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.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Conceptual model to inform Legionella-amoebae control, including the roles of extracellular vesicles in engineered water system infections.

Extracellular vesicles (EVs or exosomes) are well described for bacterial pathogens associated with our gastrointestinal system, and more recently as a novel mechanism for environmental persistence, dissemination and infection for human enteric viruses. However, the roles played by EVs in the ancient arms race that continues between amoebae and one of their prey, Legionella pneumophila, is poorly understood. At best we know of intracellular vesicles of amoebae containing a mix of bacterial prey species, which also provides an enhanced niche for bacteriophage infection/spread. Free-living amoeba-associated pathogens have recently been recognized to have enhanced resistance to disinfection and environmental stressors, adding to previously understood (but for relatively few species of) bacteria sequestered within amoebal cysts. However, the focus of the current work is to review the likely impacts of large numbers of respiratory-sized EVs containing numerous L. pneumophila cells studied in pure and biofilm systems with mixed prey species. These encapsulated pathogens are orders of magnitude more resistant to disinfection than free cells, and our engineered systems with residual disinfectants could promote evolution of resistance (including AMR), enhanced virulence and EV release. All these are key features for evolution within a dead-end human pathogen post lung infection. Traditional single-hit pathogen infection models used to estimate the probability of infection/disease and critical environmental concentrations via quantitative microbial risk assessments may also need to change. In short, recognizing that EV-packaged cells are highly virulent units for transmission of legionellae, which may also modulate/avoid human host immune responses. Key data gaps are raised and a previous conceptual model expanded upon to clarify where biofilm EVs could play a role promoting risk as well as inform a more wholistic management program to proactively control legionellosis.

Enteric pathogen reduction targets for onsite non-potable water systems: A critical evaluation.

Onsite non-potable water systems (ONWS) collect and treat local source waters for non-potable end uses such as toilet flushing and irrigation. Quantitative microbial risk assessment (QMRA) has been used to set pathogen log10-reduction targets (LRTs) for ONWS to achieve the risk benchmark of 10-4 infections per person per year (ppy) in a series of two efforts completed in 2017 and 2021. In this work, we compare and synthesize the ONWS LRT efforts to inform the selection of pathogen LRTs. For onsite wastewater, greywater, and stormwater, LRTs for human enteric viruses and parasitic protozoa were within 1.5-log10 units between 2017 and 2021 efforts, despite differences in approaches used to characterize pathogens in these waters. For onsite wastewater and greywater, the 2017 effort used an epidemiology-based model to simulate pathogen concentrations contributed exclusively from onsite waste and selected Norovirus as the viral reference pathogen; the 2021 effort used municipal wastewater pathogen data and cultivable adenoviruses as the reference viral pathogen. Across source waters, the greatest differences occurred for viruses in stormwater, given the newly available municipal wastewater characterizations used for modeling sewage contributions in 2021 and the different selection of reference pathogens (Norovirus vs. adenoviruses). The roof runoff LRTs support the need for protozoa treatment, but these remain difficult to characterize due to the pathogen variability in roof runoff across space and time. The comparison highlights adaptability of the risk-based approach, allowing for updated LRTs as site specific or improved information becomes available. Future research efforts should focus on data collection of onsite water sources.

Number of COVID-19 cases required in a population to detect SARS-CoV-2 RNA in wastewater in the province of Alberta, Canada: Sensitivity assessment.

With a unique and large size of testing results of 1,842 samples collected from 12 wastewater treatment plants (WWTP) for 14 months through from low to high prevalence of COVID-19, the sensitivity of RT-qPCR detection of SARS-CoV-2 RNA in wastewater that correspond to the communities was computed by using Probit analysis. This study determined the number of new COVID-19 cases per 100,000 population required to detect SARS-CoV-2 RNA in wastewater at defined probabilities and provided an evidence-based framework of wastewater-based epidemiology surveillance (WBE). Input data were positive and negative test results of SARS-CoV-2 RNA in wastewater samples and the corresponding new COVID-19 case rates per 100,000 population served by each WWTP. The analyses determined that RT-qPCR-based SARS-CoV-2 RNA detection threshold at 50%, 80% and 99% probability required a median of 8 (range: 4-19), 18 (9-43), and 38 (17-97) of new COVID-19 cases /100,000, respectively. Namely, the positive detection rate at 50%, 80% and 99% probability were 0.01%, 0.02%, and 0.04% averagely for new cases in the population. This study improves understanding of the performance of WBE SARS-CoV-2 RNA detection using the large datasets and prolonged study period. Estimated COVID-19 burden at a community level that would result in a positive detection of SARS-CoV-2 in wastewater is critical to support WBE application as a supplementary warning/monitoring system for COVID-19 prevention and control.

Wastewater-Based Surveillance Is an Effective Tool for Trending COVID-19 Prevalence in Communities: A Study of 10 Major Communities for 17 Months in Alberta.

The correlations between SARS-CoV-2 RNA levels in wastewater from 12 wastewater treatment plants and new COVID-19 cases in the corresponding sewersheds of 10 communities were studied over 17 months. The analysis from the longest continuous surveillance reported to date revealed that SARS-CoV-2 RNA levels correlated well with temporal changes of COVID-19 cases in each community. The strongest correlation was found during the third wave (r = 0.97) based on the population-weighted SARS-CoV-2 RNA levels in wastewater. Different correlations were observed (r from 0.51 to 0.86) in various sizes of communities. The population in the sewershed had no observed effects on the strength of the correlation. Fluctuation of SARS-CoV-2 RNA levels in wastewater mirrored increases and decreases of COVID-19 cases in the corresponding community. Since the viral shedding to sewers from all infected individuals is included, wastewater-based surveillance provides an unbiased and no-discriminate estimation of the prevalence of COVID-19 compared with clinical testing that was subject to testing-seeking behaviors and policy changes. Wastewater-based surveillance on SARS-CoV-2 represents a temporal trend of COVID-19 disease burden and is an effective and supplementary monitoring when the number of COVID-19 cases reaches detectable thresholds of SARS-CoV-2 RNA in wastewater of treatment facilities serving various sizes of populations.

Risk-based treatment targets for onsite non-potable water systems using new pathogen data.

Using local sources (roof runoff, stormwater, graywater, and onsite wastewater) to meet non-potable water demands can minimize potable water use in buildings and increase supply reliability. In 2017, an Independent Advisory Panel developed a risk-based framework to identify pathogen log reduction targets (LRTs) for onsite non-potable water systems (ONWSs). Subsequently, California's legislature mandated the development and adoption of regulations-including risk-based LRTs-for use in multifamily residential, commercial, and mixed-use buildings. A California Expert Panel was convened in 2021 to (1) update the LRT requirements using new, quantitative pathogen data and (2) propose treatment trains capable of meeting the updated LRTs. This paper presents the updated risk-based LRTs for multiple pathogens (viruses, protozoa, and bacteria) and an expanded set of end-uses including toilet flushing, clothes washing, irrigation, dust and fire suppression, car washing, and decorative fountains. The updated 95th percentile LRTs required for each source water, pathogen, and end-use were typically within 1-log10 of the 2017 LRTs regardless of the approach used to estimate pathogen concentrations. LRT requirements decreased with influent pathogen concentrations from wastewater to graywater to stormwater to roof runoff. Cost and footprint estimates provide details on the capital, operations and maintenance, and siting requirements for ONWS implementation.

Use of a Novel DNA-Loaded Alginate-Calcium Carbonate Biopolymer Surrogate to Study the Engulfment of Legionella pneumophila by Acanthamoeba polyphaga in Water Systems.

The engulfment of Legionella pneumophila by free-living amoebae (FLA) in engineered water systems (EWS) enhances L. pneumophila persistence and provides a vehicle for rapid replication and increased public health risk. Despite numerous legionellosis outbreaks worldwide, effective tools for studying interactions between L. pneumophila and FLA in EWS are lacking. To address this, we have developed a biopolymer surrogate with a similar size, shape, surface charge, and hydrophobicity to those of stationary-phase L. pneumophila. Parallel experiments were conducted to observe the engulfment of L. pneumophila and the surrogate by Acanthamoeba polyphaga in dechlorinated, filter-sterilised tap water at 30°C for 72 h. Trophozoites engulfed both the surrogate and L. pneumophila, reaching maximum uptake after 2 and 6 h, respectively, but the peak surrogate uptake was ~2-log lower. Expulsion of the engulfed surrogate from A. polyphaga was also faster compared to that of L. pneumophila. Confocal laser scanning microscopy confirmed that the surrogate was actively engulfed and maintained within vacuoles for several hours before being expelled. L. pneumophila and surrogate phagocytosis appear to follow similar pathways, suggesting that the surrogate can be developed as a useful tool for studying interactions between L. pneumophila and FLA in EWS. IMPORTANCE The internalization of L. pneumophila within amoebae is a critical component of their life cycle in EWS, as it protects the bacteria from commonly used water disinfectants and provides a niche for their replication. Intracellularly replicated forms of L. pneumophila are also more virulent and resistant to sanitizers. Most importantly, the bacteria's adaptation to the intracellular environments of amoebae primes them for the infection of human macrophages, posing a significant public health risk in EWS. The significance of our study is that a newly developed L. pneumophila biopolymer surrogate can mimic the L. pneumophila engulfment process in A. polyphaga, a free-living amoeba. With further development, the surrogate has the potential to improve the understanding of amoeba-mediated L. pneumophila persistence in EWS and the associated public health risk management.

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.

Tenets of a holistic approach to drinking water-associated pathogen research, management, and communication.

In recent years, drinking water-associated pathogens that can cause infections in immunocompromised or otherwise susceptible individuals (henceforth referred to as DWPI), sometimes referred to as opportunistic pathogens or opportunistic premise plumbing pathogens, have received considerable attention. DWPI research has largely been conducted by experts focusing on specific microorganisms or within silos of expertise. The resulting mitigation approaches optimized for a single microorganism may have unintended consequences and trade-offs for other DWPI or other interests (e.g., energy costs and conservation). For example, the ecological and epidemiological issues characteristic of Legionella pneumophila diverge from those relevant for Mycobacterium avium and other nontuberculous mycobacteria. Recent advances in understanding DWPI as part of a complex microbial ecosystem inhabiting drinking water systems continues to reveal additional challenges: namely, how can all microorganisms of concern be managed simultaneously? In order to protect public health, we must take a more holistic approach in all aspects of the field, including basic research, monitoring methods, risk-based mitigation techniques, and policy. A holistic approach will (i) target multiple microorganisms simultaneously, (ii) involve experts across several disciplines, and (iii) communicate results across disciplines and more broadly, proactively addressing source water-to-customer system management.

Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater.

Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology. In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E). Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2-3 PCR-cycle threshold (Ct) values less when compared to samples stored at -20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%). Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.

Knowledge Gaps in the Understanding of Antimicrobial Resistance in Canada.

Current limitations in the understanding and control of antimicrobial resistance (AMR) in Canada are described through a comprehensive review focusing on: (1) treatment optimization; (2) surveillance of antimicrobial use and AMR; and (3) prevention of transmission of AMR. Without addressing gaps in identified areas, sustained progress in AMR mitigation is unlikely. Expert opinions and perspectives contributed to prioritizing identified gaps. Using Canada as an example, this review emphasizes the importance and necessity of a One Health approach for understanding and mitigating AMR. Specifically, antimicrobial use in human, animal, crop, and environmental sectors cannot be regarded as independent; therefore, a One Health approach is needed in AMR research and understanding, current surveillance efforts, and policy. Discussions regarding addressing described knowledge gaps are separated into four categories: (1) further research; (2) increased capacity/resources; (3) increased prescriber/end-user knowledge; and (4) policy development/enforcement. This review highlights the research and increased capacity and resources to generate new knowledge and implement recommendations needed to address all identified gaps, including economic, social, and environmental considerations. More prescriber/end-user knowledge and policy development/enforcement are needed, but must be informed by realistic recommendations, with input from all relevant stakeholders. For most knowledge gaps, important next steps are uncertain. In conclusion, identified knowledge gaps underlined the need for AMR policy decisions to be considered in a One Health framework, while highlighting critical needs to achieve realistic and meaningful progress.

Towards risk assessment for antibiotic resistant pathogens in recycled water: a systematic review and summary of research needs.

Risk assessment is critical for identifying target concentrations of antibiotic resistant pathogens necessary for mitigating potential harmful exposures associated with water reuse. However, there is currently limited available data characterizing the concentrations of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in recycled water to support robust efforts at risk assessment. The objective of this systematic review was to identify and synthesize the existing literature documenting the presence and abundance of ARB and ARGs in recycled water. In addition, this review identifies best practices and explores monitoring targets for studying ARB and ARGs in recycled water to guide future work and identifies key research needs aimed at better supporting quantitative microbial risk assessment focused on recycled water and antibiotic resistance. Future efforts to collect data about ARB and ARG prevalence in recycled water should report concentration data per unit volume. Sample metadata should also be provided, including a description of treatment approach, a description of planned water uses (e.g., potable, irrigation), methods for conveyance to the point of use, and available physicochemical water quality data. Additional research is needed aimed at identifying recommended ARB and ARG monitoring targets and for developing approaches to incorporate metagenomic data into risk assessment.

Pathogen performance testing of a natural swimming pool using a cocktail of microbiological surrogates and QMRA-derived management goals.

In recent decades, natural swimming pools (NSPs) have gained popularity in Europe, especially in Germany and Austria. NSPs differ from swimming pools in that they utilize biological treatment processes based on wetland processes with no disinfection residual. However, data are missing on the specific log-reduction performance of NSPs to address enteric virus, bacteria, and parasitic protozoa removal considered necessary to meet the North American risk-based benchmark (<35 illnesses per 1,000 swimming events) set by the USEPA for voluntary swimming. In this study, we examined Canada's first NSP at Borden Park, Edmonton, Canada, to address the following three questions: (1) Given normal faecal shedding rates by bathers, what is the total log reduction (TLR) theoretically needed to meet the EPA benchmark? (2) what is the in-situ performance of the NSP based on spiking suitable microbial surrogates (MS2 coliphage, Enterococcus faecalis, and Saccharomyces cerevisiae [Baker's yeast])? and (3) how much time is required to reach acceptable bather risk levels under different representative volume-turnover rates? A reverse-quantitative microbial risk assessment (QMRA) revealed that of the four reference pathogens selected (Norovirus, Campylobacter, Cryptosporidium, and Giardia), only Norovirus was estimated to exceed the risk benchmark at the 50th, 75th, and 95th percentiles, while Campylobacter was the only other reference pathogen to exceed at the 95th percentile. Log-reduction values (LRVs) were similar to previous reports for bacterial indicators, and novel LRVs were estimated for the other two surrogates. A key finding was that more than 24 h treatment time would be necessary to provide acceptable bather protection following heavy bather use (378 bathers/day for main pool and 26 bathers/day for children's pool), due to the mixing dynamics of the treated water diluting out possible residual pool faecal contamination. The theoretical maximum number of people in the pool per day to be below USEPA's 35 gastro cases in 1,000 swimming events was 113, 47, and 8, at the 50th, 75th, and 95th percentiles. Further, the use of ultra-violet disinfection to the pool return flow had little effect on reducing the treatment time required.

Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses.

Human respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and immunocompromised adults worldwide. Here we report that amoebae-release respirable-sized vesicles containing high concentrations of infectious RSV that persisted for the duration of the experiment. Given the ubiquity of amoebae in moist environments, our results suggest that extracellular amoebal-vesicles could contribute to the environmental persistence of respiratory viruses, including potential resistance to disinfection processes and thereby offering novel pathways for viral dissemination and transmission.

Differential Bacterial Predation by Free-Living Amoebae May Result in Blooms of Legionella in Drinking Water Systems.

Intracellular growth of pathogenic Legionella in free-living amoebae (FLA) results in the critical concentrations that are problematic in engineered water systems (EWS). However, being amoeba-resistant bacteria (ARB), how Legionella spp. becomes internalized within FLA is still poorly understood. Using fluorescent microscopy, we investigated in real-time the preferential feeding behavior of three water-related FLA species, Willaertia magna, Acanthamoeba polyphaga, and Vermamoeba vermiformis regarding Legionella pneumophila and two Escherichia coli strains. Although all the studied FLA species supported intracellular growth of L. pneumophila, they avoided this bacterium to a certain degree in the presence of E. coli and mostly fed on it when the preferred bacterial food-sources were limited. Moreover, once L. pneumophila were intracellular, it inhibited digestion of co-occurring E. coli within the same trophozoites. Altogether, based on FLA-bacteria interactions and the shifts in microbial population dynamics, we propose that FLA's feeding preference leads to an initial growth of FLA and depletion of prey bacteria, thus increases the relative abundance of Legionella and creates a "forced-feeding" condition facilitating the internalization of Legionella into FLA to initiate the cycles of intracellular multiplication. These findings imply that monitoring of FLA levels in EWS could be useful in predicting possible imminent high occurrence of Legionella.

Screening Level Risk Assessment (SLRA) of human health risks from faecal pathogens associated with a Natural Swimming Pond (NSP).

Natural swimming ponds (NSPs) are artificially created bodies of water intended for human recreation, characterised by the substitution of chemical disinfection with natural biological processes for water purification. NSPs are growing in popularity, however little is known regarding the public health risks. A screening level risk assessment was undertaken as an initial step in assessing the first Canadian public NSP located in Edmonton, Alberta. Risk of enteric pathogens originating from pool bathers was assessed under normal conditions and following accidental faecal release events. The performance of the natural treatment train for health protection was quantified with and without the addition of UV disinfection of naturally-treated water, and compared to the US EPA benchmark to provide a reference point to consider acceptability. Estimated levels of pathogen contamination of the pond were dependant upon the discrete number of shedders present, which in turn depended upon the prevalence of infection in the population. Overall performance of the natural disinfection system was dependant upon the filtration rate of the natural treatment system or turnover time. Addition of UV disinfection reduced the uncertainty around the removal efficacy, and mitigated the impact of larger shedding events, however the impact of UV disinfection on the natural treatment biome is unknown. Further information is needed on the performance of natural barriers for pathogen removal, and therefore challenge studies are recommended. Given the identified risks, the pool is posted that there is risk from accidental faecal releases, as in any natural water body with swimmers. Screening level risk assessment was a valuable first step in understanding the processes driving the system and in identifying important data gaps.

Role of amoebae for survival and recovery of 'non-culturable' Helicobacter pylori cells in aquatic environments.

Helicobacter pylori is a fastidious Gram-negative bacterium that infects over half of the world's population, causing chronic gastritis and is a risk factor for stomach cancer. In developing and rural regions where prevalence rate exceeds 60%, persistence and waterborne transmission are often linked to poor sanitation conditions. Here we demonstrate that H. pylori not only survives but also replicates within acidified free-living amoebal phagosomes. Bacterial counts of the clinical isolate H. pylori G27 increased over 50-fold after three days in co-culture with amoebae. In contrast, a H. pylori mutant deficient in a cagPAI gene (cagE) showed little growth within amoebae, demonstrating the likely importance of a type IV secretion system in H. pylori for amoebal infection. We also demonstrate that H. pylori can be packaged by amoebae and released in extracellular vesicles. Furthermore, and for the first time, we successfully demonstrate the ability of two free-living amoebae to revert and recover viable but non-cultivable coccoid (VBNC)-H. pylori to a culturable state. Our studies provide evidence to support the hypothesis that amoebae and perhaps other free-living protozoa contribute to the replication and persistence of human-pathogenic H. pylori by providing a protected intracellular microenvironment for this pathogen to persist in natural aquatic environments and engineered water systems, thereby H. pylori potentially uses amoeba as a carrier and a vector of transmission.

Interactions between Human Reovirus and Free-Living Amoebae: Implications for Enteric Virus Disinfection and Aquatic Persistence.

Free-living amoebae (FLA) are ubiquitous protozoa in aquatic/soil habitats and known to resist various disinfection methods commonly used in drinking and wastewater treatment plants. Reoviruses are emerging as useful infectious enteric virus indicators of wastewater treatment efficacy. The possible enhanced protection FLA may provide reoviruses, however, has not been previously described. Using an infectious clinical reovirus isolate in coculture with three FLA, namely, Vermamoeba vermiformis, Acanthamoeba polyphaga, and Willaertia magna, we followed reovirus persistence (by quantitative reverse transcription polymerase chain reaction (RT-qPCR)) and infectivity (TCID50). Virions present in samples persisted over the experimental time period, with most virions remaining infectious. Surprisingly, electron microscopy revealed virions accumulated within the nucleus of amoebae. The current work appears to be the first report of reovirus being internalized within FLA and remaining infectious, providing a previously unreported environmental reservoir and potential mode of dissemination. FLA also appeared to be providing some logs in protection to internalized viruses during UV irradiation, which if not accounted for when determining UV dosage needed for sufficient disinfection may result in unintentional release of pathogens into surrounding water systems.