Identification of Naegleria fowleri in Domestic Water Sources by Nested PCR

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ABSTRACT

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.

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Ecology of free-living amoebae.

S Zaragoza (1993)

Small free-living amoebae (FLA) are the main predators controlling bacterial populations in soils. They are distributed in the rhizospheric zone and the surrounding bulk soil; however, they may spread deeper, reaching the vadose zone of groundwater systems, especially where bacterial populations get to high densities. Soil texture is the physical factor controlling the distribution of FLA because it determines the mean bore pore of soil aggregates and other important physical factors. FLA help maintain the high bacterial mineralization rate of organic matter through predation. As attachment onto a surface is necessary for feeding, the quantity of available surfaces is very important for developing this activity. However, the role of protozoa on plant growth promotion is still unclear because they may increase this effect by feeding on both fungi and bacteria. Small FLA are found in soils or sediments, as well as attached to suspended particulate matter in water columns, in the first 30 microns of water surface, or on the bodies of submerged animals and plants. These microorganisms do not distinguish between terrestrial or aquatic environments because they live in the interfaces between them. However, their importance in aquatic systems has been considered as negligible because they are outcompeted by free swimmers. The water conditions affecting amoebae survival are pH, temperature, concentration of sulfhydric acid and salinity. These factors have a strong influence on the structure of amoebae communities in aquatic environments. FLA are considered cosmopolitan as a group, and they live inside vertebrates, in soils, freshwater, marine waters, and on the aerial parts of plants and animals. These microbes, are spread by wind and water currents. Once in the air, cysts and trophozoites behave like any other suspended particulate matter. Therefore, suspension transportation, and removal depend on atmospheric dynamics rather than on their own mechanisms. Ultraviolet light and drought are the main causes of losing viability, but much needs to be learned about the effects of air contaminants on amoebal survival. Naked amoebae also live in the phyllosphere as part of phylloplane community, but their importance and participation in this environment remain unknown. Some species belonging to the genera Acanthamoebae, Naegleria, and Balamuthia cause fatal diseases in humans and are carriers of other pathogens such as Legionella pneumophilia. However, FLA communities can be of some utility in sewage treatment works based in soil filters. FLA's predatory activity in the root zone method may be of greater importance than previously thought, because this is their natural or more favorable environment.(ABSTRACT TRUNCATED AT 400 WORDS)

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Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

W Vahjen, Christoph Tebbe (1993)

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)