Year: 2013
Type: dissertation
Abstract: Background \nMycobacterium tuberculosis isolates such as the Beijing and F15/LAM4/KZN families \ndominate in patients. The emergence of extensively drug resistant (XDR) M. tuberculosis \nisolates raises concern. The need to better understand the pathogenesis of M. tuberculosis \nisolates resulted in this work. \nMethods \nM. tuberculosis clinical isolates that belonged to the Beijing and F15/LAM4/KZN \nfamilies, isolates with unique DNA fingerprints and laboratory strains were used. Isolates \nwere grown in the presence of oxygen and then exposed to A549 alveolar and BBM \nbronchial epithelial cells. The number of bacilli that adhered to the epithelial cells were \nviewed and counted using light microscopy. Isolates grown in the presence of oxygen and \nunder oxygen deprivation were used for subsequent assays. Invasion of A549 and BBM \ncells by isolates grown under these different circumstances was investigated. Based on \nthe results, the remaining assays were performed with A549 cells only. Cytotoxicity was \nquantified using the Cyto Tox96 Non-Radioactive Cytotoxicity Assay kit. \nMorphological changes in A549 cells after exposure to the isolates were observed using \nthe scanning electron microscopy (SEM). Real-time quantitative PCR was performed to \nassess the relative expression levels of four genes potentially associated with virulence \n(hbhA; mdp1; fdxA; hspX). Results were normalized against 16S rRNA and ftsZ gene \ntranscription and reported as fold difference as compared to H37Rv. \nResults \nAll isolates adhered to and invaded A549 cells in significantly higher numbers than BBM \ncells (P<0.0029). Isolates grown under oxygen deprivation displayed higher levels of \nvirulence than their aerobic phenotype. Grouped together, the isolates belonging to the \nBeijing and F15/LAM4/KZN families of strains showed greater adhesion capacity (28%) \nthan isolates with unique DNA fingerprints (5%) (P<0.05%). Three F15/LAM4/KZN \nisolates (two XDR-variants), were at least twice as invasive (>33%) as the most invasive \nBeijing isolate (15%) (P<0.05). The highest cytotoxicity level (35.7%) was produced by \nan XDR-F15/LAM4/KZN strain. SEM revealed bleb-like structures on bacterial cells \ngrown under oxygen deprivation. Beijing and XDR-F15/LAM4/KZN isolates had the \nhighest number of projections (16+5 per bacillus. The expression levels of all four genes \nwere highest in Beijing and F15/LAM4/KZN isolates grown under oxygen deprivation \nand exposed to A549 cells. \nConclusions \nBeijing and F15/LAM4/KZN strains are more virulent and their successful spread might \nbe related to their interaction with alveolar epithelium. M. tuberculosis pathogenesis \nstudies should include isolates grown under oxygen deprivation.
Author: Olubisi T. Ashiru
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1526859542