Natalie Prystajecky

Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities

Cryptosporidium and Giardia were characterized in a watershed in southern Ontario, Canada, over a 2½ year period. River samples were collected every two weeks, primarily near a municipal drinking water treatment plant intake. Cryptosporidium and Giardia were frequently detected with an overall occurrence rate of 88 and 97%, respectively. Giardia concentrations were higher than Cryptosporidium, with median values of 80 cysts 100 L(-1) and 12 oocysts 100 L(-1), respectively. Although pathogens rarely show a significant relationship with fecal or water quality indicators, this study determined that Cryptosporidium, but not Giardia, was significantly correlated with Escherichia coli, turbidity and river flow. There was no correlation between the two types of protozoa, and only Giardia showed a seasonal trend with higher concentrations at cold water temperatures. Cryptosporidium genotyping of all samples found that farm animals and wildlife were an important contributor of oocysts in the watershed, and that Cryptosporidium strains/genotypes of medium to high risk for human infection (C. hominis, C. parvum and C. ubiquitum) were detected in 16% of samples. This study was able to identify Cryptosporidium host sources and human health risk, and to identify differences between Cryptosporidium and Giardia occurrence in the watershed.

A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples

BackgroundStudies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary.ResultsWe developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels.ConclusionThis study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.

A comparative analysis of current microbial water quality risk assessment and management practices in British Columbia and Ontario, Canada☆

Bacteria, protozoa and viruses are ubiquitous in aquatic environments and may pose threats to water quality for both human and ecosystem health. Microbial risk assessment and management in the water sector is a focus of governmental regulation and scientific inquiry; however, stark gaps remain in their application and interpretation. This paper evaluates how water managers practice microbial risk assessment and management in two Canadian provinces (BC and Ontario). We assess three types of entities engaged in water management along the source-to-tap spectrum (watershed agencies, water utilities, and public health authorities). We analyze and compare the approaches used by these agencies to assess and manage microbial risk (including scope, frequency, and tools). We evaluate key similarities and differences, and situate them with respect to international best practices derived from literatures related to microbial risk assessment and management. We find considerable variability in microbial risk assessment frameworks and management tools in that approaches 1) vary between provinces; 2) vary within provinces and between similar types of agencies; 3) have limited focus on microbial risk assessment for ecosystem health and 4) diverge considerably from the literature on best practices. We find that risk assessments that are formalized, routine and applied system-wide (i.e. from source-to-tap) are limited. We identify key limitations of current testing methodologies and looking forward consider the outcomes of this research within the context of new developments in microbial water quality monitoring such as tests derived from genomics and metagenomics based research.

A comparison of the regulatory frameworks governing microbial testing of drinking water in three Canadian provinces

In Canada, the provinces have primary responsibility for water management, including drinking water management; the federal government has comparatively more limited water-related responsibilities. Currently, Canada does not have a harmonized federal drinking water strategy or law (although it does have non-binding guidelines); thus provinces have developed different approaches to drinking water management. In this study, key features of the current regulatory frameworks for microbial testing in drinking water quality management are examined in Canada’s three most populous provinces – British Columbia, Ontario and Quebec. Salient regulatory features including the types of drinking water samples, water quality standards, testing frequency and system size are described and compared. Distinct differences were found in provincial approaches to drinking water quality assessment, and microbial water quality management is variable not only among, but also within, provinces. This finding of inter- and intra-province variability in drinking water quality management shows that these three provinces are approaching similar challenges in different ways. In turn, these different approaches demonstrate that regulatory frameworks can be adapted in response to drinking water management challenges. Regulatory frameworks should be flexible and adaptable to new knowledge and scientific developments, such as molecular testing methods, in order to facilitate their translation into water management tools.

Giardia spp. Are Commonly Found in Mixed Assemblages in Surface Water, as Revealed by Molecular and Whole-Genome Characterization.

ABSTRACT Giardia is the most common parasitic cause of gastrointestinal infections worldwide, with transmission through surface water playing an important role in various parts of the world. Giardia duodenalis (synonyms: G. intestinalis and G. lamblia), a multispecies complex, has two zoonotic subtypes, assemblages A and B. When British Columbia (BC), a western Canadian province, experienced several waterborne giardiasis outbreaks due to unfiltered surface drinking water in the late 1980s, collection of isolates from surface water, as well as from humans and beavers (Castor canadensis), throughout the province was carried out. To better understand Giardia in surface water, 71 isolates, including 29 from raw surface water samples, 29 from human giardiasis cases, and 13 from beavers in watersheds from this historical library were characterized by PCR. Study isolates also included isolates from waterborne giardiasis outbreaks. Both assemblages A and B were identified in surface water, human, and beavers samples, including a mixture of both assemblages A and B in waterborne outbreaks. PCR results were confirmed by whole-genome sequencing (WGS) for one waterborne outbreak and supported the clustering of human, water, and beaver isolates within both assemblages. We concluded that contamination of surface water by Giardia is complex, that the majority of our surface water isolates were assemblage B, and that both assemblages A and B may cause waterborne outbreaks. The higher-resolution data provided by WGS warrants further study to better understand the spread of Giardia.

Year-Long Metagenomic Study of River Microbiomes Across Land Use and Water Quality.

Select bacteria, such as Escherichia coli or coliforms, have been widely used as sentinels of low water quality; however, there are concerns regarding their predictive accuracy for the protection of human and environmental health. To develop improved monitoring systems, a greater understanding of bacterial community structure, function, and variability across time is required in the context of different pollution types, such as agricultural and urban contamination. Here, we present a year-long survey of free-living bacterial DNA collected from seven sites along rivers in three watersheds with varying land use in Southwestern Canada. This is the first study to examine the bacterial metagenome in flowing freshwater (lotic) environments over such a time span, providing an opportunity to describe bacterial community variability as a function of land use and environmental conditions. Characteristics of the metagenomic data, such as sequence composition and average genome size (AGS), vary with sampling site, environmental conditions, and water chemistry. For example, AGS was correlated with hours of daylight in the agricultural watershed and, across the agriculturally and urban-affected sites, k-mer composition clustering corresponded to nutrient concentrations. In addition to indicating a community shift, this change in AGS has implications in terms of the normalization strategies required, and considerations surrounding such strategies in general are discussed. When comparing abundances of gene functional groups between high- and low-quality water samples collected from an agricultural area, the latter had a higher abundance of nutrient metabolism and bacteriophage groups, possibly reflecting an increase in agricultural runoff. This work presents a valuable dataset representing a year of monthly sampling across watersheds and an analysis targeted at establishing a foundational understanding of how bacterial lotic communities vary across time and land use. The results provide important context for future studies, including further analyses of watershed ecosystem health, and the identification and development of biomarkers for improved water quality monitoring systems.

Assessment of Giardia and Cryptosporidium spp. as a microbial source tracking tool for surface water: application in a mixed-use watershed.

ABSTRACT Knowledge of host specificity, combined with genomic sequencing of Giardia and Cryptosporidium spp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period. Cryptosporidium was detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters. Giardia was detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% of Cryptosporidium samples and 98% of Giardia samples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy. Cryptosporidium genotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared to Giardia, since 98% of Giardia isolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows that Cryptosporidium subtyping for MST purposes is superior to the use of Giardia subtyping, based on better detection limits for Cryptosporidium-positive samples than for Giardia-positive samples and on greater host specificity among Cryptosporidium species. These additional tools could be used for risk assessment in public health and watershed management decisions.

Identification of fecal contamination sources in water using host-associated markers

In British Columbia, Canada, drinking water is tested for total coliforms and Escherichia coli, but there is currently no routine follow-up testing to investigate fecal contamination sources in samples that test positive for indicator bacteria. Reliable microbial source tracking (MST) tools to rapidly test water samples for multiple fecal contamination markers simultaneously are currently lacking. The objectives of this study were (i) to develop a qualitative MST tool to identify fecal contamination from different host groups, and (ii) to evaluate the MST tool using water samples with evidence of fecal contamination. Singleplex and multiplex polymerase chain reaction (PCR) were used to test (i) water from polluted sites and (ii) raw and drinking water samples for presence of bacterial genetic markers associated with feces from humans, cattle, seagulls, pigs, chickens, and geese. The multiplex MST assay correctly identified suspected contamination sources in contaminated waterways, demonstrating that this test may have utility for heavily contaminated sites. Most raw and drinking water samples analyzed using singleplex PCR contained at least one host-associated marker. Singleplex PCR was capable of detecting host-associated markers in small sample volumes and is therefore a promising tool to further analyze water samples submitted for routine testing and provide information useful for water quality management.

Use of Lean response to improve pandemic influenza surge in public health laboratories.

These tools enabled laboratory response to the 10-fold increase in testing demands.

Characterization of Legionella Species from Watersheds in British Columbia, Canada

Many species of Legionella can cause Legionnaires’ disease, a significant cause of bacterial pneumonia. Legionella in human-made water systems such as cooling towers and building plumbing systems are the primary sources of Legionnaires’ disease outbreaks. In this temporal study of natural aquatic environments, Legionella relative abundance was shown to vary in watersheds associated with different land uses. Analysis of the Legionella sequences detected at these sites revealed highly diverse populations that included potentially novel Legionella species. These findings have important implications for understanding the ecology of Legionella and control measures for this pathogen that are aimed at reducing human disease. ABSTRACT Legionella spp. present in some human-made water systems can cause Legionnaires’ disease in susceptible individuals. Although legionellae have been isolated from the natural environment, variations in the organism’s abundance over time and its relationship to aquatic microbiota are poorly understood. Here, we investigated the presence and diversity of legionellae through 16S rRNA gene amplicon and metagenomic sequencing of DNA from isolates collected from seven sites in three watersheds with varied land uses over a period of 1 year. Legionella spp. were found in all watersheds and sampling sites, comprising up to 2.1% of the bacterial community composition. The relative abundance of Legionella tended to be higher in pristine sites than in sites affected by agricultural activity. The relative abundance levels of Amoebozoa, some of which are natural hosts of legionellae, were similarly higher in pristine sites. Compared to other bacterial genera detected, Legionella had both the highest richness and highest alpha diversity. Our findings indicate that a highly diverse population of legionellae may be found in a variety of natural aquatic sources. Further characterization of these diverse natural populations of Legionella will help inform prevention and control efforts aimed at reducing the risk of Legionella colonization of built environments, which could ultimately decrease the risk of human disease. IMPORTANCE Many species of Legionella can cause Legionnaires’ disease, a significant cause of bacterial pneumonia. Legionella in human-made water systems such as cooling towers and building plumbing systems are the primary sources of Legionnaires’ disease outbreaks. In this temporal study of natural aquatic environments, Legionella relative abundance was shown to vary in watersheds associated with different land uses. Analysis of the Legionella sequences detected at these sites revealed highly diverse populations that included potentially novel Legionella species. These findings have important implications for understanding the ecology of Legionella and control measures for this pathogen that are aimed at reducing human disease.