Assessment of sources of human pathogens and fecal contamination in a Florida freshwater lake

We investigated the potential for a variety of environmental reservoirs to harbor or contribute fecal indicator bacteria (FIB), DNA markers of human fecal contamination, and human pathogens to a freshwater lake. We hypothesized that submerged aquatic vegetation (SAV), sediments, and stormwater act as reservoirs and/or provide inputs of FIB and human pathogens to this inland water. Analysis included microbial source tracking (MST) markers of sewage contamination (Enterococcus faecium esp gene, human-associated Bacteroides HF183, and human polyomaviruses), pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses), and FIB (fecal coliforms, Escherichia coli, and enterococci). Bayesian analysis was used to assess relationships among microbial and physicochemical variables. FIB in the water were correlated with concentrations in SAV and sediment. Furthermore, the correlation of antecedent rainfall and major rain events with FIB concentrations and detection of human markers and pathogens points toward multiple reservoirs for microbial contaminants in this system. Although pathogens and human-source markers were detected in 55% and 21% of samples, respectively, markers rarely coincided with pathogen detection. Bayesian analysis revealed that low concentrations (<45 CFU × 100 ml⁻¹) of fecal coliforms were associated with 93% probability that pathogens would not be detected; furthermore the Bayes net model showed associations between elevated temperature and rainfall with fecal coliform and enterococci concentrations, but not E. coli. These data indicate that many under-studied matrices (e.g. SAV, sediment, stormwater) are important reservoirs for FIB and potentially human pathogens and demonstrate the usefulness of Bayes net analysis for water quality assessment.     

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were collected during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples collected during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both Campylobacter jejuni mapA and Salmonella invA genes detected in 10% samples collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 10² and 4.3 × 10² genomic copies per 500 ml of water Giardia lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.     

Prevalence of human pathogens and indicators in stormwater runoff in Brisbane,Australia

Elevated numbers of enteric pathogens in the receiving waters following a storm event can be a serious public health concern. The purpose of this study was to conduct a preliminary investigation into the presence of human pathogens of concern in urban stormwater runoff. The involvement of a human sewage as a potential source of contamination was also investigated by using microbial source tracking methods. Water samples (20 L) were collected after storm events and during the dry weather from six sites in Brisbane, Australia. Collected samples were analyzed for fecal indicator bacteria (FIB), and then concentrated using hollow fiber ultrafiltration followed by molecular detection of selected enteric pathogens. The levels of FIB were found to frequently exceed the upper limit of Australian guidelines for managing risks in recreational water, during the dry periods and by further several orders of magnitude in the stormwater runoff. Enterococcus spp. numbers as high as 3 × 10⁴ 100 mL^?1 were detected in the stormwater runoff at the Fitzgibbon site. Human adenovirus and polyomavirus were frequently detected from all six sampling sites during wet and dry weather conditions suggesting their wide spread presence in the urban aquatic environments. Campylobacter jejuni, Campylobacter coli and Salmonella enterica were also detected during both dry and wet weather conditions. Presence of human-specific HF183 Bacteroides marker in most of the samples tested suggests ubiquitous sewage contamination in the urban environment. Since stormwater runoff routinely contains high numbers of FIB and other enteric pathogens, some degree of treatment of captured stormwater would be required if it were to be used for non-potable purposes.     

Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays

Microbial source tracking to distinguish between human, livestock and wildlife fecal pollution using molecular techniques is a rapidly evolving approach in many developed countries, but has not previously been applied on the African continent. DNA extracts from cow, donkey, and human fecal specimens and raw domestic sewage samples collected in Kenya were tested against five existing quantitative PCR assays designed to detect universal (2), human-specific (2), and cow-specific (1) fecal Bacteroidales genetic markers. Water samples from the River Njoro in Kenya were evaluated using the five tested Bacteroidales markers and a multi-species assay for Cryptosporidium in a preliminary exploration of fecal pollution sources and health risks in this watershed. Diagnostic sensitivity on the validation set varied from 18 to 100% for the five assays while diagnostic specificity was 100%. Of the 2 universal assays, Total Bacteroidales [Dick, L.K, Field, K.G., 2004. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl. Environ. Microbiol. 70, 5695-5697] showed lower generic fecal diagnostic sensitivity, at 55%, than BacUni-UCD, at 100%, in detecting fecal markers on the 42-sample validation set. Human-specific assay HF183 demonstrated 65% sensitivity overall, and 80% on the human sewage samples, compared to 18% overall and 0% sewage for human-specific assay BacHum-UCD. Cow-specific assay BacCow-UCD had 94% sensitivity. Testing of 18 water samples indicates cows are a likely predominant source of fecal contamination in the Njoro Watershed (78% prevailing rate). Probabilistic assessment of human assay results indicates at most three of the river water samples contained human Bacteroidales. Cryptosporidium spp. markers were detected in samples from nine of the 12 sampling locations. Evidence suggesting widespread contamination by cow feces and Cryptosporidium in the Njoro watershed raises serious concerns for human and animal health.     

Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces

We measured the concentrations of four host-specific (human, dog, cow, and horse Bacteroidales), four generic fecal (16S total Bacteroidales and Escherichia coli, 23S Enterococcus and uidA E. coli,) and two universal bacterial (16S universal and rpoB universal) DNA targets by qPCR in raw sewage and pooled fecal samples from dogs, cows, horses, and Canada Geese. A spiking protocol using the non-fecal bacterium Pseudomonas syringae pph6 was developed to estimate the recovery of DNA from fecal and environmental samples. The measured fecal marker concentrations were used to calculate baseline ratios and variability of host-specific to generic indicators for each host type. The host-specific markers were found in high concentrations (8-9 log₁₀ copies/g dry wt.) in their respective hosts' samples, which were equal to or greater than the concentrations of generic E. coli and Enterococcus markers, lending support to the use of host-specific and generic Bacteroidales as sensitive indicators of fecal pollution. The host-specific markers formed a consistent percentage of total Bacteroidales in target host feces and raw sewage, with human-specific comprising 82%, dog-specific 6%, cow-specific 4% and horse-specific 2%. Based on this limited data set, the measurement of host-specific indicators by qPCR has several promising applications. These applications include determining the percentage of total Bacteroidales contributed by a specific host type, using the ratios of host-specific markers to E. coli or Enterococcus to estimate the contribution of each source to these regulated fecal indicator bacteria, and estimating the mass of feces from each host type in environmental samples.     

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker

Each year the National Resource Defense Council addresses the quality of US beaches by routine bacterial indicators. In the Great Lakes region the indicator used is Escherichia coli and for 2007 more beaches were closed and impacted than ever before. In this study, water quality was addressed at two Lake Michigan Beaches over the 2004 swimming season by monitoring infectious enteric viruses by cell culture and integrated PCR and for a human sewage marker based on the Enterococcal Surface Protein (esp). Our goals for this study were to 1) examine the occurrence and variety of human enteric viruses present during peak usage of the beaches 2) determine key variables for development of predictive models for viruses; and 3) use quantitative risk assessment to estimate the potential health impact. Our results demonstrate that for both beaches predictive models of virus pollution were best described utilizing physical parameters like wind speed, wind direction and water temperature. The esp marker was not predictive of human viruses. The daily risk of acquiring a viral infection at either of the beaches ranged from 0.2 to 2.4/1000 swimmers using a quantitative microbial risk assessment model, with three swims during a day at the beach for children and over the season, the risk was 9-15/1000 swimmers using adenovirus as the model.Conclusions: Lake Michigan recreational beaches are being adversely impacted by human fecal pollution. Monitoring for the traditional indicators of water quality does not address viral risks and models can be developed and potentially used as real-time water quality forecasting tools.     

Evaluation of multiple sewage-associated Bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for the detection of sewage pollution in environmental waters in Australia. The HuBac (i.e., 63%) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

Specificity and sensitivity evaluation of novel and existing Bacteroidales and Bifidobacteria-specific PCR assays on feces and sewage samples and their application for microbial source tracking in Ireland

Three novel ruminant-specific PCR assays, an existing ruminant-specific PCR assay and five existing human-specific PCR assays, which target 16S rDNA from Bacteroidales or Bifidobacteria, were evaluated. The assays were tested on DNA extracted from ruminant (n = 74), human (n = 59) and non-ruminant animal (n = 44) sewage/fecal samples collected in Ireland. The three novel PCR assays compared favourably to the existing ruminant-specific assay, exhibiting sensitivities of 91-100% and specificities of 95-100% as compared to a sensitivity of 95% and specificity of 94%, for the existing ruminant-specific assay. Of the five human-specific PCR assays, the assay targeting the Bifidobacterium catenulatum group was the most promising, exhibiting a sensitivity of 100% (with human sewage samples) and a specificity of 87%. When tested on rural water samples that were naturally contaminated by ruminant feces, the three novel PCR assays tested positive with a much greater percentage (52-87%) of samples than the existing ruminant-specific assay (17%). These novel ruminant-specific assays show promise for microbial source tracking and merit further field testing and specificity evaluation.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.     

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.     

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was ≥94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.     

Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: Implications for their use in assessing risk in recreational waters

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10 d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.     

Distribution of sewage input in marine sediments around a maritime Antarctic research station indicated by molecular geochemical indicators

Sediments from Admiralty Bay, Antarctica were collected during the austral summers of 2002/2003 and 2003/2004 in order to assess the distribution and concentration of sewage indicators originating from Comandante Ferraz Brazilian Antarctic Station. Fecal sterols (coprostanol + epicoprostanol) and linear alkylbenzenes (LABs) ranged from < 0.01 to 0.95 μg g⁻¹ and < 1.0 to 23 ng g⁻¹ dry weight, respectively. In general, the higher concentrations were found only locally in the vicinity of Ferraz station at Martel Inlet. Baseline values for fecal sterols and coprostanone were calculated as 0.19 and 0.40 μg g⁻¹, respectively. According to fecal sterols concentrations, sewage contribution to Martel Inlet has increased more than twice since 1997, as result of the increase in the number of researchers at the station especially during the last decade. A low correlation was found between total LABs and fecal steroids, which could be attributed to the contribution of the natural sources of steroids.     

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.     

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.     

UV inactivation and resistance of rotavirus evaluated by integrated cell culture and real-time RT-PCR assay

Rotaviruses are double-stranded RNA viruses which are among the most resistant waterborne enteric viruses to UV disinfection. An integrated cell culture and real-time RT-PCR (ICC real-time RT-PCR) assay was developed to detect the infectivity of rotaviruses in water, which uses real-time RT-PCR to detect RNA produced by infectious rotaviruses during replication in host cells. Detection of rotaviral RNA in host cells provides direct evidence of the presence of infectious rotavirus rather than just the presence of rotavirus RNA. Using this newly developed method, the inactivation and resistance of rotavirus to UV treatments at various doses was evaluated. With an initial concentration of 2 × 10⁴ PFU/ml simian rotavirus (SA11), a first-order linear relationship was obtained at UV dose range of 0-120 mJ cm⁻², and the inactivation rate constant was estimated to be 0.0343 cm² mJ⁻¹ (R² = 0.966). The dose-inactivation curve tailed off and reached plateau as the UV dose increased from 120 to 360 mJ cm⁻², indicating resistance phenomena of sub-populations of SA11 at very high UV doses. A maximal reduction of 4.8 log₁₀ was observed. Through parallel comparison with traditional culture assay, the ICC real-time RT-PCR method demonstrated more effective, sensitive and faster infectivity detection of rotavirus and, the results reveal that rotaviruses are more resistant to UV irradiation than previously reported with traditional cell culture assays.     

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.     

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory,comparative study

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10–50 gene copies or plaques × 50 ml⁻¹) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40–70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.     

Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6-25 h assay period with detection and determination limits and EC₅₀ values for 17β-estradiol of 2.8 ng L^− 1, 5.9 ng L^− 1, 33.2 ng L^− 1 (nAES-P) and 3.1 ng L^− 1, 6.7 ng L^− 1 and 39.4 ng L^− 1 (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.