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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9606 |
| Date | January 2013 |
| Creators | Ashiru, Olubisi T. |
| Contributors | Sturm, A. Willem. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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