First evidence of free-living Naegleria species in recreational lakes of Alberta, Canada.

Rising temperatures are increasing environmental habitats for thermotolerant pathogens, such as the so-called 'brain-eating amoeba', Naegleria fowleri. To the best of our knowledge, however, Naegleria species have not been reported in environmental water sources in Canada. We surveyed popular recreational lakes in Alberta, Canada during the summer bathing period to determine the presence or absence of Naegleria species. While N. fowleri was not isolated in this study, we identified other thermotolerant species, including Naegleria pagei, Naegleria gruberi, Naegleria jejuensis and Naegleria fultoni using culture-based methods, hence indicating the potential conditions to support N. fowleri. Ongoing monitoring and examination of water for pathogenic amoebae is recommended in order to assist in the public health management of water sources.

SARS-CoV-2 surrogate (Phi6) environmental persistence within free-living amoebae.

The reported persistence of SARS-CoV-2 virions in aquatic environments highlights the need to better understand potential mechanisms that may prolong its dissemination. We evaluated the possibility that amoebae might serve as transport hosts by studying the interaction of the enveloped bacteriophage Phi6, as a potential surrogated along with one of the most common amoebae in engineered aquatic environments, Vermamoeba vermiformis. Using microscopy, imaging flow cytometry and bacteriophage cell culture, our results imply that the SARS-CoV-2 surrogate triggers amoebic mitochondria and induced apoptosis to promote viral persistence in trophozoites. Furthermore, virus-infected amoebae were still infectious after 2 months within FLA cysts. These results suggest that amoebae could contribute to the environmental persistence of SARS-CoV-2, including disinfection processes. In addition, amoebae could be a successful model system for understanding respiratory virus-eukaryotic biology at the cellular and molecular levels.

Tolerance and cytotoxicity of naphthenic acids on microorganisms isolated from oil sands process-affected water.

The expansion of oil sands has made remediation of oil sands process-affected water (OSPW) critical. As naphthenic acids (NAs) are the primary contributors to toxicity, remediation is required. Bioremediation by native microorganisms is potentially effective, however, toxicity of NAs towards native microorganisms is poorly understood. The aim of this study was to isolate microorganisms from OSPW, assess tolerance to stressors, including naturally sourced NAs and examine exposure effect of NAs on cell membranes. Microorganisms were isolated from OSPW, including the first reported isolation of a fungus (Trichoderma harzianum) and yeast (Rhodotorula mucilaginosa). Isolates tolerated alkaline pH, high salinity, and NA concentrations far exceeding those typical of OSPW indicating toxic effects of OSPW are likely the result of interactions between OSPW components. Comparisons of toxicity determined that OSPW exhibited higher cytotoxicity than NAs. The fungal isolate was able to grow using commercial NAs as its sole carbon source, indicating high resistance to NAs' cytotoxic effects. Future studies will focus on the organisms' ability to degrade NAs, and subsequent effects on toxicity. Characterization of OSPW constituents should be investigated with focus on the synergistic toxic effects of dissolved compounds. A better understanding of OSPW toxicity would enable more effective and targeted bioremediation schemes by native microorganisms.

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water.

Flow cytomtery (FCM) has become a standard approach to enumerate viruses in water research. However, the nature of the fluorescent signal in flow cytometric analysis of water samples and the mechanism of its formation, have not been addressed for bacteriophages expected in wastewaters. Here we assess the behaviour of fluorescent DNA-staining dyes in aqueous solutions, as well as sensitivity and accuracy of FCM for enumeration of DNA-stained model bacteriophages λ, P1, and T4. We demonstrate that in aqueous systems fluorescent dyes form a self-stabilized (pseudolyophilic) emulsion of auto-fluorescing colloid particles. Sample shaking and addition of surfactants enhance auto-fluorescence due to increased dispersion and, in the presence of surfactants, stabilization of the dye emulsion. Bacteriophages with genome sizes <100 kbp (i.e. λ & P1) did not generate a distinct population signal to be detected by one of the most sensitive FCM instruments available (BD LSR Fortessa™ X-20), whereas the larger T4 bacteriophage was resolved as a distinct population of events. These results indicate that the use of fluorescent dyes for bacteriophage enumeration by flow cytometry can produce false positive signals and lead to wrong estimation of total virus counts by misreporting colloid particles as virions, depending on instrument sensitivity.