Pathogen performance testing of a natural swimming pool using a cocktail of microbiological surrogates and QMRA-derived management goals.

In recent decades, natural swimming pools (NSPs) have gained popularity in Europe, especially in Germany and Austria. NSPs differ from swimming pools in that they utilize biological treatment processes based on wetland processes with no disinfection residual. However, data are missing on the specific log-reduction performance of NSPs to address enteric virus, bacteria, and parasitic protozoa removal considered necessary to meet the North American risk-based benchmark (<35 illnesses per 1,000 swimming events) set by the USEPA for voluntary swimming. In this study, we examined Canada's first NSP at Borden Park, Edmonton, Canada, to address the following three questions: (1) Given normal faecal shedding rates by bathers, what is the total log reduction (TLR) theoretically needed to meet the EPA benchmark? (2) what is the in-situ performance of the NSP based on spiking suitable microbial surrogates (MS2 coliphage, Enterococcus faecalis, and Saccharomyces cerevisiae [Baker's yeast])? and (3) how much time is required to reach acceptable bather risk levels under different representative volume-turnover rates? A reverse-quantitative microbial risk assessment (QMRA) revealed that of the four reference pathogens selected (Norovirus, Campylobacter, Cryptosporidium, and Giardia), only Norovirus was estimated to exceed the risk benchmark at the 50th, 75th, and 95th percentiles, while Campylobacter was the only other reference pathogen to exceed at the 95th percentile. Log-reduction values (LRVs) were similar to previous reports for bacterial indicators, and novel LRVs were estimated for the other two surrogates. A key finding was that more than 24 h treatment time would be necessary to provide acceptable bather protection following heavy bather use (378 bathers/day for main pool and 26 bathers/day for children's pool), due to the mixing dynamics of the treated water diluting out possible residual pool faecal contamination. The theoretical maximum number of people in the pool per day to be below USEPA's 35 gastro cases in 1,000 swimming events was 113, 47, and 8, at the 50th, 75th, and 95th percentiles. Further, the use of ultra-violet disinfection to the pool return flow had little effect on reducing the treatment time required.

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.

Long-term persistence of infectious Legionella with free-living amoebae in drinking water biofilms.

Prolific growth of pathogenic Legionella pneumophila within engineered water systems and premise plumbing, and human exposure to aerosols containing this bacterium results in the leading health burden of any water-related pathogen in developed regions. Ecologically, free-living amoebae (FLA) are an important group of the microbial community that influence biofilm bacterial diversity in the piped-water environment. Using fluorescent microscopy, we studied in-situ the colonization of L. pneumophila in the presence of two water-related FLA species, Willaertia magna and Acanthamoeba polyphaga in drinking water biofilms. During water flow as well as after periods of long-stagnation, the attachment and colonization of L. pneumophila to predeveloped water-biofilm was limited. Furthermore, W. magna and A. polyphaga showed no immediate interactions with L. pneumophila when introduced to the same natural biofilm environment. A. polyphaga encysted within 5-7 d after introduction to the tap-water biofilms and mostly persisted in cysts till the end of the study period (850 d). W. magna trophozoites, however, exhibited a time delay in feeding on Legionella and were observed with internalized L. pneumophila cells after 3 weeks from their introduction to the end of the study period and supported putative (yet limited) intracellular growth. The culturable L.pneumophila in the bulk water was reduced by 2-log over 2 years at room temperature but increased (without a change in mip gene copies by qPCR) when the temperature was elevated to 40 °C within the same closed-loop tap-water system without the addition of nutrients or fresh water. The overall results suggest that L. pneumophila maintains an ecological balance with FLA within the biofilm environment, and higher temperature improve the viability of L. pneumophila cells, and intracellular growth of Legionella is possibly cell-concentration dependent. Observing the preferential feeding behavior, we hypothesize that an initial increase of FLA numbers through feeding on a range of other available bacteria could lead to an enrichment of L. pneumophila, and later force predation of Legionella by the amoeba trophozoites results in rapid intracellular replication, leading to problematic concentration of L. pneumophila in water. In order to find sustainable control options for legionellae and various other saprozoic, amoeba-resisting bacterial pathogens, this work emphasizes the need for better understanding of the FLA feeding behavior and the range of ecological interactions impacting microbial population dynamics within engineered water systems.

Evaluating Microbial and Chemical Hazards in Commercial Struvite Recovered from Wastewater.

Controlled struvite (NH4MgPO4·6H2O) precipitation has become a well-known process for nutrient recovery from wastewater treatment systems to alleviate the pressures of diminishing, finite rock phosphate reservoirs. Nonetheless, coprecipitation of potential microbial and chemical hazards is poorly understood. On the other hand, antimicrobial resistance (AMR) is a major global public health concern and wastewater is thought to disseminate resistance genes within bacteria. Fecal indicator bacteria (FIB) are typically used as measures of treatment quality, and with multiresistant E. coli and Enterococcus spp. rising in concern, the quantification of FIB can be used as a preliminary method to assess the risk of AMR. Focusing on struvite produced from full-scale operations, culture and qPCR methods were utilized to identify FIB, antibiotic resistance genes, and human enteric viruses in the final product. Detection of these hazards occurred in both wet and dry struvite samples indicating that there is a potential risk that needs further consideration. Chemical and biological analyses support the idea that the presence of other wastewater components can impact struvite formation through ion and microbial interference. While heavy metal concentrations met current fertilizer standards, the presence of K, Na, Ca, and Fe ions can impact struvite purity yet provide benefit for agricultural uses. Additionally, the quantified hazards detected varied among struvite samples produced from different methods and sources, thus indicating that production methods could be a large factor in the risk associated with wastewater-recovered struvite. In all, coprecipitation of metals, fecal indicator bacteria, antimicrobial resistance genes, and human enteric viruses with struvite was shown to be likely, and future engineered wastewater systems producing struvite may require additional step(s) to manage these newly identified public health risks.

Persistence of infectious Enterovirus within free-living amoebae - A novel waterborne risk pathway?

Free-living amoebae (FLA) are phagocytic protozoa found in natural and engineered water systems. They can form disinfectant-resistant cysts, which can harbor various human pathogenic bacteria, therefore providing them with a means of environmental persistence and dispersion through water distribution and other engineered water systems. The association of FLA with human viruses has been raised, but the limited data on the persistence of infectious virions within amoebae leaves this aspect unresolved. Enteroviruses can cause a wide range of illness and replicate in human respiratory and gastrointestinal tracts, both of which could be exposed through contact with contaminated waters if virus detection and removal are compromised by virion internalization in free-living protozoa. This is especially problematic for high-risk contaminants, such as coxsackieviruses, representative members of the Enterovirus genus that are likely infectious at low doses and cause a variety of symptoms to a vulnerable portion of the population (particularly infants). To investigate Enterovirus persistence within free-living amoebae we co-cultured an infectious clinical coxsackievirus B5 (CVB5) isolate, with the commonly reported tap water amoeba Vermamoeba vermiformis, after which we tracked virus localization and persistence in co-culture over time through a combination of advanced imaging, molecular and cell culture assays. Our results clearly demonstrate that infectious CVB5 can persist in all life stages of the amoebae without causing any visible injury to them. We also demonstrated that the amoeba generated vesicles containing virions that were expelled into the bulk liquid surroundings, a finding previously described for FLA-bacteria interactions, but not for FLA and human pathogenic viruses. Therefore, our findings suggest that the ability of CVB5 to persist in V. vermiformis could be a novel waterborne risk pathway for the persistence and dispersion of infectious human enteric viruses through water systems.

Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants.

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 ∼59% 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.

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.

UNLABELLED: Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE: The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.