Evaluation of an Automated Ultrafiltration System for Concentrating a Range of Viruses from Saline Waters.

Pathogenic viruses in environmental water are usually present in levels too low for direct detection and thus, a concentration step is often required to increase the analytical sensitivity. The objective of this study was to evaluate an automated filtration device, the Innovaprep Concentrating Pipette Select (CP Select) for the rapid concentration of viruses in saline water samples, while considering duration of process and ease of use. Four bacteriophages (MS2, P22, Phi6, and PhiX174) and three animal viruses (adenovirus, coronavirus OC43, and canine distemper virus) were seeded in artificial seawater, aquarium water, and bay water samples, and processed using the CP Select. The recovery efficiencies of viruses were determined either using a plaque assay or droplet digital PCR (ddPCR). Using plaque assays, the average recovery efficiencies for bacteriophages ranged from 4.84 ± 3.8% to 82.73 ± 27.3%, with highest recovery for P22 phage. The average recovery efficiencies for the CP Select were 39.31 ± 26.6% for adenovirus, 19.04 ± 11.6% for coronavirus OC43, and 19.84 ± 13.6% for canine distemper virus, as determined by ddPCR. Overall, viral genome composition, not the size of the virus, affected the recovery efficiencies for the CP Select. The small sample volume size used for the ultrafilter pipette of the system hinders the use of this method as a primary concentration step for viruses in marine waters. However, the ease of use and rapid processing time of the CP Select are especially beneficial when rapid detection of viruses in highly contaminated water, such as wastewater or sewage-polluted surface water, is needed.

Temporal and spatial relationships of CrAssphage and enteric viral and bacterial pathogens in wastewater in North Carolina.

Enteric disease remains one of the most common concerns for public health, particularly when it results from human exposure to surface and recreational waters contaminated with wastewater. Characterizing the temporal and spatial variation of enteric pathogens prevalent in wastewater is critical to develop approaches to mitigate their distribution in the environment. In this study, we aim to characterize pathogen variability and test the applicability of the human-associated wastewater indicator crAssphage as an indicator of enteric viral and bacterial pathogens. We conducted weekly samplings for 14 months from four wastewater treatment plants in North Carolina, USA. Untreated wastewater samples were processed using hollow fiber ultrafiltration, followed by secondary concentration methods. Adenovirus, norovirus, enterovirus, Salmonella, Shiga toxin 2 (stx2), Campylobacter, and crAssphage were measured by quantitative polymerase chain reaction (qPCR) and reverse transcriptase (rt)-qPCR. Our results revealed significant correlations between crAssphage and human adenovirus, enterovirus, norovirus, Salmonella, and Campylobacter (p<0.01). Pathogens and crAssphage concentrations in untreated wastewater showed distinct seasonal patterns, with peak concentrations of crAssphage and viral pathogens in fall and winter, while bacterial pathogens showed peaked concentrations in either winter (Campylobacter), fall (Salmonella), or summer (stx2). This study enhances the understanding of crAssphage as an alternative molecular indicator for both bacterial and viral pathogens. The findings of this study can also inform microbial modeling efforts for the prediction of the impact of wastewater pathogens on surface waters due to increased flooding events and wastewater overflows associated with climate change.

Prevalence of opportunistic pathogens in a school building plumbing during periods of low water use and a transition to normal use.

The spread of opportunistic pathogens via building water supply and plumbing is of public health concern. This study was conducted to better understand microbial water quality changes in a LEED-certified school building during low water use (Summer) and normal water use (Autumn). The copper plumbed building contained water saving devices, a hot water recirculation system, and received chloraminated drinking water from a public water system. Three separate sampling events were conducted during the summer break inside the building and another three sampling events were conducted after the school returned to session. Using quantitative PCR, Legionella spp. were detected in all water samples, followed by Mycobacterium spp. (99%). Mycobacterium avium (75%) and Acanthamoeba spp. (17.5%) throughout the building water system. Legionella pneumophila and Naegleria fowleri were not detected in any of the samples. The mean concentrations of Legionella spp., Mycobacterium spp., Mycobacterium avium, and Acanthamoeba spp. detected in water samples were 3.9, 5.7, 4.7, and 2.8 log10 gene copies per 100 ml, respectively. There was a statistically significantly difference in the mean concentrations of Legionella spp., Mycobacterium spp. and M. avium gene markers in water samples between school breaks and when school was in session. Cultivable Legionella were also detected in water samples collected during periods of low water use. This study highlights the need for routine proactive water quality testing in school buildings to determine the extent of drinking water quality problems associated with plumbing and direct action to remediate microbial colonization.

Detection of coliphages and human adenoviruses in a subtropical estuarine lake.

Fecal indicator bacteria (FIB) have been used to assess fecal contamination in recreational water. However, enteric viruses have been shown to be more persistent in the environment and resistant to wastewater treatment than bacteria. Recently, U.S Environmental Protection Agency has proposed the use of coliphages as viral indicators to better protect against viral waterborne outbreaks. This study aimed to detect and determine correlation between coliphages (F-specific and somatic), fecal indicator bacteria (enterococci and fecal coliforms), and human enteric viruses (human adenovirus) in a subtropical brackish estuarine lake. Water samples were collected from 9 estuarine recreation sites on Lake Pontchartrain in southeast Louisiana. Water samples (n = 222, collected weekly) were analyzed for coliphages and fecal indicator bacteria using culture-based methods and large volume water samples (n = 54, collected monthly) were analyzed for human adenovirus using quantitative PCR. Somatic coliphage and F-specific coliphage were found in 93.7 and 65.2% of samples with geometric mean concentrations of 30 and 3 plaque forming units (PFU) per 100 mL, respectively. Enterococci, fecal coliforms, and adenovirus were found in all samples with geometric mean concentrations of 27 most probable number (MPN), 77 MPN, and 3.0 × 104 gene copies per 100 mL, respectively. Watersheds in suburban areas exhibited significantly higher concentrations of coliphages and fecal indicator bacteria, indicating potential fecal contamination from septic systems. There was no significant correlation (p > 0.05) observed between the presence of adenoviruses and fecal indicator bacteria and coliphages. The presence of human adenovirus in Lake Pontchartrain poses a significant public health problem for both recreational use and seafood harvesting as it increases exposure risks. This study demonstrated the lack of relationship between fecal indicators and human viral pathogen in Lake Pontchartrain supporting an alternative microbial surveillance system such as direct pathogen detection.

Antibacterial and antiviral effectiveness of two household water treatment devices that use monobrominated hydantoinylated polystyrene.

Many different household water treatment (HWT) methods have been researched and promoted to mitigate the serious burden of diarrheal disease in developing countries. However, HWT methods using bromine have not been extensively evaluated. Two gravity-fed HWT devices (AquaSure™ and Waterbird™) were used to test the antimicrobial effectiveness of HaloPure® Br beads (monobrominated hydantoinylated polystyrene) that deliver bromine. As water flows over the beads, reactive bromine species are eluted, which inactivate microorganisms. To assess log10 reduction values (LRVs) for Vibrio cholerae, Salmonella enterica Typhimurium, bacteriophage MS2, human adenovirus 2 (HAdV2), and murine norovirus (MN), these organisms were added to potable water and sewage-contaminated water. These organisms were quantified before and after water treatment by the HWT devices. On average, 6 LRVs against Vibrio were attained, as well as 5 LRVs against Salmonella, 4 LRVs against MS2, 5 LRVs against HAdV2, and 3 LRVs against MN. Disinfection was similar regardless of whether sewage was present. Polymer beads delivering bromine to drinking water are a potentially effective and useful component of HWT methods in developing countries.

Metagenomic approaches for direct and cell culture evaluation of the virological quality of wastewater.

Genomic-based molecular techniques are emerging as powerful tools that allow a comprehensive characterization of water and wastewater microbiomes. Most recently, next generation sequencing (NGS) technologies which produce large amounts of sequence data are beginning to impact the field of environmental virology. In this study, NGS and bioinformatics have been employed for the direct detection and characterization of viruses in wastewater and of viruses isolated after cell culture. Viral particles were concentrated and purified from sewage samples by polyethylene glycol precipitation. Viral nucleic acid was extracted and randomly amplified prior to sequencing using Illumina technology, yielding a total of 18 million sequence reads. Most of the viral sequences detected could not be characterized, indicating the great viral diversity that is yet to be discovered. This sewage virome was dominated by bacteriophages and contained sequences related to known human pathogenic viruses such as adenoviruses (species B, C and F), polyomaviruses JC and BK and enteroviruses (type B). An array of other animal viruses was also found, suggesting unknown zoonotic viruses. This study demonstrated the feasibility of metagenomic approaches to characterize viruses in complex environmental water samples.

Detection and characterization of human pathogenic viruses circulating in community wastewater using multi target microarrays and polymerase chain reaction.

Sewage pollution remains the most significant source of human waterborne pathogens. This study describes the detection and characterization of human enteric viruses in community wastewaters using cell culture coupled with multiple target microarrays (with a total of 780 unique probes targeting 27 different groups of both DNA and RNA viruses) and polymerase chain reaction (PCR) assays. Over a 13-month sampling period, RNA viruses (astroviruses and enteroviruses) were more frequently detected compared to DNA viruses (adenoviruses, particularly type 41 and BK polyomavirus). Overall, many more viruses were shed during the winter months (December-February) compared to the summer months. Exploration of the multiple types of enteric viruses particularly in winter months identified much more significant prevalence of key viral pathogens associated with sewage pollution of the water environment than previously realized and seasonal disinfection used in some parts of the world may lead to a seeding of ambient waters. Molecular characterization of pathogenic viruses in community wastewater will improve the understanding of the potential risk of waterborne disease transmission of viral pathogens.