Thesis--M. Phil., University of Hong Kong. / Typewritten.

Drug efflux represents an important protection mechanism against antibiotics and environmental toxic compounds in bacteria. Efflux genes constitute from 6% to 18% of all transporters in bacterial genomes, yet their regulation, natural substrates, and physiological functions are poorly understood. Among the 20 chromosomally encoded efflux genes in Escherichia coli K-12, only the AcrAB-TolC efflux system is constitutively expressed under the ordinary laboratory growth of E. coli. To explore conditions and circumstances that trigger the expression of additional efflux genes as well as their physiological functions, I examined the expression of all 20 efflux genes under a physiologically relevant circumstance for E. coli, which is anaerobic condition in this study. I found that expression of an RND type efflux pump MdtEF is up-regulated more than 20 fold when E. coli is cultured under anaerobic conditions. Mutagenesis studies revealed that the anaerobically induced expression of mdtEF is subject to the regulation of the anaerobic global transcription factor ArcA. Direct drug efflux and tolerance assay showed that anaerobically grown E. coli cells display an increased efflux activity and enhanced drug tolerance in an MdtEF dependent manner, confirming the functional up-regulation of the efflux pump MdtEF in the anaerobic physiology of E. coli. Since the up-regulation of mdtEF by anaerobic growth occurs in the absence of antibiotics and drugs, I speculate that MdtEF has physiological functions under the anaerobic growth of E. coli. To explore this, I first compared the viability of ΔmdtEF and WT MG1655 strains and found that ΔmdtEF caused a decreased cell survival during prolonged anaerobic growth of E. coli. Interestingly, this defect became more pronounced when cells grow in the presence of 10 mM nitrate, but no defect was observed in ΔmdtEF strain when cells grow in the presence of 40 mM fumarate under the same anaerobic conditions, suggesting that MdtEF has physiological roles relevant to the anaerobic respiration of nitrate. I further found that E. coli cells harboring the deletion of mdtEF are susceptible to indole nitrosative derivatives, a class of toxic by-products formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions, and deletion of the genes responsible for the biosynthesis of indole, tnaAB, restores the growth defect of the ΔmdtEF strain during anaerobic respiration of nitrate. Taken together, I conclude that the multidrug efflux pump MdtEF expels the nitrosated indole derivatives out of E. coli cells under anaerobic conditions. Since the production and accumulation of nitrosyl indole derivatives is ascribed to the reactive nitrogen species elicited when E. coli consumes nitrate, I propose that the up-regulated multidrug efflux pump MdtEF functions to protect E. coli from nitrosative damage in its anaerobic ecological niches. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Increasing carbapenem resistance among clinical isolates of E. coli and K. pneumoniae has become a serious public health problem over the last decade. Molecular epidemiology studies have shown that there is a global dissemination of epidemic clones of carbapenem-resistant E. coli and K. pneumoniae. Besides, successful epidemic plasmids were reported to disseminate carbapenemase genes in Enterobacteriaceae. The wide spread of carbapenem-resistant E. coli and K. pneumoniae limits treatment options of the infection, poses severe challenges to clinical professionals and threatens our health. Recently, carbapenem-resistant E. coli and K. pneumoniae are increasingly reported in Hong Kong. In 2012, our group has documented the emergence of carbapenem-resistant clinical isolates in Hong Kong. The findings of the previous study showed that 26.1% of the Enterobacteriaceae isolates were confirmed to produce carbapenemase. Notably, a novel IncX3 plasmid was found to be involved in the dissemination of blaNDM-1 gene. However, the previous findings fail to explicate the carbapenem resistance mechanisms of the remaining non-carbapenemase producing isolates. Further investigation is needed to elucidate the situation. Firstly, we investigated the carbapenem resistance mechanism of carbapenem-resistant E. coli and K. pneumoniae isolates recovered from the Hong Kong West Cluster hospitals from 2010 to 2012. PCRs were used to detect carbapenemase genes (blaNDM, blaKPC, blaIMP, blaVIM and blaOXA-48), blaCTX-M ESBL genes and blaAmpC genes. SDS-PAGE was used to detect porin loss. Among the 92 isolates in this study, only nine (9.8 %) isolates were detected with carbapenemase genes. The blaCTX-M and/or blaAmpC β-lactamase genes plus porin loss were detected in 47 non-carbapenemase-producing isolates (16 E. coli and 31 K. pneumoniae). The resistance determinant profiles of these 16 E. coli included: blaCTX-M + porin loss (n= 10), blaCIT + porin loss (n = 1), blaCTX-M + blaCIT/DHA + porin loss (n = 5). The resistance determinant profiles of the 31 K. pneumoniae included: blaCTX-M + porin loss (n= 4), blaDHA + porin loss (n = 7), blaCTX-M + blaCIT/DHA + porin loss (n = 20). The results showed that apart from acquired carbapenemases, the production of AmpC β-lactamase and/or ESBLs plus porin loss played a main role in the carbapenem resistance mechanism of the carbapenem-resistant E. coli and K. pneumoniae isolates. Secondly, we accessed the clonal relatedness of the isolates. Multi-locus sequence typing results showed that 55 (77.5%) K. pneumoniae isolates fall into the clonal complex 37. Our results suggest that the CC37 K. pneumoniae are associated with the acquisition of DHA-1 β-lactamase, CTXM-1-group β-lactamase and porin alterations which could confer a high-level of resistance to carbapenems resulting in their predominance in this study. Finally, we characterized the plasmids that carry carbapenemase gene by S1-PFGE, Southern blot, plasmid replicon typing and whole plasmid sequencing. A novel IncX3 plasmid was found to carry blaKPC gene. Together with the previously reported blaNDM-1 carrying IncX3 plasmids, it shows that IncX3 plasmids might be new epidemic plasmids involved in the dissemination of carbapenemase genes. These novel IncX3 plasmids are worrisome. Nationwide surveillance and more epidemiological study of IncX3 plasmids are needed. (Word / published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy

As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

Pyrimethamine efficacy was investigated in vitro on the blood asexual stages, the sexual stages and exflagellation in Plasmodium falciparum. Gametocytogenesis was stimulated following the standard methods on five isolates of Plasmodium falciparum. From these five isolates, RSA 2, 3 and 5 produced gametocytes which reached maturity within seven days and the gametocytes were able to exflagellate. Isolate MW2 produced young gametocytes which disappeared within ten days. NF54 produced mature gametocytes which lasted for 24 hours only. There were no statistically significant differences between the static and the synchronization methods of gametocyte stimulation for any of the isolates. The effect of pyrimethamine was investigated by adding a known concentration of the drug (For RSA 2, MW2 and NF54, l00nmol/ℓ; RSA 3 and 5, 3000nmol/ℓ pyrimethamine) to the culture medium for seven days during gametocyte stimulation. The results of this investigation show that there was gametocytocidal activity on the isolates that were used and pyrimethamine also had a schizontocidal action on NF54 and the young gametocytes of this isolate were destroyed by the drug. At concentrations which were inhibitory to asexual parasites, the drug had a sporontocidal effect on isolate RSA 2 but not on isolate RSA 5. The pyrimethamine MIC values for asexual parasites ranged from 300nmol/ℓ to > 3000nmol/ℓ (RSA 2 and 5 were not inhibited at 3000nmol/ℓ ). These results are consistent with those found in previous studies when pyrimethamine resistance was first detected in South Africa. The chloroquine MICs indicate a good correlation with the results obtained from previous drug sensitivity tests for all the isolates examined using both the 48-hour in vitro test and isotope incorporation for growth assessment. The isobolograms constructed to determine relationship between chloroquine and pyrimethamine indicated no synergism for isolates RSA 2 and 5, but the Σ relative IC[50]s indicated a weak synergism. Both the isobolograms and the Σ relative IC[50]s for the isolates RSA 6, 9 and 14 indicated an antagonistic action between chloroquine and pyrimethamine. The results obtained from this study have important implications for malaria control in South Africa. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Antibiotic resistance has become a major problem in the United States. Approximately 2 million people are affected by hospital-acquired infections. Each year about 90,000 people are killed from them. Of the infections 70% of them are resistant to at least one drug. In order to trigger antibiotic resistance in bacteria, the antibiotics need to be detected by sensors in the bacteria. Riboswitches may act as toxin sensors in bacteria. Riboswitches are RNA aptamers that regulate gene expression via allosteric structural changes triggered by binding of a small molecule. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. When the concentration of the metabolite reaches its threshold it binds to the riboswitch causing a structural change that in most cases turns off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic, tetracycline to up-regulate expression of an efflux pump (also called ykkCD) that exports toxic drugs from the bacterial cell. In this work we present initial characterization of the previously uncharacterized ykkCD riboswitch. With the help of tetracycline derivatives and mutagenesis studies on the riboswitch we will (1) determine the substrate specificity of this riboswitch; (2) assess the importance of aromatic character and/or functional groups in antibiotic recognition. To achieve this goal we have developed a fluorescent binding assays. The binding assays will measure the binding affinity (Kd) of the riboswitch-antibiotic complex. Since substrates of the efflux pump are toxic to the bacterial cell, we posit that the ykkCD riboswitch might work as a toxin sensor and could serve as a target in the fight against bacterial pathogens. Afterwards we will evaluate how the ykkCD riboswitch sensor works in vivo. In order to do this we will have to quantify the amount of protein produced in the presence of tetracycline derivatives and mutant sensors. However quantifying the level of a particular protein in the cell is difficult so instead we replace the sequence of the efflux pump with the B-gal gene in B subtilis cell and quantify B-gal enzymatic activity using a colorimetric assay. This is a widely used technique in which the fluorescence corresponds to how much protein is produced. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Riboswitches are naturally occurring RNA aptamers that form a precise three-­dimensional structure and selectively bind to cellular target molecules. Binding of the target molecule initiates an allosteric structural change in the riboswitch that in turn regulates expression of a relevant target gene. Most riboswitches specifically recognize the metabolic product of the gene that is being regulated. Expression may be regulated at either transcription or translation stage of gene expression. Most riboswitches are off switches meaning they turn off expression of metabolite producing gene when metabolite concentration is high enough. The ykkCD putative riboswitch appears to increases production of an efflux pump that expels toxic drugs from the cell by binding to the antibiotic tetracycline. Based on previous data collected the ykkCD putative riboswitch seems to regulate the efflux pump at the transcriptional level. To confirm this hypothesis we want to map the structural change that takes place upon binding of the antibiotic tetracycline to the mRNA. Nucleic acid footprinting studies will be used to map the binding site of tetracycline and the allosteric change that takes place upon tetracycline binding. / Department of Chemistry