Yes

Yes

The thesis presents the development of functionalized electrospun nylon 6 nanofibers for the eradication of pathogenic microorganisms in drinking water. Imidazole derivatives were synthesized as the antimicrobial agents and were characterized by means of NMR spectroscopy, IR spectroscopy, elemental analysis and X-ray crystallography. The first set of compounds (2-substituted N-alkylimidazoles) consisted of imidazole derivatives substituted with different alkyl groups (methyl, ethyl, propyl, butyl, heptyl, octyl, decyl and benzyl) at the 1-position and various functional groups [carboxaldehyde (CHO), alcohol (CH2OH) and carboxylic acid (COOH)] at the 2-position. It was observed that the antimicrobial activity of the compounds increased with increasing alkyl chain length and decreasing pKa of the 2-substituent. It was also observed that the antimicrobial activity was predominantly against a Gram-positive bacterial strains [Staphylococcus aureus (MIC = 5-160 μg/mL) and Bacillus subtilis subsp. spizizenii (MIC = 5-20 μg/mL)], with the latter being the more susceptible. However, the compounds displayed poor antimicrobial activity against Gram-negative bacterial strain, E. coli (MIC = 150- >2500 μg/mL) and did not show any activity against the yeast, C. albicans. The second set of compounds consisted of the silver(I) complexes containing 2-hydroxymethyl-N-alkylimidazoles. The complexes displayed a broad spectrum antimicrobial activity towards the microorganisms that were tested and their activity [E. coli (MIC = 5-40 μg/mL), S. aureus (MIC = 20-80 μg/mL), Bacillus subtilis subsp. spizizenii (MIC = 5-40 μg/mL) and C. albicans (MIC = 40-80 μg/mL)] increased with the alkyl chain length of the 2-hydroxymethyl-N-alkylimidazole. The third set of compounds consisted of the vinylimidazoles containing the vinyl group either at the 1-position or at the 4- or 5- position. The imidazoles with the vinyl group at the 4- or 5-position contained the alkyl group (decyl) at the 1-position. For the fabrication of the antimicrobial nanofibers, the first two sets of imidazole derivatives (2-substituted N-alkylimidazoles and silver(I) complexes) were incorporated into electrospun nylon 6 nanofibers while the third set (2-substituted vinylimidazoles) was immobilized onto electrospun nylon 6 nanofibers employing the graft polymerization method. The antimicrobial nylon nanofibers were characterized by IR spectroscopy and SEM-EDAX (EDS). The electrospun nylon 6 nanofibers incorporated with 2-substituted N-alkylimidazoles displayed moderate to excellent levels of growth reduction against S. aureus (73.2-99.8 percent). For the electrospun nylon 6 nanofibers incorporated with silver(I) complexes, the levels of growth reduction were >99.99 percent, after the antimicrobial activity evaluation using the shake flask method. Furthermore, the grafted electrospun nylon 6 nanofibers showed excellent levels of growth reduction for E. coli (99.94-99.99 percent) and S. aureus (99.93-99.99 percent). The reusability results indicated that the grafted electrospun nylon 6 nanofibers maintained the antibacterial activity until the third cycle of useage. The cytotoxicity studies showed that grafted electrospun nylon 6 nanofibers possess lower cytotoxic effects on Chang liver cells with IC50 values in the range 23.48-26.81 μg/mL. The thesis demonstrated that the development of antimicrobial electrospun nanofibers, with potential for the eradication of pathogenic microoganisms in water, could be accomplished by incorporation as well as immobilization strategies.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The World Health Organisation (WHO) estimates that at least 75% of infants receive infant formula milk (IFM) either entirely or in conjunction with breast milk during the first four months after birth. The presence of the emerging pathogen Enterobacter sakazakii in IFM has been associated with rare but fatal cases of neonatal infections and deaths. There is thus a need for accurate methods for the rapid detection of E. sakazakii in foods. At present, the methods used to detect and identify this micro-organism are inadequate, controversial and contradictory. The aim of this study was to determine the most suitable method for E. sakazakii detection after evaluation of the currently available methods. A further aim was to optimise a polymerase chain reaction (PCR) method for the detection of only viable E. sakazakii cells utilising the DNA-intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA). The Food and Drug Administration (FDA) method for E. sakazakii detection was utilised to select 50 isolates from IFM and 14 from the environment, regardless of colony appearance. These isolates were identified by sequencing a 1.5 kilobase (kb) fragment of the 16S ribosomal DNA (rDNA) and by using the National Centre for Biotechnological Information (NCBI) database to confirm the closet known relatives. Seven of the 50 (14%) IFM isolates and six of the 14 (43%) environmental isolates were identified as E. sakazakii. The methods that were evaluated for accuracy in detecting and identifying these E. sakazakii isolates included yellow pigment production on tryptone soy agar (TSA), chromogenic Druggan-Forsythe-Iversen (DFI) and Enterobacter sakazakii (ES) agars and PCR using six different species-specific primer pairs described in the literature. The suitability of the FDA method was lowered by the low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively) of using yellow pigment production for E. sakazakii identification. DFI and ES agars were shown to be sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications was found to vary between 8% and 92%, with Esakf and Esakr being the most accurate of the primer pairs evaluated. The current FDA method for E. sakazakii detection requires revision in the light of the availability of more sensitive, specific and accurate detection methods. Based on the results obtained in this study, a new method is proposed for the detection of E. sakazakii in food and environmental samples. This proposed method replaces the culturing steps on violet red bile glucose agar (VRBGA) and TSA with culturing on chromogenic DFI or ES agar. For identification and confirmation of presumptive E. sakazakii isolates, the oxidase test, yellow pigment production and API biochemical profiling is replaced by DNA sequencing and/or species-specific PCR with the most accurate primer pair (Esakf and Esakr). The amendments to the current FDA method will reduce the time to detect E. sakazakii from approximately 7 days to 4 days and should prove to be more sensitive, specific and accurate for E. sakazakii detection. In this study, a novel PCR-based method was developed which was shown to be capable of discriminating between viable and dead E. sakazakii cells. This was achieved utilising the irreversible binding of bacterial DNA to photo-activated PMA or EMA in order to prevent PCR amplification from the dead cells. At concentrations of 50 and 100 μg.ml-1, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the PCR amplification from viable cells. EMA was equally effective in preventing PCR amplification from dead cells, however, it also inhibited PCR amplification from viable cells. PMA-PCR in particular, will be useful for assessing the efficacy of processing techniques, as well as for monitoring the resistance, survival strategies and stress responses of E. sakazakii. This will be an important step in the efforts to eliminate E. sakazakii from food and food production environments. / AFRIKAANSE OPSOMMING: Die Wêreld Gesondheidsorganisasie (WGO) beraam dat ten minste 75% van alle babas net baba formule melk (BFM) of BFM in kombinasie met moedersmelk in die eerste vier maande na geboorte kry. Die teenwoordigheid van die voortkomende patogeen Enterobacter sakazakii in BFM is al geassosieer met skaars maar noodlottige gevalle van neonatale infeksies en sterftes. Akkurate metodes word dus benodig vir die vinnige deteksie van E. sakazakii in voedsel. Die metodes wat huidiglik gebruik word vir die deteksie en identifikasie van hierdie mikroörganisme is onvoldoende, kontroversieël en teenstrydig. Die doel van hierdie studie was om die beste metode vir die deteksie van E. sakazakii te bepaal, na 'n evaluasie van die metodes wat huidiglik beskikbaar is. 'n Verdere doel was om 'n polimerase ketting reaksie (PKR) metode vir die deteksie van slegs lewensvatbare E. sakazakii selle te optimiseer deur gebruik te maak van die DNSbindende kleurstowwe, etidium mono-asied (EMA) en propidium mono-asied (PMA). Die Voedsel en Medisyne Administrasie (VMA) se metode vir E. sakazakii deteksie is gebruik om, ongeag van die kolonie kleur, 50 isolate vanuit BFM en 14 isolate vanuit die omgewing te kies. Hierdie isolate is geïdentifiseer deur die DNS volgorde van 'n 1.5 kilo-basis (kb) fragment van die 16S ribosomale DNS (rDNS) te bepaal en die Nationale Sentrum vir Biotegnologiese Informasie (NSBI) databasis te gebruik om die mees verwante spesie te bevestig. Sewe van die 50 (14%) BFM isolate en ses van die 14 (43%) omgewings isolate is geïdentifiseer as E. sakazakii. Die metodes wat geëvalueer is in terme van akkuraatheid vir deteksie en identifikasie van hierdie E. sakazakii isolate het PKR met ses verskillende spesie-spesifieke peiler pare soos beskryf in die literatuur, geel-pigment produksie op triptoon soja agar (TSA) en chromogeniese Druggan-Forsythe-Iversen (DFI) en Enterobacter sakazakii (ES) agars ingesluit. Die geskiktheid van die VMA metode is verlaag deur die lae sensitiwiteit, spesifisiteit en akkuraatheid (87%, 71% en 74% onderskeidelik) van geel pigment produksie vir E. sakazakii identifikasie. Chromogeniese DFI en ES agars was sensitief, spesifiek en akkuraat (100%, 98% en 98% onderskeidelik) vir die identifikasie van E. sakazakii. Die spesifisiteit van die PKR produkte het gewissel tussen 8% en 92%, en Esakf en Esakr is as die akkuraatste geëvalueerde peiler paar geidentifiseer. Die huidige VMA metode vir E. sakazakii deteksie vereis hersiening aangesien meer sensitiewe, spesifieke en akkurate deteksiemetodes voortdurend beskikbaar word. 'n Nuwe metode, gebaseer op die resultate van hierdie studie, word voorgestel vir die deteksie van E. sakazakii in voedsel- en omgewingsmonsters. Die voorgestelde metode vervang die kwekingsstap op violet rooi gal glukose agar (VRGGA) en TSA deur kweking op chromogeniese DFI of ES agars. Verder word die oksidase toets, geel pigment produksie en API biochemiese profiele van vermoeidelike E. sakazakii isolate vervang deur DNS volgorde bepaling en/of spesie-spesifieke PKR met die mees spesifieke peiler paar (Esakf and Esakf) vir die identifikasie en bevestiging van E. sakazakii. Die voorgestelde wysigings van die VMA metode sal die tydsduur van E. sakazakii identifikasie van 7 dae na 4 dae verminder, en behoort ook meer sensitief, spesifiek en akkuraat te wees vir die deteksie van E. sakazakii. 'n Nuwe PKR-gebaseerde metode wat tussen lewensvatbare en dooie E. sakazakii selle kan onderskei is in hierdie studie ontwikkel. Dit is bereik deur die onomkeerbare binding van bakteriële DNS aan lig-geaktiveerde EMA of PMA om die PKR amplifisering van dooie selle te voorkom. Konsentrasies van 50 en 100 μg.ml-1 PMA het PKR amplifikasie heeltemal geïnhibeer, terwyl geen inhibisie van lewensvatbare selle bespeur kon word nie. EMA was ook suksesvol in die voorkoming van die PKR amplifikasie van dooie selle, alhoewel daar ook 'n mate van DNS inhibisie was tydens die amplifikasie van lewensvatbare selle. PMA-PKR kan ook van nut wees vir die assessering van die doeltreffendheid van prosesseringstegnieke, en ook vir die waarneming van die weerstandigheid, oorlewingsstrategieë en stresresponse van E. sakazakii. Dit sal 'n belangrike stap wees in pogings om E. sakazakii van voedsel en voedsel produksieomgewings te elimineer.

Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2014. / Antibiotic resistance amongst bacterial pathogens is a crisis that has been worsening over recent decades, resulting in serious and often fatal infections that cannot be treated by conventional means. Diseases caused by these drug resistant agents result in protracted illnesses, greater mortality rates and increases in treatment costs. Improvements to existing therapies and the development of novel treatments are urgently required to deal with this escalating threat to human health. One of the more promising strategies to combat antibiotic resistance is the use of metallic nanoparticles. Research into this area has shown that the binding of antibiotics to nanoparticles enhances their antimicrobial effects, reduces side-effects due to requirement of lower dosages of the drug, concentrates the drug at the interaction site with bacterial cells and in certain cases, has re-introduced susceptibility into bacterial strains that have developed drug resistance. Furthermore, these nanoparticles can be used in cancer treatment in similar drug delivery roles. Based on the promising data that demonstrated the synergistic effects of antimicrobial agents with nanoparticles, the aim of our research is to determine the effect of ciprofloxacin-conjugated gold nanoparticles as antimicrobial agents. To achieve this aim our objectives were: (i) to synthesize citrate-capped and ciprofloxacin-conjugated gold nanoparticles; (ii) to determine the physical and chemical characteristics of the ciprofloxacin-nanoparticle hybrid molecule; (iii) to investigate the antimicrobial activity of the conjugated nanoparticles against various species of common pathogens and (iv) to investigate the anti-cancer potential of the citrate-capped nanoparticles against a Caco-2 cell line. In this study, citrate-capped gold nanoparticles were conjugated to the antibiotic, ciprofloxacin, and their antibacterial and anti-cancer activity was evaluated. Initial experiments involved the synthesis and characterization of gold nanoparticles and ciprofloxacin conjugated nanoparticles. The gold nanoparticles were synthesized using the Turkevich citrate reduction technique which has been extensively used in studies thus far. The synthesized nanoparticles were characterized for specific absorbance using a UV-Spectrophotometer. The bond between the nanoparticles and ciprofloxacin was characterized by FTIR. Ultra structural details of the gold nanoparticles were established by TEM. The colloidal stability of the nanoparticles was determined by spectroscopic analysis. The antibacterial activity of the ciprofloxacin-conjugated gold nanoparticles was studied by exposure to pathogenic bacteria (Staphyloccocus aureus, E. coli, Klebsiella pneumoniae, Enterocococcus spp., Enterobacter spp., and Psuedomonas spp.). MIC values were measured to give indication of antimicrobial effect. These bactericidal properties of the conjugate nanoparticles were further investigated by electron microscopy. To evaluate the action of the citrate capped gold nanoparticles on cancer cells, we exposed Caco-2 cells to various concentrations of the nanoparticles and its effect was evaluated by measuring the viability of the cells. The results showed that 0.5 mM trisodium citrate reduced gold chloride to yield gold nanoparticles, which were spherical and 15 to 30 nm (by TEM characterization) and had an absorption maxima of 530 nm. The ciprofloxacin conjugated nanoparticles had an absorption maxima of 667nm. The colloidal stability, which is used to assess whether the synthesized particles will retain their integrity in solution showed that citrate-capped GNPs were most stable at 37°C over a 14 day storage period while ciprofloxacin-conjugated GNPs were found to be most stable at 4°C over a 14 day period. The FTIR results showed that chemical bonding in the conjugated nanoparticles occurs between the pyridone moiety of ciprofloxacin and the nanoparticle surface. The antimicrobial results of ciprofloxacin-conjugated GNPs had a significantly improved killing response compared to ciprofloxacin on both Gram positive and Gram negative bacteria. The citrate-capped GNPs are shown to exert a similar cytotoxic effect to gemcitabine on the Caco-2 cell line at a concentration of 0.5 mM. These results indicate that combining gold nanoparticles and ciprofloxacin enhances the antimicrobial effect of the antibiotic. The conjugate nanoparticles increase the concentration of antibiotics at the site of bacterium-antibiotic interaction, and thus enhance the binding and entry of antibiotics into bacteria. This has great implications for treatment of infection, as these antibiotic-conjugated nanoparticles can be incorporated into wound dressings, be administered intravenously as drug delivery agents, be engineered to possess multiple functionalities in addition to antibacterial activity and act as dual infection tracking and antimicrobial agents. Likewise, in this study, gemcitabine, an anticancer drug and gold nanoparticles were shown to kill cancer cells. In addition to their use in photothermal therapy and as drug delivery agents, the nanoparticles themselves possess anti-cancer activity against the Caco-2 cells. Thus, they have potential to act alone as a form of cancer treatment if functionalized with certain targeting agents that are specific to cancer cells, reducing the side-effects that come with regular chemotherapeutic drugs. It can be concluded that ciprofloxacin-conjugated gold nanoparticles enhance antibacterial effects of the antibiotic ciprofloxacin against bacterial cells and citrate-capped gold nanoparticles have anti-cancer activity against the Caco-2 cell line.

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban Institute of Technology, 2005 xx, 175, [14] leaves : ill. ; 30 cm / Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.

Thesis (MCur (Nursing Science))--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: The purpose of this study was to determine the knowledge of Universal Precautions Policy by Registered Nurses at Kenyatta National Hospital (Kenya) and their perception of occupational risk of exposure to blood-borne pathogens. The study also assessed management of blood and body fluids of patients and identified the types and frequency of occupational exposure common among these Registered Nurses. A structured 24-item, self-administered questionnaire was distributed to 185 randomly sampled Registered Nurses in selected departments at this hospital. Compliance with Universal Precautions practices was also observed using a checklist. Data analysis was done by use of a computer software package, Statistical Package for Social Sciences (SPSS) version 11.0. The study findings suggest: 1) lack of continuous education demonstrated by a high level of non-response about knowledge of Universal Precautions Policy with only 19% of the respondents having attended an in-service course in Universal Precautions Policy, and 2) inaccurate understanding of transmission modes of blood-borne pathogens. The majority of nurses surveyed were using Universal Precautions; with indications that nurses were not as familiar with Universal Precautions as they think they were. Respondents admitted modifying personal protection habits based on subjective judgment regarding patient’s perceived blood-borne infectious state. Non-compliant behaviours with barrier precautions were identified, which included failure to use gloves, gowns and protective eyewear, failure to wash hands, and recapping used needles. Compliance with barrier precautions was associated with patients’ perceived blood-borne status. The study revealed a high level of occupational exposures, of which the majority went unreported. Although respondents were aware of the risk of occupationally acquired blood-borne infections, their irregular practice of Universal Precautions Policy is likely to perpetuate the risks. The findings suggest a need for more educational interventions, which may result into integration of concepts into practice. Educational programmes should focus on the epidemiology of occupationally acquired blood-borne pathogens and their modes of transmission, risk of occupationally acquired blood-borne infections at work place, and with emphasis on the principle and practice of Universal Precautions Policy and current protocol of reporting mechanisms in Kenya.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The increased consumption of fresh produce has been shown to be related to increases in foodborne disease outbreaks and these have in many cases been ascribed directly to carry-over of pathogens from contaminated irrigation water. In South Africa, rivers are the main source of irrigation water but many have been found to be unsuitable for irrigation of fresh produce because of the unacceptably high levels of faecal contamination. The main aim of this study was to do a baseline evaluation of the microbiological quality of the Plankenburg and Eerste Rivers and to determine which bacterial contaminants are present. Two sampling sites were selected for the Plankenburg (Plank-1 and -3) and one for the Eerste River (Eerste-1). The microbiological analysis included aerobic colony count (ACC), aerobic and anaerobic sporeformers, Staphylococcus, Salmonella, Listeria, enterococci, coliforms, faecal coliforms and E. coli using standard methods. The faecal contamination levels for both rivers exceeded the DWAF and WHO guidelines of <1 000 E. coli per 100 mL water for irrigation of fresh produce intended to be consumed raw. The Plankenburg River sites always had higher coliform contamination levels (1 200 - 13 000 000 MPN per 100 mL water) than the Eerste River site (230 - 79 000 MPN per 100 mL water). There was also a high incidence of index organisms including Salmonella, Staphylococcus, Listeria and endosporeformers. The isolation of intestinal enterococci suggested the presence of potential pathogens that can cause disease outbreaks. The baseline data also showed large variations in microbial loads over the 15 month study with the faecal coliform counts ranging for Plank-1 from 1 200 to 7 000 000 MPN.100mL-1, Plank-3 from 10 to 460 000 MPN.100mL-1 and Eerste-1 from 28 to 79 000 MPN.100mL-1. The water temperatures at all three sites ranged from 12.1° to 21.7°C with COD values in most cases below 100 mg.L-1. As the baseline study showed large variations in microbial loads over the 15 month study period an assessment using the Colilert-18 system of the weekly, daily and hourly variations, for 6 weeks over a period of 4 months was conducted at site Plank-2. This site was specifically used as it is an irrigation source point for nearby fresh produce farmers and is about 2 km further downstream from an informal settlement. The weekly variation trend for total coliforms (TC) showed a decrease over the entire sampling period with the highest count of 3 200 000 MPN.100 mL-1 during the warmer period. The E.coli (Ec) counts showed a similar trend with the highest count of 440 000 MPN.100 mL-1 also in March. The daily variation trends were the same for both the TC and Ec and counts found to increase from Monday to Thursday followed by a decrease to Sunday. The highest counts were on Thursday with average TC and Ec counts of 1 900 000 and 160 000 MPN.100 mL-1, respectively. The hourly variation trends were similar for both TC and Ec with counts increasing from 06h00 to 12h00 followed by a decrease to 18h00. The increases in TC and Ec counts found during the weekly, daily and hourly variation trend studies clearly suggests that the 15 month sampling that was done once a month on Mondays at 08h00 could be considered an underestimation of the contamination levels of the Plankenburg and Eerste Rivers. The overall weekly variation trend for the water temperature showed a decrease over the sampling period while the daily and hourly variation trends showed an increase from 06h00 to 18h00. The overall weekly trend for pH differed from that of the temperature with an increase over the sampling period. The analysis of covariance showed no correlation (p < 0.05) between the physico-chemical (temperature and pH) and the microbial variables (TC and Ec). Therefore it was concluded that temperature and pH had no direct impact on either the total coliform or E. coli counts. Both the Plankenburg and Eerste Rivers were found to be unsuitable for the irrigation of fresh produce intended to be consumed raw due to the high levels of faecal contamination that exceeded DWAF and WHO guidelines. Irrigation with such water could pose a health risk because of presence of potential pathogens that could be carried-over to fresh produce. / AFRIKAANSE OPSOMMING: Die toenemende gebruik van vars produkte hou direk verband met die toename in voedseloordraagbare siektes. Alte dikwels kan dit toegeskryf word aan die teenwoordigheid van patogene in besproeiingswater. In Suid Afrika is riviere die hoofbron van besproeiingswater maar dit is al gevind dat meeste ongeskik is vir gebruik as besproeïngsbron as gevolg van die onaanvaarbare hoe vlakke van fekale besmetting. Die hoofdoel van hierdie studie was om ‘n basislyn evaluasie van die mikrobiologiese kwaliteit van die Plankenburg en Eerste Riviere te doen en ook vas te stel watter bakteriese kontaminante teenwoordig is. Twee bemonsteringpunte is geselekteer vir die Plankenburg (Plank- 1 en -3) en een vir die Eerste Rivier (Eerste-1). Mikrobiologiese analises met standaard metodes het die volgende ingesluit: aërobe kolonie telings (AKT), aërobe en anaërobe spoorevormers, Staphylococcus, Salmonella, Listeria, enterococci, koliforms, fekale koliforms en E. coli met gebruik van standaard metode. Die fekale besmettingsvlakke vir beide riviere het die DWAF en WHO leistreep van <1 000 E. coli per 100 mL water vir besproeiing van vars produkte wat rou geëet kan word oorskry. Die Plankenburg Rivier bemonsteringspunte het in alle gevalle ‘n hoër kolivorm besmettingsvlak (1 200 - 13 000 000 MPN per 100 mL water) as die Eerste Rivier punt (230 - 79 000 MPN per 100 mL water) gehad. Daar was ook ‘n hoër voorkoms van indeksorganismes insluitend Salmonella, Staphylococcus, Listeria en endosporevormers. Die voorkoms van ingewand enterococci was ‘n addisionele aanduiding van die voorkoms van patogene wat ernstige gesondheidsrisikos vir die verbruiker kan inhou. Die basislyn data het groot variasies in die mikrobe vlakke oor die 15 maand van studie getoon. Die faecal koliforms vir Plank- 1 het gewissel van 1 200 tot 7 000 000 MPN.100mL-1, vir Plank-3 van 10 tot 460 000 MPN.100mL-1 en vir Eerste-1 van 28 tot 79 000 MPN.100mL-1. Die water temperature het gewissel van 12.1° tot 21.7°C met die CSB waardes in meeste gevalle minder as 100 mg.L-1. Aangesien daar sulke groot variasies in mikrobe ladings oor die 15 maande tydperk voorgekom het, is die Colilert-18 sisteem gebruik om die weeklikse, daaglikse en uurlikse variasies vas te stel vir 6 weke oor ‘n periode van 4 maande by die Plank-2 bemonsteringspunt. Daar is spesifiek op die bemonsteringspunt gefokus omdat dit as ‘n besproeiingsbron gebruik word deur groente produsente. Dit is ook gelee ongeveer 2 km stroomaf van ‘n informele nedersetting. Die weeklikse variasies in totaal koliforms (TC) het ‘n afname oor die hele bemonsteringsperiode getoon, met die hoogstes telling van 3 200 000 MPN.100 mL-1 gedurende die warmer tydperk. Die E.coli (Ec) tellings het ‘n soortgelyke neiging getoon, met die hoogste telling van 440 000 MPN.100 mL-1 ook in Maart. Die daaglikse neigings was dieselfde vir beide die TC en Ec en die tellings het vermeerder van Maandag tot Donderdag, met ‘n afname tot Sondag. Die hoogste telling was op Donderdag met gemiddelde TC and Ec tellings van 1 900 000 and 160 000 MPN.100 mL-1, respektiewelik. Die uurlikse variasie profiel was soortgelyk vir beide TC and Ec met tellings wat vermeerder het van 06h00 tot 12h00 gevolg deur ‘n afname tot 18h00. Die toename in TC en Ec getalle soos vasgestel gedurende die weeklikse, daaglikse en uurlikse variasie het duidelik getoon dat die bemonsterings wat een maal per maand op Maandae om 08h00 gedurende die 15 maande tydperk uitgevoer is, tot ‘n erg onderskatting van die besmettings vlakke in die Plankenburg en Eerste Riviere gelei het. Die algehele weeklikse variasies vir die water temperatuur het ‘n verlaging oor die bemonsteringstydperk getoon terwyl die daaglikse en uurlikse variasie neigings ‘n verhoging van 06h00 tot 18h00 getoon het. Die weeklikse neigings vir pH het van die van die temperatuur verskil. Die analises van kovariante het geen korrelasie (p < 0.05) tussen die fisiese-chemiese (temperature en pH) parameters en die mikrobe veranderlikes (TC en Ec) getoon nie. Dus is daar afgelei dat temperatuur en pH geen direkte impak op die totale kolivorm of E. coli tellings gehad nie. Die data van die studie het duidelik getoon dat water van beide die Plankenburg en Eerste Riviere nie geskik is vir gebruik vir besproeiing van vars produkte wat rou geëet gaan word nie. In beide gevalle het die fekale besmettingsvlakke die DWAF en WHO leistreep oorskry. Besproeiing met sulke water hou ‘n gesondheidsgevaar in as gevolg van die teenwoordigheid van potensiële patogene wat oorgedra kan word na vars produkte.

This thesis provides a capabilities-based approach to assessing the bioterrorism threat from non-state actors. Through comparative case study, prior bioterrorism attacks are analyzed to assess capability in the three areas necessary to complete a biological weapons attack: obtaining or isolating a pathogen, weaponizing the agent, and employing or disseminating the weapon. The three cases are the Rajneeshee cult in 1984, the Aum Shinrikyo cult in the early 1990's, and the United States Postal System anthrax attacks of 2001. In contrast to current wisdom that employing biological weapons is too difficult for non-state actors, this thesis reveals a broad spectrum of capability in all studies in the areas necessary to culminate an attack. Applications of these findings must be used to assess risk generally rather than against specific groups because capability is deemed to be exptremely difficult to track. The these finds that a significant threat exists but not large enough to be over-hyped above other national security concerns. In light of this, recommendations are provided for U.S. biodefense policy emphasis in the areas of the nonproliferation regime, attribution capabilities, and defending against the changing nature of future attacks with a particular emphasis on the public health system.