Detecta Mycobacterium tuberculosis en muestras de heces de niños del Instituto Nacional de Salud del Niño, mediante la técnica molecular de reacción en cadena de la polimerasa (PCR). Para esto se toman muestras biológicas de heces de 236 niños menores de 12 años de edad pertenecientes al Instituto Nacional de Salud del Niño, estas muestras son sometidas a dos métodos de extracción de ADN una denominado “FastDNA® SPIN Kit for Soil” (Qbiogene) y el otro “Chelex 100”. Este estudio demuestra que se puede estandarizar la técnica de PCR para detección de M. tuberculosis en muestras de heces, y sus posibles aplicaciones como método rápido de diagnóstico, en una población en donde la naturaleza paucibacilar de las muestras convencionales no permite la detección inmediata del bacilo. / Tesis

A l’instar des D,D-transpeptidases de la famille des protéines de liaison à la pénicilline (PLP), les L,D-transpeptidases catalysent la formation des ponts interpeptidiques du peptidoglycane. Chez un mutant de Enterococcus faecium, la totalité du peptidoglycane est synthétisée par les L,D-transpeptidases entrainant une résistance à toutes les β-lactamines à l’exception des carbapénèmes. Le peptidoglycane de Mycobacterium tuberculosis étant majoritairement synthétisé par des L,D-transpeptidases, ces enzymes sont une cible potentielle pour le développement de nouveaux traitements de la tuberculose. Les objectifs de cette thèse sont de comprendre la spécificité des L,D­transpeptidases pour les carbapénèmes et d’identifier les sites de fixation des précurseurs du peptidoglycane à ces enzymes. Les résultats montrent que l’oxyanion formé lors de l’attaque du cycle β-lactame des carbapénèmes par la cystéine active est stabilisé dans le site actif des L,D­transpeptidases. Cette stabilisation combinée à l’absence d’hydrolyse de l’acylenzyme conduisent à l’inactivation rapide, totale et irréversible des L,D­transpeptidases par les carbapénèmes. La structure de l’acylenzyme indique que ces propriétés sont indépendantes de la nature de la chaine latérale des antibiotiques qui pourrait être optimisée. La localisation de l’accepteur d’acyle dans la Poche II de Ldtfm ainsi que des interactions supplémentaires avec les chaines glycanes du peptidoglycane montrent que d’autres sites que celui ciblé par les β­lactamines, mimes du donneur d’acyle, sont des cibles pour le développement de nouveaux antibiotiques qui pourraient agir en synergie avec les β­lactamines. / L,D-transpeptidases, as D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family, catalyse the last cross-links step of peptidoglycan biosynthesis. The peptidoglycan of an Enterococcus faecium mutant is exclusively cross-linked by L,D-transpeptidases leading to resistance to all β-lactams except the carbapenems. Since peptidoglycan cross-links are predominantly synthesized by L,D-transpeptidases in Mycobacterium tuberculosis these enzymes are potential targets for chemotherapy of tuberculosis. The aims of the thesis are to identify the kinetic features that account for the specificity of L,D-transpeptidases for carbapenems and to characterise the binding sites for the peptidoglycan precursors in these enzymes. Our results show that the oxyanion resulting from nucleophilic attack of carbapebems by the catalytic cysteine is stabilized into the active site of L,D-transpeptidases. This stabilisation, combined to the absence of hydrolysis of the acylenzyme, leads to the rapid, total and irreversible inactivation of L,D-transpeptidases by carbapenems. Resolution of the acylenzyme structure shows that these kinetic features are independent from the carbapenem side chain that could be modified to optimize the antibiotics. The binding of the acyle acceptor has been identified in Pocket II of Ldtfm that is distinct from the binding site for β-lactams (Pocket I), which mimic the acyle donor. This site and additional peptidoglycan binding sites reveal additional targets for development of new antibiotics that might act in synergy with β-lactams.

Magister Pharmaceuticae - MPharm / Many studies have reported low cure rates for multidrug-resistant tuberculosis (MDRTB) patients and MDR-TB patients co-infected with human immunodeficiency virus (HIV). However, little is known about the effect of HIV infection and antiretroviral therapy on the treatment outcomes of MDR-TB in South Africa. Therefore, the objectives of the study are: to find out whether HIV infection and interactions between ARVs and second line anti-TB drugs have an impact on the following MDR-TB treatment outcomes: cure rate and treatment failure at Brewelskloof Hospital. MDR-TB patients were treated for 18-24 months. The study was designed as a case-control retrospective study comparing MDR-TB treatment outcomes between HIV positive (cases) and HIV negative patients (controls). Patients were included in the study only if they complied with the following criteria: sensitivity to second line anti-TB drugs, MDR-TB infection, co-infection with HIV (for some of them), male and female patients, completion of treatment between 1 January 2006 and 31 December 2008. Any patients that presented with extreme drug-resistant tuberculosis (XDR-TB) were excluded from the study. Data were retrospectively collected from each patient’s medical records. There were a total of 336 patients of which 242 (72%) were MDR-TB patients and 94 (27.9%) MDRTB co-infected with HIV patients. Out of the 242 MDR-TB patients, 167 (69.2%) were males and 75 (30.7%) were females. Of the 94 patients with MDR-TB co-infected with HIV, 51 (54.2%) males and 43 (45.7%) females. Patients with multidrug-resistant tuberculosis co-infected with HIV who qualify for antiretroviral therapy were treated with stavudine, lamivudine and efavirenz while all MDR-TB patients were given kanamycin, ethionamide, ofloxacin, cycloserine and pyrazinamide. The cure rate of MDR-TB in HIV (+) patients and in HIV (-) patients is 34.5% and 30 % respectively. There is no significant difference between both artes (pvalue = 0.80). The MDR-TB cure rate in HIV (+) patients taking antiretroviral drugs and in HIV (+) patients without antiretroviral therapy is 35% and 33% respectively. The difference between both rates is not statistically significant. The study shows that 65 (28.0%) patients completed MDR-TB treatment but could not be classified as cured or failure, 29 (12.5%) patients failed, 76 (32.7%) defaulted, 18 (7.7%) were transferred out and 44 (18.9%) died. As far as treatment completed and defaulted is concerned, there is no significant statistical difference between HIV (+) and HIV (-) The number of patients who failed the MDR-TB treatment and who were transferred out is significantly higher in the HIV (-) group than in the HIV (+) group. Finally the number of MDR-TB patients who died is significantly higher in the HIV (+) group). The median (range) duration of antiretroviral therapy before starting anti-tuberculosis drugs is 10.5 (1-60) months. According to this study results, the MDR-TB treatment cure rate at Brewelkloof hospital is similar to the cure rate at the national level. The study also hows that HIV infection and antiretroviral drugs do not influence any influence on MDR-TB treatment outcomes. / South Africa

Magister Scientiae - MSc / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). TB mainly affects lungs of patients but other parts of the body can also be affected. It kills approximately 2 million people annually. HIV/AIDS and drug resistance make TB difficult to control. Mtb CarD protein forms a physiological complex with Ribonucleic Acid Polymerase (RNAP). This complex causes Mtb to undergo dormancy rendering it difficult to control using current antibiotics. CarD and a size-reduced subunit β1 (denoted β1m for “minimized”) of Thermus thermophilus RNAP, in which the central domain has been replaced by a Gly-Gly linker, were produced and purified using affinity nickel nitrilotriaceticacid and glutathione-Stransferase (GST) affinity chromatography techniques respectively. CarD N terminal domain (CarDN) was generated from CarD by inserting a stop codon by site directed mutagenesis. CarD was stabilised by adding 5 % (v/v) glycerol to PBS pH 7.4 ensuring protein stability of up to 67 days rather than 2 days without glycerol. CarDN was stable in PBS pH 7.4 without addition of glycerol. This suggests that the CarD C terminal domain may be responsible for CarD instability. To further purify the proteins both anion exchange and gel permeation chromatography techniques were used. CarD and CarDN degrade immediately after anion exchange potentially because of the high ion concentration which partially unfolds the protein making it prone to proteolytic cleavage. GST-pull down assays were used to demonstrate complex formation between RNAP β1m and both CarD and CarDN confirming that complex formation is dependent on the N-terminal domain of CarD.

Philosophiae Doctor - PhD / Mycobacterium tuberculosis, the causative agent of tuberculosis, is estimated to infect approximately one-third of the world’s population and is responsible for around 2 million deaths per year. The disease is endemic in South Africa which has one of the world’s highest tuberculosis incidence and death rates. The M. tuberculosis Beijing genotype are characterised by having an enhanced virulence capability over other M. tuberculosis strains and are the predominant strain observed in the Western Cape of South Africa. DNA methylation is a largely untapped area of research in M.tuberculosis and has been poorly described in the literature especially given its connection to virulence despite it being well characterised along with its role in virulence in other pathogenic bacteria such as E.coli. The overall aim was to characterise a global DNA methylation profile for two M. tuberculosis Beijing strains, hyper-virulent and hypo-virulent, using single molecule real time sequencing data technology. Moreover, to determine if adenine methylation in promoter regions has a possible functional role. This study identified and characterised the DNA methylation profile at the single nucleotide resolution in these strains using Pacific Biosciences single molecule real time sequencing data. A computational approach was used to discern DNA methylation patterns between the hyper and hypo-virulent strains with a view of understanding virulence in the hyper-virulent strain. Methylated motifs, which belong to known Restriction Modification (RM) systems of the H37Rv referencegenome were also identified. N6-methyladenine (m6A) and N4-methlycytosine (m4C) loci were identified in both strains. m6A were idenitified in both strains occuring within the following sequence motifs CACGCAG (Type II RM system), GATNNNNRTAC/GTAYNNNNATC (Type I RM system), while the CTGGAGGA motif was found to be uniquley methylated in the hyper-virulentstrain.Interestingly, the CACGCAG motif was significantly methylated (p = 9.9 x10 -63) at a higher proportion in intergenic regions (~70%) as opposed to genic regions in both the hyper-virulent and hypo-virulent strains suggesting a role in gene regulation. There appeared to be a higher proportion of m6A occuring in intergenic regions compared to within genes for hyper-virulent (61%) and hypo-virulent (62%) strains. The genic proportion revealed that 35% of total m6A occurred uniquely within genes for the hyper-virulent strain while 27.9% for uniquely methylated genes in hypo-virulent strain.

>Magister Scientiae - MSc / Next generation sequencing (NGS) technology platforms have accelerated ability to produce completed genome assemblies. Recently, collaborators at Tygerberg Medical School outsourced the sequencing of Oryx bacillus, a member of the Mycobacterium tuberculosis complex (MTC). A total of 31,271,059 short reads were generated and required filtering, assembly and annotation using bioinformatics algorithms. In this project, an NGS assembly pipeline was implemented, tailored specifically for SOLiD sequence data. The raw reads were aligned to seven fully sequenced and annotated MTC members, namely, Mycobacterium tuberculosis H37Rv, H37Ra, CDC1551, F11, KZN 1435, Mycobacterium bovis AF2122/97 and Mycobacterium bovis BCG str. Pasteur 1173P2 using NovoalignCS. Depth and breadth of sequence coverage across each base of the reference genome was calculated using BEDTools, and structural variation. Structural variation at the nucleotide level including deletions, insertions and single nucleotidepolymorphisms (SNPs) were called using three tools, GATK, SAMtools and Nesoni. These variations were further filtered using in-house PERL scripts. Putative functional roles for the alterations at the DNA level were extrapolated from the overlap with essential genes present in annotated MTC members. Approximately 20,730,631 short reads (59.78%) out of a total of 31,271,059 reads aligned to the seven reference genomes. The per base sequence coverage calculations revealed an average of 1,243 unaligned regions. These unaligned regions overlapped with mycobacterial regions of difference (RD) and genetic phage elements acquired by the MTC through horizontal gene transfer and are genes prevalent in the clinical isolates of M. tuberculosis. A total of 2,680 genetic variations were identified and categorised into 845 synonymous and 1,724 non-synonymous SNPs together with 44 insertions and 67 deletions. Some of the variant alleles overlapped known genes to be involved in TB drug resistance. While the biological significance of our findings remain to be elucidated, it nonetheless deserves further attention, because SNPs have the potential to impact on strain phenotype by gene disruption. Therefore, any hypotheses generated from these large-scale analyses will be tested by our collaborators at Tygerberg medical school.

The isolation and antimycobacterial activity of several naphthoquinones from Euclea natalensis were previously reported and initiated this study into the occurrence, chemistry and biological activity of this class of compounds. The structure activity relationship of the isolated naphthoquinones, and commercially available derivatives were also studied. Several plant species were investigated to establish a possible link between their traditional use for chest related symptoms (including tuberculosis infection) and the occurrence of 7-methyljuglone in these plants. The plants were extracted and tested qualitatively with the use of three analytical tools for the presence of 7-methyljuglone or related naphthoquinones. Due to its commercial unavailability, the chemical synthesis of two of these naphthoquinones, 7-methyljuglone and diospyrin, was attempted with varying degrees of success. The Friedel-Crafts acylation method was used to synthesise 7-methyljuglone from m-cresol and maleic anhydride as starting material. The optimisation of the synthesis was also investigated. Through a two-step pathway of epoxidation and steam distillation, diospyrin was successfully synthesised albeit in small quantities. During the attempts to synthesise diospyrin, two other related compounds were also synthesised. These compounds, neodiospyrin and mamegakinone, are structural isomers of diospyrin. The stability of some of the naphthoquinones was tested in various carriers in an attempt to explain the influence this will have on the obtained antituberculosis and toxicity data. The BACTEC vial solution, which is widely used to determine potency against Mycobacterium tuberculosis, was analysed with HPLC to determine the stability of these compounds in it. In addition the stability in organic solvents especially DMSO, was also tested as this is the solvent of choice for hydrophobic compounds in almost all bioassays. The antituberculosis activity and/or toxicity of 7-methyljuglone was investigated with three bioassays, to broaden our knowledge on the mechanism of action of naphthoquinones. Vero cells were employed to determine the inhibitory concentration (IC50) of most of the naphthoquinones. Mice experiments were carried out to determine the toxicity of 7-methyljuglone and diospyrin in vivo. In addition the lead compound, 7-methyljuglone, was tested on Musca domestica (house fly) to establish its toxicity on this organism. In order to find the pharmacophore of this class of compounds, a preliminary structure-activity relationship was conducted. During this study the active site in the compounds which confers potency and toxicity was partly established. The mode of action of some of the naphthoquinones was investigated and it was established that the compounds might interfere with the mycobacterial electron transport chain. A fluorinated 7-methyljuglone stops the production of menaquinone which transports electrons from the NADH dehydrogenase complex to the cytochrome bc complex and effectively kills the mycobacterium. / Thesis (PhD (Botany))--University of Pretoria, 2007. / Plant Science / unrestricted

Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaC. / Mycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC.

Tuberculosis (TB) is a disease caused by the infection of Mycobacterium tuberculosis, which is progressively becoming multi-drug resistant (MDR). Understanding the mechanism by which the organism interacts with host lipids, infect macrophages and how components redistribute within the host could open the investigation of new ways of inhibiting and eradicating the infection suffered by patients world wide. Flow fluorometry of liposomes containing mycolic acids, which are â-hydroxy fatty acids with a long á-alkyl side chain of mycobacteria, may be useful to determine the dynamics of interaction of these lipids with the host membrane lipids and with cholesterol. This will increase the understanding about the structure-function relationship of mycolic acids in M.tb. It was shown in this thesis that natural mycolic acids had a unique property, it could exchange rapidly between liposomes in the presence and absence of cholesterol even at low temperatures. Rapid exchange of mycolic acids within the host could be the mechanism by which trafficking of mycobacterial lipids comes about, ultimately leading to immune response modulation beyond the infected cell. It also provides direction for future investigation to bring about new serodiagnostic tests based on lipid antigens. Although flow fluorometry as a modern technique was unable to resolve the exchange of mycolic acids in relation with other lipids, a unique property of mycolic acids was demonstrated for the first time, that of rapid exchange. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

The emergence and spread of multi-drug resistant (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) are a major medical and public problem threatening the global health. The objectives of this study were to (i) determine the prevalence of MDR-TB and XDR-TB in the Eastern Cape; (ii) analyze patterns of gene mutations in MDR-TB and (iii) identify gene mutations associated with resistance to second line injectable drugs in XDR-TB isolates. A total of 1520 routine sputum specimens sequentially received within a period of 12 months i.e. February 2012 to February 2013 from all MDR-TB and XDR-TB patients treated by Hospitals and clinics in the Eastern Cape were included in this study, of which 1004 had interpretable results. Samples were analyzed with the Genotype MTBDRplus VER 2.0 assay kit (Hain Lifescience) for detection of resistance to Rifampicin and Isoniazid while solid and liquid culture drug susceptibility tests were used for ethambutol, streptomycin, ethionamide, ofloxacin, capreomycin and amikacin. PCR and sequence analysis of short regions of target genes gyrA, (encode subunit of DNA topoisomerase gyrase), rrs (16S rRNA) and tlyA (encodes a 2’-O-methyltransferase) were performed on 20 XDR-TB isolates. MTBDRplus kit results and drug susceptibility tests identified 462 MDR-TB, 284 pre-XDR and 258 XDR-TB isolates from 267 clinics and 25 hospitals in the Eastern Cape. There was a high frequency of resistance to streptomycin, ethionamide, amikacin, ofloxacin and capreomycin. Mutation patterns indicated differences between the health districts as well as differences between the facilities within the health districts. The most common mutation patterns observed were: (i) ΔWT3, ΔWT4, MUT1 [D516V+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG), ΔWT1 [C15T] (inhA) [39 MDR, 204 XDR-TB and 214 pre XDR-TB isolates], (ii) ΔWT8, MUT3 [L533P+S531L] (rpoB), ΔWT, MUT1 [S315T1] [145 MDR, 18 pre-XDR and 3 XDR-TB solates] and (iii) ΔWT3, WT4 [D516Y+del515] (rpoB), ΔWT, MUT1 [S315T1] (katG) [75 MDR, 1 pre-XDR and 7 XDR-TB isolates]. Mutations in inhA promoter regions were strongly associated with XDR-TB isolates. Two thirds (66.6 percent (669/1004) of the isolates had inhA mutations present with 25.4 percent (170/669) found among the MDR isolates, 39.2 percent (262/669) among the pre-XDR isolates and 35.4 percent (237/669) among the XDR-TB isolates, which implies that these resistant isolates are being spread by transmission within the community and circulating in the province. There was good correlation between XDR-TB drug susceptibility test results and sequence analyses of the gyrA and rrs genes. The majority of XDR-TB isolates contained mutations at positions C269T (6/20) and 1401G (18/20) in gyrA and rrs genes respectively. Sequence analysis of short regions of gyrA and rrs genes may be useful for detection of fluoroquinolone and amikacin/ kanamycin resistance in XDR-TB isolates but the tlyA gene is not a sensitive genetic marker for capreomycin resistance. This study highlighted the urgent need for the development of rapid diagnostics for XDR-TB and raised serious concerns regarding ineffective patientmanagement resulting in ongoing transmission of extremely resistant strains of XDRTB in the Eastern Cape suggesting that the Eastern Cape could be fast becoming the epicenter for the development of Totally Drug-resistant Tuberculosis (TDR-TB) in South Africa.