RESUMEN
Climate change is already impacting the North American Great Lakes ecosystem and understanding the relationship between climate events and public health, such as waterborne acute gastrointestinal illnesses (AGIs), can help inform needed adaptive capacity for drinking water systems (DWSs). In this study, we assessed a harmonized binational dataset for the effects of extreme precipitation events (≥90th percentile) and preceding dry periods, source water turbidity, total coliforms, and protozoan AGIs - cryptosporidiosis and giardiasis - in the populations served by four DWSs that source surface water from Lake Ontario (Hamilton and Toronto, Ontario, Canada) and Lake Michigan (Green Bay and Milwaukee, Wisconsin, USA) from January 2009 through August 2014. We used distributed lag non-linear Poisson regression models adjusted for seasonality and found extreme precipitation weeks preceded by dry periods increased the relative risk of protozoan AGI after 1 and 3-5 weeks in three of the four cities, although only statistically significant in two. Our results suggest that the risk of protozoan AGI increases with extreme precipitation preceded by a dry period. As extreme precipitation patterns become more frequent with climate change, the ability to detect changes in water quality and effectively treat source water of varying quality is increasingly important for adaptive capacity and protection of public health.
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Agua Potable , Ciudades , Ecosistema , Lagos , América del Norte , Ontario , LluviaRESUMEN
Areas of concern (AOCs) around the Great Lakes are characterized by historic and ongoing problems with microbial water quality, leading to beneficial use impairments (BUIs) such as beach postings and closures. In this study, we assessed river and beach sites within the Rouge River watershed, associated stormwater outfalls, and at Rouge Beach. The concentrations of Escherichia coli as well as human- and gull-specific qPCR microbial source tracking (MST) markers were assessed at all sites. A preliminary comparison of digital PCR (dPCR) methodologies for both MST markers was conducted regarding sensitivity and specificity. Within the watershed, the outfalls were found to be a prominent source of human fecal contamination, with two outfalls particularly affected by sewage cross-connections. However, the occurrence of human fecal contamination along Rouge Beach and in the lower portions of the watershed was largely dependent on rain events. Gull fecal contamination was the predominant source of contamination at the beach, particularly during dry weather. The multiplex human/gull dPCR methodology used in this study tended to be more sensitive than the individual quantitative PCR (qPCR) assays, with only a slight decrease in specificity. Both dPCR and qPCR methodologies identified the same predominance of human and gull markers in stormwater and beach locations, respectively; however, the dPCR multiplex assay was more sensitive and capable of detecting fecal contamination that was undetected by qPCR assays. These results demonstrate the dPCR assay used in this study could be a viable tool for MST studies to increase the ability to identify low levels of fecal contamination.IMPORTANCE Fecal contamination of recreational water poses a persistent and ongoing problem, particularly in areas of concern around the Great Lakes. The identification of the source(s) of fecal contamination is essential for safeguarding public health as well as guiding remediation efforts; however, fecal contamination may frequently be present at low levels and remain undetectable by certain methodologies. In this study, we utilized microbial source tracking techniques using both quantitative and digital PCR assays to identify sources of contamination. Our results indicated high levels of human fecal contamination within stormwater outfalls, while lower levels were observed throughout the watershed. Additionally, high levels of gull fecal contamination were detected at Rouge Beach, particularly during drier sampling events. Furthermore, our results indicated an increased sensitivity of the digital PCR assay to detect both human and gull contamination, suggesting it could be a viable tool for future microbial source tracking studies.
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Bacterias/aislamiento & purificación , Monitoreo del Ambiente/métodos , Heces/microbiología , Lagos/microbiología , Aguas del Alcantarillado/microbiología , Microbiología del Agua , Animales , Bacterias/genética , Playas , Charadriiformes , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Great Lakes Region , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Recreación , Contaminación del Agua/análisis , Calidad del AguaRESUMEN
Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4â¯400â¯000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2-8.0 marker equivalents (ME) 100 mL-1) and biologically treated wastewater samples (median log10 4.6-6.0 ME 100 mL-1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.
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Aguas Residuales , Contaminación del Agua , Animales , Monitoreo del Ambiente , Heces , Marcadores Genéticos , Humanos , Microbiología del AguaRESUMEN
Storm water runoff is a major source of pollution, and understanding the components of storm water discharge is essential to remediation efforts and proper assessment of risks to human and ecosystem health. In this study, culturable Escherichia coli and ampicillin-resistant E. coli levels were quantified and microbial source tracking (MST) markers (including markers for general Bacteroidales spp., human, ruminant/cow, gull, and dog) were detected in storm water outfalls and sites along the Humber River in Toronto, Ontario, Canada, and enumerated via endpoint PCR and quantitative PCR (qPCR). Additionally, chemical source tracking (CST) markers specific for human wastewater (caffeine, carbamazepine, codeine, cotinine, acetaminophen, and acesulfame) were quantified. Human and gull fecal sources were detected at all sites, although concentrations of the human fecal marker were higher, particularly in outfalls (mean outfall concentrations of 4.22 log10 copies, expressed as copy numbers [CN]/100 milliliters for human and 0.46 log10 CN/100 milliliters for gull). Higher concentrations of caffeine, acetaminophen, acesulfame, E. coli, and the human fecal marker were indicative of greater raw sewage contamination at several sites (maximum concentrations of 34,800 ng/liter, 5,120 ng/liter, 9,720 ng/liter, 5.26 log10 CFU/100 ml, and 7.65 log10 CN/100 ml, respectively). These results indicate pervasive sewage contamination at storm water outfalls and throughout the Humber River, with multiple lines of evidence identifying Black Creek and two storm water outfalls with prominent sewage cross-connection problems requiring remediation. Limited data are available on specific sources of pollution in storm water, though our results indicate the value of using both MST and CST methodologies to more reliably assess sewage contamination in impacted watersheds. IMPORTANCE: Storm water runoff is one of the most prominent non-point sources of biological and chemical contaminants which can potentially degrade water quality and pose risks to human and ecosystem health. Therefore, identifying fecal contamination in storm water runoff and outfalls is essential for remediation efforts to reduce risks to public health. This study employed multiple methods of identifying levels and sources of fecal contamination in both river and storm water outfall sites, evaluating the efficacy of using culture-based enumeration of E. coli, molecular methods of determining the source(s) of contamination, and CST markers as indicators of fecal contamination. The results identified pervasive human sewage contamination in storm water outfalls and throughout an urban watershed and highlight the utility of using both MST and CST to identify raw sewage contamination.
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Monitoreo del Ambiente/métodos , Heces/microbiología , Ríos , Aguas del Alcantarillado , Microbiología del Agua , Agua/análisis , Animales , Bacteroidetes/genética , Bacteroidetes/aislamiento & purificación , Bovinos/microbiología , Charadriiformes/microbiología , Perros/microbiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Humanos , Ontario , Reacción en Cadena de la Polimerasa , Ríos/química , Ríos/microbiología , Aguas del Alcantarillado/química , Aguas del Alcantarillado/microbiología , Agua/química , Contaminación del Agua , Calidad del AguaRESUMEN
UNLABELLED: Escherichia coli has been proposed to have two habitats-the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and â¼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS30) located within the uspC-flhDC intergenic region. The positional location of the IS30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates (n = 1,177). Phylogenetics clustered the IS30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli We developed an endpoint PCR targeting the IS30 element within the uspC-flhDC intergenic region, and all raw sewage samples (n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater. IMPORTANCE: The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.
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Adaptación Biológica , Escherichia coli/clasificación , Escherichia coli/fisiología , Genotipo , Estrés Fisiológico , Aguas Residuales/microbiología , Técnicas de Tipificación Bacteriana , Cloro/metabolismo , Análisis por Conglomerados , Elementos Transponibles de ADN , Desinfectantes/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Viabilidad Microbiana/efectos de los fármacos , Filogenia , Polimorfismo de Nucleótido Simple , Purificación del AguaRESUMEN
Several studies have demonstrated that E. coli appears to display some level of host adaptation and specificity. Recent studies in our laboratory support these findings as determined by logic regression modeling of single nucleotide polymorphisms (SNP) in intergenic regions (ITGRs). We sought to determine the degree of host-specific information encoded in various ITGRs across a library of animal E. coli isolates using both whole genome analysis and a targeted ITGR sequencing approach. Our findings demonstrated that ITGRs across the genome encode various degrees of host-specific information. Incorporating multiple ITGRs (i.e., concatenation) into logic regression model building resulted in greater host-specificity and sensitivity outcomes in biomarkers, but the overall level of polymorphism in an ITGR did not correlate with the degree of host-specificity encoded in the ITGR. This suggests that distinct SNPs in ITGRs may be more important in defining host-specificity than overall sequence variation, explaining why traditional unsupervised learning phylogenetic approaches may be less informative in terms of revealing host-specific information encoded in DNA sequence. In silico analysis of 80 candidate ITGRs from publically available E. coli genomes was performed as a tool for discovering highly host-specific ITGRs. In one ITGR (ydeR-yedS) we identified a SNP biomarker that was 98% specific for cattle and for which 92% of all E. coli isolates originating from cattle carried this unique biomarker. In the case of humans, a host-specific biomarker (98% specificity) was identified in the concatenated ITGR sequences of rcsD-ompC, ydeR-yedS, and rclR-ykgE, and for which 78% of E. coli originating from humans carried this biomarker. Interestingly, human-specific biomarkers were dominant in ITGRs regulating antibiotic resistance, whereas in cattle host-specific biomarkers were found in ITGRs involved in stress regulation. These data suggest that evolution towards host specificity may be driven by different natural selection pressures on the regulome of E. coli among different animal hosts.
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Biomarcadores/metabolismo , ADN Intergénico/genética , Escherichia coli/genética , Genoma Bacteriano , Especificidad del Huésped/genética , Animales , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Bases de Datos Genéticas , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Variación Genética , Humanos , Modelos Logísticos , Filogenia , Polimorfismo de Nucleótido Simple , Selección Genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Foreshore beach sands and pore water may act as a reservoir and nonpoint source of fecal indicator bacteria (FIB) to surface waters. This paper presents data collected at a fine sand beach on Lake Huron, Canada over three field events. The data show that foreshore sand erosion as wave height increases results in elevated Escherichia coli concentrations in surface water, as well as depletion of E. coli from the foreshore sand and pore water. E. coli initially attached to foreshore sand rather than initially residing in the pore water was found to be the main contributor to elevated surface water concentrations. Surface water E. coli concentrations were a function of not only wave height (and associated sand erosion) but also the time elapsed since a preceding period of high wave intensity. This finding is important for statistical regression models used to predict beach advisories. While calculations suggest that foreshore sand erosion may be the dominant mechanism for releasing E. coli to surface water during intensified wave conditions at a fine sand beach, comparative characterization of the E. coli distribution at a coarse sand-cobble beach suggests that interstitial pore water flow and discharge may be more important for coarser sand beaches.
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Playas , Escherichia coli , Agua Dulce/microbiología , Dióxido de Silicio , Agua , Microbiología del AguaRESUMEN
Several beaches within the Toronto region area of concern have persistent issues with fecal contamination, causing a beach beneficial use impairment (BUI). In this study, Escherichia coli, including ampicillin-resistant strains, were enumerated via culturable and quantitative polymerase chain reaction (qPCR) methods. Microbial source tracking (MST) markers (for general Bacteroidales, human, ruminant/cow, gull, and dog) were detected and enumerated via PCR and qPCR to identify sources of fecal contamination at Sunnyside Beach and in the Humber River. Human, cow, and dog markers had good host-specificity, while gull markers sometimes amplified a few other bird species. The ruminant endpoint PCR marker amplified a variety of other animal species rendering it less useful. Both human and gull fecal contamination were prevalent in the Humber River, while Sunnyside Beach was predominantly impacted by gull fecal contamination. Human sewage impacts were more prevalent in the lower Humber River, particularly in Black Creek. However, to reduce Sunnyside beach postings, reducing bird fecal contamination in the river and at the beach would be necessary. When there are high levels of E. coli throughout a beachshed, an MST toolbox approach can add value to discriminate source(s) of E. coli contamination and guide decisions relating to public health risk and remediation strategies.
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Monitoreo del Ambiente/métodos , Heces/microbiología , Aguas del Alcantarillado/microbiología , Microbiología del Agua , Contaminación del Agua/análisis , Animales , Bacterias/clasificación , Bacterias/aislamiento & purificación , Playas , Aves , Perros , Humanos , Ganado , OntarioRESUMEN
Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.
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Bacterias/aislamiento & purificación , Biomarcadores/química , Monitoreo del Ambiente , Ríos/microbiología , Ríos/virología , Virus/aislamiento & purificación , Agricultura , Animales , Bacterias/genética , Humanos , Ríos/química , Estaciones del Año , Virus/genética , Microbiología del AguaRESUMEN
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.
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Monitoreo del Ambiente , Lagos , Humanos , Monitoreo del Ambiente/métodos , Contaminación del Agua/análisis , Escherichia coli/genética , Aguas del Alcantarillado , Microbiología del Agua , Heces , AguaRESUMEN
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.
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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.
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Cianobacterias , Monitoreo del Ambiente , Floraciones de Algas Nocivas , Lagos , Microcistinas , Microcistinas/genética , Microcistinas/análisis , Lagos/microbiología , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Cianobacterias/clasificación , Monitoreo del Ambiente/métodos , Ecosistema , Metagenómica , Recreación , Microbiología del Agua , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
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.
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Escherichia coli , Microbiología del Agua , Calidad del Agua , Escherichia coli/genética , Escherichia coli/clasificación , Proteínas de Escherichia coli/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Shigella/genética , Shigella/clasificación , Shigella/aislamiento & purificación , Secuencia Conservada , Monitoreo del Ambiente/métodos , Reacción en Cadena de la Polimerasa/métodos , Heces/microbiologíaRESUMEN
This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.
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Campylobacter coli/aislamiento & purificación , Campylobacter jejuni/aislamiento & purificación , Campylobacter lari/aislamiento & purificación , Heces/microbiología , Agua Dulce/microbiología , Técnicas Microbiológicas/métodos , Aguas Residuales/microbiología , Animales , Aves , ADN Espaciador Ribosómico/genética , Ontario , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , TemperaturaRESUMEN
Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.
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Bacteroidetes/aislamiento & purificación , Cryptosporidium/aislamiento & purificación , Ríos/microbiología , Ríos/virología , Virus/aislamiento & purificación , Contaminación del Agua , Animales , Bacteroidetes/clasificación , Bovinos , Humanos , Ríos/parasitología , Virus/clasificaciónRESUMEN
Nearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and Escherichia coli O157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) for Giardia spp., Campylobacter spp., and Salmonella spp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium since most Cryptosporidium genotypes classed as wildlife in this study (e.g., muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish should not be rejected solely on the basis of their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated with Cryptosporidium and other zoonotic pathogens.
Asunto(s)
Cryptosporidium/clasificación , Cryptosporidium/aislamiento & purificación , Variación Genética , Filogeografía , Agua/parasitología , Animales , Animales Salvajes/parasitología , Bacterias/aislamiento & purificación , Criptosporidiosis/epidemiología , Criptosporidiosis/transmisión , Cryptosporidium/genética , Genotipo , Giardia/aislamiento & purificación , Humanos , Ontario , Carga de Parásitos , Medición de Riesgo , Análisis Espacio-Temporal , Factores de TiempoRESUMEN
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.
RéSUMé: 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 = 0,410,56) 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.
Asunto(s)
Enterococcus , Calidad del Agua , Humanos , Enterococcus/genética , Escherichia coli/genética , Salud Pública , Estudios Retrospectivos , Playas , Microbiología del Agua , Heces , Monitoreo del Ambiente/métodosRESUMEN
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.
RESUMEN
Lake Erie is subject to recurring events of cyanobacterial harmful algal blooms (cHABs), but measures of nutrients and total phytoplankton biomass seem to be poor predictors of cHABs when taken individually. A more integrated approach at the watershed scale may improve our understanding of the conditions that lead to bloom formation, such as assessing the physico-chemical and biological factors that influence the lake microbial community, as well as identifying the linkages between Lake Erie and the surrounding watershed. Within the scope of the Government of Canada's Genomics Research and Development Initiative (GRDI) Ecobiomics project, we used high-throughput sequencing of the 16S rRNA gene to characterize the spatio-temporal variability of the aquatic microbiome in the Thames River-Lake St. Clair-Detroit River-Lake Erie aquatic corridor. We found that the aquatic microbiome was structured along the flow path and influenced mainly by higher nutrient concentrations in the Thames River, and higher temperature and pH downstream in Lake St. Clair and Lake Erie. The same dominant bacterial phyla were detected along the water continuum, changing only in relative abundance. At finer taxonomical level, however, there was a clear shift in the cyanobacterial community, with Planktothrix dominating in the Thames River and Microcystis and Synechococcus in Lake St. Clair and Lake Erie. Mantel correlations highlighted the importance of geographic distance in shaping the microbial community structure. The fact that a high proportion of microbial sequences found in the Western Basin of Lake Erie were also identified in the Thames River, indicated a high degree of connectivity and dispersal within the system, where mass effect induced by passive transport play an important role in microbial community assembly. Nevertheless, some cyanobacterial amplicon sequence variants (ASVs) related to Microcystis, representing less than 0.1% of relative abundance in the upstream Thames River, became dominant in Lake St. Clair and Erie, suggesting selection of those ASVs based on the lake conditions. Their extremely low relative abundances in the Thames suggest additional sources are likely to contribute to the rapid development of summer and fall blooms in the Western Basin of Lake Erie. Collectively, these results, which can be applied to other watersheds, improve our understanding of the factors influencing aquatic microbial community assembly and provide new perspectives on how to better understand the occurrence of cHABs in Lake Erie and elsewhere.
RESUMEN
Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.