Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2016. / The reference phenotypic methods for Mycobacterium tuberculosis drug susceptibility testing are qualitative and based on drug critical concentrations. Limitations include lack of standardization and variations in laboratory preparation of drug stock solutions. The recommended critical concentrations are determined by consensus and experience rather than scientific data. Consequently incorrect and inadequate susceptibility breakpoints are used and patients receive ineffective antimicrobial therapy. The determination of wild-type minimal inhibitory concentration distribution is an important tool used by European Committee for antimicrobial susceptibility Testing (EUCAST) to establish clinical breakpoints in Europe. This could be applicable in South Africa. Aim To determine wild-type minimal inhibitory concentration distributions of first and secondline drugs against Mycobacterium tuberculosis complex clinical isolates and compare these with the recommended critical concentration in Limpopo province. Methods A sample of 101 Mycobacterium tuberculosis complex positive cultures were collected from National Health Laboratory Services in Polokwane (Limpopo province) and subcultured on BACTEC MGIT 960 system. The isolates were inoculated on MYCOTB MIC plates to determine the wild-type MIC distributions of first and second-line drugs. The data were compared with currently recommended critical concentrations. DNA was extracted and amplified by PCR. Genotypic drug susceptibility testing was performed using GenoType MTBDRplus version 2.0 and GenoType MTBDRsl version 2.0 for the first- and second-line drugs, respectively. Genotyping of clinical isolates was performed to determine M. tuberculosis strain families using spoligotyping. vi Results Wild-type MIC distributions range reported in this study are as follows rifampin (≤ 0.12 - 0.5 μg/μg/ml), isoniazid (≤ 0.3 - 2.00 μg/ml), rifabutin (≤ 0.12 - 0.25 μg/ml), ethionamide (≤ 0.12 - 5 μg/ml), ethambutol (≤ 0.5 - 2 μg/ml), streptomycin (≤ 0.25 - 0.5 μg/ml), paraaminosalicylic (≤ 0.5 - 4.0 μg/ml), cycloserine (≤ 2 -16 μg/ml), amikacin (≤ 0.12 - 0.5 μg/ml), kanamycin (≤ 0.6 -2.5 μg/ml), moxifloxacin (≤ 0.6 - 0.5 μg/ml), ofloxacin (≤ 0.25 - 1 μg/ml). GenoType MTBDRplus detected (n= 68, 67%) rifampin resistance (MUT 3=26, MUT 2=18, MUT 2B=8) on the rpoB gene. Isoniazid resistant (n=20, 19.8%) was detected katG MUT (n=20, 19.8%) on katG gene (S315T1). Genotypic resistance to second-line drugs determined by GenoType MTBRsl detected no mutations in (n= 98, 97%) isolates on gyrA, gyrB rrs and eis gene and (n=3, 2.9%) isolates non mycobacterium tuberculosis complex were detected. The frequency and percentage of Mycobacterium tuberculosis family strain were identified in (n= 81, 80%) of the clinical isolates which matched 18 pre-existing shared types. The results showed high genotype diversity with the Beijing strain (n= 30, 29.7%) and T family (n= 19, 18.8%) dominating. Twenty isolates (19.8%) had no shared types thus reported as orphan. Conclusion The findings obtained in this study suggest wild-type Minimal Inhibitory Concentration distributions may be considered when setting clinical breakpoints. Discordant results were observed between phenotypic and genotypic DST for rifampin, isoniazid, streptomycin, rifabutin and ethambutol, suggesting that breakpoint concentrations for some drugs are set too high while others are too low. The Mycobacterium tuberculosis clinical isolates displayed diverse family strain with Beijing and T strain predominate breakpoints for first-line and second-line drugs used in Mycobacterium tuberculosis treatments. Poster Presentations Poster presented at faculty of Health science first annual research day on Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using MYCOTB MIC plate. University of Limpopo Tiro hall 16th to 17th September 2014. Poster presented at National Health Laboratory Service Pathology Research and Development Congress (PathReD) on Determination of families strains of Mycobacterium tuberculosis circulating in Limpopo Province, South Africa. Emperors Palace 14th April-16th April 2015.

by Carrie Au-Yeang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 45-54). / Abstract also in Chinese. / Chapter I. --- Abstract --- p.iii / Chapter II. --- Acknowledgements --- p.iv / Chapter III. --- Table of Contents --- p.v / Chapter IV. --- List of Abbreviations --- p.vii / Chapter V. --- List of Figures --- p.viii / Chapter VI. --- List of Tables --- p.ix / Chapter VII. --- Introduction --- p.1 / Chapter VIII. --- Literature Review --- p.3 / Chapter A. --- Mycobacterium tuberculosis Infections - clinical importance --- p.3 / Chapter B. --- Treatment of M. tuberculosis Infections - the past & present --- p.3 / Chapter C. --- Laboratory Supports for Treatment of Tuberculosis --- p.5 / Chapter D. --- The Postantibiotic Effects (PAE) --- p.6 / Chapter E. --- PAE of Antituberculosis Drugs Against Mycobacteria --- p.15 / Chapter F. --- Radiometric Measurement of growth --- p.16 / Chapter IX. --- Materials and Methods --- p.17 / Chapter A. --- Bacterial Strains and Their Maintenance --- p.17 / Chapter B. --- Antimicrobial Agents --- p.17 / Chapter C. --- Antimicrobial Susceptibility Testing --- p.18 / Chapter D. --- Assessment of PAE and KI In Vitro --- p.20 / Chapter E. --- Assessment of PAE and KI Ex Vivo --- p.22 / Chapter F. --- Determination of Drug Uptake --- p.25 / Chapter X. --- Results --- p.29 / Chapter A. --- In Vitro Susceptibility Testing of the M. tuberculosis Strains --- p.29 / Chapter B. --- PAE In Vitro - the Classical Viable Count Method --- p.29 / Chapter C. --- PAE Measured by the Bactec Method --- p.30 / Chapter D. --- PAE In Vitro - the Bactec Method --- p.30 / Chapter E. --- Postantibiotic Effects Ex Vivo by the Bactec Method --- p.31 / Chapter F. --- Bactericidal Activities In Vitro and Ex Vivo --- p.32 / Chapter XI. --- Discussion --- p.34 / Chapter A. --- Selection of M. tuberculosis isolates and Drug Susceptibility --- p.34 / Chapter B. --- PAE and KI In Vitro & Ex Vivo - the study methods --- p.34 / Chapter C. --- PAE In Vitro & Ex Vivo - single drug --- p.36 / Chapter D. --- PAE Ex Vivo - drug combinations --- p.38 / Chapter E. --- KI In vitro & Ex vivo --- p.39 / Chapter F. --- PAE and Clinical Therapeutic Regimens --- p.41 / Chapter G. --- Conclusion & Future Studies --- p.44 / Chapter XII. --- Literature Cited --- p.45 / Chapter XIII. --- Figures --- p.55 / Chapter IVX --- Tables --- p.68

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and poses a global threat to human health. A third of the world’s population is infected with Mtb. Multi-drug resistant and extensively drug resistant Mtb strains are widespread and development of new drugs is urgently needed to treat drug resistant TB. This thesis focuses on the Mtb cytochrome P450 (P450) enzymes CYP126A1 and CYP143A1. P450s are heme-binding enzymes that catalyse activation of molecular oxygen and the oxidation of substrates bound close to the heme. CYP126A1 and CYP143A1 are “orphans” in terms of their functional characterization, but potential drug targets in view of ability of azole-based P450 inhibitors to inhibit growth and viability of Mtb. The CYP126A1 and CYP143A1 genes were cloned and expressed in Escherichia coli. Expression conditions and strains were optimised to maximise soluble protein production and methods were developed to purify the P450s using affinity, ion exchange and size exclusion chromatography. Both P450s were shown to bind heme b, and heme was shown to be axially coordinated by a cysteine thiolate and a water molecule in both cases using UV-visible and electron paramagnetic resonance (EPR) spectroscopy. Both P450s bound carbon monoxide (CO) in their reduced forms to produce heme Fe2+-CO complexes with absorption maxima at ~450 nm – characteristic of P450s. CYP126A1 and CYP143A1 bound avidly to a range of inhibitors, including several azole drugs. As examples, binding constant (Kd) values of 13.8 µM and 21.9 µM were determined for clotrimazole and econazole with CYP143A1; while ketoconazole bound CYP126A1 with a Kd of 0.20 µM. Each of these drugs is very effective in inhibiting Mtb growth. EPR confirmed inhibitory coordination of both P450s by azole drug nitrogen atoms; though indirect coordination via a retained axial water ligand may also occur in some cases. Extinction coefficients were determined as έ420 = 125 mM-1 cm-1 (CYP126A1) and έ415 = 105 mM-1 cm-1 (CYP143A1). CYP126A1’s heme iron redox potential was shown to be unusually positive (E°’ = -80 mV). Light scattering studies showed CYP126A1 to be a monodisperse, monomeric protein. CYP143A1 is also mainly a monomer, but with a small proportion of an oligomeric form. Despite its polydispersity, CYP143A1 was crystallized and its structure solved by X-ray diffraction to a resolution of 1.9 Å, using molecular replacement with the Mtb P450 CYP142A1. A limited compound screen of typical P450 substrates failed to provide “hits” to identify CYP143A1 substrate selectivity, but the presence of polyethylene glycol in the CYP143A1 active site in crystals suggests fatty acids as potential substrates. CYP126A1 was crystallized for studies to identify binding modes of small molecules (“fragments”) identified to interact with CYP126A1 by NMR. Crystal structures of CYP126A1 in complex with two such fragments (NMR401 and NMR343) were determined to ~2.0 Å resolution in ongoing research to build Mtb P450 isoform-specific inhibitors. Compounds identified as CYP126A1 substrates/inhibitors identified by high-throughput screening were validated by UV-visible titrations with the P450, and binding modes and affinity established. In conclusion, this thesis provides novel insights into the biochemical, biophysical and structural properties of two novel Mtb P450s that are potential targets for new anti-TB drugs.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis (MScMedSc)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: In this thesis, research results are reported on the role of dipalmitoyl phosphatidyl choline (DPPC) and DPPC-liposomes on the in vitro permeability characteristics of various antimycobacterial drugs across porcine bronchial tissue. The permeability flux values of the different compounds (isoniazid, ofloxacin and moxifloxacin) and their relevant DPPC formulations were determined using a continuous flow through perfusion system. Mean steady state flux values were compared statistically by means of a t-test at a significance level of 5% as well as an F-test using whole curve comparisons. The results indicated that the different formulations of drug and their DPPC combinations retard the permeation of drug through bronchial tissue. However, moxifloxacin permeation was significantly enhanced when in a DPPC-liposomal formulation. These results demonstrate the important role that molecular weight, electrostatic charge, partitioning of the molecules in DPPC and DPPC-liposomes play in transmembrane diffusion. In addition, the effect of individual drugs and their DPPC combinations on the surface tension lowering property of DPPC was evaluated. The results obtained showed minimal decreases in the surface tension lowering capability of DPPC; however, the minimal increases in surface tension do not alter the integrity of DPPC to a large extent. Drug susceptibility testing of Mycobacterium tuberculosis cultures against the individual antitubercular drugs and their DPPC combinations was done by using the Radiometric BACTEC 460TB™ system. Drug-entrapped DPPC liposomes were tested at concentrations comparable to their relative minimum inhibitory concentrations (MIC). The results for the BACTEC assay indicated that the mycobacteria were susceptible to the developed drug entrapped liposomes; of which their encapsulation efficiencies for the relevant drugs were approximately ± 50%. It was concluded that drug-entrapped DPPC liposomes could fulfill the dual role of pulmonary drug delivery and alveolar stabilization due to antiatelectatic effect of DPPC which can improve the distribution of anti-tubercular drugs in the lung / AFRIKAANSE OPSOMMING: Hierdie tesis doen verslag oor navorsingsresultate met betrekking tot die rol van dipalmitoïel-fosfatidiel-cholien (DPPC) en DPPC-liposome in die in vitro-permeasiekenmerke van verskeie antimikobakteriese middels oor vark- brongiale weefsel. Die permeasievloedwaardes van die verskillende verbindings (isoniasied, ofloksasien en moksifloksasien) en hul betrokke DPPC-formules is met behulp van ’n deurlopende-deurvloei-perfusiestelsel bepaal. Gemiddelde vloedwaardes in ’n bestendige staat is statisties vergelyk met behulp van ’n t-toets op ’n beduidendheidsvlak van 5%, sowel as ’n F-toets met behulp van heelkurwevergelykings. Die resultate dui daarop dat die verskillende middelformules en hul DPPC-kombinasies middelpermeasie oor brongiale weefsel vertraag. Tog is die permeasie van moksifloksasien aansienlik versterk in ’n DPPC-liposomale formule. Hierdie resultate bevestig die belangrike rol van molekulêre gewig, elektrostatiese lading, die verdeling van molekules in DPPC sowel as DPPC-liposome in transmembraandiffusie. Daarbenewens is die uitwerking van individuele middels en hul DPPC-kombinasies op die oppervlakspanningsverligtingsvermoë van DPPC beoordeel. Die resultate toon minimale afnames in die oppervlakspanningsverligtingsvermoë van DPPC. Die minimale toenames in oppervlakspanning het egter meestal geen noemenswaardige effek op die integriteit van DPPC gehad nie. Voorts is die vatbaarheid van Mycobacterium tuberculosis-kwekings vir die individuele anti-tuberkulêre middels en hul DPPC-kombinasies met behulp van die radiometriese BACTEC 460TB™-stelsel getoets. Middel-ingeslote DPPC-liposome is getoets in konsentrasies wat met hul relatiewe minimum inhibisiekonsentrasies (MIK) vergelyk kan word. Die resultate van die BACTEC-toets toon dat die mikobakterieë vatbaar was vir die ontwikkelde middel-ingeslote liposome, met ’n enkapsuleringsdoeltreffendheid van ongeveer 50% vir die betrokke middels. Die studie kom tot die gevolgtrekking dat middel-ingeslote DPPC-liposome die dubbele rol van pulmonêre middel-lewering en alveolêre stabilisering kan vervul weens die anti-atelektatiese werking van DPPC, wat die verspreiding van anti-tuberkulêre middels in die long kan verbeter.