Norovirus risk at a golf course irrigated with reclaimed water: Should QMRA doses be adjusted for infectiousness?

About 25 golf courses in Ontario, Canada have environmental compliance approvals to use reclaimed water for irrigation, where disinfection is confirmed through E. coli limits. A previous study at five Ontario municipal wastewater treatment plants (WWTPs) confirmed that enteric viruses are less susceptible to disinfection than E. coli, when plants provided conventional (secondary or tertiary) treatment and routine (chlorine or UV) disinfection. Here we query whether these four treatment-disinfection scenarios plus 60-day lagoon storage of disinfected effluent would be sufficient to reduce norovirus genogroups I and II (NoV GI and GII) risk of infection to tolerable levels for a golfer who incidentally ingests NoV after handling wet golf balls at a golf course irrigated with reclaimed water. We used our RT-qPCR NoV enumeration datasets from the four treatment-disinfection scenarios above and modeled detected and non-detected NoV by Bayesian inference in 'R'. Monte Carlo simulation included pre-disinfection NoV GI and GII gene copy densities; Ontario WWTP-derived chlorine and UV log10 reductions; literature-derived effluent storage decay parameters and golfer ingested volumes, followed by six different NoV dose-response (DR) models. Quantitative Microbial Risk Assessment (QMRA) results suggest that there is an unacceptable NoV infection risk when using the conservative assumption that all detected NoV particles (RT-qPCR gene copies) are infectious, in both aggregated or disaggregated form. However, after adjusting for PCR target sequences and for infectiousness using data from recently published studies on cultivation of human NoV in human intestinal enteroids, we noted a significant reduction of infection risk. However, this less conservative (i.e., less protective) assumption for water reuse applications such as golf course irrigation may not be corroborated until human NoV are efficiently and routinely grown in cell cultures. In addition, further studies on drivers of NoV risk estimation by DR models are needed, e.g., the extent of NoV particle aggregation resulting from wastewater treatment, as well as the role of immunity. Meantime, regulatory agencies could consider more stringent treatment-disinfection requirements that target enteric viruses rather than E. coli and testing of actual reclaimed irrigation waters.

Enteric viruses in municipal wastewater effluent before and after disinfection with chlorine and ultraviolet light.

In Ontario, Canada, information is lacking on chlorine and ultraviolet (UV) light disinfection performance against enteric viruses in wastewater. We enumerated enteroviruses and noroviruses, coliphages, and Escherichia coli per USEPA methods 1615, 1602, and membrane filtration, respectively, in pre- and post-disinfection effluent at five wastewater treatment plants (WWTPs), with full-year monthly sampling, and calculated log10 reductions (LRs) while WWTPs complied with their monthly geometric mean limit of 200 E. coli/100 mL. Modeling of densities by left-censored estimation and Bayesian inference gave very similar results. Polymerase chain reaction (PCR)-detected enteroviruses and noroviruses were abundant in post-disinfection effluent (mean concentrations of 2.1 × 10+4-7.2 × 10+5 and 2.7 × 10+4-3.6 × 10+5 gene copies (GC)/L, respectively). Chlorine or UV disinfection produced modest LRs for culture- (0.3-0.9) and PCR-detected enteroviruses (0.3-1.3), as well as noroviruses GI + GII (0.5-0.8). Coliphages and E. coli were more susceptible, with LRs of 0.8-3.0 and 2.5, respectively. Sand-filtered effluent produced significantly higher enteric virus LRs (except cultured enteroviruses). Coliphage and human enteric virus densities gave significantly positive correlations using Kendall's Tau test. Enteric viruses are abundant in wastewater effluent following routine chlorine or UV disinfection processes that target E. coli. Coliphages appear to be good indicators for evaluating wastewater disinfection of enteric viruses.

Pathogen Survey of Pulp and Paper Mill Biosolids Compared with Soils, Composts, and Sewage Biosolids.

Regulatory policies to manage land application of organic materials are risk based, with focus on the quality of these residuals. The Ontario Ministry of the Environment and Climate Change (MOECC) determined that limited information was available on pulp and paper biosolids (PPB) with respect to human enteric pathogens. To address this data gap, MOECC conducted an extensive survey (2005-2006) across Ontario to characterize the microbiological quality of PPB. Quantitative testing was performed for fecal indicators (, enterococci, ) and enteric pathogens (, , , and ) using matrix-validated methods. Comparative benchmark materials (soils and soil amendments) were analyzed concurrently for risk comparison. Results showed that detection rates in PPB were low, 5 to 25% for pathogens and <55% for . , and were found at low frequency (6-8% of samples) and at low mean concentrations (2 most probable number g dry wt., 9 oocysts g dry wt., and 7 cells g dry wt., respectively). was more frequently observed (19% of samples), with a mean of 30 cysts g dry wt. Pathogen concentrations in PPB were generally equivalent to or higher than those in soils, composts, and pelletized sewage biosolids but significantly lower than in sewage biosolids. levels exceeded standards (1000 colony-forming units g dry wt.) in one-third of samples, most often in fresh PPB rather than stored and lagoon solids. Microbial quality of PPB across all surveyed mills tended to be variable and sector- and/or site-specific but in many cases would not consistently meet Canadian federal fertilizers standards. These findings were important to inform Ontario's nutrient management regulations, supporting classification of PPB as higher pathogen risk than compost and commercial fertilizers.

Pathogen Characterization of Fresh and Stored Mesophilic Anaerobically Digested Biosolids.

Culturable bacterial pathogens (Campylobacter, Salmonella, Listeria, Yersinia) and indicators (E. coli, enterococci, Clostridium perfringens) were quantified at six water resource recovery facilities that land apply anaerobically digested biosolids in Ontario, Canada. Cryptosporidium parvum and Giardia lamblia were also quantified by polymerase chain reaction (PCR). Salmonella and Listeria were frequently detected in sludge and liquid biosolids (70-100% of samples) but less often in fresh dewatered cake biosolids (50-60%); with low levels in fresh cake (<100 cells/g dw). Yersinia were in 20 to 30% of samples, typically at very low levels (<10 cell/g dw). Giardia and Cryptosporidium were detected in 80 and 20% of cake biosolids at geometric means of 270 cysts/g dw and 70 oocysts/g dw, respectively. E. coli reduction was typically >2-log10 while pathogen reduction was variable. "Sudden increase" of pathogens was not observed, however, Salmonella and E. coli showed regrowth (at 1 to 3 orders of magnitude) after 2- to 3-day storage at 30 °C.

Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli.

Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by realtime polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5-1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.

Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration.

Sewage sludge is the solid, organic material remaining after wastewater is treated and discharged from a wastewater treatment plant. Sludge is treated to stabilize the organic matter and reduce the amount of human pathogens. Once government regulations are met, including material quality standards (e.g., E. coli levels and heavy metal content) sludge is termed "biosolids", which may be disposed of by land application according to regulations. Live-culture techniques have traditionally been used to enumerate select pathogens and/or indicator organisms to demonstrate compliance with regulatory requirements. However, these methods may result in underestimates of viable microorganisms due to several problems, including their inability to detect viable but non-culturable (VBNC) cells. Real-time quantitative polymerase chain reaction (qPCR) is currently under investigation as a fast, sensitive, and specific molecular tool for enumeration of pathogens in biosolids. Its main limitation is that it amplifies all target DNAs, including that from non-viable cells. This can be overcome by coupling qPCR with propidium monoazide (PMA), a microbial membrane-impermeant dye that binds to extracellular DNA and DNA in dead or membrane-compromised cells, inhibiting its amplification. PMA has successfully been used to monitor the presence of viable pathogens in several different matrices. In this review the use of PMA-qPCR is discussed as a suitable approach for viable microbial enumeration in biosolids. Recommendations for optimization of the method are made, with a focus on DNA extraction, dilution of sample turbidity, reagent concentration, and light exposure time.

A novel selective growth medium-PCR assay to isolate and detect Sphingomonas in environmental samples.

Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin-piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 microg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt-PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt-PCR test. None of the unpigmented isolates was positive to the spt-PCR assay. The 16S rDNA of 17% of the spt+ve and -ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin-piperacillin selective growth medium in combination with the colour pigmentation and the positive spt-PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.