Land use and rainfall influences on bacterial levels and sources in stormwater ponds.

Urban stormwater runoff is a known source of microbial contamination of stormwater ponds. However, less is known about the influences of land use and rainfall on microbial quality over time in these receiving waters. In this study, two fecal indicator bacteria (FIB), namely Escherichia coli and thermotolerant coliforms, were monitored in three stormwater ponds in Calgary, Alberta, Canada. The stormwater ponds were selected due to their potential as water sources for beneficial uses such as irrigation, which requires lower water quality than drinking water, thereby alleviating the pressure on the city's potable water demands. The selected stormwater ponds vary in size and shape, contribution catchment size, and percentages of several primary land use types. Microbial source tracking for human, dog, seagull, Canada goose, ruminant, and muskrat was also conducted to determine sources of bacterial contamination in the stormwater ponds. Sampling was conducted near the pond surface and adjacent to the shoreline, specifically near the outfalls that discharge stormwater runoff into the ponds and the inlets that convey water out of the ponds. Overall, the FIB concentrations in the vicinity of pond outfalls were significantly or relatively higher than those near pond inlets. The contamination in the McCall Lake and the Country Hills stormwater ponds showed higher amounts of human markers (40 to 60%) compared to the Inverness stormwater pond (< 20%), which coincided with their higher FIB concentration medians. The results revealed that stormwater drained from catchments with a higher percentage of commercial land use was more contaminated than those with primary residential land use, while the impacts of residential development on the FIB levels in the Inverness stormwater pond were not obvious. Furthermore, FIB concentrations in the ponds increased in response to both rain events and inter-event dry periods, with human-specific markers being predominant despite the high levels of animal markers during inter-event dry periods. Human-origin sources might be among the main microbial loading contributors in the pond catchments in general. All these findings can inform the development or improvement of measures for mitigating microbial pollution, strategies for reusing stormwater, and maintenance programs.

A scoping review of factors associated with antimicrobial-resistant Campylobacter species infections in humans.

Human infection with antimicrobial-resistant Campylobacter species is an important public health concern due to the potentially increased severity of illness and risk of death. Our objective was to synthesise the knowledge of factors associated with human infections with antimicrobial-resistant strains of Campylobacter. This scoping review followed systematic methods, including a protocol developed a priori. Comprehensive literature searches were developed in consultation with a research librarian and performed in five primary and three grey literature databases. Criteria for inclusion were analytical and English-language publications investigating human infections with an antimicrobial-resistant (macrolides, tetracyclines, fluoroquinolones, and/or quinolones) Campylobacter that reported factors potentially linked with the infection. The primary and secondary screening were completed by two independent reviewers using Distiller SR®. The search identified 8,527 unique articles and included 27 articles in the review. Factors were broadly categorised into animal contact, prior antimicrobial use, participant characteristics, food consumption and handling, travel, underlying health conditions, and water consumption/exposure. Important factors linked to an increased risk of infection with a fluoroquinolone-resistant strain included foreign travel and prior antimicrobial use. Identifying consistent risk factors was challenging due to the heterogeneity of results, inconsistent analysis, and the lack of data in low- and middle-income countries, highlighting the need for future research.

Naturalized Escherichia coli in Wastewater and the Co-evolution of Bacterial Resistance to Water Treatment and Antibiotics.

Antibiotic resistance represents one of the most pressing concerns facing public health today. While the current antibiotic resistance crisis has been driven primarily by the anthropogenic overuse of antibiotics in human and animal health, recent efforts have revealed several important environmental dimensions underlying this public health issue. Antibiotic resistant (AR) microbes, AR genes, and antibiotics have all been found widespread in natural environments, reflecting the ancient origins of this phenomenon. In addition, modern societal advancements in sanitation engineering (i.e., sewage treatment) have also contributed to the dissemination of resistance, and concerningly, may also be promoting the evolution of resistance to water treatment. This is reflected in the recent characterization of naturalized wastewater strains of Escherichia coli-strains that appear to be adapted to live in wastewater (and meat packing plants). These strains carry a plethora of stress-resistance genes against common treatment processes, such as chlorination, heat, UV light, and advanced oxidation, mechanisms which potentially facilitate their survival during sewage treatment. These strains also carry an abundance of common antibiotic resistance genes, and evidence suggests that resistance to some antibiotics is linked to resistance to treatment (e.g., tetracycline resistance and chlorine resistance). As such, these naturalized E. coli populations may be co-evolving resistance against both antibiotics and water treatment. Recently, extraintestinal pathogenic strains of E. coli (ExPEC) have also been shown to exhibit phenotypic resistance to water treatment, seemingly associated with the presence of various shared genetic elements with naturalized wastewater E. coli. Consequently, some pathogenic microbes may also be evolving resistance to the two most important public health interventions for controlling infectious disease in modern society-antibiotic therapy and water treatment.

Development of a qPCR assay for the detection of naturalized wastewater E. coli strains.

We recently demonstrated the presence of naturalized populations of Escherichia coli in municipal sewage. We wanted to develop a quantitative polymerase chain reaction (qPCR) assay targeting the uspC-IS30-flhDC marker of naturalized wastewater E. coli and assess the prevalence of these naturalized strains in wastewater. The limit of detection for the qPCR assay was 3.0 × 10-8 ng of plasmid DNA template with 100% specificity. This strain was detected throughout the wastewater treatment process, including treated effluents. We evaluated the potential of this marker for detecting municipal sewage/wastewater contamination in water by comparing it to other human and animal markers of fecal pollution. Strong correlations were observed between the uspC-IS30-flhDC marker and the human fecal markers Bacteroides HF183 and HumM2, but not animal fecal markers, in surface and stormwater samples. The uspC-IS30-flhDC marker appears to be a potential E. coli-based marker for human wastewater contamination.

The prevalence and levels of enteric viruses in groundwater of private wells in rural Alberta, Canada.

The prevalence and levels of enteric viruses in untreated groundwater of private wells used for drinking and/or agricultural practices in rural Alberta were studied using the qPCR panel assay, integrated cell culture with qPCR and cell culture in the volume of 500 liters per sample through serial sampling. Seven viruses were assessed including adenovirus, rotavirus, norovirus, astrovirus, sapovirus, reovirus and JC virus. Five viruses were detected with an overall positive detection rate of 6.33 % (45 of 711 samples). The most frequently detected virus was adenovirus (48.9%, 22/45) followed by rotavirus (44.4%, 20/45), reovirus (20%, 9/45), JC virus (6.7%, 3/45) and norovirus (6.7%, 3/45). There was no significant difference in the positive detection rates, ranging from 1.1% to 3.4% by various well settings used for broiler farms, cow/calf farms, feedlots and rural acreages. Effects of well characteristics (aquifer type, well depth, static level of water, well seal) and well completion lithology on potential viral contamination of groundwater of private wells were also analyzed upon available data. The findings demonstrate that occurrence of enteric viruses is low and viral contamination is sporadic in groundwater of private wells in rural Alberta. Conventional fecal bacterial indicators (coliform and/or E. coli) were not a representative marker for viral contamination in groundwater wells in rural Alberta.

Phenotypic and Genetic Determination of Biofilm Formation in Heat Resistant Escherichia coli Possessing the Locus of Heat Resistance.

Despite the effectiveness of thermal inactivation processes, Escherichiacoli biofilms continue to be a persistent source of contamination in food processing environments. E. coli strains possessing the locus of heat resistance are a novel food safety threat and raises the question of whether these strains can also form biofilms. The objectives of this study were to determine biofilm formation in heat resistant E. coli isolates from clinical and environmental origins using an in-house, two-component apparatus and to characterize biofilm formation-associated genes in the isolates using whole genome sequencing. Optimal conditions for biofilm formation in each of the heat resistant isolates were determined by manipulating inoculum size, nutrient concentration, and temperature conditions. Biofilm formation in the heat resistant isolates was detected at temperatures of 24 °C and 37 °C but not at 4 °C. Furthermore, biofilm formation was observed in all environmental isolates but only one clinical isolate despite shared profiles in biofilm formation-associated genes encoded by the isolates from both sources. The circulation of heat resistant E. coli isolates with multi-stress tolerance capabilities in environments related to food processing signify that such strains may be a serious food safety and public health risk.

A review of the taxonomy, genetics, and biology of the genus Escherichia and the type species Escherichia coli.

Historically, bacteriologists have relied heavily on biochemical and structural phenotypes for bacterial taxonomic classification. However, advances in comparative genomics have led to greater insights into the remarkable genetic diversity within the microbial world, and even within well-accepted species such as Escherichia coli. The extraordinary genetic diversity in E. coli recapitulates the evolutionary radiation of this species in exploiting a wide range of niches (i.e., ecotypes), including the gastrointestinal system of diverse vertebrate hosts as well as non-host natural environments (soil, natural waters, wastewater), which drives the adaptation, natural selection, and evolution of intragenotypic conspecific specialism as a strategy for survival. Over the last few years, there has been increasing evidence that many E. coli strains are very host (or niche)-specific. While biochemical and phylogenetic evidence support the classification of E. coli as a distinct species, the vast genomic (diverse pan-genome and intragenotypic variability), phenotypic (e.g., metabolic pathways), and ecotypic (host-/niche-specificity) diversity, comparable to the diversity observed in known species complexes, suggest that E. coli is better represented as a complex. Herein we review the taxonomic classification of the genus Escherichia and discuss how phenotype, genotype, and ecotype recapitulate our understanding of the biology of this remarkable bacterium.

A Velocity Meter for Quantifying Advection Velocity Vectors in Large Water Bodies.

A velocity meter was designed and built in order to meet market needs for an affordable instrument that measures the range of velocity magnitudes and direction experienced in medium- to large-sized water bodies. The velocity meter consists of a graduated plate with an injector protruding from the center and a camera held downward above the plate. Once the Dye Injection Velocity (DIV) meter is in the flow, dye is injected and the camera records the dye fluid transport. The recorded video is analyzed to determine the local flow velocity and direction. The DIV was calibrated for a range of velocities between 0.0094 m/s and 0.1566 m/s using particle image velocimetry (PIV) in a flow visualization flume. The accuracy of the instrument was found to be +6.3% and -9.8% of full scale. The coefficient of determination of the calibration curve was equal to 98%. Once calibrated, the DIV was deployed to the Inverness Stormwater pond in Calgary, Canada, for validation tests against an Acoustic Doppler Velocity (ADV) meter. During the validation tests, both flow velocity magnitude and direction were measured at several spatial points. The velocity magnitude results showed good agreement and the Mann-Whitney test showed no statistically significant difference (p-value > 0.05). At two spatial points, the differences between direction data were significant, which could be caused by the random errors involved in the validation test. However, the averaged data showed good agreement.

Characterization of water treatment-resistant and multidrug-resistant urinary pathogenic Escherichia coli in treated wastewater.

A growing body of evidence has demonstrated that extraintestinal pathogenic E. coli (ExPEC), such as the urinary pathogenic E. coli (UPEC), are common constituents of treated wastewater, and therefore represent a potential public health risk. However, no single virulence gene, or set of virulence genes, can be used to conclusively identify this genetically diverse pathotype. As such we sought to identify and characterize the public health relevance of potential UPEC found in treated sewage/wastewater using a comparative genomics approach. Presumptive wastewater UPEC (W-UPEC) were initially identified by virulence gene screening against 5 virulence genes, and for which isolates containing ≥3 virulence genes were whole genome sequenced (n = 24). Single nucleotide polymorphic (SNP) spanning tree analysis demonstrated that many of these wastewater UPEC (WUPEC) were virtually identical at the core genome (0.4 Mbp) when compared to clinical UPEC (C-UPEC) sequences obtained from NCBI, varying by as little as 1 SNP. Remarkably, at the whole genome level, W-UPEC isolates displayed >96% whole genome similarity to C-UPEC counterparts in NCBI, with one strain demonstrating 99.5% genome similarity to a particular C-UPEC strain. The W-UPEC populations were represented by sequence types (ST) known to be clinically important, including ST131, ST95, ST127 and ST640. Many of the W-UPEC carried the exact same complement of virulence genes as their most closely related C-UPEC strains. For example, O25b-ST131 W-UPEC strains possessed the same 80 virulence genes as their most closely related C-UPEC counterparts. Concerningly, W-UPEC strains also carried a plethora of antibiotic resistance genes, and O25b-ST131strains were designated as extended spectrum beta-lactamase (ESBL) producing E. coli by both genome profiling and phenotypic resistance testing. W-UPEC ST131 strains were found in the effluents of a single treatment plant at different times, as well as different wastewater treatment plants, suggesting a differentially ability to survive wastewater treatment. Indeed, in sewage samples treated with chlorine doses sufficient for inducing a ∼99.99% reduction in total E. coli levels, UPEC represented a significant proportion of the chlorine-resistant population. By contrast, no Shiga toxin-producing E. coli were observed in these chlorinated sewage libraries. Our results suggest that clinically-relevant UPEC exist in treated wastewater effluents and that they appear to be specifically adapted to survive wastewater treatment processes.

Molecular characterization of Giardia spp. and Cryptosporidium spp. from dogs and coyotes in an urban landscape suggests infrequent occurrence of zoonotic genotypes.

Giardia spp. and Cryptosporidium spp. are common gastrointestinal parasites with the potential for zoonotic transmission. This study aimed to (1) determine the genotypes occurring in dogs and coyotes occupying a similar urban area; (2) determine if these hosts were infected with potentially zoonotic genotypes; (3) provide baseline molecular data. In August and September 2012, 860 dog owners living in neighborhoods bordering six urban parks in Calgary, Alberta, Canada, provided faecal samples from their dogs. From March 2012 through July 2013, 193 coyote faeces were also collected from five of six of the same parks. Direct immunofluorescence microscopy (DFA) indicated that Giardia spp. and Cryptosporidium spp. infected a total of 64 (7.4%) and 21 (2.4%) dogs, as well as 15 (7.8%) and three (1.6%) coyotes, respectively. Semi-nested, polymerase chain reactions targeting the 16S small-subunit ribosomal ribonucleic acid (SSU rRNA) and 18S SSU rRNA genes of Giardia spp. and Cryptosporidium spp., respectively, were conducted on samples that screened positive by DFA, and products were sequenced and genotyped. Dogs were infected with Giardia intestinalis canid-associated assemblages C (n = 14), D (n = 13), and Cryptosporidium canis (n = 3). Similarly, G. intestinalis assemblages C (n = 1), D (n = 1) and C. canis (n = 1), were detected in coyotes, as well as G. intestinalis assemblage A (n = 1) and Cryptosporidium vole genotype (n = 1). Dogs and coyotes were predominantly infected with host-specific genotypes and few potentially zoonotic genotypes, suggesting that they may not represent a significant risk for zoonotic transmission of these parasites in urban areas where these hosts are sympatric.

Evaluating the risks associated with Shiga-toxin-producing Escherichia coli (STEC) in private well waters in Canada.

Shiga-toxin-producing Escherichia coli (STEC) represent a major concern for waterborne disease outbreaks associated with consumption of contaminated groundwater. Over 4 million people rely on private groundwater systems as their primary drinking water source in Canada; many of these systems do not meet current standards for water quality. This manuscript provides a scoping overview of studies examining STEC prevalence and occurrence in groundwater, and it includes a synopsis of the environmental variables affecting survival, transport, persistence, and overall occurrence of these important pathogenic microbes in private groundwater wells used for drinking purposes.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Evidence for the evolution, clonal expansion and global dissemination of water treatment-resistant naturalized strains of Escherichia coli in wastewater.

We previously demonstrated the existence of naturalized strains of E. coli in wastewater and herein perform an in-depth comparative whole genome analysis of these strains (n = 17). Fourteen of the Canadian E. coli strains, isolated from geographically separated wastewater treatment plants, were virtually identical at the core genome and were ≥96% similar at the whole genome level, suggesting clonal-relatedness among these isolates. Remarkably, these strains were shown to be extremely similar to the genome of an E. coli isolated from wastewater in Switzerland, suggesting a global distribution of these strains. The genomes of three other Canadian wastewater strains were more diverse but very similar to the genomes of E. coli isolates collected from U.S. wastewater samples. Based on maximum likelihood phylogenetic analysis, wastewater strains from Canada, the U.S. and Switzerland formed a clade separate from other known enteric phylogroups (i.e., A, B1, B2, D, E) and the cryptic clades. All Canadian, Swiss and U.S. wastewater strains possessed a common SNP biomarker pattern across their genomes, and a sub-population (i.e., 14 Canadian and 1 Swiss strain) also possessed a previously identified wastewater-specific marker known as uspC-IS30-flhDC element. Biochemical heat mapping of 518 categories of genes recapitulated phylogeny, with wastewater strains phenotypically clustering separately from enteric and cryptic clades. Wastewater strains were enriched for stress-response genes (i.e., nutrient acquisition/deprivation, DNA repair, oxidative stress, and UV resistance) - elements reflective of their environmental survival challenges. Wastewater strains were shown to carry a plethora of known antibiotic resistance (AR) genes, the patterns of which were remarkably similar among all Canadian, U.S. and Swiss wastewater strains. Virulence gene composition was also similar among all the wastewater strains, with an abundant representation of virulence genes commonly associated with urinary pathogenic E. coli (UPEC) as well as enterohemorrhagic (EHEC) E. coli. The remarkable degree of similarity between all wastewater strains from Canada, Switzerland and the U.S. suggests the evolution and global-dissemination of water treatment-resistant clone of E. coli. These finding, along with others, raise some important concerns about the potential for emergence of E. coli pathotypes resistant to water-treatment.

Prevalence, levels and seasonal variations of human enteric viruses in six major rivers in Alberta, Canada.

The prevalence and seasonal variation of 7 viruses in 6 major rivers in Alberta were assessed using a combination of qPCR, cell culture and integrated cell culture with qPCR (ICC-qPCR). Water samples were collected monthly from rivers at different sites upstream and downstream of major urban centers. Seven viruses including rotavirus, adenovirus, astrovirus, norovirus, sapovirus, JC virus and enterovirus, were detected in at least one of the water samples at each site using qPCR. Rotavirus was most common with concentration ranging from 2.3 to 4.5 log10 genomic equivalent (GE) copies/L. Norovirus, sapovirus, astrovirus, adenoviruses and JC virus peaked during the winter (November to March). Viruses were most prevalent at the Bow River sampling site downstream of the City of Calgary, followed by the North Saskatchewan River site downstream of the City of Edmonton and the Red Deer River site downstream of the City of Red Deer. The detection rates and quantity of viruses had significant difference in the sampling sites between upstream and downstream of major urban centers (p < 0.001). 14% of the samples tested positive using viral culture indicating the presence of infectious viruses in river. Sequencing analysis identified human rotavirus in 75% of the samples collected from downstream versus 37% of the samples collected from upstream sites (p < 0.02). Multivariate binary regression showed that human activity in watersheds is a significant determinant of viruses in Alberta's Rivers. The discharge from wastewater treatment plants may be the possible sources of viral contamination. Seasonal coincidence of acute viral gastroenteritis outbreaks and monthly peak occurrence of enteric viruses in river water implies potential impact of waterborne viruses on human health.

UV inactivation of human infectious viruses at two full-scale wastewater treatment plants in Canada.

Ultraviolet (UV) disinfection is widely used to inactivate microorganisms prior to release of treated municipal wastewater. However, limited data are available for in situ inactivation of infectious enteric viruses by UV treatment at full-scale. In this study, a total of 51 pre-UV and 50 post-UV samples were collected over a two-year period from two wastewater treatment plants (WWTPs) and analyzed for noroviruses, rotavirus, reovirus, sapovirus, astrovirus, enteroviruses, adenoviruses and JC virus. Both pre-UV and post-UV samples had relatively high concentrations of these viruses determined by qPCR. Infectious viruses were also observed in 98% of pre-UV samples and 76% of post-UV samples by cell culture, using either cytopathic effect (CPE) or integrated cell culture with qPCR (ICC-qPCR). Reovirus was the most common virus detected by ICC-qPCR, present in 92% of pre-UV and 48% of post-UV samples. Infectious enterovirus and adenovirus were detected by ICC-qPCR in 33% and 31% of pre-UV samples, 14% and 20% of post-UV samples, respectively. Mean log10 reduction estimates for infectious reovirus was 1.2 and 1.8 log for the two WWTPs as assessed by ICC-qPCR, which was similar to the reduction of total infectious viruses (1.5 and 1.7 log) as assessed by CPE in cells culture. Overall, quantification of infectious reovirus appears to provide a useful index of enteric virus inactivation during wastewater treatment at full-scale. To our knowledge, this is the first comprehensive study to assess UV inactivation of human enteric viruses at full-scale in WWTPs using both molecular and cell culture techniques, providing important information for quantitative microbial risk assessment of UV inactivation of human viruses in municipal wastewater.

Zoonotic Fecal Pathogens and Antimicrobial Resistance in Canadian Petting Zoos.

This study aimed to better understand the potential public health risk associated with zoonotic pathogens in agricultural fairs and petting zoos in Canada. Prevalence of Salmonella, Shiga toxin-producing Escherichia coli (STEC) O157:H7, and top six non-O157 STEC serogroups in feces (n = 88), hide/feather (n = 36), and hand rail samples (n = 46) was assessed, as well as distributions of antimicrobial resistant (AMR) broad and extended-spectrum ß-lactamase (ESBL)-producing E. coli. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in pig nasal swabs (n = 4), and Campylobacter, Cryptosporidium, and Giardia in feces was also assessed. Neither Salmonella nor MRSA were detected. Campylobacter spp. were isolated from 32% of fecal samples. Cryptosporidium and Giardia were detected in 2% and 15% of fecal samples, respectively. Only one fecal sample was positive for STEC O157, whereas 22% were positive for non-O157 STEC. Multi-drug resistance (MDR) to antibiotics classified as critically and highly important in human medicine was proportionally greatest in E. coli from cattle feces. The ß-lactamase-producing E. coli from pig, horse/donkey feces, and hand rail samples, as well as the STEC E. coli from handrail swabs were MDR. The diversity and prevalence of zoonotic pathogens and AMR bacteria detected within agricultural fairs and petting zoos emphasize the importance of hygienic practices and sanitization with respect to reducing associated zoonotic risks.

Clonal expansion of environmentally-adapted Escherichia coli contributes to propagation of antibiotic resistance genes in beef cattle feedlots.

Livestock wastewater lagoons represent important environmental reservoirs of antibiotic resistance genes (ARGs), although factors contributing to their proliferation within these reservoirs remain poorly understood. Here, we characterized Escherichia coli from feedlot cattle feces and associated wastewater lagoons using CRISPR1 subtyping, and demonstrated that while generic E. coli were genetically diverse, populations were dominated by several 'feedlot-adapted' CRISPR types (CTs) that were widely distributed throughout the feedlot. Moreover, E. coli bearing beta-lactamase genes, which confer reduced susceptibility to third-generation cephalosporin's, predominantly belonged to these feedlot-adapted CTs. Remarkably, the genomic region containing the CRISPR1 allele was more frequently subject to genetic exchange among wastewater isolates compared to fecal isolates, implicating this region in environmental adaptation. This allele is proximal to the mutS-rpoS-nlpD region, which is involved in regulating recombination barriers and adaptive stress responses. There were no loss-of-function mutS or rpoS mutations or beneficial accessory genes present within the mutS-rpoS-nlpD region that would account for increased environmental fitness among feedlot-adapted isolates. However, comparative sequence analysis revealed that protein sequences within this region were conserved among most feedlot-adapted CTs, but not transient fecal CTs, and did not reflect phylogenetic relatedness, implying that adaptation to wastewater environments may be associated with genetic variation related to stress resistance. Collectively, our findings suggest adaptation of E. coli to feedlot environments may contribute to propagation of ARGs in wastewater lagoons.

Total coliform and Escherichia coli contamination in rural well water: analysis for passive surveillance.

With increasing stress on our water resources and recent waterborne disease outbreaks, understanding the epidemiology of waterborne pathogens is crucial to build surveillance systems. The purpose of this study was to explore techniques for describing microbial water quality in rural drinking water wells, based on spatiotemporal analysis, time series analysis and relative risk mapping. Tests results for Escherichia coli and coliforms from private and small public well water samples, collected between 2004 and 2012 in Alberta, Canada, were used for the analysis. Overall, 14.6 and 1.5% of the wells were total coliform and E. coli-positive, respectively. Private well samples were more often total coliform or E. coli-positive compared with untreated public well samples. Using relative risk mapping we were able to identify areas of higher risk for bacterial contamination of groundwater in the province not previously identified. Incorporation of time series analysis demonstrated peak contamination occurring for E. coli in July and a later peak for total coliforms in September, suggesting a temporal dissociation between these indicators in terms of groundwater quality, and highlighting the potential need to increase monitoring during certain periods of the year.

Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR.

Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

Enterobius vermicularis as a Novel Surrogate for the Presence of Helminth Ova in Tertiary Wastewater Treatment Plants.

Significant effort has gone into assessing the fate and removal of viruses, bacteria, and protozoan parasites during wastewater treatment to provide data addressing potential health risks associated with reuse options. Comparatively less is known about the fate of parasitic worm species ova in these complex systems. It is largely assumed that these helminths settle, are removed with the sludge, and consequently represent a relatively low risk for wastewater reuse applications. However, helminths are a highly diverse group of organisms that display a wide range of physical properties that complicate the application of a single treatment for helminth reduction during wastewater treatment. Moreover, their diverse biological and physical properties make some ova highly resistant to both disinfection (i.e., with chlorine or UV treatment) and physical removal (settling) through the wastewater treatment train, indicating that there may be reason to broaden the scope of our investigations into whether parasitic worm eggs can be identified in treated wastewater. The ubiquitous human parasitic nematode Enterobius vermicularis (pinworm) produces small, buoyant ova. Utilizing a novel diagnostic quantitative PCR (qPCR), this study monitored E. vermicularis presence at two full-scale wastewater treatment plants over the course of 8 months and demonstrated incomplete physical removal of E. vermicularis ova through tertiary treatment, with removal efficiencies approximating only 0.5 and 1.6 log10 at the two wastewater treatment plants based on qPCR. These findings demonstrate the need for more-diverse surrogates of helminthic ova to fully assess treatment performance with respect to reclaimed wastewaters.IMPORTANCE Helminths, despite being a diverse and environmentally resistant class of pathogens, are often underestimated and ignored when treatment performance at modern wastewater treatment plants is considered. A one-size-fits-all surrogate for removal of helminth ova may be inappropriate to adequately assess risk and ensure public safety when treated and partially treated wastewaters are encountered. This study argues for the use of human pinworm as a conservative indicator of the presence of helminth ova due to its small size, buoyancy, prevalence in humans, and environmental resistance.