with unique DNA fingerprints (5%) (P<0.05%). Three F15/LAM4/KZN isolates (two XDR-variants), were at least twice as invasive (>33%) as the most invasive Beijing isolate (15%) (P<0.05). The highest cytotoxicity level (35.7%) was produced by an XDR-F15/LAM4/KZN strain. SEM revealed bleb-like structures on bacterial cells grown under oxygen deprivation. Beijing and XDR-F15/LAM4/KZN isolates had the highest number of projections (16+5 per bacillus. The expression levels of all four genes were highest in Beijing and F15/LAM4/KZN isolates grown under oxygen deprivation and exposed to A549 cells. Conclusions Beijing and F15/LAM4/KZN strains are more virulent and their successful spread might be related to their interaction with alveolar epithelium. M. tuberculosis pathogenesis studies should include isolates grown under oxygen deprivation.