The spread of antibiotic-resistant microorganisms in the environment is recognized widely as an important public health issue, with concerns about future ability to treat infectious diseases. The main risk to public health is that the resistance genes are transferred from environmental bacteria to human pathogens. Safe water is one of the most important needs in public health in the twenty first century. The major health threat posed by drinking unsafe water is the transmission of infectious diseases, which are the leading causes of mortality and morbidity for children under the age of 5 and it is estimated to cause 1.5 million deaths annually in developing countries. In addition to the wide spread cases of water-borne diseases resulting from the contamination of water sources, concerns have been raised when these diseases fail to be cured due to development of resistance to most prescribed antibiotics by the contaminating microorganisms. It is now a well-established fact that E.coli is a significant cause of diarrheal illnesses both in infants and adults in many parts of the world. Data on clinical isolates is plenty while less attention has been given to environmental isolates of these enteric pathogens. Samples from the environment such as water may serve as probable reservoirs of these pathogens; this is compounded by the entry of functional compounds of antibiotics into waterways, through humans and animals that have ingested antibiotics. This is because antibiotics are not completely metabolized and may enter waterways through the waste products of these humans or animals.Studies on antimicrobial resistance is important in order to detect changes in patterns of resistance, implement control measures on the use of antimicrobial agents, and to prevent the spread of multidrug-resistant strains of bacteria. It also provides surveillance data for antibiotic resistances, necessary to define or update guidelines for empirical treatment, as well as a guide for appropriate antibiotic supplies. Study objectives: The objectives of this research were: (i) to determine the total and faecal coliform status of drinking water sources, as an indication of quality; (ii) to determine the bacteriological profile of bacteria flora in the drinking water sources; (iii) to determine prevalence and susceptibility profiles of antibiotic resistant water-borne E.coli; (iv) to investigate the virulence genes associated with multiple antibiotic resistant E. coli isolates; (v) to compare three laboratory based techniques: PCR, API 20E, and Culture based methods used for detection of E.coli and (vi) to determine the association between multiple antibiotic resistance and radiation sensitivity (D10). © University of South Africa 2014 VII Methodology: Four hundred and sixty four (464) water samples were collected for assessment between June 2011 and May 2012. The samples were collected from 57 sampling sites, from six different water sources including: boreholes (10), a canal (1), dams (15), hand-dug wells (15), a river (1), and streams (15). Total coliforms, faecal coliforms, and E. coli analysis were done by the MPN method. Bacteria isolation and identification were done using API 20E, conventional methods, and a PCR based DNA STRIP technology that allows simultaneous detection of virulence genes and confirmation of E. coli isolates. Antibiotic susceptibility testing was also conducted using the Kirby-bauer method. Radiation sensitivity was done using a cobalt 60 source. Results: The results obtained indicated that all the water sources were of poor quality in terms of microbial distributions with total coliform and faecal coliform counts ranging between 0 to 2.4x103 MPN/100ml. E. coli counts ranged between 10 to 7.9x101MPN/100ml. Disease risk assessment of the various water sources indicated that dam water sources presented a high disease risk, while borehole water sources had a low disease risk. A total of five hundred and twenty bacterial isolates (520) were obtained during the period of study. Three hundred and five (305) isolates representing 58.65% of the total were obtained during the dry season, as against (205) representing 41.35% in the rainy season. The most commonly occurring bacteria in the water samples was Klebsiella spp constituting 20%. The next most occurring organism was E. coli (18.7%). This was followed by Pseudomonas aeruginosa (15.61%), Enterobacter spp. (15.4%), Proteus vulgaris (13.1%), and Enterococcus faecalis (9.2%). The least isolated bacteria were Vibrio cholerae (1.2%) and Shigella spp. (1.2%). The prevalence of multi drug resistance E. coli was 49.48 %. E. coli isolates showed a high sistance patterns to the tested antibiotics. They were most resistant to penicillin (32.99%), cefuroxime (28.87%), erythromycin (23.71%), and tetracycline (21.45%). In contrast, they were susceptible to nitrofurantoin (93.8%), cefotaxime and amikacin (91.75%), gentamicin (90.7%), nalidixic acid (89.65%), ciprofloxacin (74.2%), chloramphenicol (69.07%), pipemidic acid (65.97%) and cefuroxime (52.58%). Sixty-three percent (63%) of the multidrug resistant E. coli strains recorded a multiple antibiotic resistance (MAR) index value of >0.2. Six (6%) percent of he multiple antibiotic resistant were eae virulence genes producing however, none of the E. coli isolates produced the stx1 and stx2 virulent gene. The analytical profile index (API) recorded specificity and sensitivity of 99.7% and 98.50 % respectively for the detection of E. coli. The © University of South Africa 2014 VIII culture/ biochemical based methods for detection of E coli recorded specificity of 81.82% and a sensitivity of 96.91%. There was no association (P> 0.05) between radiation sensitivity (D10) and antibiotic resistances. Conclusion: The study has confirmed that majority of the water sources used for drinking and domestic purposes in the study area are highly contaminated with high levels of faecal coliforms above the recommended standards. There were also resence of bacteria of public health importance in the water sources. Both animals and humans could be sources of faecal bacteria contamination of the drinking water sources. The study confirmed a high prevalence of multiple antibiotic resistances in E. coli isolates. The eae virulence gene was associated with some of the multiple resistant E. coli isolates. The study also concludes that API 20E has a high specificity and sensitivity close to that of the PCR. Lastly, There is no association between multiple antibiotic resistant indexes and radiation sensitivity (D10) of antibiotic resistant E. coli. / School of Environmental Sciences / D. Phil. (Environmental Science)

Systems biology methods can provide novel insight into the responses of an organism to a suboptimal environment, an infection or exposure to a xenobiotic. In the interaction of salmon and salmon lice, there are several areas requiring further research. These include the impacts of lice infection on wild salmon, response mechanisms of different salmon species or life stages to lice infections, effects of environmental conditions on lice stress, and mechanisms underlying the emergence of resistance to important parasiticidal chemicals. Here, I combine global gene expression analyses with phenotypic and physiological responses of salmon or salmon lice to further our understanding of these topics. In the first chapter, I introduce the work by discussing relevant background material on the current knowledge of salmon and salmon lice interactions, salmon immunity, the state of salmon and louse genomics and the emerging field of ecological genomics. I also discuss how these approaches are applied to the study of non-model organisms and sustainable aquaculture development and fisheries conservation. In the second chapter, I present the first large-scale transcriptome profiling of a Pacific salmon to a salmon lice infection, identifying transcript signatures associated with an infection in a sensitive life stage of pink salmon Oncorhynchus gorbuscha. In the third chapter, I present the results of multiple co-habitation infections of three species of Pacific and Atlantic salmon to compare physiological and transcriptomic responses at the local (skin) and systemic levels (anterior kidney). In the fourth chapter, I explore louse transcriptome functioning during temperature and salinity perturbations to characterize the molecular stress response and coping strategies of lice, as well as provide stressor context to response genes. In the fifth chapter, I evaluate sensitive Pacific and resistant and sensitive Atlantic lice responses to emamectin benzoate, an important compound for louse control which has recently been evaded by the louse through resistance development in multiple regions worldwide. In the sixth and final chapter, I conclude with a synthesis of what was learned about knowledge gaps discussed above and how to best apply this information by providing some approaches for future research to address remaining challenges. / Graduate / 0369 / 0792 / 0718 / bensutherland7@gmail.com

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state of the art technology available in the field of proteomics as a complementary tool to characterize the proteome of members of the Mycobacterium tuberculosis Beijing lineage which have been linked to outbreaks and drug resistance of Tuberculosis (TB). Our label-free comparative analysis of two closely related M. tuberculosis strains with different transmission patterns and levels of virulence highlighted numerous factors that may alter metabolic pathways leading to hyper-virulence whereby the strain was able to rapidly replicate in the host and cause extensive disease. This comparative analysis clearly demonstrated that both instrumentation and analysis software impacts on the number of proteins identified and thereby the interpretation of the proteomic data. These proteomes also served as substrates for the discovery of phosphorylation sites, a field of research that reflects a significant knowledge gap in the field of M. tuberculosis. By using differential separation techniques in combination with the state of the art mass spectrometry we described the phosphorylation sites on 286 proteins. This was the first study to document phosphorylation of tyrosine residues in M. tuberculosis. By this means, our data set further extend and complement previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. Using advanced mass spectrometry methods we further investigated the impact of the in vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistant strain containing a S531L rpoB mutation. We identified the presence of overabundant proteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Our findings suggest that proteins involved in a stress response may relate to an altered physiology enabling the pathogen to tolerate and persist when exposed to anti-TB drugs. Together this suggests that structural changes in the RNA polymerase precipitated a cascade of events leading to alterations of metabolic pathways. In addition, we present the first comprehensive analysis of the effect of rifampicin on the proteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicin continues to influence the biology of M. tuberculosis despite the presence of an rpoB mutation. Our analysis showed alterations in the cell envelope composition and allowing the bacterium to survive in a metabolically dormant/persistent growth state. The results presented in this study illustrate the full potential of using a proteomic approach as a complementary molecular technique to select promising candidate molecules and genes for further characterization using the tools of molecular biology. / AFRIKAANSE OPSOMMING: Die huidige studie is ‘n eerste van sy soort, deur die nuutste gevorderde tegnologie in die proteomika veld te gebruik. Die proteoom van lede van die Mycobacterium tuberculosis Beijing stam, wat die oorsaak is van tuberkulose (TB) uitbrake en ook weerstandige TB, is gekarakteriseer. Ons merkervrye vergelykende analise van twee naby verwante M. tuberculosis stamme met verskillende vlakke van oordraagbaarheid en virulensie, beklemtoon verskeie faktore wat metaboliese paaie mag verander, wat kan ly tot hiper-virulensie, wat die TB-stam in staat stel om vinniger te repliseer in die gasheer en ‘n uitgebreide siektetoestand kan veroorsaak. Die analise het duidelik gewys dat die toerusting wat gebruik word, sowel as die sagteware ‘n invloed kan hê op die hoeveelheid proteïne wat geïdentifiseer kan word en daardeur intrepretasie van proteomika data kan beïnvloed. Hierdie proteome dien as substrate vir die ondekking van fosforilasie setels, ‘n veld van navorsing wat dui op ‘n gaping in ons kennis van M. tuberculosis. Deur gebruik te maak van differensiële skeidingstegnieke en moderne spektrometrie beskryf ons fosforileringsetels in 286 proteine. Hierdie is die eerste studie wat fosforilasie van tirosien residue in M. tuberculosis beskryf. Hierdeur komplimenteer en brei ons data die huidige kennis oor gefosforileerde peptiede en fosforilasie setels in M. tuberculosis uit. Deur gebruik te maak van gevorderde massa spektrometriese tegnieke het ons verder ook die impak van in vivo evolusie van rifampicin weerstandigheid op die proteoom van ‘n rifampicin weerstandige TB-stam met die algemene S531L rpoB mutasie ondersoek. Ons het proteïne geïdentifiseer wat in groot hoeveelhede voorkom en kan nuwe insigte gee tot potensiele kompenserende meganismes wat deur die bacillus gebruik word om vatbaarheid vir anti-TB middels te verminder. Ons bevindings dui daarop dat proteïene betrokke in ‘n stresreaksie mag lei tot ‘n verandering in fisologie wat die patogeen in staat stel om anti-TB middels te verdra en te volhard in die teenwoordigheid van sulke middels. Saam impliseer dit dat ‘n ketting van gebeure wat lei tot veranderinge in metaboliese paaie, word vooraf gegaan deur strukturele veranderinge in die RNS polimerase. Tesame hiermee bied ons ook die eerste omvattende analise aan van die effek wat rifampicin op die proteoom van ‘n rifampicin weerstandige M. tuberculosis isolaat het, en wat aan die hand doen dat rifampicin voordurend die biologie van M. tuberculosis beïnvloed, ten spyte van die teenwoordigheid van ‘n rpoB mutasie. Ons analise dui op veranderinge in die samestelling van die selomhulsel wat die bakterie toelaat om te oorleef in ‘n metabolies dormante staat. Die resultate wat in hierdie studie aangebied word illustreer die volle potensiaal van ‘n proteomiese benadering as komplementêre molekulêre tegniek om belowende kandidaat molekules en gene te kies vir verdere karakterisering, deur gebruik te maak van molekulêre tegnieke. / The National Research Foundation (RSA), / Norwegian Research Council (Norway) / National Institute of Health –Forgarty (USA) / Southern Africa Consortium for Research Excellence-Welcome Trust (SACORE) (United Kingdom) / Kwazulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) (USA)

The aim of this thesis was to isolate the compounds responsible for the antibacterial activity of cell extracts of the marine diatom, Phaeodactylum tricornutum. Marine microalgae are not only important primary producers but, due to their phylogenetic diversity, they are also a potential source of novel bioactive compounds. The marine diatom, P. tricornutum, was selected for study because its cell extracts are known to be antibacterial but the compounds responsible have not been isolated. In this thesis, the compounds responsible for the antibacterial activity are isolated from aqueous methanol P. tricornutum cell extracts by column chromatography and reverse phase high-performance liquid chromatography using a bioassay-guided approach. The compounds in three active fractions were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as the unsaturated fatty acids (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, (9Z)-hexadecenoic acid and (6Z, 9Z, 12Z)-hexadecatrienoic acid. The fatty acids were found to be antibacterial against Staphylococcus aureus at micromolar concentrations. P. tricornutum exists in different cell morphs and, interestingly, extracts prepared from cultures in the fusiform morph were found to have greater antibacterial activity than extracts from oval cultures. This is explained by greater levels of the three antibacterial fatty acids in the fusiform cell extracts. The antibacterial fatty acids are proposed to be released by enzyme action when the diatom cells lose their integrity. The release of free fatty acids by diatoms is suggested to be a simple, very low cost population-level activated defence mechanism against potential pathogenic bacteria triggered when the cell loses its integrity. Further, this pathway may act against multiple threats to the microalga, including grazers, as fatty acids exhibit activity in diverse biological assays. Finally, whilst two of the fatty acids, (9Z)-hexadecenoic acid and (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, inhibited the growth of MRSA their usefulness as therapeutic compounds may be limited due to their instability and their broad biological activity.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

<p>Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %.</p><p>In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML.</p><p>The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved.</p><p>Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS.</p><p>Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL).</p><p>Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.</p>

Statins, common cholesterol control drugs, are appreciated to have promising anticancer activity through inhibition of the mevalonate pathway. Several lines of evidence suggest that certain tumours are susceptible to statins, but the underlying molecular features arbitrating this sensitivity remain unknown. We hypothesize that (i) not all statins will behave equivalently in the context of anticancer therapy, and (ii) a molecularly-defined subset of tumours are intrinsically sensitive to statins. My objectives have therefore been to further our understanding of functional differences between statins influencing their anticancer effects, and to investigate molecular features associated with statin sensitivity in breast cancer. Specifically, this thesis addresses two aims: (i) to characterize differential interactions between four statins and the xenobiotic transporter P-glycoprotein (P-gp; also known as ABCB1), and (ii) to identify molecular features associated with fluvastatin and lovastatin sensitivity in breast tumour cell lines. We first characterized the interactions of statins with P-gp in vitro and in multidrug-resistant (MDR) tumour cells. While lovastatin could directly bind to P-gp and modulate MDR, no significant interactions were observed with fluvastatin. Fluvastatin may therefore be appropriate for use in unselected patients, to avoid adverse drug interactions with coadministered P-gp substrate chemotherapeutics. Fluvastatin has also shown promise in breast cancer treatment, where molecular features predictive of statin sensitivity would be particularly valuable. A panel of 19 immortalized breast cell lines was therefore characterized for sensitivity to fluvastatin and lovastatin. Relatively statin-sensitive cells underwent apoptosis upon statin treatment, and were more likely to have an estrogen receptor alpha (ERα)-negative, basal-like phenotype. By mining available baseline gene expression data, a candidate 10-gene signature predictive of fluvastatin sensitivity was also generated. Taken together, this research provides insight into molecular markers of statin sensitivity that may facilitate fast-tracking of these drugs to clinical trials in subsets of cancer patients most likely to respond.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT&gt;MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV&lt;15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in the majority of triple negative breast cancer patients (TNBC). However, the molecular determinants behind their limited response to EGFR-targeted therapies are poorly understood. Here, both the acute and chronic responses of TNBC to the EGFR-targeted therapy, cetuximab (CTX), have been investigated. The expression of EGFR has been analyzed in a cohort of 2000 breast cancer tumours from the public dataset as well as in a panel of breast cancer cell lines. Furthermore, the response of TNBC cell lines to CTX has been investigated using conventional biochemical methods. Finally, a comprehensive transcriptomic profiling of an acquired CTX-resistant TNBC model by RNA sequencing has been performed to understand the molecular determinants of acquired CTX resistance. The results confirmed that EGFR is highly expressed in TNBC in comparison to non-TNBC breast cancer tumours and cell lines, which was associated with adverse clinical outcomes. Targeting EGFR in TNBC cell lines using CTX failed to completely inhibit the EGFR signalling pathway and was associated with an increase in ADAMs-mediated release of endogenous EGFR ligands, EGF and TGFα. Inhibition of ADAMs (ADAM10 and ADAM17) significantly enhanced the anti tumour efficacy of CTX both in vitro and in vivo. Furthermore, transcriptomic profiling of the acquired CTX-resistant TNBC cell line (MDA-MB-468CR) revealed an activation of several key oncogenic pathways and genes, including the TGFβ/BMP pathway. Blocking BMP receptors (BMPRs) restored the sensitivity of resistant cells to CTX treatment. Collectively, current findings offer alternative strategies that could enhance the CTX response in TNBC. We further reported that simultaneous targeting of both EGFR and BMPR pathways could overcome CTX resistance, which might have important implications for the treatment of TNBC.