Whole-Genome Sequencing and Virulome Analysis of Escherichia coli Isolated from New Zealand Environments of Contrasting Observed Land Use.

Generic Escherichia coli is commonly used as an indicator of fecal contamination to assess water quality and human health risk. Where measured E. coli exceedances occur, the presence of other pathogenic microorganisms, such as Shiga toxin-producing E. coli (STEC), is assumed, but confirmatory data are lacking. Putative E. coli isolates (n = 709) were isolated from water, sediment, soil, periphyton, and feces samples (n = 189) from five sites representing native forest and agricultural environments. Ten E. coli isolates (1.41%) were stx2 positive, 19 (2.7%) were eae positive, and stx1-positive isolates were absent. At the sample level, stx2-positive E. coli (5 of 189, 2.6%) and eae-positive isolates (16 of 189, 8.5%) were rare. Using real-time PCR, these STEC-associated virulence factors were determined to be more prevalent in sample enrichments (stx1, 23.9%; stx2, 31.4%; eae, 53.7%) and positively correlated with generic E. coli isolate numbers (P < 0.05) determined using culture-based methods. Whole-genome sequencing (WGS) was undertaken on a subset of 238 isolates with assemblies representing seven E. coli phylogroups (A, B1, B2, C, D, E, and F), 22 Escherichia marmotae isolates, and 1 Escherichia ruysiae isolate. Virulence factors, including those from extraintestinal pathogenic E. coli, were extremely diverse in isolates from the different locations and were more common in phylogroup B2. Analysis of the virulome from WGS data permitted the identification of gene repertoires that may be involved in environmental fitness and broadly align with phylogroup. Although recovery of STEC isolates was low, our molecular data indicate that they are likely to be widely present in environmental samples containing diverse E. coli phylogroups. IMPORTANCE This study takes a systematic sampling approach to assess the public health risk of Escherichia coli recovered from freshwater sites within forest and farmland. The New Zealand landscape is dominated by livestock farming, and previous work has demonstrated that "recreational exposure to water" is a risk factor for human infection by Shiga toxin-producing Escherichia coli (STEC). Though STEC isolates were rarely isolated from water samples, STEC-associated virulence factors were identified more commonly from water sample culture enrichments and were associated with increased generic E. coli concentrations. Whole-genome sequencing data from both E. coli and newly described Escherichia spp. demonstrated the presence of virulence factors from E. coli pathotypes, including extraintestinal pathogenic E. coli. This has significance for understanding and interpreting the potential health risk from E. coli where water quality is poor and suggests a role of virulence factors in survival and persistence of E. coli and Escherichia spp.

Comparison of faecal indicator and viral pathogen light and dark disinfection mechanisms in wastewater treatment pond mesocosms.

This study compared light and dark disinfection of faecal bacteria/viral indicator organisms (E. coli and MS2 (fRNA) bacteriophage) and human viruses (Echovirus and Norovirus) in Wastewater Treatment Pond (WTP) mesocosms. Stirred pond mesocosms were operated in either outdoor sunlight-exposed or laboratory dark conditions in two experiments during the austral summer. To investigate wavelength-dependence of sunlight disinfection, three optical filters were used: (1) polyethylene film (light control: transmitting all solar UV and visible wavelengths), (2) acrylic (removing most UVB <315 nm), and (3) polycarbonate (removing both UVB and UVA <400 nm). To assess different dark disinfection processes WTP effluent was treated before spiking with target microbes, by (a) 0.22 µm filtration to remove all but colloidal particles, (b) 0.22 µm filtration followed by heat treatment to destroy enzymes, and (c) addition of Cytochalasin B to supress protozoan grazing. Microbiological stocks containing E. coli, MS2 phage, Echovirus, and Norovirus were spiked into each mesocosm 10 min before the experiments commenced. The light control exposed to all sunlight wavelengths achieved >5-log E. coli and MS2 phage removal (from ~1.0 × 106 to <1 PFU/mL) within 3 h compared with up to 6 h in UV-filtered mesocosms. This result confirms that UVB contributes to inactivation of E. coli and viruses by direct sunlight inactivation. However, the very high attenuation with depth of UVB in WTP water (99% removal in the top 8 cm) suggests that UVB disinfection may be less important than other removal processes averaged over time and full-scale pond depth. Dark removal was appreciably slower than sunlight-mediated inactivation. The dark control typically achieved higher removal of E. coli and viruses than the 0.22 µm filtered (dark) mesocosms. This result suggests that adsorption of E. coli and viruses to WTP particles (e.g., algae and bacteria bio-flocs) is an important mechanism of dark disinfection, while bacteria and virus characteristics (e.g. surface charge) and environmental conditions can influence dark disinfection processes.

Fecal Bacteria, Bacteriophage, and Nutrient Reductions in a Full-Scale Denitrifying Woodchip Bioreactor.

Denitrifying bioreactors using woodchips or other slow-release carbon sources can be an effective method for removing nitrate (NO) from wastewater and tile drainage. However, the ability of these systems to remove fecal microbes from wastewater has been largely uninvestigated. In this study, reductions in fecal indicator bacteria () and viruses (F-specific RNA bacteriophage [FRNA phage]) were analyzed by monthly sampling along a longitudinal transect within a full-scale denitrifying woodchip bioreactor receiving secondary-treated septic tank effluent. Nitrogen, phosphorus, 5-d carbonaceous biochemical oxygen demand (CBOD), and total suspended solids (TSS) reduction were also assessed. The bioreactor demonstrated consistent and substantial reduction of (2.9 log reduction) and FRNA phage (3.9 log reduction) despite receiving highly fluctuating inflow concentrations [up to 3.5 × 10 MPN (100 mL) and 1.1 × 10 plaque-forming units (100 mL) , respectively]. Most of the removal of fecal microbial contaminants occurred within the first meter of the system (1.4 log reduction for ; 1.8 log reduction for FRNA phage). The system was also efficient at removing NO (>99.9% reduction) and TSS (89% reduction). There was no evidence of consistent removal of ammonium, organic nitrogen, or phosphorus. Leaching of CBOD occurred during initial operation but decreased and stabilized at lower values (14 g O m) after 9 mo. We present strong evidence for reliable microbial contaminant removal in denitrifying bioreactors, demonstrating their broader versatility for wastewater treatment. Research on the removal mechanisms of microbial contaminants in these systems, together with the assessment of longevity of removal, is warranted.

Microbial Transport, Retention, and Inactivation in Streams: A Combined Experimental and Stochastic Modeling Approach.

Long-term survival of pathogenic microorganisms in streams enables long-distance disease transmission. In order to manage water-borne diseases more effectively we need to better predict how microbes behave in freshwater systems, particularly how they are transported downstream in rivers. Microbes continuously immobilize and resuspend during downstream transport owing to a variety of processes including gravitational settling, attachment to in-stream structures such as submerged macrophytes, and hyporheic exchange and filtration within underlying sediments. We developed a stochastic model to describe these microbial transport and retention processes in rivers that also accounts for microbial inactivation. We used the model to assess the transport, retention, and inactivation of Escherichia coli in a small stream and the underlying streambed sediments as measured from multitracer injection experiments. The results demonstrate that the combination of laboratory experiments on sediment cores, stream reach-scale tracer experiments, and multiscale stochastic modeling improves assessment of microbial transport in streams. This study (1) demonstrates new observations of microbial dynamics in streams with improved data quality than prior studies, (2) advances a stochastic modeling framework to include microbial inactivation processes that we observed to be important in these streams, and (3) synthesizes new and existing data to evaluate seasonal dynamics.

High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments.

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Makirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.

Workplace stressor factors, profiles and the relationship to career stage in UK veterinarians, veterinary nurses and students.

BACKGROUND: Veterinary professionals experience higher psychological distress and lower wellbeing compared with the general population. Identifying workplace stressors is key to understanding and alleviating these difficulties. OBJECTIVE: Identify the severity of workplace stressors in veterinary professions across different levels of professional experience. METHOD: A cross-sectional quantitative design was utilised via administration of a veterinary stressor questionnaire that measured the severity of 93 generic and veterinary-specific workplace stressors. RESULTS: A total of 658 participants reported stressor severity score. Factor analysis revealed four main stressor categories: workload and job demands, client relations stress, performance and accountability, patient care challenges. An inverse relationship was found between experience level and stressor severity scores. Furthermore, the most severe stressors varied as a function of career stage. FINDINGS AND IMPLICATIONS: Stressor severity tends to decrease with increasing experience levels. Stressors can be categorised by theme and unique stressor profiles can be derived for practitioners according to experience level. This knowledge can assist in the design and delivery of workplace support initiatives. CONCLUSIONS: Identify key stressors across different levels of experience is important in focusing efforts to support people in the workplace.

Draft genome sequences of Escherichia spp. isolates from New Zealand environmental sources.

Escherichia coli is often used as a fecal indicator bacterium for water quality monitoring. We report the draft genome sequences of 500 Escherichia isolates including newly described Escherichia species, namely Escherichia marmotae, Escherichia ruysiae, and Escherichia whittamii, obtained from diverse environmental sources to assist with improved public health risk assessments.

Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples.

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/µL and 0.005 pg/µl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/µL of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.

Preferential feeding by the ciliates Chilodonella and Tetrahymena spp. and effects of these protozoa on bacterial biofilm structure and composition.

Protozoa are important components of microbial food webs, but protozoan feeding preferences and their effects in the context of bacterial biofilms are not well understood. The feeding interactions of two contrasting ciliates, the free-swimming filter feeder Tetrahymena sp. and the surface-associated predator Chilodonella sp., were investigated using biofilm-forming bacteria genetically modified to express fluorescent proteins. According to microscopy, both ciliates readily consumed cells from both Pseudomonas costantinii and Serratia plymuthica biofilms. When offered a choice between spatially separated biofilms, each ciliate showed a preference for P. costantinii biofilms. Experiments with bacterial cell extracts indicated that both ciliates used dissolved chemical cues to locate biofilms. Chilodonella sp. evidently used bacterial chemical cues as a basis for preferential feeding decisions, but it was unclear whether Tetrahymena sp. did also. Confocal microscopy of live biofilms revealed that Tetrahymena sp. had a major impact on biofilm morphology, forming holes and channels throughout S. plymuthica biofilms and reducing P. costantinii biofilms to isolated, grazing-resistant microcolonies. Grazing by Chilodonella sp. resulted in the development of less-defined trails through S. plymuthica biofilms and caused P. costantinii biofilms to become homogeneous scatterings of cells. It was not clear whether the observed feeding preferences for spatially separated P. costantinii biofilms over S. plymuthica biofilms resulted in selective targeting of P. costantinii cells in mixed biofilms. Grazing of mixed biofilms resulted in the depletion of both types of bacteria, with Tetrahymena sp. having a larger impact than Chilodonella sp., and effects similar to those seen in grazed single-species biofilms.

Differential behaviour of Escherichia coli and Campylobacter spp. in a stream draining dairy pasture.

The faecal indicator bacterium Escherichia coli and thermotolerant Campylobacter spp., which are potentially pathogenic, were investigated in the Toenepi Stream draining a pastoral catchment dominated by dairying. Bacteria concentrations were monitored routinely at fortnightly intervals over 12 months and intensively during storm events to compare the transport dynamics of bacterial indicator and pathogen under varying hydro-meteorological conditions. Routine monitoring indicated median concentrations of 345 E. coli MPN 100 ml(-1) and relatively low concentrations of 2.3 Campylobacter MPN 100 ml(-1). The bacterial flux was three orders of magnitude greater under elevated stream flow compared with base-flow. E. coli peak concentrations occurred very close to the turbidity peak and consistently ahead of the Campylobacter spp. peak (which was close to the hydrograph peak). We postulate that, under flood conditions, the E. coli peak reflects the entrainment and mobilisation of in-stream stores on the flood wave front. In contrast, Campylobacter spp. are derived from wash-in from land stores upstream and have travelled at the mean water velocity which is slower than the speed of the flood wave. Our findings of different dynamics for E. coli and Campylobacter spp. suggest that mitigation to reduce faecal microbial impacts from farms will need to take account of these differences.

Near real-time notification of water quality impairments in recreational freshwaters using rapid online detection of ß-D-glucuronidase activity as a surrogate for Escherichia coli monitoring.

Waterborne disease outbreaks associated with recreational waters continue to be reported around the world despite existing microbiological water quality monitoring frameworks. Most regulations resort to the use of culture-based enumeration of faecal indicator bacteria such as Escherichia coli to protect bathers from gastrointestinal illness risks. However, the long sample-to-result time of standard culture-based assays (minimum 18-24 h) and infrequent regulatory sampling (weekly or less) do not enable detection of episodic water quality impairments and associated public health risks. The objective of this study was to assess the suitability of an autonomous online technology measuring ß-D-glucuronidase (GLUC) activity for near real-time monitoring of microbiological water quality in recreational waters and for the resulting beach management decisions. GLUC activity and E. coli concentrations were monitored at three freshwater sites in Quebec, Canada (sites Qc1-3) and one site in New Zealand (site NZ) between 2016 and 2018. We found site-dependent linear relationships between GLUC activity and E. coli concentrations and using confusion matrices, we developed site-specific GLUC activity beach action values (BAVs) matching the regulatory E. coli BAVs. Using the regulatory E. coli BAV as the gold standard, rates of false alarms (unnecessary beach advisories using GLUC activity BAV) and failures to act (failure to trigger advisories using GLUC activity) ranged between 0 and 32% and between 3 and 10%, respectively, which is comparable to the rates reported in other studies using qPCR-defined BAVs. However, a major benefit of the autonomous enzymatic technology is the real-time reporting of threshold exceedances, while temporal trends in GLUC activity can assist in understanding the underlying dynamics of faecal pollution and potential health risks. Our study is the first to describe the applicability of online near real-time monitoring of microbiological water quality as a tool for improved beach management and public health protection.

Relative diversity and community structure of ciliates in stream biofilms according to molecular and microscopy methods.

Ciliates are an important component of aquatic ecosystems, acting as predators of bacteria and protozoa and providing nutrition for organisms at higher trophic levels. Understanding of the diversity and ecological role of ciliates in stream biofilms is limited, however. Ciliate diversity in biofilm samples from four streams subject to different impacts by human activity was assessed using microscopy and terminal restriction fragment length polymorphism (T-RFLP) analysis of 18S rRNA sequences. Analysis of 3' and 5' terminal fragments yielded very similar estimates of ciliate diversity. The diversity detected using microscopy was consistently lower than that suggested by T-RFLP analysis, indicating the existence of genetic diversity not apparent by morphological examination. Microscopy and T-RFLP analyses revealed similar relative trends in diversity between different streams, with the lowest level of biofilm-associated ciliate diversity found in samples from the least-impacted stream and the highest diversity in samples from moderately to highly impacted streams. Multivariate analysis provided evidence of significantly different ciliate communities in biofilm samples from different streams and seasons, particularly between a highly degraded urban stream and less impacted streams. Microscopy and T-RFLP data both suggested the existence of widely distributed, resilient biofilm-associated ciliates as well as ciliate taxa restricted to sites with particular environmental conditions, with cosmopolitan taxa being more abundant than those with restricted distributions. Differences between ciliate assemblages were associated with water quality characteristics typical of urban stream degradation and may be related to factors such as nutrient availability and macroinvertebrate communities. Microscopic and molecular techniques were considered to be useful complementary approaches for investigation of biofilm ciliate communities.

Effect of pharmacist-led task force to reduce opioid prescribing in the emergency department.

PURPOSE: Results of a study to determine the effect of a pharmacist-led opioid task force on emergency department (ED) opioid use and discharge prescriptions are presented. METHODS: An observational evaluation was conducted at a large tertiary care center (ED volume of 115,000 visits per year) to evaluate selected opioid use outcomes before and after implementation of an ED opioid reduction program by interdisciplinary task force of pharmacists, physicians, and nurses. Volumes of ED opioid orders and discharge prescriptions were evaluated over the entire 25-month study period and during designated 1-month preimplementation and postimplementation periods (January 2017 and January 2018). Opioid order trends were evaluated using linear regression analysis and further investigated with an interrupted time series analysis to determine the immediate and sustained effects of the program. RESULTS: From January 2017 to January 2018, ED opioid orders were reduced by 63.5% and discharge prescriptions by 55.8% from preimplementation levels: from 246.8 to 90.1 orders and from 85.3 to 37.7 prescriptions per 1,000 patient visits, respectively. Over the entire study period, there were significant decreases in both opioid orders (ß, -78.4; 95% confidence interval [CI], -88.0 to -68.9; R2, 0.93; p < 0.0001) and ED discharge prescriptions (ß, -24.4; 95% CI, -27.9 to -20.9; R2, 0.90; p < 0.001). The efforts of the task force had an immediate effect on opioid prescribing practices; results for effect sustainability were mixed. CONCLUSION: A clinical pharmacist-led opioid reduction program in the ED was demonstrated to have positive results, with a more than 50% reduction in both ED opioid orders and discharge prescriptions.

Molecular characterization of ciliate diversity in stream biofilms.

Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

Quantifying treatment system resilience to shock loadings in constructed wetlands and denitrification bioreactors.

Wastewater treatment ecotechnologies such as constructed wetlands and denitrifying bioreactors are commonly perceived as robust and resilient to shock loading, but this has proved difficult to quantify, particularly when comparing different systems. This study proposes a method of quantifying and comparing performance resilience in response to a standard disturbance. In a side-by-side study we compare the treatment performance of four different configurations of wetlands and denitrifying bioreactors subjected to hydraulic shock loads of five times the standard inflow rate of primary treated sewage for five days. The systems consist of: horizontal-flow gravel-bed wetlands (HG); single pass vertical-flow sand or gravel media wetlands followed by carbonaceous denitrifying bioreactors (VS + C and VG + C respectively); and a recirculating anoxic attached-growth bioreactor and vertical sand media wetland followed by carbonaceous denitrifying bioreactors (R(A + VS)+C). Resilience was quantified for Total Suspended Solids (TSS), Five-day Biochemical Oxygen Demand (BOD5) and Total Nitrogen (TN) by time integration of Relative Disturbance in Performance relative to pre-shock loading performance (days equivalent Performance Reduction), and by the Recovery Time after shock loading ceased. The quantification method allowed an unbiased comparison of the four different systems. It highlighted important differences in the resilience for different removal mechanisms associated with the configuration of the wetlands/bioreactor systems. Relative Disturbances in Performance were expressed in comparison to percent daily removal under standard loading, and, for the different pollutants were equivalent to loss of between 0.08 and 2.51 days of removal capacity. Average Recovery Times ranged from zero to three days, with all systems exhibiting substantial recovery even during the five-day shock loading period. This study demonstrated that both the horizontal gravel wetland and the vertical flow wetland systems combined with carbonaceous bioreactors tested are generally resilient to shock loading of five times hydraulic and organic loading for periods of up to five days. Standard quantification of performance resilience to shock loadings or other perturbations has potential application across a wide range of technologies and research fields.

Discharge-based QMRA for estimation of public health risks from exposure to stormwater-borne pathogens in recreational waters in the United States.

This study is the first to report a quantitative microbial risk assessment (QMRA) on pathogens detected in stormwater discharges-of-concern, rather than relying on pathogen measurements in receiving waters. The pathogen concentrations include seven "Reference Pathogens" identified by the U.S. EPA: Cryptosporidium, Giardia, Salmonella, Norovirus, Rotavirus, Enterovirus, and Adenovirus. Data were collected from 12 sites representative of seven discharge types (including residential, commercial/industrial runoff, agricultural runoff, combined sewer overflows, and forested land), mainly during wet weather conditions during which times human health risks can be substantially elevated. The risks calculated herein therefore generally apply to short-term conditions (during and just after rainfall events) and so the results can be used by water managers to potentially inform the public, even for waters that comply with current criteria (based as they are on a 30-day mean risk). Using an example waterbody and mixed source, pathogen concentrations were used in QMRA models to generate risk profiles for primary and secondary water contact (or inhalation) by adults and children. A number of critical assumptions and considerations around the QMRA analysis are highlighted, particularly the harmonization of the pathogen concentrations measured in discharges during this project with those measured (using different methods) during the published dose-response clinical trials. Norovirus was the most dominant predicted health risk, though further research on its dose-response for illness (cf. infection) is needed. Even if the example mixed-source concentrations of pathogens had been reduced 30 times (by inactivation and mixing), the predicted swimming-associated illness rates - largely driven by Norovirus infections - can still be appreciable. Rotavirus generally induced the second-highest incidence of risk among the tested pathogens while risks for the other Reference Pathogens (Giardia, Cryptosporidium, Adenovirus, Enterovirus and Salmonella) were considerably lower. Secondary contact or inhalation resulted in considerable reductions in risk compared to primary contact. Measurements of Norovirus and careful incorporation of its concentrations into risk models (harmonization) should be a critical consideration for future QMRA efforts. The discharge-based QMRA approach presented herein is particularly relevant to cases where pathogens cannot be reliably detected in receiving waters with detection limits relevant to human health effects.