Thesis (M.Sc.) from the Department of Zoology, University of Adelaide, 1975.

Naegleria fowleri are free-living ameboflagellates found in soil and freshwater habitats throughout the world that cause a fatal disease in humans called Primary Amoebic Meningoencephalitis (PAM). Mechanisms of host resistance or susceptibility to infection have not been fully elucidated, and possible treatment methods are still sub optimal. The disease is diagnosed using specific laboratory tests available in only a few laboratories in the United States. Because of the rarity of infection and difficulty in initial detection, more than often PAM is misdiagnosed. Therefore, it is very important to find causative marker for early detection of an infection. The purpose of this study is to create a proteomic signature map using two-dimensional gel electrophoresis (2-D gel) and recommend a subset of proteins that may be directly linked to the pathogenic state of N. fowleri. Replicates of 2-D Gels were created for both strains of N. fowleri and the proteomic templates from these gels were compared with each other. Scatter Plots were generated measuring the density of protein spots from 2-D gels being analyzed for each study. For each strains of N. fowleri, the 2-D gels from each study were compared within and compared between the two studies for reproducibility in data. The resulting correlation values for all of the Scatter Plots were greater or equal to 0.90. Finally, the representative proteomic template for axenically grown N. fowleri and mouse passaged N. fowleri were compared and the correlation value of 0.60 was observed. This confirmed our theory that these two strains or states of N. fowleri have very different protein expressions, and we were able to identify a subset of proteins, both over expressed and newly synthesized, that may be linked to the highly pathogenic state of N. fowleri.

The free-living amoeba and causative agent of Primary Amoebic Meningoencephalitis, Naegleria fowleri, has three life stages: the trophozoite, the flagellate, and the cyst. This study examined the ability of the cyst to attach to, excyst upon, and destroy cell cultures grown to confluent monolayers, and to cause Primary Amoebic Meningoencephalitis in a murine animal model. The co-culture of cysts with P388D.1, CHME3, Vero, human nasal epithelial, and rat primary mixed glial cells resulted in destruction of the monolayer of all cell types once the cysts attached and excysted. One day post exposure to cysts, the mixed glial cells exhibited a two-fold increase in lactate dehydrogenase (LDH) release compared to cells without cysts, and on day eight post exposure, showed a nearly four-fold increase in LDH. In this study, the cysts of N. fowleri were shown not to be infective in vivo in a murine model using B6C3F1 male mice. The mediation of the encystment process by the intracellular concentration of the secondary messenger cAMP, as described in other closely related genera and species of amoeba, was also investigated. Encystment of N. fowleri was shown to be mediated at least in part by the secondary messenger cAMP by treating cultures of the trophozoite with 100 uM dipyridamole, an inhibitor of cAMP-specific phosphodiesterases. Dipyridamole (100 μM) increased the rate of encystment by nearly two-fold compared to 0.1% DMSO by the end of a five day period of observation. This suggests that cAMP is an essential mediator of the encystment process within Naegleria fowleri.

Emerging pathogens are organisms whose incidence has increased within the past two decades. In the last 40 years, several pathogens have emerged to cause infectious waterborne and foodborne diseases, thus causing a significant public health concern. Enterobacter sakazakii and Naegleria fowleri are emerging pathogens that have been documented to cause fatal infections. E. sakazakii is an emerging foodborne pathogen that represents a significant health risk by causing infections resulting in septicemia, meningitis and necrotizing enterocolitis in neonates, premature infants and also elderly immunocompromised individuals. Naegleria fowleri is a water-based protozoan flagellate that is the cause of primary amoebic meningoencephalitis; a fatal disease that mostly infects children and young adults through water-related recreational activities. The focus of this dissertation is to identify environmental reservoirs of Enterobacter sakazakii and to determine inactivation strategies to control Naegleria fowleri by chlorine and ultraviolet disinfection. In Appendix A, samples from various household kitchens were collected to determine the presence of E.sakazakii. The highest percentage of E.sakazakii was isolated from kitchen sponges (8%; n=50) and dishrags (10%; n=50). This study provided information on the presence of E.sakazakii on environmental surfaces in the kitchen. In Appendix B, our recent research has determined that N. fowleri is present in 8% (n=143) of municipal drinking water wells in central and southern Arizona. Therefore, guidelines need to be established for treatment of water with various disinfectants to control the growth and proliferation of N.fowleri. In Appendix C, the Ct values (concentration (mg/l) × exposure time) for chlorine inactivation of N. fowleri trophozoites and cysts were determined using the Efficiency Hom Kinetic Model (EHM). The Ct values for 99% inactivation of trophozoites and cysts were estimated to be 9 and 31, respectively. The ultraviolet light dose required for the 99% inactivation of N.fowleri trophozoites and cysts was determined to be 63 mW.sec/cm² and 13 mW.sec/cm², respectively.

No description available.

Dans l'objectif d'anticiper et de réduire la prolifération de l'amibe pathogène Naegleria fowleri dans les circuits de refroidissement de certaines centrales électriques, notre travail vise à mieux comprendre l'écologie de cette amibe dans des environnements complexes tels que les biofilms d'eau douce récemment reconnus comme niche écologique préférentielle des amibes libres. Des essais de laboratoire ont été réalisés pour déterminer l'impact des facteurs environnementaux naturels et anthropiques: température, nature du matériau support de la formation du biofilm, charge nutritionnelle et monochloramination sur le comportement et le devenir de Naegleria fowleri dans le biofilm. Ces travaux ont permis de démontrer que la survie, l'implantation, la croissance, le maintien et le déclin de Naegleria fowleri dans les biofilms sont principalement gouvernés par la concomitance des facteurs température et ressource nutritive. Les autres facteurs: nature du matériau, désinfection à la monochloramine et compétition amibienne, se présentent plutôt comme des paramètres de perturbation ou d'inhibition de cette dynamique. Par ailleurs, les résultats obtenus sur la colonisation du biofilm par les amibes confortent le rôle prépondérant de cet habitat comme réservoir naturel des amibes libres et Naegleria fowleri / This study is aiming at preventing and reducing the proliferation of the pathogenic free-living amoeba Naegleria fowleri in several power plant cooling circuits. This work contributes to provide a better understanding of the ecology of this amoeba in complex environments such as freshwater biofilms, which recently has been recognized as privileged ecological niche for free-living amoebae. Laboratory tests were conducted to determine the impact of environmental factors such as temperature, type of support material for the biofilm formation, nutritional resources and monochloramination treatment on the behavior and the fate of Naegleria fowleri in the biofilm. This work has demonstrated that the survival, implantation, maintain, growth and decline of Naegleria fowleri in biofilms are mainly governed by a combination of the temperature and nutritional resource factors. The other factors: type of support material, monochloramination treatment, and amoebic competition, appeared rather as disruptive or inhibitory parameters of this dynamic. Moreover, the obtained results for the amoebic colonization of the biofilm matrix confirm the crucial role of this habitat as natural reservoir for free-living amoebae and Naegleria fowleri

The amoebae Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are free-living amoebae found in both water and soil. They are opportunistic pathogens in humans. Acanthamoeba is the most common cause of illness, usually infecting the eyes and sometimes causing a sight-threatening keratitis. Acanthamoeba spp. and B. mandrillaris can cause granulomatous amoebic encephalitis, in addition to infections of the lungs and skin. N. fowleri causes primary amoebic meningoencephalitis . There is little known regarding the ecology and occurrence of these organisms. A total of 36 high-use recreational surface waters in Arizona were surveyed over a period of two years to assess the occurrence of N. fowleri and seasonal and environmental factors. Overall, 9.3% of the warm weather samples collected were positive for N. fowleri, whereas 16.3% of the samples were positive during cold weather. Although the presence of N. fowleri could not be significantly correlated with physical and chemical parameters such as temperature, pH, turbidity, conductivity, and the presence of heterotrophic bacteria, total coliforms, and Escherichia coli, a weak correlation (0.52) with live amoebic activity was observed. Five lakes to the north and northeast of Phoenix tested positive for the N. fowleri on more than one occasion over multiple seasons. Finished drinking water samples (n= 785) from a municipal potable distribution system were evaluated for the presence of N. fowleri, B. mandrillaris and Acanthamoeba spp. from 18 different regions during three different sampling periods. Physical and chemical parameters were also evaluated but provided no significant correlations with the occurrence of amoebae or indicator organisms. A total of 138 samples (17.9%) were positive for viable amoebae in distribution water with more than an adequate chlorine residual (average of 0.86 mg/L). Microorganisms that are typically used to monitor microbial water quality such as coliforms and E. coli would likely not be found under these circumstances. Clusters with three or more samples testing positive for viable amoebae per region were observed during all three periods. Viable amoebae may not only provide a better assessment of the microbial quality of water, but such clustering could reveal areas with potential water quality issues within the distribution system.

abstract: The deterioration of drinking-water quality within distribution systems is a serious cause for concern. Extensive water-quality deterioration often results in violations against regulatory standards and has been linked to water-borne disease outbreaks. The causes for the deterioration of drinking water quality inside distribution systems are not yet fully understood. Mathematical models are often used to analyze how different biological, chemical, and physical phenomena interact and cause water quality deterioration inside distribution systems. In this dissertation research I developed a mathematical model, the Expanded Comprehensive Disinfection and Water Quality (CDWQ-E) model, to track water quality changes in chloraminated water. I then applied CDWQ-E to forecast water quality deterioration trends and the ability of Naegleria fowleri (N.fowleri), a protozoan pathogen, to thrive within drinking-water distribution systems. When used to assess the efficacy of substrate limitation versus disinfection in controlling bacterial growth, CDWQ-E demonstrated that bacterial growth is more effectively controlled by lowering substrate loading into distribution systems than by adding residual disinfectants. High substrate concentrations supported extensive bacterial growth even in the presence of high levels of chloramine. Model results also showed that chloramine decay and oxidation of organic matter increase the pool of available ammonia, and thus have potential to advance nitrification within distribution systems. Without exception, trends predicted by CDWQ-E matched trends observed from experimental studies. When CDWQ-E was used to evaluate the ability N. fowleri to survive in finished drinking water, the model predicted that N. fowleri can survive for extended periods of time in distribution systems. Model results also showed that N. fowleri growth depends on the availability of high bacterial densities in the 105 CFU/mL range. Since HPC levels this high are rarely reported in bulk water, it is clear that in distribution systems biofilms are the prime reservoirs N. fowleri because of their high bacterial densities. Controlled laboratory experiments also showed that drinking water can be a source of N. fowleri, and the main reservoir appeared to be biofilms dominated by bacteria. When introduced to pipe-loops N. fowleri successfully attached to biofilms and survived for 5 months. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2010

The aim of this study was to compare the effects of arginine vasotocin (AVT) administration in the endangered boreal toad (Bufo boreas boreas) and common Fowler’s toad (Bufo fowleri). The objectives of this study were to determine if AVT could elicit: 1) calling, and 2) amplexus behaviors. Toads were paired into single male:female groups and administered AVT at varying concentrations: 0.1, 1.0, 5.0, 10.0 and 25.0 μg/g and in different combinations: 1) only male was treated; 2) only female was treated, and 3) both male and female treated. AVT failed to stimulate any breeding behavior in the boreal toad, but the administration of AVT to both B. Fowleri genders significantly affected the duration of amplexus (p<0.0347). Also, the concentration of AVT significantly affected the length of amplexus (p<0.0429) and call frequency (p<0.0294). These results will be valuable for breeding programs where animals are failing to show natural reproductive behavior.

Clinical genomics/metagenomics is a rapidly developing field and it makes genomic, transcriptomic, and epigenomic evaluations of clinically relevant samples possible due to decreasing sequencing costs and large volumes of sequence datasets. Applications of comprehensive protocols for clinical metagenomic analysis is rapidly moving from the research laboratories to the clinical laboratories in healthcare settings. It has not only improved medical interventions but also continue to contribute to precision treatments. In this dissertation, I am going to discuss (i) the applications of clinical genomics/metagenomics protocols in several clinical cases of infections and (ii) the application of comparative genomics in Mycobacterium riyadhense clinical isolates which provide insights into ancestry and adaptive evolution in MTBC (Mycobacterium tuberculosis complex); The research questions are systematically addressed in four chapters. Briefly, Chapter 1 provides a brief introduction of conventional clinical microbiology and clinical metagenomics and a research summary of the thesis; Chapter 2 presents an analysis of Mycobacterium riyadhense from an evolutionary genomics point of view, followed by functional genomics experiments to look for clues obtained from the comparative genome analysis of M. riyadhense and other mycobacteria including members of the MTBC. The third chapter describes an imported case of Mycobacterium leprae found in Riyadh, Saudi Arabia, revealed by metagenomic sequencing and bioinformatic analysis, which was challenging for the clinicians to treat due to lack of timely diagnosis and occurrence of drug resistance during the course of treatment. The fourth chapter describes how applications of NGS facilitate rapid pathogen discovery in an imported case of Naegleria fowleri from the Cerebrospinal fluid (CSF) of a resident in KSA with recent travel history to Pakistan. The fifth chapter presents how we have monitored an ongoing outbreak by drug-resistant strains of the human pathogenic yeast Candida auris in Saudi Arabia from King Faisal Medical City, Riyadh, Saudi Arabia. In this thesis, I have shown several applications of NGS and bioinformatic analysis protocols in the clinical genomics/metagenomics fields. I believe some of the main clinical applications of NGS will lead to the adoption of these methodologies in clinical settings in Saudi Arabia in the forthcoming future.