No description available.

The utility of focal-plane-array Fourier transform infrared (FPA-FTIR) spectroscopy as a rapid method for the differentiation of antibiotic resistant foodborne pathogens was studied. / Optimum spectral acquisition and processing parameters as well as appropriate film thickness of bacterial films were empirically established for the discrimination between two Shigella species (S. flexneri and S sonnei) in order to optimize the scanning parameters of an FPA-FTIR spectrometer. A detailed study of the potential of FPA-FTIR spectroscopy for the discrimination between antibiotic resistant and sensitive strains from two Salmonella species (S. Typhimurium and S. Heidelberg) was subsequently undertaken. The results of these studies demonstrated that the infrared spectra recorded by an FPA-FTIR spectrometer contained sufficient information to differentiate between antibiotic resistant and sensitive strains of Salmonella. Accordingly, FPA-FTIR spectroscopy may potentially serve as a high-throughput technique for the identification of foodborne as well as antibiotic resistant bacteria. / Interpretation of the regions selected in relation to the different resistance mechanisms would require more detailed studies. However, the identification of specific biochemical markers based on such spectral interpretation is generally not feasible owing to the complexity of the FTIR spectra of microorganisms.

Ivermectin (IVM) is the only safe drug for mass-treatment of onchocerciasis. IVM-resistance has been reported in gastrointestinal nematode parasites of animals. A reduction in response to IVM in Onchocerca volvulus could have significant consequences for the onchocerciasis control programs. Over the last few years, studies have reported genetic selection or reduced responses to IVM in some O. volvulus populations. The risk of a recrudescence of the disease was recently reported with the emergence of resistant adult parasite population in Ghana. It is important to understand the effects of IVM on O. volvulus populations to be able to identify genetic markers to follow IVM selection in the field. In this study, O. volvulus samples were derived from a clinical trial in Cameroon, in which patients were sampled before, and following three years (1994-1997) of IVM treatments. There were four treatment groups: 150mug/kg (1xp.a. or 4xp.a.) and 800mug/kg (1xp.a. or 4xp.a.). DNA from macrofilariae was genotyped for beta-tubulin and P-glycoprotein-like protein (PLP) gene, as well as two control genes and other loci. Reproductive organs of female worms were analyzed by microscopy. A correlation was established between the reproductive status of the female worms and beta-tubulin genotype with the beta-tubulin heterozygous female worms being less fertile than the homozygous female worms. This disadvantage in fertility seemed to disappear after repeated exposure with IVM. We have found evidence that repeated IVM treatment selects for specific alleles of beta-tubulin and PLP. We observed that IVM selection pressure was higher in the female worms than in the male worms. Additionally, loss of polymorphism and selection pressure were higher following thirteen three-monthly doses of IVM compared to annual doses of IVM. Moreover, we found evidence of excess of homozygosity in O. volvulus population, that may be caused by non-random mating and/or subdivision population, which may have implication for the control program. PLP and beta-tubulin genes appear to be promising DNA markers for field use to follow IVM selection. In this perspective, alternative control measures could be considered locally in regions where gene selection is apparent, reducing the likelihood that IVM resistance would develop further and spread.

Onchocerciasis, commonly known as "River blindness" is a disease affecting over 37 million people, worldwide. It is caused by the parasitic nematode Onchocerca volvulus and transmitted by the blackfly vector of the genus Simulium. The drug ivermectin (IVM) is the principal means of controlling the disease. As a result of recent reports on sub-optimal response to ivermectin and genetic selection in O. volvulus, we carried out a 21 month epidemiological study to investigate the response of O. volvulus to repeated rounds of IVM treatments in 2501 subjects from 19 Ghanaian communities that have received between 6 and 18 annual treatments and one IVM naive community. Skin microfilaria (mt) assessments were done before IVM treatment and at days 30, 90, 180 and 364 post-IVM treatment. At day 90 after the second IVM treatment, nodulectomies were carried out on 140 patients and embryogrammes constructed on female worms. We found IVM is still an effective microfilaricide, with efficacy of 98-100%. However, its effect on adult worm fertility has been reduced. Day 90 and 180 post-treatment showed significantly higher (p<0.05) skin mf repopulation of 7.1% to 53.9%, and >100% of pretreatment counts at day 364 post-treatment in four communities compared with the other six communities, which had <80% of pretreatment mf counts on day 364. From these results we classified the 10 communities into good IVM response (four communities), intermediate IVM response (two communities), poor IVM response (three communities), and the previously IVM naive community. Nodule and worm viability and worm densities were significantly higher (p<0.05) in the poor response communities compared with the good response communities, with the intermediate falling between the two. Embryogramme analysis showed significantly higher reproductive activity and output in worms from poor response communities with up to 41% of females having live stretched mf in utero compared with good response communities which had no intra-uterine stretched mf. These results show evidence of lack of sustained response of adult O. volvulus to IVM in the poor response communities, manifested as a rapid return to fertility after IVM treatment. We conclude that IVM resistance is emerging in onchocerciasis and is manifested as a loss of effect of IVM on suppression of parasite reproduction. / Beta tubulin isotype 1 gene has been shown to be linked to IVM treatment and selection in O. volvulus and veterinary nematodes. Genetic analysis of the full length genomic DNA sequence of beta-tubulin from worms obtained in the three IVM response categories and IVM naive community showed single nucleotide polymorphisms (SNPs) at 24 sites on the entire 3696 bp. The frequencies of eight SNPs were significantly different (p< 0.05) between the poor response communities and the good response/naive communities. Four SNPs, 183 T/G, 1188 T/C, 1309 CIT and 1545 A/G resulting in a genotype configuration GG/CC/TT/GG (183/1188/1309/1545) was significantly higher in the poor IVM response communities than the other communities. The phenotypic and genotypic analyses are consistent with a conclusion that IVM resistance has been selected. These four SNPs could be used to develop a genetic marker for early detection of IVM resistance. This study has shown for the first time that IVM resistance is emerging in Onchocerca volvulus and that there are genetic changes associated with IVM resistance which could be used for epidemiological monitoring for emerging resistance.

Infections caused by resistant pathogens fail to respond to treatment, resulting in increased costs due to prolonged illness and hospitalization. Determining the extent of resistance in animal populations is thus of great importance to public health. In this work, we first showed that asymptomatic populations of Salmonella in pigs present greater genotypic and phenotypic diversity than disease-associated populations. Second, we identified a clonal population structure associated with asymptomatic Salmonella found in the Canadian swine industry and we confirmed that food-producing pigs are a significant reservoir of Salmonella enterica, more particularly the clinically important serotype Typhimurium DT104. Finally, we identified the possible independent evolution of multidrug-resistance in serotypes Typhimurium, Derby and Heidelberg. Our work on asymptomatic Salmonella enterica stresses the importance of linking ecology and evolutionary biology to public health in order to understand and predict the response of pathogenic bacteria to selective pressure imposed by host immunity, whether naturally or artificially induced.

There is an increasing number of patients failing second line highly active antiretroviral therapy (AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR). Therefore, it is important to collect data on subtype C protease and gag sequences from patients as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens. In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM resistance genotyping kit and the gag region from these isolates were then characterised. These sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and subtype B, C and group M naïve sequences that were downloaded from the Los Alamos Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To investigate the effect of CS mutations on replication capacity, a mutation was introduced by site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit). Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23 in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro was found to have a lower replication capacity compared to the wild type. These findings emphasize the need for further investigation of gag mutations and their contribution to the evolution of HIV resistance to PIs. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2010.

Onchocerca volvulus is a parasitic filarial nematode responsible for human onchocerciasis, a disease commonly known as "River Blindness". Although there are no well documented cases of ivermectin resistance in O. volvulus, reports of suboptimal responses to ivermectin have appeared. The purpose of this thesis was to examine genetic polymorphisms in O. volvulus and to determine whether there was genetic evidence of ivermectin selection on O. volvulus genes. Analysis of 17 genes from O. volvulus was undertaken in two populations of worms, either from ivermectin-naive patients or from patients who had been repeatedly treated with ivermectin annually. In 14 of the genes no differences in genetic polymorphism were found (although polymorphisms were identified). However, chi square analysis (chi2=0.05) indicated significant differences in allele frequencies for a P-glycoprotein, a beta-tubulin and a putative dyf=8 gene. Analysis of the O. volvulusbeta-tubulin alleles identified three amino acid substitutions in the H3 region with ivermectin selection. Microtubules play a key structural role in the formation of neurons, and in ivermectin-resistant Haemonchus contortus, amphidial neurons show distorted microtubule bundles. Polymerization and depolymerization assays of the recombinant O. volvulus beta-tubulin alleles showed interesting differences between the polymerized tubulin using the two different alleles. It is speculated that similar differences could cause the disorganization of the microtubules identified in the amphidial neurons in ivermectin resistant H. contortus. In addition to the coding mutations, a 24 bp deletion in the adjacent intron to the H3 was detected. A PCR diagnostic assay was developed to genotype individual macro- and microfilariae. Further analyses were conducted to investigate the possibility of a direct relationship between ivermectin and beta-tubulin. Data obtained from equilibrium dialysis experiments indicated that BODIPY FL ivermectin bound to purified O. volvulus alpha- and beta-tubulins. More interesting, non-fluorescent ivermectin and taxol competed with the BODIPY FL ivermectin. The work presented in this thesis provides evidence of genetic selection by ivermectin on O. volvulus and suggests a putative binding site for ivermectin on tubulin. These data provide novel information on ivermectin selection in O. volvulus and on the possible involvement of tubulin in ivermectin resistance.

Background: Although glucocorticoids are the most effective treatment for asthma, some patients show a poor response. In such patients with steroid resistant asthma, this has been ascribed to altered glucocorticoid receptor (GR) ligand-binding affinity induced by IL-2 combined with IL-4 or IL-13 alone- all of which can also modulate glucocorticoid function in vitro. Objective: We sought to assess the ligand-binding affinity in a distinct group of oral steroid-dependent asthmatic subjects and examine the mechanisms by which IL-2 and IL-4 (or IL-13) modify the ligand-binding affinity of the GR. Methods: Using dexamethasone-binding assays, we examined PBMCs ex vivo from healthy subjects, subjects with controlled asthma, and oral steroiddependent subjects with severe asthma. In addition, IL-2 and IL-4 were used to alter GR affinity in vitro. We used mediators or inhibitors of signal transduction to investigate the mechanisms of resistance. We also determined cytokine production of PBMC's by means of ELISA. Results: GR ligand-binding affinity was significantly reduced in the nucleus but not in the cytoplasm of oral steroid-dependent asthmatic subjects compared with that seen in steroid-sensitive and healthy subjects (dissociation constant, 41.37 ± 17.83 vs. 25.36 ± 2.63 nmol/L vs. 9.40 ± 4.01 nmol/L, respectively [p<.05 for both in comparison to normals] ). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 co-treatment and was blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. PBMC's rendered resistant in vitro demonstrated lower IL-10 and increased GM-CSF production following LPS or PMA & PHA stimulation compared to cells with normal GR affinity. Resistant cells also showed reduced dexamethasone repression of LPSstimulated IL-10 release. These effects were also reversed by SB203580. Inhibition of the ERK MAPK pathway by PD098059 (10 mol/L), phosphoinositol 3 kinase by wortmannin (5 nmol/L) or treatment with IL-10 (10 ng/mL) failed to modulate the effect of IL-2 and IL-4 on receptor affinity. Ro318220 (10 nmol/L), a specific protein kinase C inhibitor and theophylline, similarly, had no effect on affinity. Conclusion: GR ligand binding affinity is tiered; compared to normal subjects; steroid responsive asthmatics have a mild reduction in ligand binding whereas oral steroid dependent asthmatics have greater reductions. When mononuclear cells are rendered resistant in vitro, cytokine production (low IL-10 and high GM-CSF) favours a pro-inflammatory state. Our data do not support the ERK MAPK, phosphoinositol 3 kinase, protein kinase C pathways in steroid resistance. Treatment with IL-10 and theophylline also failed to modulate the effect of IL-2 and IL-4 on receptor affinity. However, P38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and re-establishing the beneficial effects of glucocorticoids in patients with severe asthma. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

The use of antibiotics and antimicrobial drugs has played a large role in the elimination of many infectious diseases, however the wide spread use of such drugs has given rise to the phenomenon of antimicrobial resistance and has rendered antibiotics ineffective to a broad range of bacteria. The aim of the study was to ascertain the differences if any in the phenotypic and genotypic resistance profiles of K. pneumoniae isolated from a single tertiary hospital in two surveillance studies undertaken at different times, viz., 2001 and 2007 with special emphasis on ESBLs. A correlation with antibiotic use was also undertaken. ESBL positives were identified and phenotypic resistance profiles were generated based on the resistance profiles of individual isolates by means of their MIC data. The molecular detection of ESBLs was carried out using representative isolates and sequencing was based on the phenotypic expression of the most common ESBL genes. The data was summarized using median values and interquartile ranges. Antibiotic use and susceptibility in 2000 was compared to that in 2007 using a Wilcoxon signed rank test for paired data since the same drugs were tested in both years. Of the isolates that were tested, sequencing revealed that TEM – 1 was identified in all isolates and SHV-1 and SHV-2 were identified in 60 % in the isolates collected in 2000 and 77 % and 11 % respectively in the isolates collected in 2007. SHV – 11 was present in 67% of isolates from 2007 and 55% of those were in combination with SHV – 1. Sequencing also revealed CTXM-15 present in one of the isolates collected in 2007. There was 100% susceptibility to cefoxitin and only one isolate in 2007 showing an intermediate result to imipenem. No novel β-lactamases were identified in this study; however the decrease in susceptibility over time is proof of bacterial evolution. The variety of β-lactamases and diversity of plasmid profiles in these two small populations provides proof to the claim that dissemination of resistance in Klebsiella pneumonia is effortless. Statistical analysis showed an increase in resistance from the year 2000 to 2007 however the correlation between overall antibiotic use and the increase in resistance did not reach statistical significance. It was observed that resistance increased despite only a slight increase in the use of a few antibiotics to which we attributed co-carriage of resistance genes. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.

The employment of HIV-1 protease (PR) inhibitors (PIs) in antiviral therapy has been successful in reducing mortality of HIV/AIDS patients. However, the long-term efficacy of PIs is challenged by the rapid emergence of drug-resistant mutants of PR. To understand the underlying mechanism of drug resistance, structures and activities of HIV-1 PR and its drug resistant mutants have been extensively studied. Here, PR mutants PRR8Q, PRD30N, PRI47V, PRI50V, PRI54M, PRV82A, and PRN88D/S bearing single substitutions have been investigated by crystallography and kinetics. GRL-0519 is a potent new antiviral inhibitor of HIV-1 PR that possesses tris-tetrahydrofuran (tris-THF) as the P2 ligand. The crystal structures of GRL-0519 were determined at resolutions of 1.06-1.49 Å in complex with the mutants PRR8Q, PRD30N, PRI50V, PRI54M, and PRV82A. I50V lost its interaction with inhibitor while V82Aand I54M compensated for the mutation through the main chain shift and flexibility of 80’s loop (residues 78-82), respectively. The structural changes may account for the worst inhibition of GRL-0519 for PRI50V (60-fold decrease relative to wild-type enzyme)and moderate inhibition for PRI54M and PRV82A (6-7-fold decrease). The large tris-THF group at P2 provides a good fit in the S2 subsite and may be effective against resistant virus with mutations of residues in this subsite. SQV and DRV are two clinical inhibitors that were designed to target the wild type PR and its drug resistant mutants, respectively. The crystal structures of PR mutants PRI47V, PRN88D/s in complex with DRV and mutants PRI47V and PRN88D in complex with SQV with resolutions of 1.13-1.72 Å were also analyzed. Mutation I47V gained more hydrophobic interactions with DRV and SQV. Interestingly, the structural changes did not affect the inhibition of both inhibitors for PRI47V (relative Ki is 0.7 and 1 for DRV and SQV, respectively). DRV and SQV showed 8-fold increase in Ki for PRN88D and only very subtle local changes have been observed on the structures. DRV induced 0.3 fold reduction in Ki for PRN88S and the distal structural changes have been transferred to the active site. This study provided fundamental information for understanding drug resistance and future design of potential antiviral drugs.