Preferential feeding in Naegleria fowleri; intracellular bacteria isolated from amoebae in operational drinking water distribution systems.

The amoeba Naegleria fowleri is the causative agent of the highly fatal disease, primary amoebic meningoencephalitis, and estimated to cause 16 deaths per year in the United States alone. Colonisation of drinking water distribution systems (DWDSs) by the N. fowleri is a significant public health issue. Understanding the factors which enable this pathogen to colonise and thrive in DWDSs is critical for proper management. The microbial ecology within DWDSs may influence the ability of N. fowleri to colonise DWDSs by facilitating the availability of an appropriate food source. Using biofilm samples obtained from operational DWDSs, 16S rRNA amplicon metabarcoding was combined with genus-specific PCR and Sanger sequencing of intracellular associated bacteria from isolated amoeba and their parental biofilms to identify Meiothermus chliarophilus as a potential food source for N. fowleri. Meiothermus was confirmed as a food source for N. fowleri following successful serial culturing of axenic N. fowleri with M. chliarophilus or M. ruber as the sole food source. The ability to identify environmental and ecological conditions favourable to N. fowleri colonisation, including the detection of appropriate food sources such as Meiothermus, could provide water utilities with a predictive tool for managing N. fowleri colonisation within the DWDS.

Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater.

BACKGROUND: Legionella spp. employ multiple strategies to adapt to stressful environments including the proliferation in protective biofilms and the ability to form associations with free-living amoeba (FLA). The aim of the current study was to identify Legionella spp., Acanthamoeba spp., Vermamoeba (Hartmannella) vermiformis and Naegleria fowleri that persist in a harvested rainwater and solar pasteurization treatment system. METHODS: Pasteurized (45 °C, 65 °C, 68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples were screened for Legionella spp. and the heterotrophic plate count was enumerated. Additionally, ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) was utilized for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri in pasteurized (68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples, respectively. RESULTS: Of the 82 Legionella spp. isolated from unpasteurized tank water samples, Legionella longbeachae (35 %) was the most frequently isolated, followed by Legionella norrlandica (27 %) and Legionella rowbothamii (4 %). Additionally, a positive correlation was recorded between the heterotrophic plate count vs. the number of Legionella spp. detected (ρ = 0.710, P = 0.048) and the heterotrophic plate count vs. the number of Legionella spp. isolated (ρ = 0.779, P = 0.0028) from the tank water samples collected. Solar pasteurization was effective in reducing the gene copies of viable V. vermiformis (3-log) and N. fowleri (5-log) to below the lower limit of detection at temperatures of 68-93 °C and 74-93 °C, respectively. Conversely, while the gene copies of viable Legionella and Acanthamoeba were significantly reduced by 2-logs (P = 0.0024) and 1-log (P = 0.0015) overall, respectively, both organisms were still detected after pasteurization at 93 °C. CONCLUSIONS: Results from this study indicate that Acanthamoeba spp. primarily acts as the vector and aids in the survival of Legionella spp. in the solar pasteurized rainwater as both organisms were detected and were viable at high temperatures (68-93 °C).

Counting Legionella cells within single amoeba host cells.

Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.

Cocultivation of the amoeba Naegleria fowleri and the amoebicin- producing strain Bacillus licheniformis M-4.

Antagonism between Bacillus licheniformis M-4 and the pathogenic amoeba Naegleria fowleri HB-1 during cocultivation was influenced by the composition of the medium and the initial amoeba/bacterium ratio. While a ratio of 50 caused complete lysis of amoebae in soil extract with 0.3% glucose (SEG) before 72 h, this ratio had to be at least 12-fold lower in order to obtain similar results in Cline medium. Sporulation of B. licheniformis M-4 took place much earlier in SEG. Amoebicin production was stimulated by the presence of amoebae by either shortening the time of production (as in SEG) or increasing the amount of amoebicins released (as in Cline medium). Electron microscopy showed that amoebae cocultivated in the Cline medium contained bacteria enclosed in digestive vacuoles, while amoebae from SEG cocultures did not.