Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants

Hospital wastewater has been described as an important source of spreading pathogenic microorganisms in the environment. However, there are few studies reporting the presence and concentrations of gastroenteric viruses and hepatitis A viruses in these environmental matrices. The aim of this study was to assess the contamination by viruses responsible for acute gastroenteritis and hepatitis derived from hospital wastewater treatment plants (WWTPs). Rotavirus A (RV-A), human adenoviruses (HAdV), norovirus genogroup I and II (NoV GI/GII) and hepatitis A viruses (HAV) were detected and quantified in sewage samples from two WWTPs located in Rio de Janeiro (Brazil) that operates different sewage treatments. WWTP-1 uses an Upflow Anaerobic Sludge Blanket (UASB reactor) and three serial anaerobic filters while WWTP-2 uses aerobic processes, activated sludge with extended aeration and final chlorination of the effluents. Viruses’ detection was investigated by using conventional PCR/RT-PCR, quantitative real-time PCR (qPCR) and partial sequencing of the genome of the viruses detected. Rate of viruses detection ranged from 7% (NoV GI in WWTP-1) to 95% (RV-A in WWTP-2) and genome from all viruses were detected. The most prevalent genotypes were RV-A SG I, HAdV species D and F, NoV GII/4 and HAV subgenotype IA. Mean values of viral loads (genome copies (GC)/ml) obtained in filtered effluents from anaerobic process was 1.9 × 10³ (RV-A), 2.8 × 10³ (HAdV) and 2.4 × 10³ (NoV GII). For chlorinated effluents from activated sludge process, the mean values of viral loads (GC/ml) was 1.2 × 10⁵ (RV-A), 1.4 × 10³ (HAdV), 8.1 × 10² (NoV GII) and 2.8 × 10⁴ (HAV). Data on viral detection in treated effluents of hospital WWTPs confirmed the potential for environmental contamination by viruses and could be useful to establish standards for policies on wastewater management.     

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water

A reliable indicator is needed to predict and reduce the risk of infection associated with fecal contamination of surface water. Since Pepper mild mottle virus (PMMoV), human picobirnaviruses (hPBV) and Torque teno virus (TTV) have been detected at substantial levels in human feces, we explored whether detection of nucleic acids of these viruses is a suitable indicator of fecal contamination in river water. From September 2008 to December 2009, water samples (n = 111) were collected from the Ruhr and Rhine rivers and from the influents and effluents of a wastewater plant (n = 12). Quantitative real time (RT-) PCR was used to determine the abundance of PMMoV, hPBV, and TTV in comparison to human adenoviruses (HAdV) and human polyomaviruses (HPyV) that are frequently detected in surface water and were previously proposed as indicators. While PMMoV was detected in all river water samples, the other viruses were detected less frequently. The concentration of the studied viruses in positive river water ranged from 5 × 10¹ to 1.07 × 10⁶ genome equivalents per liter (gen.equ./l). All wastewater samples were positive for PMMoV, HAdV and HPyV, while TTV and hPBV were detected in 6/12 and 3/12 of samples, respectively. To determine if PMMoV is specific to human-derived fecal waste, fecal samples from human (n = 20) and animal (n = 53) were also tested. In contrast to the ubiquity of PMMoV in human feces (19/20) the virus was only detected at low concentration in a minority of the animal fecal samples tested (7/15 from chicken, 1/10 from Geese and 1/6 from cows). Therefore, in this setting TTV and hPBV do not seem to be suitable indicators of fecal contamination in water. Whereas, the high excretion level and dissemination of PMMoV in human sewage and river water suggest that PMMoV could be a promising indicator of fecal pollution in surface water.     

Release of infectious human enteric viruses by full-scale wastewater utilities

In the United States, infectious human enteric viruses are introduced daily into the environment through the discharge of treated water and the digested sludge (biosolids). In this study, a total of 30 wastewater and 6 biosolids samples were analyzed over five months (May-September 2008-2009) from five full-scale wastewater treatment plants (WWTPs) in Michigan using real-time PCR and cell culture assays. Samples were collected from four different locations at each WWTP (influent, pre-disinfection, post-disinfection and biosolids) using the 1MDS electropositive cartridge filter. Adenovirus (HAdV), enterovirus (EV) and norovirus genogroup II (NoV GGII) were detected in 100%, 67% and 10%, respectively of the wastewater samples using real-time PCR. Cytopathic effect (CPE) was present in 100% of the cell culture samples for influent, pre- and post-disinfection and biosolids with an average log concentration of 4.1 (2.9-4.7, range) 1.1 (0.0-2.3, range) and 0.5 (0.0-1.6, range) MPN/100 L and 2.1 (0.5-4.1) viruses/g, respectively. A significant log reduction in infectious viruses throughout the wastewater treatment process was observed at an average 4.2 (1.9-5.0, range) log units. A significant difference (p-value <0.05) was observed using real-time PCR data for HAdV but not for EV (p-value >0.05) removal in MBR as compared to conventional treatment. MBR treatment was able to achieve an additional 2 and 0.5 log reduction of HAdV and EV, respectively. This study has demonstrated the release of infectious enteric viruses in the final effluent and biosolids of wastewater treatment into the environment.     

Detection of the human specific Bacteroides genetic marker provides evidence of widespread sewage contamination of stormwater in the urban environment

Human sewage contamination of surface waters is a major human health concern. We found urban stormwater systems that collect and convey runoff from impervious surfaces act as a conduit for sewage originating from breeches in sanitary sewer infrastructure. A total of 828 samples at 45 stormwater outfalls were collected over a four-year period and assessed by culture based methods, PCR, and quantitative PCR (qPCR) to test for traditional and alternative indicators of fecal pollution. All outfalls had the HF183 (human) Bacteroides genetic marker detected in at least one sample, suggesting sewage contamination is nearly ubiquitous in the urban environment. However, most outfalls were intermittently positive, ranging from detection in 11%-100% of the samples. Positive results did not correlate with seasonality, rainfall amounts, or days since previous rainfall. Approximately two-thirds of the outfalls had high (>5000 copy number, i.e. CN, per 100 ml) or moderate levels (1000-5000 CN per 100 ml) of the human Bacteroides genetic marker. Escherichia coli (E. coli) and enterococci levels did not correlate to human Bacteroides. A total of 66% of all outfall samples had standard fecal indicator levels above 10,000 CFU per 100 ml. A tiered assessment using this benchmark to identify high priority sites would have failed to flag 35% of the samples that had evidence of sewage contamination. In addition, high fecal indicators would have flagged 33% of samples as priority that had low or no evidence of sewage. Enteric virus levels in one outfall with high levels of the human Bacteroides genetic marker were similar to untreated wastewater, which illustrates stormwater can serve as a pathway for pathogen contamination. The major source of fecal pollution at four of five river sites that receive stormwater discharge appeared to be from sewage sources rather than non-human sources based on the ratios of human Bacteroides to total Bacteroides spp. This study shows the feasibility and benefits of employing molecular methods to test for alternative indicators of fecal pollution to identify sewage sources and potential health risks and for prioritization of remediation efforts.     

Analysis of adenoviruses and polyomaviruses quantified by qPCR as indicators of water quality in source and drinking-water treatment plants

Three drinking-water treatment plants were analyzed for the presence of human adenoviruses (HAdV) and JC polyomavirus (JCPyV), previously suggested as viral contamination indicators, in order to define their water quality in relation to the presence of viral pathogens and the efficiency of the treatments applied.The 90% of the river water samples had positive results of HAdV (10¹-10⁴ genome copies (GC)/L); and 48%, of JCPyV (10⁰-10³ GC/L). Lower concentrations of HAdV and JCPyV were found in different treatment steps of the plants in absence of bacterial standards. Virus removal efficiencies were higher than 5 logs in plants 1 and 3 and could be quantified as >2 logs in plant 2. However, three post-chlorinated samples from plants 2 and 3 (11%) were found to be positive for HAdV by qPCR, but did not show infectivity in the cell cultures assayed. Simple methods based on the adsorption-elution of viruses from glass wool give low-cost and efficient virus recovery from source water and large-volume water samples. Quantification of JCPyV and HAdV using qPCR is useful for evaluating virus removal efficiency in water treatment plants, identification of Hazard Analysis and Critical Control Points (HACCP) and as a molecular index of the virological quality of water. Though infectivity is not guaranteed when using qPCR techniques in water treated with disinfection processes, the quality of source water, where viruses have proved to have infective capabilities, and the removal efficiency of viral particles may be efficiently quantified.     

Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.     

Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment

Fecal indicators such as Escherichia coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods. This critical uncertainty has furthered water pollution science toward new molecular approaches. Members of Bacteroides genus, such as Bacteroides thetaiotaomicron are found to have features that allow their use as alternative fecal indicators and for Microbial Source Tracking (MST). The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility. A large number of samples were collected and tested for E. coli and enterococci by both cultivation and qPCR assays. B. thetaiotaomicron qPCR equivalent cells (mean: 1.8 × 10⁷/100 mL) were present in significantly higher concentrations than E. coli or enterococci in raw sewage and at the same levels in raw septage. The removal of B. thetaiotaomicron target qPCR signals was similar to E. coli and enterococci DNA during the treatment of these wastes and ranged from 3 to 5 log₁₀ for wastewater and was 7 log₁₀ for the septage. A significant correlation was found between B. thetaiotaomicron marker and each of the conventional indicators throughout the waste treatment process for both raw sewage and septage. A greater variability was found with enterococci when compared to E. coli, and CFU and equivalent cells could be contrasted by various treatment processes to examine removal and inactivation via septage and wastewater treatment. These results are compared and contrasted with other qPCR studies and other targets in wastewater samples providing a view of DNA targets in such environments.     

Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and have been recently isolated from environmental water samples. Although enterophages have not been conclusively linked to human fecal pollution, we are currently characterizing enterophages to propose them as viral indicators and possible surrogates of enteric viruses in recreational waters. Little is known about the morphological or genetic diversity which will have an impact on their potential as markers of human fecal contamination. In the present study we are determining if enterophages can be grouped by their ability to replicate at different temperatures, and if different groups are present in the feces of different animals. As one of the main objectives is to determine if these phages can be used as indicators of the presence of enteric viruses, the survival rate under different conditions was also determined as was their prevalence in sewage and a large watershed. Coliphages were used as a means of comparison in the prevalence and survival studies. Results indicated that the isolates are mainly DNA viruses. Their morphology as well as their ability to form viral plaques at different temperatures indicates that several groups of enterophages are present in the environment. Coliphage and enterophage concentrations throughout the watershed were lower than those of thermotolerant coliforms and enterococci. Enterophage concentrations were lower than coliphages at all sampling points. Enterophages showed diverse inactivation rates and T₉₀ values across different incubation temperatures in both fresh and marine waters and sand. Further molecular characterization of enterophages may allow us to develop probes for the real-time detection of these alternative indicators of human fecal pollution.     

Occurrence of enteric viruses and faecal indicators in submarine groundwater discharges in the coastal environment of the Mexican Caribbean

The occurrence of faecal microorganisms such as total coliforms (TCs), Escherichia coli (E. coli), coliphages (somatic and F+ specific) and the genomes of faecal viruses: human adenovirus (HAdV), norovirus (NoV) genogroup I (GI) and II (GII), enterovirus (EV), aichi virus (AiV) and Pepper mild mottle virus (PMMoV), was explored in submarine groundwater discharges (SGDs) in the Mexican Caribbean. Ultra-filtrated water samples were subjected to nucleic acid extraction and real-time quantitative polymerase chain reaction (PCR). Results showed the presence of TC bacteria at all sites, whereas the detection of E. coli occurred only during the dry season. Coliphages occurred during the rainy and dry seasons, with concentrations ranging from 10 to 2560 plaque forming units (PFU/100 ml). HAdV and AiV were detected in 50% and 41% of the samples, at concentrations ranging from 10² to 10⁴ genome copies per litre (GC/L) and from 10² to 10⁶ GC/L, respectively. NoV GI and II were detected in 25% and 16% of the samples, at concentrations of 10¹ GC/L and 10²–10³ GC/L, respectively. PMMoV was detected in 50% of the samples at concentrations ranging from 10¹ to 10³ GC/L. The EV genomes were not detected. These findings demonstrate that faecal microorganisms can be transported through SGDs in the Mexican Caribbean, potentially contributing to human health risks for recreation.     

Assessment of human adenovirus removal in a full-scale membrane bioreactor treating municipal wastewater

Human adenoviruses (HAdVs) in wastewater samples taken from four different treatment stages of a full-scale municipal wastewater treatment plant (i.e., incoming raw sewage, primary sedimentation effluent, membrane bioreactor (MBR) influent, and MBR effluent) were quantified by real-time PCR assays to further estimate removal efficiency of the HAdVs. Based on hexon gene sequence comparisons, HAdV species A, C, and F were consistently found in the wastewater samples. In general, all three identified HAdV species were detected in most of the wastewater samples using the real-time PCR assays. Overall HAdV concentrations were rather stable over the entire 8-month study period (January-August, 2008) (approximately 10⁶-10⁷ viral particles/L of wastewater for the raw sewage and primary effluent; 10⁸-10⁹ viral particles/L for the MBR influent; and, 10³-10⁴ viral particles/L for the MBR effluent). No significant seasonal differences were noticed for the HAdV abundances. Removal efficiencies of the viral particles in the full-scale MBR process were assessed and showed an average HAdV removal of 5.0 ± 0.6 logs over the study period. The removal efficiencies for F species (average log removal of 6.5 ± 1.3 logs) were typically higher (p-value <0.05) than those of the other two species (average of 4.1 ± 0.9 and 4.6 ± 0.5 logs for species A and C, respectively). These results demonstrate that the full-scale MBR system efficiently removed most HAdV from the wastewater leaving about 10³ viral particles/L in the MBR effluent.     

Surveillance of adenoviruses and noroviruses in European recreational waters

Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.     

Illicit drugs, a novel group of environmental contaminants

It is now well established that residues from therapeutic drugs consumed by humans can end up, through the sewage system, in the surface water of populated areas. Given that the global production of major illicit drugs is comparable to that of widely used pharmaceuticals, we tested for the presence of drugs of abuse (cocaine, opioids, amphetamines and cannabis derivatives), some related opioid pharmaceuticals (codeine and methadone) and/or their metabolites in Italian and British surface waters. Having identified residues of all major drugs of abuse in raw and treated urban wastewater, we now measured their levels in several rivers and lakes by a selective multi-residue assay based on liquid chromatography-tandem mass spectrometry. Recoveries in surface water were generally higher than 80%, with overall variability of the method lower than 10%. LODs were generally lower than 0.2 ng/L, and LOQs were lower than 0.6 ng/L, with few exceptions. Many of the tested substances were found in both rivers and lakes, at concentrations ranging from high pg/L to high ng/L, with loads in rivers in the range of tenths to hundreds of grams per day. Our data indicate that residues of drugs of abuse have become widespread surface water contaminants in populated areas. Since most of these residues still have potent pharmacological activities, their presence in the aquatic environment may have potential implications for human health and wildlife.     

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic Escherichia coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.     

A simple method for the measurement of organic iodine in wastewater and surface water

Total adsorbable organic iodine (TAOI) has been used as a surrogate parameter for X-ray contrast media in municipal wastewater. Available methods require specialized equipment for pyrolyzing these compounds to convert the covalently bound iodine into iodide, which is then measured by ion chromatography. In this study we describe a simple method for liberating iodide from these compounds with Cu(II) and hydrogen peroxide. Concentrations of TAOI were measured in wastewater effluent, surface waters that were expected to be impacted by wastewater, and unimpacted surface waters. TAOI concentrations ranged between 1.9 and 16.3 microg/L I in wastewater with a median of 6.5 microg/L I. The lowest TAOI concentrations consistently were detected in a wastewater treatment plant that apparently had few hospitals within its collection area. In comparison, TAOI concentrations in surface water that was unimpacted by sewage were between 0.3 and 3.5 microg/L I. TAOI concentrations in surface waters that were impacted by sewage were consistent with the expected conservative behavior of TAOI.     

Occurrence and suitability of sucralose as an indicator compound of wastewater loading to surface waters in urbanized regions

Urban watersheds are susceptible to numerous pollutant sources and the identification of source-specific indicators can provide a beneficial tool in the identification and control of input loads, often times needed for a water body to achieve designated beneficial uses. Differentiation of wastewater flows from other urban wet weather flows is needed in order to more adequately address such environmental concerns as water body nutrient impairment and potable source water contamination. Anthropogenic compounds previously suggested as potential wastewater indicators include caffeine, carbamazepine, N,N-diethyl-meta-toluamide (DEET), gemfibrozil, primidone, sulfamethoxazole, and TCEP. This paper compares the suitability of a variety of anthropogenic compounds to sucralose, an artificial sweetener, as wastewater indicators by examining occurrence data for 85 trace organic compounds in samples of wastewater effluents, source waters with known wastewater point source inputs, and sources without known wastewater point source inputs. The findings statistically demonstrate the superior performance of sucralose as a potential indicator of domestic wastewater input in the U.S. While several compounds were detected in all of the wastewater effluent samples, only sucralose was consistently detected in the source waters with known wastewater discharges, absent in the sources without wastewater influence, and consistently present in septic samples. All of the other compounds were prone to either false negatives or false positives in the environment.     

Evaluating the importance of faecal sources in human-impacted waters

Quantitative microbial risk assessment (QMRA) was used to evaluate the relative contribution of faecal indicators and pathogens when a mixture of human sources impacts a recreational waterbody. The waterbody was assumed to be impacted with a mixture of secondary-treated disinfected municipal wastewater and untreated (or poorly treated) sewage, using Norovirus as the reference pathogen and enterococci as the reference faecal indicator. The contribution made by each source to the total waterbody volume, indicator density, pathogen density, and illness risk was estimated for a number of scenarios that accounted for pathogen and indicator inactivation based on the age of the effluent (source-to-receptor), possible sedimentation of microorganisms, and the addition of a non-pathogenic source of faecal indicators (such as old sediments or an animal population with low occurrence of human-infectious pathogens). The waterbody indicator density was held constant at 35 CFU 100 mL⁻¹ enterococci to compare results across scenarios. For the combinations evaluated, either the untreated sewage or the non-pathogenic source of faecal indicators dominated the recreational waterbody enterococci density assuming a culture method. In contrast, indicator density assayed by qPCR, pathogen density, and bather gastrointestinal illness risks were largely dominated by secondary disinfected municipal wastewater, with untreated sewage being increasingly less important as the faecal indicator load increased from a non-pathogenic source. The results support the use of a calibrated qPCR total enterococci indicator, compared to a culture-based assay, to index infectious human enteric viruses released in treated human wastewater, and illustrate that the source contributing the majority of risk in a mixture may be overlooked when only assessing faecal indicators by a culture-based method.     

Lack of correlation between enterococcal counts and the presence of human specific fecal markers in Mississippi creek and coastal waters

The objective of this study was to determine whether statistically valid correlations could be shown between enterococcal counts of samples from creek and coastal sites and the presence of two molecular, library-independent markers that specify human and/or sewage pollution. Four hundred ninety samples were collected between August 2007 and April 2009 to determine enterococcal counts and the presence of genetic markers for the sewage indicator organisms Methanobrevibacter smithii and Bacteroidales. The presence of human/sewage markers and enterococcal counts were higher in creek samples than coastal samples, but the higher creek levels did not statistically correlate with the either enterococcal count or the presence of the markers present in coastal samples. Furthermore, there was no correlation between enterococcal counts in coastal samples and either marker at any of the beach sites tested. The results of this investigation in Mississippi coastal waters suggest that human/sewage markers are unlikely to correlate with enterococci counts in the nearshore environment and that enterococcal counts may be indicative of other animal or environmental sources. Additionally, a study comparing conventional gel electrophoresis with capillary electrophoresis did not convincingly establish that one method was better than the other in regard to the results obtained. The capillary method does allow reproducibility of results and the ability to analyze multiple samples in a short period of time; however, the operational expenditures exceed the cost of traditional gel electrophoresis.