Sanitary quality of recreational waters worldwide is assessed using fecal indicator bacteria (FIB), such as Escherichia coli and enterococci. However, fate and transport characteristics of FIB in ...aquatic habitats can differ from those of viral pathogens which have been identified as main etiologic agents of recreational waterborne illness. Coliphages (bacteriophages infecting E. coli) are an attractive alternative to FIB because of their many morphological and structural similarities to viral pathogens.
In this in situ field study, we used a submersible aquatic mesocosm to compare decay characteristics of somatic and F+ coliphages to those of infectious human adenovirus 2 in a freshwater lake. In addition, we also evaluated the effect of ambient sunlight (and associated UV irradiation) and indigenous protozoan communities on decay of somatic and F+ coliphage, as well as infectious adenovirus.
Our results show that decay of coliphages and adenovirus was similar (p = 0.0794), indicating that both of these bacteriophage groups are adequate surrogates for decay of human adenoviruses. Overall, after 8 days the greatest log
reductions were observed when viruses were exposed to a combination of biotic and abiotic factors (2.92 ± 0.39, 4.48 ± 0.38, 3.40 ± 0.19 for somatic coliphages, F+ coliphages and adenovirus, respectively). Both, indigenous protozoa and ambient sunlight, were important contributors to decay of all three viruses, although the magnitude of that effect differed over time and across viral targets.
While all viruses studied decayed significantly faster (p < 0.0001) when exposed to ambient sunlight, somatic coliphages were particularly susceptible to sunlight irradiation suggesting a potentially different mechanism of UV damage compared to F+ coliphages and adenoviruses. Presence of indigenous protozoan communities was also a significant contributor (p value range: 0.0016 to < 0.0001) to decay of coliphages and adenovirus suggesting that this rarely studied biotic factor is an important driver of viral reductions in freshwater aquatic habitats.
The U.S. Environmental Protection Agency's information collection rule requires the use of 1MDS electropositive filters for concentrating enteric viruses from water, but unfortunately, these filters ...are not cost-effective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the NanoCeram filter, was evaluated for its ability to concentrate enteroviruses and noroviruses from large volumes of water. Seeded viruses were concentrated using the adsorption-elution procedure. The mean percent retention of seeded polioviruses by NanoCeram filters was 84%. To optimize the elution procedure, six protocols, each comprising two successive elutions with various lengths of filter immersion, were evaluated. The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 minute during the first elution and for 15 min during the second elution. The recovery efficiencies of poliovirus, coxsackievirus B5, and echovirus 7 from 100-liter samples of seeded tap water were 54%, 27%, and 32%, respectively. There was no significant difference in virus recovery from tap water with a pH range of 6 to 9.5 and a water flow rate range of 5.5 liters/min to 20 liters/min. Finally, poliovirus and Norwalk virus recoveries by NanoCeram filters were compared to those by 1MDS filters, using tap water and Ohio River water. Poliovirus and Norwalk virus recoveries by NanoCeram filters from tap and river water were similar to or higher than those by the 1MDS filters. These data suggest that NanoCeram filters can be used as an inexpensive alternative to 1MDS filters for routine viral monitoring of water.
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•Ultrafiltration and single agar layer method was developed for coliphage concentration.•Method effectively concentrated somatic and F+ coliphages from diverse surface waters.•Percent ...recoveries were 40–79% for river, 45–63% for lake and 70–94% for marine water.•Both coliphages were consistently and reliably detected in highly diluted wastewater.•Autochthonous coliphages were detected in 76% (somatic) and 57% (F+) of samples.
Somatic and F+ coliphages are promising alternative fecal indicators, but current detection methods are hindered by lower levels of coliphages in surface waters compared to traditional bacterial fecal indicators. We evaluated the ability of dead-end hollow fiber ultrafiltration (D- HFUF) and single agar layer (SAL) procedure to concentrate and enumerate coliphages from 1L and 10L volumes of ambient surface waters (lake, river, marine), river water with varying turbidities (3.74–118.7 NTU), and a simulated combined sewer overflow (CSO) event. Percentage recoveries for surface waters were 40–79% (somatic) and 35–94% (F+). The method performed equally well in all three matrices at 1L volumes, but percent recoveries were significantly higher in marine waters at 10L volumes when compared to freshwater. Percent recoveries at 1L and 10L were similar, except in river water where recoveries were significantly lower at higher volume. In highly turbid waters, D-HFUF-SAL had a recovery range of 25–77% (somatic) and 21–80% (F+). The method produced detectable levels of coliphages in diluted wastewater and in unspiked surface waters, emphasizing its applicability to CSO events and highlighting its utility in recovery of low coliphage densities from surface waters. Thus D-HFUF-SAL is a good candidate method for routine water quality monitoring of coliphages.
Ultrafiltration concentration of microorganisms in large volume water samples containing high levels of particulate matter was evaluated in a proof of concept study. The organisms tested were ...Bacillus atrophaeus subspecies globigii spores and MS2 bacteriophage. To produce the large volume samples, fresh water sediment of a known particle size was added to 51 l of tap water. Five different concentrations of particulate matter were studied: 0, 50, 100, 150 and 750 mg solids/l. The concentration procedure used a dialysis filter as the ultrafilter configured for axial flow, either with or without recirculation. The target number of organisms spiked was 1 × 105 of either spores or bacteriophage per 51 l. After concentration, the filters were dissected to retrieve the fibers which were then washed using surfactant solution which was then analyzed for the target organisms. Two washes of the filter fibers were carried out sequentially. For axial flow with recirculation, the first wash produced statistically greater recovery of B. globigii spores (26–40% of spike) compared to the second wash (8–13% of spike). Total recovery (the sum of the recoveries for the first and second washes) ranged from 35 to 53%. Recovery increased as the solids level increased from 0 to 150 mg solids/l. Recovery at the 100 and 150 mg solids/L loadings was statistically higher at the P < .05 level than recovery at 0 mg/L solids. At 150 mg solids/L, axial flow without recirculation (dead end) yielded lower recovery than axial flow with recirculation, however the difference was not significant at the P < .05 level. Recovery of B. globigii at 750 mg solids/L averaged 38% using dead end axial flow. The average recovery of MS2 bacteriophage was 45% at a solids concentration of 150 mg/L using axial flow with recirculation. PhiX174 and Phi8 were also studied, however these bacteriophage appeared to be inactivated in the matrix of concentrated wash water. One hundred liters of water containing 750 mg solids/L was concentrated using dead end axial flow, and only minimal problems with filter clogging were observed. Results described herein suggest axial flow ultrafiltration is an effective concentration method for microorganisms in water containing high levels of particulate matter.
•Hollow-fiber ultrafiltration concentrates viruses and bacteriophage from water.•Celite secondary method efficiently concentrates viruses from water following HFUF.•The complete concentration method ...resulted in 60.6% recoveries from tap water.•The complete concentration method resulted in 60.0% recoveries from river water.
The collection of waterborne pathogen occurrence data often requires the concentration of microbes from large volumes of water due to the low number of microorganisms that are typically present in environmental and drinking waters. Hollow-fiber ultrafiltration (HFUF) has shown promise in the recovery of various microorganisms. This study has demonstrated that the HFUF primary concentration method is effective at recovering bacteriophage φX174, poliovirus, enterovirus 70, echovirus 7, coxsackievirus B4 and adenovirus 41 from large volumes of tap and river water with an average recovery of all viruses of 73.4% and 81.0%, respectively. This study also evaluated an effective secondary concentration method using celite for the recovery of bacteriophage and enteric viruses tested from HFUF concentrates of both matrices. Overall, the complete concentration method (HFUF primary concentration plus celite secondary concentration) resulted in a concentration factor of 3333 and average recoveries for all viruses from tap and river waters of 60.6% and 60.0%, respectively.
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► Virus recovery was best at low filtration rates. ►
C. parvum tested at high filtration rates and had an average recovery was 105%. ► Beef extract–celite method and PBS elution is ...ideal for concentration of poliovirus. ► Complete concentration method recovered poliovirus at 97% or 89%.
Tangential flow hollow-fiber ultrafiltration (HFUF) was evaluated for virus and
Cryptosporidium parvum concentration from water. Recovery of viruses at a low filtration rate was found to be significantly greater than at a higher filtration rate, with the recoveries of bacteriophage MS2 at high and low filtration rates shown to be 64.7% and 98.7%, respectively. Poliovirus recoveries from tap water were similar to MS2, with recoveries of 62.9% and 104.5% for high and low filtration rates, respectively.
C. parvum, which was only tested at high filtration rates, had an average recovery was 105.1%. In addition to the optimization of the primary concentration technique, this study also compared several secondary concentration procedures. The highest recovery (89.5%) of poliovirus from tap water concentrates was obtained when a beef extract–celite method was used and the virus was eluted from the celite with phosphate buffered saline, pH 9.0. When HFUF primary concentration and the optimal secondary concentration methods were combined, an average recovery of 97.0
±
35.6% or 89.3
±
19.3%, depending on spike level, was achieved for poliovirus. This study demonstrated that HFUF primary concentration method is effective at recovering MS2, poliovirus and
C. parvum from large volumes of water and that beef extract–celite method is an effective secondary concentration method for the poliovirus tested.
•Primary effluent of wastewater is a rich source of diverse enteric viruses.•Isolation of these viruses allows for use in many research applications.•Celite (diatomaceous earth) facilitates ...concentration of viruses from wastewater.•Celite and a large volume extraction method allow sufficient recovery of viruses.•Celite with centrifugal concentration and extraction show reduced virus recovery.
Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater, making wastewater a source of a potentially diverse group of enteric viruses. In this study, two procedures were evaluated to concentrate noroviruses, adenoviruses and enteroviruses from primary effluent of wastewater. In the first procedure, indigenous enteroviruses, noroviruses and adenoviruses were concentrated using celite (diatomaceous earth) followed by centrifugation through a 30K MWCO filter and nucleic acid extraction. The second procedure used celite concentration followed by nucleic acid extraction only. Virus quantities were measured using qPCR. A second set of primary effluent samples were seeded with Coxsackievirus A7, Coxsackievirus B1, poliovirus 1 or enterovirus 70 before concentration and processed through both procedures for recovery evaluation of enterovirus species representatives. The pairing of the single step extraction procedure with the celite concentration process resulted in 47–98% recovery of examined viruses, while the celite concentration process plus additional centrifugal concentration before nucleic acid extraction showed reduced recovery (14–47%). The celite concentration process followed by a large volume nucleic acid extraction technique proved to be an effective procedure for recovering these important human pathogens from wastewater.
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 Cryptosporidium, Giardia, 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 (x¯ = 2.5 × 103 genome copies dry g−1) and Giardia (x¯ = 4.14 × 103 cysts dry g−1) were in all biosolids samples regardless of treatment processes, location, or season. Cryptosporidium oocysts were also detected (38% positive; range: 0−1.9 × 103 oocysts dry g−1), 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., Cryptosporidium, Giardia, 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.
•Performance comparison of three coliphage methods with 1 L surface water samples.•D-HFUF-SAL significantly outperformed M-SAL and DMF methods.•Overall, frequency of non-detects ranged from 65.6% ...(DMF) to 10.8% (D-HFUF-SAL).•D-HFUF-SAL yielded the highest coliphage concentrations.
Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique. Overall, D-HFUF-SAL outperformed other methods as it yielded the lowest frequency of non-detects (ND); 10.8% and the highest average concentrations of recovered coliphage for positive samples (2.51 ± 1.02 standard deviation, SD log10 plaque forming unit/liter (PFU/L) and 0.79 ± 0.71 (SD) log10 PFU/L for somatic and F+, respectively). M-SAL yielded 29.7% ND and average concentrations of 2.26 ± 1.15 (SD) log10 PFU/L (somatic) and 0.59 ± 0.82 (SD) log10 PFU/L (F+). DMF performance was inferior to D-HFUF-SAL and M-SAL methods (ND of 65.6%; average somatic coliphage concentration 1.52 ± 1.32 SD log10 PFU/L, no F+ detected), indicating this procedure is unsuitable for 1 L surface water sample volumes. This study represents an important step toward the use of a coliphage method for recreational water quality criteria purposes.
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