Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BACKGROUND: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. RESULTS: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. CONCLUSIONS: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Application of untargeted metabolomics for the detection of pathogenic Naegleria fowleri in an operational drinking water distribution system.

Found in drinking water distribution systems (DWDSs), swimming pools, and recreational waters, N. fowleri, is the causative agent of primary amoebic meningoencephalitis (PAM). Although cases of N. fowleri infections are rare, the fatality is comparatively high (>95%) and surveillance is essential to minimize N. fowleri infections. However, conventional N. fowleri detection methods are less satisfying owing to their time-consuming and lab intensive characteristics as well as the lack of the ability to determine viability. As a result, an alternative detection approach capable of determining viability as well as species identification is required to better ensure public health. Based on our previous research focusing on distinguishing laboratory cultured N. fowleri from N. lovaniensis and N. italica, this study applies untargeted metabolomics methods to field samples from operational DWDSs. A list of diagnostic features was found to preliminarily discriminate the N. fowleri positive from N. fowleri negative and N. lovaniensis positive field samples with satisfying predictive accuracy. The results outlined in this manuscript further validate and improve the metabolite-based N. fowleri detection approach, potentially aiding water utilities in the detection and management of N. fowleri in drinking water.

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.

Competition between Naegleria fowleri and Free Living Amoeba Colonizing Laboratory Scale and Operational Drinking Water Distribution Systems.

Free living amoebae (FLA), including pathogenic Naegleria fowleri, can colonize and grow within pipe wall biofilms of drinking water distribution systems (DWDSs). Studies on the interactions between various FLA species in biofilms are limited. Understanding the interaction between FLA and the broader biofilm ecology could help better predict DWDS susceptibility to N. fowleri colonization. The aim of this study was to determine if N. fowleri and other FLAs ( Naegleria, Vermamoeba, Willaertia, and Vahlkampfia spp.) cocolonize DWDS biofilm. FLAs commonly isolated from DWDSs ( N. fowleri, V. vermiformis, and N. lovaniensis) were introduced into laboratory-scale biomonitors to determine the impact of these amoebae on N. fowleri's presence and viability. Over 18 months, a single viable amoebae ( N. fowleri, N. lovaniensis, or V. vermiformis) was detected in each biofilm sample, with the exception of N. lovaniensis and N. fowleri, which briefly cocolonized biofilm following their coinoculation. The analysis of biofilm and bulk water samples from operational DWDSs revealed a similar lack of cocolonization with a single FLA detected in 99% ( n = 242) of samples. Interestingly, various Naegleria spp. did colonize the same DWDS locations but at different times. This knowledge furthers the understanding of ecological factors which enable N. fowleri to colonize and survive within operational DWDSs and could aid water utilities to control its occurrence.

Development of Untargeted Metabolomics Methods for the Rapid Detection of Pathogenic Naegleria fowleri.

Despite comparatively low levels of infection, primary amoebic meningoencephalitis (PAM) induced by Naegleria fowleri is extremely lethal, with mortality rates above 95%. As a thermophile, this organism is often found in moderate-to-warm climates and has the potential to colonize drinking water distribution systems (DWDSs). Current detection approaches require days to obtain results, whereas swift corrective action can maximize the benefit of public health. Presently, there is little information regarding the underlying in situ metabolism for this amoeba but the potential exists to exploit differentially expressed metabolic signatures as a rapid detection technique. This research outlines the biochemical profiles of selected pathogenic and nonpathogenic Naegleria in vitro using an untargeted metabolomics approach to identify a panel of diagnostically meaningful compounds that may enable rapid detection of viable pathogenic N. fowleri and augment results from traditional monitoring approaches.

Elimination of Naegleria fowleri from bulk water and biofilm in an operational drinking water distribution system.

Global incidence of primary amoebic meningoencephalitis cases associated with domestic drinking water is increasing. The need for understanding disinfectant regimes capable of eliminating the causative microorganism, Naegleria fowleri, from bulk water and pipe wall biofilms is critical. This field study demonstrated the successful elimination of N. fowleri from the bulk water and pipe wall biofilm of a persistently colonised operational drinking water distribution system (DWDS), and the prevention of further re-colonisation. A new chlorination unit was installed along the pipe line to boost the free chlorine residual to combat the persistence of N. fowleri. Biofilm and bulk water were monitored prior to and after re-chlorination (RCl), pre-rechlorination (pre-RCl) and post-rechlorination (post-RCl), respectively, for one year. A constant free chlorine concentration of > 1 mg/L resulted in the elimination of N. fowleri from both the bulk water and biofilm at the post-RCl site. Other amoeba species were detected during the first two months of chlorination, but all amoebae were eliminated from both the bulk water and biofilm at post-RCl after 60 days of chlorination with free chlorine concentrations > 1 mg/L. In addition, a dynamic change in the biofilm community composition and a four log reduction in biofilm cell density occurred post-RCl. The pre-RCl site continued to be seasonally colonised by N. fowleri, but the constant free chlorine residual of > 1 mg/L prevented N. fowleri from recolonising the bulk and pipe wall biofilm at the post-RCl site. To our knowledge, this is the first study to demonstrate successful removal of N. fowleri from both the bulk and pipe wall biofilm and prevention of re-colonisation of N. fowleri in an operational DWDS. The findings of this study are of importance to water utilities in addressing the presence of N. fowleri and other amoeba in susceptible DWDSs.

Reduced Efficiency of Chlorine Disinfection of Naegleria fowleri in a Drinking Water Distribution Biofilm.

Naegleria fowleri associated with biofilm and biological demand water (organic matter suspended in water that consumes disinfectants) sourced from operational drinking water distribution systems (DWDSs) had significantly increased resistance to chlorine disinfection. N. fowleri survived intermittent chlorine dosing of 0.6 mg/L for 7 days in a mixed biofilm from field and laboratory-cultured Escherichia coli strains. However, N. fowleri associated with an attached drinking water distribution biofilm survived more than 30 times (20 mg/L for 3 h) the recommended concentration of chlorine for drinking water. N. fowleri showed considerably more resistance to chlorine when associated with a real field biofilm compared to the mixed laboratory biofilm. This increased resistance is likely due to not only the consumption of disinfectants by the biofilm and the reduced disinfectant penetration into the biofilm but also the composition and microbial community of the biofilm itself. The increased diversity of the field biofilm community likely increased N. fowleri's resistance to chlorine disinfection compared to that of the laboratory-cultured biofilm. Previous research has been conducted in only laboratory scale models of DWDSs and laboratory-cultured biofilms. To the best of our knowledge, this is the first study demonstrating how N. fowleri can persist in a field drinking water distribution biofilm despite chlorination.