Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Assessment of two volumetrically different concentration approaches to improve sensitivities for SARS-CoV-2 detection during wastewater monitoring.

Wastewater monitoring for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic, has highlighted the need for methodologies capable of assessing viral prevalence during periods of low population infection. To address this need, two volumetrically different, methodologically similar concentration approaches were compared for their abilities to detect viral nucleic acid and infectious SARS-CoV-2 signal from primary influent samples. For Method 1, 2 L of SARS-CoV-2 seeded wastewater was evaluated using a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration. For Method 2, 100 mL of SARS-CoV-2 seeded wastewater was evaluated using the CP Select™ procedure. Following D-HFUF concentration (Method 1), significantly lower levels of infectious SARS-CoV-2 were lost (P value range: 0.0398-0.0027) compared to viral gene copy (GC) levels detected by the US Centers for Disease Control (CDC) N1 and N2 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. Subsamples at different steps in the concentration process were also taken to better characterize the losses of SARS-CoV-2 during the concentration process. During the centrifugation step (prior to CP Select™ concentration), significantly higher losses (P value range: 0.0003 to <0.0001) occurred for SARS-CoV-2 GC levels compared to infectious virus for Method 1, while between the methods, significantly higher infectious viral losses were observed for Method 2 (P = 0.0002). When analyzing overall recovery of endogenous SARS-CoV-2 in wastewater samples, application of Method 1 improved assay sensitivities (P = <0.0001) compared with Method 2; this was especially evident during periods of lower COVID-19 case rates within the sewershed. This study describes a method which can successfully concentrate infectious SARS-CoV-2 and viral RNA from wastewater. Moreover, we demonstrated that large volume wastewater concentration provides additional sensitivity needed to improve SARS-CoV-2 detection, especially during low levels of community disease prevalence.

Understanding Microbial Loads in Wastewater Treatment Works as Source Water for Water Reuse.

Facing challenges in water demands and population size, particularly in the water-scarce regions in the United States, the reuse of treated municipal wastewater has become a viable potential to relieve the ever-increasing demands of providing water for (non-)potable use. The objectives of this study were to assess microbial quality of reclaimed water and to investigate treatability of microorganisms during different treatment processes. Raw and final treated effluent samples from three participating utilities were collected monthly for 16 months and analyzed for various microbial pathogens and fecal indicator organisms. Results revealed that the detectable levels of microbial pathogens tested were observed in the treated effluent samples from all participating utilities. Log10 reduction values (LRVs) of Cryptosporidium oocysts and Giardia cysts were at least two orders of magnitude lower than those of human adenovirus and all fecal indicator organisms except for aerobic endospores, which showed the lowest LRVs. The relatively higher LRV of the indicator organisms such as bacteriophages suggested that these microorganisms are not good candidates of viral indicators of human adenovirus during wastewater treatment processes. Overall, this study will assist municipalities considering the use of wastewater effluent as another source of drinking water by providing important data on the prevalence, occurrence, and reduction of waterborne pathogens in wastewater. More importantly, the results from this study will aid in building a richer microbial occurrence database that can be used towards evaluating reuse guidelines and disinfection practices for water reuse practices.

Performance evaluation of a dead-end hollowfiber ultrafiltration method for enumeration of somatic and F+ coliphage from recreational waters.

Dead-end hollow fiber ultrafiltration combined with a single agar layer assay (D-HFUF-SAL) has potential use in the assessment of sanitary quality of recreational waters through enumeration of coliphage counts as measures of fecal contamination. However, information on applicability across a broad range of sites and water types is limited. Here, we tested the performance of D-HFUF-SAL on 49 marine and freshwater samples. Effect of method used to titer the spiking suspension (SAL versus double agar layer [DAL]) on percent recovery was also evaluated. Average somatic coliphage recovery (72 % ± 27) was significantly higher (p < 0.0001) compared to F+ (53 % ± 19). This was more pronounced for marine (p ≤ 0.0001) compared to freshwaters (p = 0.0134). Neither method affected somatic coliphage, but DAL (28 % ± 12) significantly (p < 0.0001) underestimated F + coliphage recoveries compared to SAL (53 % ± 19). Overall, results indicate that, while D-HFUF-SAL performed well over a wide variety of water types, F + coliphage recoveries were significantly reduced for marine waters suggesting that some components unique to this habitat may interfere with the assay performance. More importantly, our findings indicate that choice of spike titer method merits careful consideration since it may under-estimate method percent recovery.

Development of a large volume concentration method for recovery of coronavirus from wastewater.

Levels of severe acute respiratory coronavirus type 2 (SARS CoV 2) RNA in wastewater could act as an effective means to monitor coronavirus disease 2019 (COVID-19) within communities. However, current methods used to detect SARS CoV 2 RNA in wastewater are limited in their ability to process sufficient volumes of source material, inhibiting our ability to assess viral load. Typically, viruses are concentrated from large liquid volumes using two stage concentration, primary and secondary. Here, we evaluated a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration from 2 L volumes of primary treated wastewater. Various amendments to each concentration procedure were investigated to optimally recover seeded OC43 (betacoronavirus) from wastewater. During primary concentration, the D-HFUF recovered 69 ± 18% (n = 29) of spiked OC43 from 2 L of wastewater. For secondary concentration, the CP Select™ system using the Wastewater Application settings was capable of processing 100 mL volumes of primary filter eluates in <25 min. A hand-driven syringe elution proved to be significantly superior (p = 0.0299) to the CP Select™ elution for recovering OC43 from filter eluates, 48 ± 2% compared to 31 ± 3%, respectively. For the complete method (primary and secondary concentration combined), the D-HFUF and CP select/syringe elution achieved overall 22 ± 4% recovery of spiked OC43 through (n = 8) replicate filters. Given the lack of available standardized methodology confounded by the inherent limitations of relying on viral RNA for wastewater surveillance of SARS CoV 2, it is important to acknowledge these challenges when interpreting this data to estimate community infection rates. However, the development of methods that can substantially increase sample volumes will likely allow for reporting of quantifiable viral data for wastewater surveillance, equipping public health officials with information necessary to better estimate community infection rates.

A Gallus gallus Model for Determining Infectivity of Zoonotic Campylobacter.

To better understand public health implications of waterfowl as reservoirs for zoonotic sources of Campylobacter in recreational waters, we developed a Gallus gallus (chick) model of infection to assess the pathogenicity of environmental isolates of Campylobacter. This method involved exposure of 1-day-old chicks through ingestion of water, the natural route of infection. Viable Campylobacter from laboratory-infected animals were monitored by using a modified non-invasive sampling of fresh chick excreta followed by a passive polycarbonate-filter migration culture assay. The method was used to evaluate the infectivities of three laboratory strains of Campylobacter spp. (Campylobacter coli, Campylobacter jejuni, and Campylobacter lari), three clinical isolates of C. jejuni, and four environmental Campylobacter spp. isolated from California gulls (Larus californicus). The results revealed that chicks were successfully infected with all laboratory and clinical isolates of Campylobacter spp. through ingestion of Campylobacter-spiked water, with infection rates ranging from <10 to >90% in a dose-dependent manner. More importantly, exposure of chicks with Campylobacter spp. isolated from Gallus gallus excreta also resulted in successful establishment of infection (≤90%). Each monitored Campylobacter spp. contained ≥7.5 × 104 CFU⋅g-1 of feces 7 days post-exposure. These results suggest that a G. gallus model can be used to assess infectivity of Campylobacter isolates, including gull and human clinical isolates. Use of an avian animal model can be applied to assess the importance of birds, such as the G. gallus, as potential contributors of waterborne-associated outbreaks of campylobacteriosis.

A Constructed Wetland for Treatment of an Impacted Waterway and the Influence of Native Waterfowl on its Perceived Effectiveness.

A constructed, variable-flow treatment wetland was evaluated for its ability to reduce microbial loads from the Banklick Creek, an impacted recreational waterway in Northern Kentucky. For this study, levels of traditional (Escherichia coli and enterococci measured by culture and molecular techniques) and alternative fecal indicators (infectious somatic and F+ coliphage, Clostridium spp. and Clostridium perfringens by culture), potential pathogens (molecular signal of Campylobacter spp.) as well as various microbial source tracking (MST) markers (human fecal marker HF183 and avian fecal marker GFD) were monitored during the summer and early fall through five treatment stages within the Banklick Creek Wetland. No difference in concentrations of traditional or alternative fecal indicators were observed in any of the sites monitored. Microbial source tracking markers were employed to identify sources of fecal contamination within the wetland. Human marker HF183 concentrations at beginning stages of treatment were found to be significantly higher (P value range: 0.0016-0.0003) than levels at later stages. Conversely, at later stages of treatment where frequent bird activity was observed, Campylobacter and avian marker (GFD) signals were detected at significantly higher frequencies (P value range: 0.024 to <0.0001), and both signals were strongly correlated (P = 0.0001). Our study suggests constructed wetlands are an effective means for removal of microbial contamination in ambient waters, but reliance on general fecal indicators is not ideal for determining system efficacy or assessing appropriate remediation efforts.

Genome Sequences of Escherichia Bacteriophages Isolated from Raw Wastewater.

Somatic coliphages are alternative indicators of fecal pollution and attractive surrogates for viral pathogens. Here, we report the draft genome sequences of three replicate plaques from a novel Myoviridae bacteriophage isolated from raw wastewater. These genomes were similar to felix01virus phage and are predicted to contain up to 148 protein-coding genes.

Viability of Pseudocapillaria tomentosa Eggs Exposed to Heat, Ultraviolet Light, Chlorine, Iodine, and Desiccation.

Pseudocapillaria tomentosa is an important pathogen in zebrafish facilities. We investigated heat, ultraviolet (UV) light, chlorine, iodine, and dessciation for killing the parasite's eggs. Eggs released with feces larvate in about 5-10 days, and treatments were evaluated by exposing fresh eggs and subsequently comparing larvation to untreated eggs as an indication of survival. Collectively, untreated eggs in all trials showed high levels of survival. Eggs were exposed to elevated temperatures (40°C, 45°C and 50°C) for 1, 8, or 24 h, which resulted in substantial reduction in viability of eggs. UV radiation was effective, with no larvation at 50-300 mWs/cm2 and <2% at 20 mWs/cm2. Three chlorine products (JT Baker, Clorox®, and Bi-Mart) were tested at 25, 50, 100, 500, and 3,000 ppm (pH 7.0-7.3) with 10 min exposure. All were effective at 500 or 1,000 ppm. There was variability between three products and trials at lower concentrations, but overall chlorine was not very effective at 25-100 ppm except for Bi-Mart brand at 100 ppm. Povidone-iodine was not effective at 25 or 50 ppm for 10 min, but was effective at 200 ppm for 1 h. Desiccation was effective, and no eggs larvated after 2 h drying.

Propagation of Giardia duodenalis cysts in immunosuppressed CF-1 mice.

This study developed and evaluated Giardia duodenalis cyst propagation using a dexamethasone immunosuppressed CF-1 mouse model as an alternative to a previously described Mongolian gerbil model. The CF-1 mouse model shed significantly more cysts per animal during a 16-18 h collection period compared to the gerbil (averages: 7.8 × 106 cysts/CF-1 mouse and 2.5 × 106 cysts/gerbil). In addition, the patency period for this model differed from both G. muris in mice and G. duodenalis in gerbils in that cysts were shed continuously for over 20 days. Results further showed that the ß-giardin gene sequences from gerbil derived and mouse derived G. duodenalis were identical, after 34 serial passages through the CF-1 mouse model. Overall, the CF-1 mouse model produced higher concentrations of cysts per animal, and were genetically and phenotypically stable based on ß-giardin gene sequences.

Reducing inherent biases introduced during DNA viral metagenome analyses of municipal wastewater.

Metagenomics is a powerful tool for characterizing viral composition within environmental samples, but sample and molecular processing steps can bias the estimation of viral community structure. The objective of this study is to understand the inherent variability introduced when conducting viral metagenomic analyses of wastewater and provide a bioinformatic strategy to accurately analyze sequences for viral community analyses. A standard approach using a combination of ultrafiltration, membrane filtration, and DNase treatment, and multiple displacement amplification (MDA) produced DNA preparations without any bacterial derived genes. Results showed recoveries in wastewater matrix ranged between 60-100%. A bias towards small single stranded DNA (ssDNA; polyomavirus) virus types vs larger double stranded DNA (dsDNA; adenovirus) viruses was also observed with a total estimated recovery of small circular viruses to be as much as 173-fold higher. Notably, ssDNA abundance decreased with sample dilution while large dsDNA genomes (e.g., Caudovirales) initially increased in abundance with dilution before gradually decreasing with further dilution in wastewater samples. The present study revealed the inherent biases associated with different components of viral metagenomic methods applied to wastewater. Overall, these results provide a well-characterized approach for effectively conducting viral metagenomics analysis of wastewater and reveal that dilution can effectively mitigate MDA bias.

Nationwide reconnaissance of contaminants of emerging concern in source and treated drinking waters of the United States.

When chemical or microbial contaminants are assessed for potential effect or possible regulation in ambient and drinking waters, a critical first step is determining if the contaminants occur and if they are at concentrations that may cause human or ecological health concerns. To this end, source and treated drinking water samples from 29 drinking water treatment plants (DWTPs) were analyzed as part of a two-phase study to determine whether chemical and microbial constituents, many of which are considered contaminants of emerging concern, were detectable in the waters. Of the 84 chemicals monitored in the 9 Phase I DWTPs, 27 were detected at least once in the source water, and 21 were detected at least once in treated drinking water. In Phase II, which was a broader and more comprehensive assessment, 247 chemical and microbial analytes were measured in 25 DWTPs, with 148 detected at least once in the source water, and 121 detected at least once in the treated drinking water. The frequency of detection was often related to the analyte's contaminant class, as pharmaceuticals and anthropogenic waste indicators tended to be infrequently detected and more easily removed during treatment, while per and polyfluoroalkyl substances and inorganic constituents were both more frequently detected and, overall, more resistant to treatment. The data collected as part of this project will be used to help inform evaluation of unregulated contaminants in surface water, groundwater, and drinking water.

Human infective potential of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in urban wastewater treatment plant effluents.

Cryptosporidiosis, giardiasis, and microsporidiosis are important waterborne diseases. In the standard for wastewater treatment plant (WWTP) effluents in China and other countries, the fecal coliform count is the only microbial indicator, raising concerns about the potential for pathogen transmission through WWTP effluent reuse. In this study, we collected 50 effluent samples (30 L/sample) from three municipal WWTPs in Shanghai, China, and analyzed for Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi by microscopy and/or polymerase chain reaction (PCR). Moreover, propidium monoazide (PMA)-PCR was used to assess the viability of oocysts/cysts. The microscopy and PCR-positive rates for Cryptosporidium spp. were 62% and 40%, respectively. The occurrence rates of G. duodenalis were 96% by microscopy and 92-100% by PCR analysis of three genetic loci. Furthermore, E. bieneusi was detected in 70% (35/50) of samples by PCR. Altogether, 10 Cryptosporidium species or genotypes, two G. duodenalis genotypes, and 11 E. bieneusi genotypes were found, most of which were human-pathogenic. The chlorine dioxide disinfection employed in WWTP1 and WWTP3 failed to inactivate the residual pathogens; 93% of the samples from WWTP1 and 83% from WWTP3 did not meet the national standard on fecal coliform levels. Thus, urban WWTP effluents often contain residual waterborne human pathogens.

Microbial pathogens in source and treated waters from drinking water treatment plants in the United States and implications for human health.

An occurrence survey was conducted on selected pathogens in source and treated drinking water collected from 25 drinking water treatment plants (DWTPs) in the United States. Water samples were analyzed for the protozoa Giardia and Cryptosporidium (EPA Method 1623); the fungi Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus (quantitative PCR [qPCR]); and the bacteria Legionella pneumophila (qPCR), Mycobacterium avium, M. avium subspecies paratuberculosis, and Mycobacterium intracellulare (qPCR and culture). Cryptosporidium and Giardia were detected in 25% and in 46% of the source water samples, respectively (treated waters were not tested). Aspergillus fumigatus was the most commonly detected fungus in source waters (48%) but none of the three fungi were detected in treated water. Legionella pneumophila was detected in 25% of the source water samples but in only 4% of treated water samples. M. avium and M. intracellulare were both detected in 25% of source water, while all three mycobacteria were detected in 36% of treated water samples. Five species of mycobacteria, Mycobacterium mucogenicum, Mycobacterium phocaicum, Mycobacterium triplex, Mycobacterium fortuitum, and Mycobacterium lentiflavum were cultured from treated water samples. Although these DWTPs represent a fraction of those in the U.S., the results suggest that many of these pathogens are widespread in source waters but that treatment is generally effective in reducing them to below detection limits. The one exception is the mycobacteria, which were commonly detected in treated water, even when not detected in source waters.

The development and implementation of a method using blue mussels (Mytilus spp.) as biosentinels of Cryptosporidium spp. and Toxoplasma gondii contamination in marine aquatic environments.

Surveillance monitoring for microbial water quality typically involves collecting single discrete grab samples for analyzing only one contaminant. While informative, current approaches suffer from poor recoveries and only provide a limited snapshot of the microbial contaminants only at the time of collection. To overcome these limitations, bivalves have been proposed as effective biosentinels of water quality particularly for their ability to efficiently concentrate and retain microbial contaminants for long periods of time. In this study, we examined the use of indigenous blue mussels (Mytilus spp.) as biosentinels to monitor for the presence of Toxoplasma gondii and Cryptosporidium water. An efficient method to extract oocyst DNA from various mussel tissues followed by PCR-based detection of these pathogens was developed, which resulted in the detection down to 10 oocysts. This method was then used to conduct a small survey in Point Lobos and Morro Bay, California to determine prevalence T. gondii and Cryptosporidium. Results revealed that mussels from Morro Bay were contaminated with T. gondii (33 %), while mussels from Point Lobos were contaminated with T. gondii (54 %) and Cryptosporidium (26.9 %) oocysts. Phylogenetic analysis using the SSU rRNA gene identified two novel Cryptosporidium parvum-like genotypes. Overall, this study demonstrated the application of using native California Mytilus spp. as biosentinels for pathogen contamination along the central California shorelines. More importantly, T. gondii and Cryptosporidium were found at higher prevalence rates in Morro Bay and in Point Lobos, an area not previously reported to be contaminated with these pathogens.

Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water.

The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.

Determining pathogen and indicator levels in class B municipal organic residuals used for land application.

Biosolids are nutrient-rich organic residuals that are currently used to amend soils for food production. Treatment requirements to inactivate pathogens for production of Class A biosolids are energy intensive. One less energy intensive alternative is to treat biosolids to Class B standards, but it could result in higher pathogen loads. Quantitative microbial risk assessments models have been developed on land application of Class B biosolids but contain many uncertainties because of limited data on specific pathogen densities and the use of fecal indicator organisms as accurate surrogates of pathogen loads. To address this gap, a 12-mo study of the levels and relationships between , , and human adenovirus (HAdV) with fecal coliform, somatic, and F-RNA coliphage levels in Class B biosolids from nine wastewater treatment plants throughout the United States was conducted. Results revealed that fecal coliform, somatic, and F-RNA coliphage densities were consistent throughout the year. More important, results revealed that HAdV ( = 2.5 × 10 genome copies dry g) and ( = 4.14 × 10 cysts dry g) were in all biosolids samples regardless of treatment processes, location, or season. oocysts were also detected (38% positive; range: 0-1.9 × 10 oocysts dry g), albeit sporadically. Positive correlations among three fecal indicator organisms and HAdV, but not protozoa, were also observed. Overall, this study reveals that high concentrations of enteric pathogens (e.g., , , and HAdV) are present in biosolids throughout the United States. Microbial densities found can further assist management and policymakers in establishing more accurate risk assessment models associated with land application of Class B biosolids.