Please read the abstract in the front section of this document / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2005. / Medical Microbiology / unrestricted

Rapidly diagnosing the precise drug resistance present in sepsis-inducing bacteria is a continuing need to maintain the efficacy of our medical systems. Diagnostics currently being developed for such scenarios are either sensitive or rapid, but not both. Sequence-specific single DNA molecule analysis could fill this gap if it could be adapted to work on smaller targets, similar to those produced by classical biological methods. In this work, I demonstrate that immobilized ssDNA in the appropriate hybridization buffer can rapidly pull its complementary sequence out of solution. I also demonstrate that such systems in a microfluidic chip can be used to capture bacterial plasmids as a step toward subsequent multiplexed analysis. Finally, I demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be modified with multiple fluorophores and used to label captured targets in a sequence-specific manner. This system shows that it is possible to label bacterial plasmids in a manner that can bridge the technological gap between single molecule counting and small oligonucleotide targets. Such a system can achieve lower limits of detection for clinically relevant samples while maintaining rapid processing times.

The emergence of drug resistance is a serious threat to the long-term virologic success and durability of HIV-1 therapy. Adherence has been shown to be a major determinant of drug resistance; however, each pharmacologic class of antiretroviral drugs has a unique adherence–resistance relationship. We develop an immunological model of the HIV-1 infected human immune system that integrates the unique mechanisms of action of reverse transcriptase and protease inhibiting drugs. A system of impulsive differential equations is used to examine the drug kinetics within CD4⁺ T cells. Stability analysis was preformed to determine the long-term dynamics of the model. Using the endpoints of an impulsive periodic orbit in the drug levels, the maximal length of a drug holiday while avoiding drug resistance is theoretically determined; the minimum number of doses that must be subsequently taken to return to pre-interruption drug levels is also established. Heterogeneity in inter-individual differences on drug-holiday length is explored using sensitivity analysis based on Latin Hypercube Sampling and Partial Rank Correlation Coefficient analysis. Extremely short drug holidays are acceptable, as long as they are followed by a period of strict adherence. Numerical simulations demonstrate that if the drug holiday exceeds these recommendations, the cost in virologic rebound is unacceptably high. These theoretical predictions are in line with clinical results and may also help form the basis of future clinical trials.

Tuberculosis is a major public health problem in Ethiopia. The aims of this study were (i) to investigate the recovery rate of M. tuberculosis from smear positive single morning sputum specimens subjected to long-term storage at -20°C, (ii) to assess the level and risk factors for first- and second-line anti-TB drug resistance, (iii) to evaluate the performance of the GenoType®MTBDRplus and GenoType®MTBDRsl assays for drug susceptibility testing compared to the BacT/ALERT 3D system as reference method, (iv) to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among M. tuberculosis isolates, and (v) to study the population structure and transmission dynamics of M. tuberculosis isolates from patients in Amhara region, Northwest Ethiopia. The median specimen storage time was 132 days. Of 319 specimens, 90.0% were culture positive. The length of time of sputum storage had no significant effect on the recovery rate of M. tuberculosis. Of 260 M. tuberculosis isolates, 15.8% were resistant to at least one first-line drug, 5.0% were multidrug resistant (MDR) and 3.5% were resistant to all first-line drugs. Any resistance to INH, RMP, streptomycin (STM), EMB and pyrazinamide (PZA) was 13.8%, 5.8%, 10.0%, 7.3% and 4.6%, respectively. All isolates were susceptible to second-line drugs. The GenoType®MTBDRplus assay had a sensitivity of 92% and specificity of 99% to detect INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType®MTBDRsl assay had a sensitivity of 42% and specificity of 100% to detect EMB resistance. According to the molecular methods, mutations conferring resistance to INH, RMP, or EMB were detected in 13.5%, 5.8%, and 3.1% of the isolates, respectively, while mutation conferring MDR was present in 5.0% of the isolates. Of 244 M. tuberculosis isolates, 59.0% were classified as known lineages; Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%) lineage. Interestingly, 31.6% of the isolates were grouped in to four previously undefined phylogenetic lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating high rate of recent transmission. Similarly, 66.7% of MDR strains were grouped in clusters.

Antimonpräparate sind seit über 60 Jahren der Standard zur Behandlung der Leishmaniose. Immer häufiger kommt es jedoch zum Therapieversagen durch resistente Erreger. In Indien sprechen über 60 % der erstmalig mit Antimonpräparaten behandelten Patienten nicht mehr auf die Therapie an (Sundar et al., 2000). Obwohl dies ein großes Problem darstellt, ist bisher wenig über die Resistenzmechanismen der Parasiten bekannt. Durch die Aufklärung dieser Mechanismen könnten zwei Hauptziele erreicht werden: i) Es könnten optimierte Medikamente entwickelt werden, die die Resistenzmechanismen der Parasiten umgehen. ii) Es könnten diagnostische Maßnahmen ergriffen werden, um vor Beginn einer Therapie deren Erfolgschancen zu kalkulieren. So würde man dem Patienten die starken Nebenwirkungen sowie die Kosten der Antimontherapie ersparen. Desweiteren könnte sofort mit einer wirkungsvollen Therapie begonnen und somit die Zeitspanne verkürzt werden, in der der infizierte Patient ein Reservoir für die weitere Transmission der Parasiten darstellt. In Vorarbeiten wurde durch genetische Komplementation das Gen LbrM20_V2.0210 (Lbr_0210) vorläufig identifiziert, das bei Überexpression Antimonresistenz vermittelt (Dissertation A. Nuehs, 2010). Diese Arbeiten wurden mit Leishmania braziliensis durchgeführt. Direkt benachbart befindet sich ein strukturell sehr ähnliches Gen, LbrM20_V2.0200 (Lbr_0200). Beide Gene wurden bei den vorangegangenen Sb(III)-Selektionen untersucht. Hierbei konnte ausschließlich Lbr_0210 als resistenzvermittelnd identifiziert werden. Datenbankrecherchen ergaben, dass es zu Lbr_0210 je ein direktes orthologes Gen in Leishmania infantum und Leishmania major gibt. Das Ziel des ersten Teils dieser Doktorarbeit war es, die resistenzvermittelnde Funktion des zu Lbr_0210 orthologen Gens aus L. infantum, LinJ34.0220, in unterschiedlichen Leishmania-Spezies zu verifizieren. Es war vor allem wichtig die Frage zu beantworten, ob das Gen auch im pathogenen Stadium des Parasiten, also in intrazellulären Amastigoten, Resistenz gegenüber Pentostam®, einem Standardmedikament, vermittelt. Im zweiten Teil dieser Arbeit sollte das Protein strukturell und zellbiologisch charakterisiert werden, um Hinweise auf den Resistenzmechanismus zu erhalten. Durch den Vergleich mit dem zu Lb_0200 orthologen Gen aus L. infantum, LinJ34.0210, sollten Hinweise auf die unterschiedlichen Aufgaben der Proteine gesammelt werden.

No description available.

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

Mycobacterium tuberculosis stably adapts to respiratory limited environments by entering into a nongrowing but metabolically active state termed quiescence. This state is inherently tolerant to antibiotics due to a reduction in growth and activity of associated biosynthetic pathways. Understanding the physiology of the quiescent state, therefore, may be useful in developing new strategies to improve drug efficiency. Here, we used an established in vitro model of respiratory stress, hypoxia, to induce quiescence. We utilized metabolomic and genetic approaches to identify essential and active pathways associated with nongrowth. Our metabolomic profile of hypoxic M. tuberculosis revealed an increase in several free fatty acids, metabolite intermediates in the oxidative pathway of the tricarboxylic acid (TCA) cycle, as well as, the important chemical messenger, cAMP. In tandem, a high-throughput transposon mutant library screen (TnSeq) revealed that a cAMP-regulated protein acetyltransferase, MtPat, was conditionally essential for survival in the hypoxic state. Via 13C-carbon flux tracing we show an MtPat mutant is deficient in re-routing hypoxic metabolism away from the oxidative TCA cycle and that MtPat is involved in inhibiting fatty-acid catabolism in hypoxia. Additionally, we show that reductive TCA metabolism is required for survival of hypoxia by depletion of an essential TCA enzyme, malate dehydrogenase (Mdh) both in in vitro hypoxia and in vivo mouse infection. Inhibition of Mdh with a novel compound resulted in a significantly greater killing efficiency than the first-line anti-M. tuberculosis drug isoniazid (INH). In conclusion, we show that understanding the physiology of the quiescent state can lead to new drug targets for M. tuberculosis.

No description available.

Methicillin-resistant Staphylococcus Aureus (MRSA) is now the most challenging bacterial pathogen affecting patients in hospitals and in care centers, and has brought on the need to develop new drugs for MRSA. This thesis centers on studies of N-thiolated β-lactams, a new family of potent antibacterial compounds that selectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Chapter 1 describes MRSA in more detail. Chapter 2 outlines experiments on the effect of a fatty ester group (CO2R) on the C4-phenyl ring of N-methylthio β-lactams, expecting that attachment of long chain ester moieties might increase the hydrophobicity, and thus enhance the drugs ability to penetrate through the cell membrane. However, the results indicate that antibacterial activity drops off rapidly when more than seven carbon atoms are in the chain. These results led to the idea about examining a β-lactam conjugated polymer as a possible pro-drug delivery method, which is the focus of Chapter 3. To synthesize the initial drug-polymer candidate, microemulsion polymerization of an acrylate-substituted lactam was done in aqueous solution to form hydrophilic polymeric nanoparticles containing the highly water-insoluble solid antibiotic, N-methylthio fO-lactam. This method has advantages over the conventional emulsion polymerization methods because a solid co-monomer (β-lactam drug) can be utilized. SEM studies show that these polymeric nanoparticles have a microspherical morphology with nano-sizes of 40-150 nm. The N-thiolated fO-lactam containing nanoparticles display potent anti-MRSA activity at much lower drug amounts compared with free lactam drug, penicillin G or vancomycin. Although at this time the relationship between particle size and activity is not clear and the mode of action is unknown, the Nthiolated β-lactam containing nanoparticles dramatically enhance bioactivity, possibly due to increased bioavailability of the antibiotic via endocytosis. In chapter 4, Fluorescence-active emulsified nanoparticles containing naphthyl or anthracenyl side chains were also successfully prepared by microemulsion polymerization for possible use in fluorescence studies to determine if the drug enters the cell of MRSA through endocytosis, and where possible bioaccumulation site are located.