Background: Beaches are popular summertime destinations in Canada. However, they can be affected by specific fecal pollution sources, increasing the risk of recreational water illness. Objectives: This study was conducted to determine the risks of acute gastrointestinal illness (AGI) among Canadian beachgoers and to evaluate the influence of different fecal indicator bacteria (FIB) and other water quality measures on assessing these risks. Methods: In a prospective cohort design, beachgoers were recruited at sites across Canada from 2023 to 2025. Sociodemographic characteristics and exposures were determined through an on-site survey, with a 7-day follow-up survey to determine risks of AGI. Bayesian mixed-effects logistic regression models were fitted to evaluate the effects of an ordinal water contact variable (no contact, minimal contact, body immersion, and swallowed water) on the incident risk of AGI, with an interaction included for water quality indicators. The levels of six FIB and water quality measures were assessed: Escherichia coli, enterococci DNA, three microbial source tracking DNA markers (human HF183/BacR287, human mitochondria, seagull Gull4), and turbidity. Results: A total of 4085 participants were recruited, with 67.6% completing the follow-up survey. The overall incident risk of AGI was 2.6%. Both swallowing water and body immersion increased AGI risks compared to no water contact: median of 20 excess cases (95% Credible Interval [CrI]: 4, 64) and 5 excess cases (95% CrI: 1, 19) of AGI predicted per 1000 beachgoers, respectively. Escherichia coli and seagull DNA marker levels were associated with AGI among those who had water contact, particularly among those who reported swallowing water. Discussion: While the overall burden of AGI due to beach water contact in Canada was low, increased risks are associated with E. coli levels particularly among those who swallow water. This could be related to fecal contamination from seagulls. However, there is substantial uncertainty in the predicted effect sizes. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Protocols ### Funding Statement This work was supported by the Canadian Institutes of Health Research (CIHR), grant numbers PJT 185894 and PJT 192023 (P.I. Young) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Toronto Metropolitan University Research Ethics Board gave ethical approval for this work (REB# 2023-043). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [https://github.com/iany33/Beach\_cohort\_AGI][1] [1]: https://github.com/iany33/Beach_cohort_AGI

Climate-driven permafrost thaw and altered precipitation, combined with reliance on limited water sources and treatment infrastructure, challenge the microbiological safety of northern drinking water. However, the interplay between water bacterial composition, treatment methods, and dissolved organic matter (DOM) remains overlooked in these systems. We investigated bacterial community composition from source to tap in eight northern communities across Nunavut, Nunavik, and the Northwest Territories, and its association with DOM and nutrient composition, to provide a comprehensive understanding of bacterial ecology and potential water safety vulnerabilities in northern drinking water systems. We hypothesized that: (1) source water bacterial composition varies with DOM composition; (2) bacterial composition in supply systems reflects source conditions and; (3) shifts with treatment, reflecting community-specific procedures. Our study reveals that drinking water overall bacterial composition is driven by DOM and nutrient composition, and shifts along supply systems, with community-specific patterns of reduction/increase in bacterial abundance from source to tap. These results underscore the necessity of regular monitoring to identify microbiological risks linked to environmental changes affecting DOM and the need to address community-specific vulnerabilities, such as bacterial regrowth, related to treatment processes. These findings enhance our understanding of northern microbiological water quality dynamics, informing targeted public health interventions.

Conventional recreational water quality monitoring is based on fecal indicator measurements for public health-related water postings or long-term remediation strategies. However, interpretation of recreational water quality is often limited by delayed culture results and the inability of traditional fecal indicators to distinguish contamination sources, which may differ across diverse recreational water environments. To address these limitations, we tested rapid qPCR for enterococci in parallel with tests for microbial source-tracking markers and Escherichia coli culturing at geographically distinct Canadian marine and freshwater beaches. Samples were collected from 10 beaches in Vancouver, Grand Beach Provincial Park, the Niagara region, Halifax, and Toronto during the 2023 to 2025 bathing seasons and analyzed for culture-based E. coli, qPCR-based enterococci, and microbial source-tracking markers. The enterococci qPCR performed well analytically and showed a strong positive association with the same-day retrospective E. coli measure, supporting the operational feasibility of qPCR-based beach monitoring throughout different regions. However, the correlation and agreement in Beach Action Value (BAV) exceedances differed among beaches, suggesting that fecal indicators may not exhibit uniform trends across sites. Source-tracking results revealed that the gull-associated molecular marker (Gull4) was prominent at most beaches and showed consistent positive correlation with fecal indicators overall. The human-associated HF183 marker was prominent only at beaches in larger urban centers. Additionally, Gull4 was the most prevalent marker and showed the strongest and most consistent association with BAV exceedances, whereas HF183 only showed an association with BAV exceedances at one urban beach region. This study demonstrates that rapid molecular monitoring can complement standard culture-based assessment in recreational water monitoring, while MST markers provide source-specific information to support adaptive beach risk management and identify data needs for future Quantitative Microbial Risk Assessment.

Cryptosporidium is a major waterborne parasite that causes gastrointestinal illness. Conventional assays, including microscopy and immunological identification, often suffer from false positives or negatives due to non-specific binding or morphological differences between Cryptosporidium species. We developed a novel qPCR assay based on a Cryptosporidium-specific Conserved Signature Protein (CSP) to address the limitations of testing complex samples, including those from recreational waters. The CSP (hypothetical protein (cgd2_3830)) was identified as taxonomically unique to Cryptosporidium species. The CSP sequence and designed qPCR assay primers/probe demonstrated high specificity for the targeted Cryptosporidium species when tested against NCBI RefSeq databases. qPCR assay efficiency was determined as 95% and an R2 value of 0.99, with a slope and intercept of −3.4 and 40.1, respectively. Additionally, the Lower Limit of Detection (ALLOD) was determined as three gene copies, suggesting the potential to detect even a single oocyst. No non-specific amplification products or primer dimers were observed when the qPCR assay was evaluated using recreational water, fecal solution, and wastewater, while spike-in-control tests indicated minimal interference with the sensitivity of the assay, highlighting application for testing complex environmental DNA extracts. These findings highlight the application of the novel CSP-based qPCR assay for the rapid and sensitive detection of Cryptosporidium sp., thereby circumventing the sequence variability and multi-copy limitations associated with existing molecular markers. This proof-of-concept study presents a diagnostic framework utilizing CSP-based markers for developing water quality monitoring strategies, with scope for expansion to other microbial pathogens and potential applications in clinical and food safety settings.

The genus Pseudalkalibacillus, created by the reclassification of specific deep-branching Alkalihalobacillus species, exhibits polyphyletic branching. The Genome Taxonomy Database (GTDB) also assigns Pseudalkalibacillus species into two families and three genera. To clarify the evolutionary relationships and classification of Pseudalkalibacillus species, we report detailed investigations using phylogenomic and molecular signature-based approaches. In phylogenomic trees, Pseudalkalibacillus species are distributed within two family-level lineages. One of these clades, containing the type species of Pseudalkalibacillus (viz. Pseudalkalibacillus decolorationis), represents the genus Pseudalkalibacillus, groups within the family Fictibacillaceae. Ten novel conserved signature indels (CSIs) identified in this study are specific for this clade, providing a robust means for the differentiation of the emended genus Pseudalkalibacillus. The remaining Pseudalkalibacillus species form a separate family-level clade, designated as f_HBI72195 in the GTDB. Within this clade, all species except Pseudalkalibacillus caeni form a robust clade designated as Pseudalkalibacillus clade -2 in our work and g_Anaerobacillus_A in the GTDB. We have also identified 15 novel CSIs specific to this clade. As the Pseudalkalibacillus clade -2 is distinct from Pseudalkalibacillus, we propose transferring species from this clade into a new genus, Guptibacillus gen. nov. The species P. caeni branches distinctly from other Pseudalkalibacillus species, and the GTDB considers it a novel genus (g_Bacillus_BR). Six newly identified CSIs are specific to this species, and we are proposing the transfer of this species into a new genus, Exobacillus gen. nov. Two additional identified CSIs are shared by members of the novel family-level taxon (f_HBI72195) comprising the proposed genera Guptibacillus and Exobacillus, for which we are proposing the name Guptibacillaceae fam. nov. Lastly, the results presented here also show that 'Pseudalkalibacillus hemicentroti' and 'Pseudalkalibacillus macyae' are later heterotypic synonyms of Guptibacillus hwajinpoensis. These changes, which reliably depict the evolutionary relationships among Pseudalkalibacillus species, should be helpful in future studies of these organisms.

A major goal of fecal pollution monitoring in the environment is to identify point sources of fecal contamination that may pose potential health risks due to animal- and human-specific pathogens. Ideal source tracking markers should have high host specificity and can be employed for the unambiguous identification of the host/fecal point sources. Conserved signature proteins (CSPs) are a class of unique, phylogenetically coherent indicators that are specific to a given taxon (e.g., genus or species). In this study, we report the identification and characterization of a new CSP, whose gene (designated as CSP-DV) is present in a single copy, and for whom homologs showing a high degree of sequence similarity are found only in genomes of Phocaeicola dorei and Phocaeicola vulgatus, two commensal species commonly found in the human gut and feces. We developed a qPCR method targeting this CSP gene to explore its usefulness as a human source tracking marker. We confirmed that the CSP-DV marker showed an absolute human sensitivity (100%) but some cross-reactivities in chicken, cats, dogs, rabbits, and rodents. In recreational water, the CSP-DV marker gene levels were well correlated with those of HF183, a well-validated human marker that predominantly targets the 16S rRNA gene of P. dorei, suggesting that it can be a new potential source tracking tool for human fecal contamination in specific environmental waters. In summary, our CSP-DV marker targets Phocaeicola clade-specific microbes and can provide an additional approach independent of the 16S rRNA gene to detect human sources of fecal pollution.

Freshwater beach quality is routinely tested by measuring fecal indicator bacteria, which can assess water quality but cannot identify sources of fecal contamination.

BackgroundRecreational water activities at beaches are popular among Canadians. However, these activities can increase the risk of recreational water illnesses (RWI) among beachgoers. Few studies have been conducted in Canada to determine the risk of these illnesses. This protocol describes the methodology for a study to determine the risk and burden of RWI due to exposure to fecal pollution at beaches in Canada.MethodsThis study will use a mixed-methods approach, consisting of a prospective cohort study of beachgoers with embedded qualitative research. The cohort study involves recruiting and enrolling participants at public beaches across Canada, ascertaining their water and sand contact exposure status, then following-up after seven days to determine the incidence of acute RWI outcomes. We will test beach water samples each recruitment day for culture-based E. coli, enterococci using rapid molecular methods, and microbial source tracking biomarkers. The study started in 2023 and will continue to 2025 at beaches in British Columbia, Manitoba, Ontario, and Nova Scotia. The target enrollment is 5000 beachgoers. Multilevel logistic regression models will be fitted to examine the relationships between water and sand contact and RWI among beachgoers. We will also examine differences in risks by beachgoer age, gender, and beach location and the influence of fecal indicator bacteria and other water quality parameters on these relationships. Sensitivity analyses will be conducted to examine the impact of various alternative exposure and outcome definitions on these associations. The qualitative research phase will include focus groups with beachgoers and key informant interviews to provide additional contextual insights into the study findings. The study will use an integrated knowledge translation approach.DiscussionInitial implementation of the study at two Toronto, Ontario, beaches in 2023 confirmed that recruitment is feasible and that a high completion rate (80%) can be achieved for the follow-up survey. While recall bias could be a concern for the self-reported RWI outcomes, we will examine the impact of this bias in a negative control analysis. Study findings will inform future recreational water quality guidelines, policies, and risk communication strategies in Canada.

Keywords: precision vaccinology, infectious diseases, global health, immunity, vulnerable populations

Harmful algal bloom (HAB) formation leads to the eutrophication of water ecosystems and may render recreational lakes unsuitable for human use. We evaluated the applicability and comparison of metabarcoding, metagenomics, qPCR, and ELISA-based methods for cyanobacteria/cyanotoxin detection in bloom and non-bloom sites for the Great Lakes region. DNA sequencing-based methods robustly identified differences between bloom and non-bloom samples (e.g., the relative prominence of Anabaena and Planktothrix). Shotgun sequencing strategies also identified the enrichment of metabolic genes typical of cyanobacteria in bloom samples, though toxin genes were not detected, suggesting deeper sequencing or PCR methods may be needed to detect low-abundance toxin genes. PCR and ELISA indicated microcystin levels and microcystin gene copies were significantly more abundant in bloom sites. However, not all bloom samples were positive for microcystin, possibly due to bloom development by non-toxin-producing species. Additionally, microcystin levels were significantly correlated (positively) with microcystin gene copy number but not with total cyanobacterial 16S gene copies. In summary, next-generation sequencing-based methods can identify specific taxonomic and functional targets, which can be used for absolute quantification methods (qPCR and ELISA) to augment conventional water monitoring strategies.

Background Fecal bacterial densities are proxy indicators of beach water quality, and beach posting decisions are made based on Beach Action Value (BAV) exceedances for a beach. However, these traditional beach monitoring methods do not reflect the full extent of microbial water quality changes associated with BAV exceedances at recreational beaches (including harmful cyanobacteria). This proof of concept study evaluates the potential of metagenomics for comprehensively assessing bacterial community changes associated with BAV exceedances compared to non-exceedances for two urban beaches and their adjacent river water sources. Results Compared to non-exceedance samples, BAV exceedance samples exhibited higher alpha diversity (diversity within the sample) that could be further differentiated into separate clusters (Beta-diversity). For Beach A, Cyanobacterial sequences (resolved as Microcystis and Pseudanabaena at genus level) were significantly more abundant in BAV non-exceedance samples. qPCR validation supported the Cyanobacterial abundance results from metagenomic analysis and also identified saxitoxin genes in 50% of the non-exceedance samples. Microcystis sp and saxitoxin gene sequences were more abundant on non-exceedance beach days (when fecal indicator data indicated the beach should be open for water recreational purposes). For BAV exceedance days, Fibrobacteres, Pseudomonas , Acinetobacter, and Clostridium sequences were significantly more abundant (and positively correlated with fecal indicator densities) for Beach A. For Beach B, Spirochaetes (resolved as Leptospira on genus level) Burkholderia and Vibrio sequences were significantly more abundant in BAV exceedance samples. Similar bacterial diversity and abundance trends were observed for river water sources compared to their associated beaches. Antibiotic Resistance Genes (ARGs) were also consistently detected at both beaches. However, we did not observe a significant difference or correlation in ARGs abundance between BAV exceedance and non-exceedance samples. Conclusion This study provides a more comprehensive analysis of bacterial community changes associated with BAV exceedances for recreational freshwater beaches. While there were increases in bacterial diversity and some taxa of potential human health concern associated with increased fecal indicator densities and BAV exceedances (e.g. Pseudomonas ), metagenomics analyses also identified other taxa of potential human health concern (e.g. Microcystis ) associated with lower fecal indicator densities and BAV non-exceedances days. This study can help develop more targeted beach monitoring strategies and beach-specific risk management approaches.

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.

Cyanobacteria (blue-green algae) are a diverse group of prokaryotic microorganisms that impact global biogeochemical cycles. Under eutrophic conditions, cyanobacterial species can produce cyanotoxins, resulting in harmful algal blooms (cHABs) that degrade water quality and result in economic and recreational losses. The Laurentian Great Lakes, a key global freshwater source, are increasingly affected by these blooms. To understand the underlying mechanisms in cHAB formation, we investigated microcystin levels, cyanotoxin genes/transcripts, and taxonomic/microcystin metabarcoding across three sampling locations in the Canadian Great Lakes region, including Hamilton Harbour, Bay of Quinte, and Three Mile Lake (Muskoka), to observe the regional and longitudinal cyanobacterial dynamics. The results revealed a positive correlation between microcystin levels, the occurrence of cyanobacterial taxonomic/cyanotoxin molecular markers, and the relative widespread abundance of specific dominant cyanobacterial taxa, including Planktothrix, Microcystis, and Dolichospermum. The Cyanobium genus was not observed in Hamilton Harbor samples during late summer (August to September), while it was consistently observed in the Three Mile Lake and Bay of Quinte samples. Notably, Dolichospermum and saxitoxin genes were predominantly higher in Three Mile Lake (an inland lake), suggesting site-specific characteristics influencing saxitoxin production. Additionally, among the potential microcystin producers, in addition to Microcystis, Hamilton Harbour and Bay of Quinte samples showed consistent presence of less dominant microcystin-producing taxa, including Phormidium and Dolichospermum. This study highlights the complexity of cHAB formation and the variability in cyanotoxin production in specific environments. The findings highlight regional and site-specific factors that can influence cyanobacterial taxonomic and molecular profiles, necessitating the integration of advanced molecular technologies for effective monitoring and targeted management strategies.

Rapid water-quality monitoring methods for beach water and sand can be helpful for public health authorities to develop comprehensive beach monitoring programs. In this study, we evaluated the performance of the Enterococcus qPCR (USEPA 1609.1) method for quality monitoring of beach water and sand-porewater at two Niagara region beaches in Ontario, Canada (Lakeside and Sunset Beaches). While the USEPA 1609.1 method has been validated for beach water, its potential for assessing associated beach sands (which may function as a microbial reservoir) has not been fully explored. All beach water samples (n = 168) passed the qPCR quality control (QC). However, only 20 out of 48 (41.7%) sand-porewater samples passed the qPCR QC, potentially due to interference by soluble inhibitors. The proportion of the sand-porewater samples passing QC improved slightly to 63 out of 120 (52.5%) with a prefiltration step to remove sand and other large particles. The faecal indicator density in the sand-porewater, tested in parallel, did not correlate with the beach water faecal indicator density. Comparing beach water data for the same-day Enterococcus qPCR threshold with the previous-day E. coli culturing-based threshold across all beach days tested, Enterococcus qPCR analyses identified 3 (7%) and 7 (16%) false positive/lost beach days for Lakeside and Sunset Beaches, respectively. Additionally, of the total beach days tested, Enterococcus qPCR analyses identified 2 (5%) and 1 (2%) false negative/health-risk days for Lakeside and Sunset Beaches, respectively. Sand-porewater testing analyses identified days when faecal indicators (in the sand) exceeded beach water quality thresholds. Compared with conventional E. coli culturing, use of the same-day qPCR method would result in fewer beach postings and could identify several additional health-risk days (when the beaches may not be posted). Future studies could include additional prefiltration steps or modifications in the Enterococcus qPCR protocol to improve the method’s applicability for sand quality monitoring.

Infectious diseases continue to be a major cause of morbidity and mortality worldwide. Diseases cause perturbation of the host's immune system provoking a response that involves genes, proteins and metabolites. While genes are regulated by epigenetic or other host factors, proteins can undergo post-translational modification to enable/modify function. As a result, it is difficult to correlate the disease phenotype based solely on genetic and proteomic information only. Metabolites, however, can provide direct information on the biochemical activity during diseased state. Therefore, metabolites may, potentially, represent a phenotypic signature of a diseased state. Measuring and assessing metabolites in large scale falls under the omics technology known as "metabolomics". Comprehensive and/or specific metabolic profiling in biological fluids can be used as biomarkers of disease diagnosis. In addition, metabolomics together with genomics can be used to differentiate patients with differential treatment response and development of host targeted therapy instead of pathogen targeted therapy where pathogens are more prone to mutation and lead to antimicrobial resistance. Thus, metabolomics can be used for patient stratification, personalized drug formulation and disease control and management. Currently, several therapeutics and in vitro diagnostics kits have been approved by US Food and Drug Administration (FDA) for personalized treatment and diagnosis of infectious diseases. However, the actual number of therapeutics or diagnostics kits required for tailored treatment is limited as metabolomics and personalized medicine require the involvement of personnel from multidisciplinary fields ranging from technological development, bioscience, bioinformatics, biostatistics, clinicians, and biotechnology companies. Given the significance of metabolomics, in this review, we discussed different aspects of metabolomics particularly potentials of metabolomics as diagnostic biomarkers and use of small molecules for host targeted treatment for infectious diseases, and their scopes and challenges in personalized medicine.

Cyanobacteria (blue-green algae) can accumulate to form harmful algal blooms (HABs) on the surface of freshwater ecosystems under eutrophic conditions. Extensive HAB events can threaten local wildlife, public health, and the utilization of recreational waters. For the detection/quantification of cyanobacteria and cyanotoxins, both the United States Environmental Protection Agency (USEPA) and Health Canada increasingly indicate that molecular methods can be useful. However, each molecular detection method has specific advantages and limitations for monitoring HABs in recreational water ecosystems. Rapidly developing modern technologies, including satellite imaging, biosensors, and machine learning/artificial intelligence, can be integrated with standard/conventional methods to overcome the limitations associated with traditional cyanobacterial detection methodology. We examine advances in cyanobacterial cell lysis methodology and conventional/modern molecular detection methods, including imaging techniques, polymerase chain reaction (PCR)/DNA sequencing, enzyme-linked immunosorbent assays (ELISA), mass spectrometry, remote sensing, and machine learning/AI-based prediction models. This review focuses specifically on methodologies likely to be employed for recreational water ecosystems, especially in the Great Lakes region of North America.

The performance of on-site wastewater treatment systems (OWTSs) can be improved by altering digester design and by manipulating environmental variables that affect microbial community composition. Community composition can potentially be assessed using high-throughput DNA sequencing, but the two most common methods of community DNA sequencing (16S and shotgun sequencing) generally yield different taxonomic identification profiles and can perform differently according to the sampled environment. To evaluate the use of these two approaches in monitoring OWTS operation, we conducted a comparative parallel analysis using both 16S rDNA and shotgun sequencing in a controlled field study. Results indicate that when examining microorganisms above 0.1% relative abundance, 16S and shotgun sequencing produced similar results in terms of individual sample species richness and between-sample community similarity. However, shotgun sequencing provided comparatively higher taxonomic richness for the bacterial communities with lower abundance in the OWTSs. In addition, 16S sequencing resolved only 48 out of 188 bacterial communities identified by shotgun sequencing (using a 0.1% abundance cutoff). Three key bacterial genera (Desulfomicrobium, Simplicispira, and Phenylobacterium) in anaerobic digestion processes were differentially abundant for both sequencing methods. These data indicate that both sequencing methods provide similar overall profiles for bacterial communities in anaerobic digestor systems. However, shotgun sequencing provides significantly (p-value < 0.01) higher taxonomic richness overall. Thus, shotgun sequencing provides a more robust taxonomic and functional profile that can be used for the optimization of anaerobic digestor systems.

EDITORIAL article Front. Microbiol., 06 October 2023Sec. Microbial Physiology and Metabolism Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1291172

CrAssphage or crAss-like phage ranks as the most abundant phage in the human gut and is present in human feces-contaminated environments. Due to its high human specificity and sensitivity, crAssphage is a potentially robust source tracking indicator that can distinguish human fecal contamination from agricultural or wildlife sources. Its suitability in the Great Lakes area, one of the world's most important water systems, has not been well tested. In this study, we tested a qPCR-based quantification method using two crAssphage marker genes (ORF18-mod and CPQ_064) at Toronto recreational beaches along with their adjacent river mouths. Our results showed a 71.4 % (CPQ_064) and 100 % (ORF18-mod) human sensitivity for CPQ_064 and ORF18-mod, and a 100 % human specificity for both marker genes. CrAssphage was present in 57.7 % or 71.2 % of environmental water samples, with concentrations ranging from 1.45 to 5.14 log10 gene copies per 100 mL water. Though concentrations of the two marker genes were strongly correlated, ORF18-mod features a higher human sensitivity and higher positive detection rates in environmental samples. Quantifiable crAssphage was mostly present in samples collected in June and July 2021 associated with higher rainfall. In addition, rivers had more frequent crAssphage presence and higher concentrations than their associated beaches, indicating more frequent and greater human fecal contamination in the rivers. However, crAssphage was more correlated with E. coli and Enterococcus at the beaches than in the rivers, suggesting human fecal sources may be more predominant in driving the increases in E. coli and Enterococcus at the beaches when impacted by river plumes.

Objectives We evaluated the potential impacts from using a rapid same-day quantitative polymerase chain reaction (qPCR) monitoring method for beach posting outcomes at two Toronto beaches. Methods In total, 228 water samples were collected at Marie Curtis Park East and Sunnyside Beaches over the 2021 summer season. Water samples were processed using the USEPA 1609.1 Enterococcus qPCR-based method. Escherichia coli ( E. coli ) culture data and daily beach posting decisions were obtained from Toronto Public Health. Results No significant correlation was observed between previous-day and same-day (retrospective) E. coli enumeration results at any Sunnyside Beach transect, and only relatively low ( R  = 0.41–0.56) or no significant correlation was observed at sampling transects for Marie Curtis Park East Beach. Comparing our same-day Enterococcus qPCR data to Toronto’s 2-day E. coli geometric mean beach posting decisions, we noted the need for additional postings for 1 (2%) and 3 (8%) missed health-risk days at Sunnyside and Marie Curtis Park East Beaches, respectively. The qPCR data also pointed to incorrect postings for 12 (31%) and 6 (16%) lost beach days at Sunnyside and Marie Curtis Park East Beaches, respectively. Conclusion Application of a rapid Enterococcus qPCR method at two Toronto beaches revealed 5% of beach posting decisions were false negatives that missed health-risk days, while 23% of decisions were false positives resulting in lost beach days. Deployment of the rapid same-day qPCR method offers the potential to reduce both health risks and unnecessary beach postings. Objectifs Nous avons évalué, à deux plages de Toronto, l’effet possible de l’utilisation d’une méthode de surveillance rapide par PCR quantitative (qPCR) le même jour sur les avis de fermeture ou d’ouverture des plages. Méthode En tout, 228 échantillons d’eau ont été prélevés aux plages Marie Curtis Park East et Sunnyside au cours de la saison estivale 2021. La présence d’ Enterococcus dans les échantillons a été détectée par la méthode USEPA 1609.1, utilisant la qPCR. Les données sur les cultures d’ Escherichia coli (E. coli) et les avis quotidiens de fermeture ou d’ouverture des plages ont été obtenus auprès du Bureau de santé de Toronto. Résultats Aucune corrélation significative n’a été observée entre les résultats (rétrospectifs) du dénombrement de E. coli obtenus la veille et le même jour dans les transects de la plage Sunnyside, et une corrélation significative faible (R = 0,41–0,56) ou nulle a été observée dans les transects d’échantillonnage de la plage Marie Curtis Park East. En comparant nos données sur Enterococcus obtenues le même jour par qPCR à la moyenne géométrique des avis de fermeture ou d’ouverture des plages sur deux jours liés à E. coli émis par le Bureau de santé de Toronto, nous avons remarqué qu’il aurait fallu émettre des avis de fermeture pour 1 jour de risques pour la santé manqué (2 %) à la plage Sunnyside et pour 3 jours de risques pour la santé manqués (8 %) à la plage Marie Curtis Park East. Les données de la qPCR ont aussi fait état d’avis de fermeture incorrects ayant entraîné la perte de 12 jours de plage (31 %) à Sunnyside et de 6 jours de plage (16 %) à Marie Curtis Park East. Conclusion L’application d’une méthode de surveillance rapide d’ Enterococcus par qPCR à deux plages de Toronto a montré que 5 % des avis étaient des faux négatifs qui n’ont pas détecté des jours de risques pour la santé, et que 23 % étaient des faux positifs qui ont entraîné des jours de plage perdus. Le déploiement de la méthode rapide par qPCR le même jour offre la possibilité de réduire à la fois les risques pour la santé et les avis de fermeture de plages inutiles.