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    Experimental evolution under predation in P. fluorescens SBW25 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand
    (Massey University, 2022) Sadeghpour Golzar, Farhad
    Predator-prey interactions are recognised to change the pace of evolution in microbial communities, but investigations into how selection for predation accelerates antagonistic behaviours, genomic evolution and the process of coadaptation are scarce. Here we performed a 20-day and an extended 90-day evolution experiment to investigate the adaptive traits that arise in prey bacterium Pseudomonas fluorescens SBW25 on solid media in the presence and absence of a wild Acanthamoeba sp. Coevolution led to bacterial diversity, resistance to predation in coevolved bacterial lineages and evolution of predators. We show evidence of reciprocal adaptation, strong phenotypic and genotypic parallelism among prey lineages undergoing predation. We observed evolution of new colony morphotypes such as Wrinkly Spreader, Volcano and Mountain. Evolved morphotypes conferred grazing resistance and an increase in relative prey fitness that resulted in increased encystment and reduced replication of the protozoan populations. Mutation profiles of the coevolved phenotypes were associated with altered gene function in amrZ, wspF, fadD1, fadD2 and putative hypothetical protein upstream of RND transporter. RNA sequencing results of the mutants also revealed a significant increase in the number of genes that up or downregulated while interacting with Acanthamoeba sp. We investigated the degree to which these mutations affect biofilm formation, capsulation, motility, mucoid and fatty acid degradation pathways. Some of these traits are associated with virulence in pathogenic organisms. We further found evidence of mutualisms where both prey and predator increased their survival relative to their respective ancestors. On the other side, we show promoted killing performance and higher generations upon feeding on WT bacteria in coevolved Acanthamoeba compared to their ancestors. Together, our findings demonstrate the emergence of divergent colony morphologies and molecular parallelism that arise as an adaptation to predation and notably affects the fitness and evolution of predators suggesting Red Queen co-evolutionary dynamics between predators and prey. These findings suggest that protozoan predation can profoundly influence the course of genetic and phenotypic evolution in short and long-time scales.
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    Identification and drug sensitivities of Acanthamoeba species causing keratitis : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1997) Johnston, Sally Vanessa
    Acanthamoebic keratitis is a distinct, vision-threatening ophthalmological condition, the incidence of which is increasing with increased usage of contact lenses. Diagnosis can be difficult and chemotherapeutic treatment is prolonged and often ineffective. It is therefore desirable to discover a quick and accurate means of diagnosing acanthamoebic keratitis, and to gain knowledge regarding which chemotherapeutic agents are most effective against acanthamoebic keratitis. The first goal of this thesis was to examine ten DNA extraction procedures and determine their effectiveness in extracting DNA from Acanthamoeba cells. Of these ten methods, four (2, 7, 8, 10) could be performed in less than one day and showed consistent results in PCR reactions. The second goal of this thesis involved the application of arbitrarily primed polymerase chain reaction (AP-PCR) in an attempt to type and group strains of Acanthamoeba species. Examination of 16 isolates with primers AP0l, AP02, AM1, AM2, P1 and P2, showed each of the banding patterns, resulting from AP-PCR analysis, were unique to the isolate tested. Further, there were few bands which occurred in more than one isolate, with insufficient similarities to form groupings of isolates. Two chemotherapeutic agents were selected for a preliminary study into drug sensitivities in Acanthamoeba species and strains. The first of these was Baquacil (20% polyhexamethylene biguanide (PHMB)), and the second was Brolene (0.1% propamidine isethionate). Within 48 hours 97% of all isolates tested reached zero viability at a concentration of 0.05% PHMB, and 100% of isolates tested reached zero viability at 0.1% PHMB. The results of this study would suggest that the concentration of PHMB be at least 0.05% when used to treat acanthamoebic keratitis. Within 48 hours of exposure to 0.1% propamidine isethionate (Brolene), only 30% of all isolates tested reached zero viability. However, 60% of isolates tested showed at least 80% reduction in viability within 48 hours of exposure to 0.1% propamidine isethionate. The ultimate goal of this thesis was to form groups of isolates using PCR and drug sensitivities and to discover any correlation between these groups. The results of AP- PCR analysis however suggests a high genetic heterogeneity within the Acanthamoeba genus, thus preventing any correlation with drug sensitivity tests.
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    Disinfection and environmental studies on pathogenic free-living amoebae : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1982) Dawson, Mark William
    Over the last fifteen years, there has been an increasing awareness of sporadic cases of Primary Amoebic Meningoencephalitis (PAM), affecting primarily younger age groups and appearing in an acute fulminant form. The earliest positive case known, may have been in England in 1909. The pathogenic free-living amoebae (PFLA), which comprises the genera Naegleria and Acanthamoeba, are the causative organisms of PAM and Granulomatous Amoebic Encephalitis (GAE) respectively. PAM is a rapidly fatal disease affecting the central nervous system (CNS), and GAE although essentially confined to the CNS, may also take the form of granulomata in the liver, spleen, uterus and kidneys. A study on the disinfecting potential of Baquacil in axenic conditions, for comparison with the disinfecting potential of the chlorine, chlorine dioxide, ozone and Deciquam 222, showed that the order of effectiveness as amoebicides was Baquacil, chlorine, chlorine dioxide, ozone and Deciquam 222 in hard water. In soft water the order is Deciquam 222, Baquacil, chlorine, chlorine dioxide and ozone. Further study on the effect of Baquacil, chlorine and chlorine dioxide on amoebae, in conditions involving the use of a known Biochemical Oxygen Demand (BOD), a known bacteria concentration, and a combination of BOD and bacteria, confirmed Baquacil as a more effective amoebicide than chlorine, which in turn was more effective than chlorine dioxide. The concentrations of each disinfectant required were increased by the presence of a BOD, and of bacteria. The bacteria were preferentially destroyed over the amoebae with all three disinfectants. Baquacil resistant clones of Naegleria fowleri were isolated, although it is not known whether this resistance is due to genetic or physiological variation. Axenically and Monoxenically cultured amoebae were used, the latter to increase the resemblance of the amoebae to those found in the environment. Differences in survival rates were observed, the monoxenically cultured amoebae invariably having higher survival rates. Competition studies were done with Naegleria spp. and T.pyriformis on three bacteria species, after preliminary studies on the ability of the two protozoa to grow on eight species of bacteria. Of the three bacteria used in the competiton studies, Escherichia coli and Enterobacter cloacae were shown to support both Naegleria spp. and T.pyriformis, with the ciliate increasing in numbers by up to 3 fold over the controls, but the amoebae were affected only slightly, with a small decrease in numbers compared to the controls. A synergistic relationship was evident on the third bacteria species, Pseudomonas fluorescens, between Naegleria spp. and T.pyriformis, where as in the controls, this bacterium was not a good growth support bacteria for either protozoan.