A perspective on the prevalence of DNA enteric virus genomes in anaerobic-digested biological wastes

The major goal of this study is to gain a perspective on the prevalence of DNA enteric virus genomes in mesophilic anaerobic-digested (MAD) sewage sludge and manure by comparing their quantitative PCR (qPCR) concentrations and removals with traditional fecal indicators (Escherichia coli, enterococci, and Bacteroidetes). In addition, relationships between qPCR and culture measurements of fecal indicators (FIs) were determined. There was no significant difference between the qPCR concentrations of human adenovirus and E. coli/enterococci in MAD sewage sludge; however, the qPCR concentrations of bovine adenovirus were significantly lower than FIs and bovine polyomavirus (BPyV) in MAD manure. The qPCR concentrations of human polyomavirus were slightly lower than E. coli and enterococci (p ≤ 0.05), but no significant difference was observed between the qPCR concentrations of BPyV and FIs. The digestion treatment achieved higher genome removal of bovine DNA enteric viruses than FIs (p ≤ 0.05). Significant correlations were observed between qPCR and culture measurements of FIs, but the concentrations and removals of FIs determined by qPCR assays were still significantly different than those determined by culture assays. Overall, we determined that the prevalence of DNA enteric virus genomes in MAD biological wastes was high due to their comparable in qPCR concentrations to FIs, indicating that mesophilic anaerobic digestion treatment alone may not be effective enough to remove DNA viral pathogens in biological wastes.     

Fecal indicator bacteria (FIB) levels during dry weather from Southern California reference streams

High levels of fecal indicator bacteria (FIB) in surface waters is a common problem in urban areas that often leads to impairment of beneficial uses such as swimming. Once impaired, common management and regulatory solutions include development of total maximum daily loads (TMDLs) and other water quality management plans. A critical element of these plans is establishment of a "reference" level of exceedances against which to assess management goals and TMDL compliance. The goal of this study was to provide information on indicator bacteria contributions from natural streams in undeveloped catchments throughout southern California during dry weather, non-storm conditions. To help establish a regional reference data set, bacteria levels [i.e. Escherichia coli (E. coli), enterococci and total coliforms] were measured from 15 unimpaired streams in 11 southern California watersheds weekly for one full year. Concentrations measured from reference areas were typically between one to two orders of magnitude lower than levels found in developed watersheds. Nearly 82% of the time, samples did not exceed daily and monthly bacterial indicator thresholds. E. coli had the lowest daily percent exceedance (1.5%). A total of 13.7% of enterococci exceeded daily thresholds. Indicator bacteria levels fluctuated seasonally with an average of 79% of both enterococci and total coliforms exceedances occurring during summer months (June to August). Temperature, at all sites, explained about one-half the variation in total coliforms density suggesting that stream temperatures regulated bacterial populations. Accounting for natural background levels will allow for management targets that are more reflective of the contributions from natural sources.     

Sources and fate of Salmonella and fecal indicator bacteria in an urban creek

This research aimed to understand the sources and fate of Salmonella and fecal bacteria in urban surface waters. An urban creek (San Pedro Creek, California, USA) that had unusually high levels of Salmonella and fecal bacteria relative to other nearby waterbodies was chosen as a model field site. State of the art microbiological methods were used in concert with modeling to investigate Salmonella and fecal bacteria sources, and determine field-relevant dark inactivation and photoinactivation rates. Three along-creek surveys that spanned reaches adjacent to both urban and forested land covers were conducted to measure Salmonella, enterococci, Escherichia coli, and horse- and human-specific Bacteroidales. Salmonella were detected adjacent to and downstream of urban land cover, but not adjacent to forested land cover. No human or horse-specific Bacteroidales fecal markers were detected implicating other urban animal sources of bacteria. Two locations along the creek where Salmonella was consistently detected were sampled hourly for 25 hours and a mass-balance model was applied to determine field-relevant light and dark inactivation rates for Salmonella, enterococci, and E. coli. Sunlight inactivation did not appear to be important in modulating concentrations of Salmonella, but was important in modulating both enterococci and E. coli concentrations. Dark inactivation was important for all three organisms. This is the first study to quantitatively examine the fate of Salmonella within an urban surface water. Although the work is carried out at a single site, the methodologies are extendable to source tracking in other waterbodies. Additionally, the rate constants determined through the modeling will be useful for modeling these organisms in other surface waters, and represent useful benchmarks for comparison to laboratory-derived inactivation rates.     

PCR技术对水中病原体的检测

水中有病毒、细菌、原生动物、蠕虫等多种病原体。常规的检测方法是用大肠菌作为病原体的指示生物,但是大肠菌的生存特性与病毒、原生动物差异较大,难以指示病毒、原生动物的存在与否。对每一种病原体单独进行检测,费时、费力。由于PCR技术具有快速、灵敏、特异的特点,使得它对于水中病原体的检测具有较高的应用价值。     

A Comparison of Bacterial Indicators and Methods in Rural Surface Waters

The United States Environmental Protection Agency (USEPA) recommends the use of Escherichia coli (E. coli) and enterococci as indicators of enteric pathogens in fresh waters; however, fecal coliform analyses will remain important by virtue of the large amount of historic data collected in prior years. In this study, we attempted, in a real-world situation (i.e., a rural inland watershed in the Piedmont of South Carolina) to compare different bacterial indicators and methods to one another. We compared fecal coliforms, enumerated by membrane filtration with E. coli, enumerated by a commercialized enzyme substrate method and observed E. coli/fecal coliform ratios of 1.63 and 1.2 for two separate tests. In the same watershed, we observed an E. coli/fecal coliform ratio of 0.84 when we used the commercialized enzyme substrate method for both enumerations. Given these results, users of such data should exercise care when they make comparisons between historic membrane filtration data and data acquired through the use of the more modern enzymatic methods. Some sampling and side-by-side testing between methods in a specific watershed may be prudent before any conversion factors between old and new datasets are applied.     

Two-year monitoring of Cryptosporidium parvum and Giardia lamblia occurrence in a recreational and drinking water reservoir using standard microscopic and molecular biology techniques

Starting in 2006, a monitoring of Giardia lamblia and Cryptosporidium parvum occurrence was conducted for 2 years in the largest drinking water reservoir of Luxembourg (Esch-sur-S?re reservoir) using microscopy and qPCR techniques. Parasite analyses were performed on water samples collected from three sites: site A located at the inlet of the reservoir, site B located 18 km downstream site A, at the inlet of the drinking water treatment plant near the dam of the reservoir and site C where the finished drinking water is injected in the distribution network. Results show that both parasites are present in the reservoir throughout the year with a higher occurrence of G. lamblia cysts compared to C. parvum oocysts. According to our results, only 25% of the samples positive by microscopy were confirmed by qPCR. (Oo)cyst concentrations were 10 to 100 times higher at site A compared to site B and they were positively correlated to the water turbidity and negatively correlated to the temperature. Highest (oo)cyst concentrations were observed in winter. In contrast, no relationship between the concentrations of (oo)cysts in the reservoir and rain events could be established. Though a correlation has been observed between both parasites and faecal indicators in the reservoir, some discrepancies highlight that the latter do not represent a reliable tool to predict the presence/absence of these pathogenic protozoa. In summer 2007, the maximal risk of parasite infection per exposure event for swimmers in the reservoir was estimated to be 0.0015% for C. parvum and 0.56% for G. lamblia. Finally, no (oo)cysts could be detected in large volumes of finished drinking water.     

水质标准中指示微生物的发展及现状

饮用水病原微生物污染是公共卫生面临的主要威胁之一,微生物监测在水质监测中的必要性日益受到人们的认可。在实际工作中,一般是通过检测指示微生物间接反映病原微生物的存在。通过调研国内外各组织、机构颁布的水质标准发现,近年来,我国环境质量标准和污染物排放标准中对于指示微生物的选择有从总大肠菌群向粪大肠菌群和大肠埃希氏菌(E.coli)转变的趋势,而美国环保署、欧盟、世界卫生组织、澳大利亚国家健康与医疗研究委员会等根据最新的流行病学证据,强调了大肠埃希氏菌(E.coli)、肠球菌(Enterococci)与粪便污染的相关性更强,可用于替代大肠菌群。建议我国在后续水质标准中对微生物指标进行增补修订时,参考国外经验,形成集成多种指示微生物与多种特定病原体的监测指标体系,以更好地保护环境功能和民众健康。     

Applicability of universal Bacteroidales genetic marker for microbial monitoring of drinking water sources in comparison to conventional indicators

Water quality monitoring is essential for the provision of safe drinking water. In this study, we compared a selection of fecal indicators with universal Bacteroidales genetic marker to identify fecal pollution of a variety of drinking water sources. A total of 60 samples were collected from water sources. The microbiological parameters included total coliforms, fecal coliforms, Escherichia coli and fecal streptococci as the fecal indicator bacteria (FIB), Clostridium perfringens and H₂S bacteria as alternative indicators, universal Bacteroidales genetic marker as a promising alternative fecal indicator, and Salmonella spp., Shigella spp., and E. coli O157 as pathogenic bacteria. From 60 samples analyzed, Bacteroidales was the most frequently detected indicator followed by total coliforms. However, the Bacteroidales assay failed to detect the marker in nine samples positive for FIB and other alternative indicators. The results of our study showed that the absence of Bacteroidales is not necessarily an evidence of fecal and pathogenic bacteria absence and may be unable to ensure the safety of the water. Further research, however, is required for a better understanding of the use of a Bacteroidales genetic marker as an indicator in water quality monitoring programs.     

Microfluidic quantification of multiple enteric and opportunistic bacterial pathogens in roof-harvested rainwater tank samples

Potable and non-potable uses of roof-harvested rainwater (RHRW) are increasing due to water shortages. To protect human health risks, it is important to identify and quantify disease-causing pathogens in RHRW so that appropriate treatment options can be implemented. We used a microfluidic quantitative PCR (MFQPCR) system for the quantitative detection of a wide array of fecal indicator bacteria (FIB) and pathogens in RHRW tank samples along with culturable FIB and conventional qPCR analysis of selected pathogens. Among the nine pathogenic bacteria and their associated genes tested with the MFQPCR, 4.86 and 2.77% samples were positive for Legionella pneumophila and Shigella spp., respectively. The remaining seven pathogens were absent. MFQPCR and conventional qPCR results showed good agreement. Therefore, direct pathogen quantification by MFQPCR systems may be advantageous for circumstances where a thorough microbial analysis is required to assess the public health risks from multiple pathogens that occur simultaneously in the target water source.     

Simultaneous detection of enteric bacteria from surface waters by QPCR in comparison with conventional bacterial indicators

A rapid quantitative polymerase chain reaction (QPCR) method was developed for simultaneous detection of enteric bacteria from surface waters by utilizing a pair of universal primers which targeted four bacteria strains, namely Shigella dysenteriae, Vibrio cholerae, Salmonella typhimurium, and Escherichia coli. It was estimated that the QPCR method had a 94% confidence, and a detection limit as 2.7 E. coli cells per sample in undiluted DNA extracts. The QPCR method was applied for the bacteriological examination of several surface waters in the urban area of Xi'an, China and comparison was made with the conventional bacteria indicators determined by conventional membrane filter (MF) method. As a result, the calibrator cell equivalents (CCE) determined by QPCR was 2.2 to five times of the total coliform CFU, and the characteristics of the bacterial quality of different waters could be well presented by the QPCR results with a higher sensitivity. The coefficient of variation (CV) of data obtained by QPCR was smaller than that by traditional MF method, indicating a more stable analysis result. The QPCR method could thus be used as a supplement of the conventional culture method for more sensitive detection of pathogenic enteric bacteria from water.     

Origin and spatial–temporal distribution of faecal bacteria in a bay of Lake Geneva, Switzerland

The origin and distribution of microbial contamination in Lake Geneva's most polluted bay were assessed using faecal indicator bacteria (FIB). The lake is used as drinking water, for recreation and fishing. During 1 year, water samples were taken at 23 points in the bay and three contamination sources: a wastewater treatment plant (WWTP), a river and a storm water outlet. Analyses included Escherichia coli, enterococci (ENT), total coliforms (TC), and heterotrophic plate counts (HPC). E. coli input flux rates from the WWTP can reach 2.5 x 10(10) CFU/s; those from the river are one to three orders of magnitude lower. Different pathogenic Salmonella serotypes were identified in water from these sources. FIB levels in the bay are highly variable. Results demonstrate that (1) the WWTP outlet at 30 m depth impacts near-surface water quality during holomixis in winter; (2) when the lake is stratified, the effluent water is generally trapped below the thermocline; (3) during major floods, upwelling across the thermocline may occur; (4) the river permanently contributes to contamination, mainly near the river mouth and during floods, when the storm water outlet contributes additionally; (5) the lowest FIB levels in the near-surface water occur during low-flow periods in the bathing season.     

An assessment of fecal indicator and other bacteria from an urbanized coastal lagoon in the City of Los Angeles, California, USA

A study was performed in Del Rey Lagoon, City of Los Angeles, to determine if the lagoon was as a source or sink for fecal indicator bacteria (FIB: total coliforms, Escherichia coli, enterococci) and to screen for the presence of other potentially pathogenic bacteria. The lagoon receives tidal flows from the adjacent Ballona Estuary whose water usually is contaminated with FIB originating from the highly urbanized Ballona Creek Watershed. During 16 sampling events from February 2008 through March 2009, replicate water samples (n?=?3) were collected 1 h prior to the high tide and 1 h prior to the following low tide. FIB concentrations were measured by the defined substrate method (IDEXX, Westbrook, Me) followed by culturing of bacterial isolates sampled from positive IDEXX Quanti-Tray wells and were identified using the Vitek 2 Compact (bioMérieux, Durham, NC). Mean concentrations of FIB often differed by an order of magnitude from flood to ebb flow conditions. The lagoon tended to act as a sink for total coliforms based on the ratio of mean flood to ebb densities (R _F/E) >1.0 during 56 % of the sampling events and during ebb flows, as a source for E. coli and enterococci (R _F/E <1.69 % of events). Approximately 54 species were identified from 277 isolates cultured from the IDEXX Quanti-Trays. Of these, 54 % were species known to include pathogenic strains that can be naturally occurring, introduced in runoff, or originated from other sources. Diversity and cluster analyses indicated a dynamic assemblage that changes in species composition with day-to-day fluctuations as well as tidal action. The concept of monitoring the lagoon and estuary as a sentinel habitat for pathogenic assemblages is discussed.     

E. coli kinetics--effect of temperature on the maintenance and respectively the decay phase

The knowledge of enteric bacteria survival kinetic is very important for environmental scientists. Enteric bacteria andspecifically the fecal indicator bacteria are typically used to measure the sanitary quality of water for recreational, industrial, agricultural and water supply purposes. They are released into the environment with feces, and are then exposedto a variety of environmental conditions that eventually causetheir death. In general, it is believed that the fecal indicatorcannot grow in natural environments, since they are adapted to live in the gastrointestinal tract. Studies have shown that fecalindicator bacteria survive from a few hours up to several daysin surface water, but may survive for days or months in lake-sediments, where they may be protected from sunlight andpredators. We assume that pathogens similar to the fecal indicator bacteria die at the same rate as fecal indicator bacteria. Therefore, if we find relatively high numbers of fecalindicator bacteria in an environment, we assume that there is anincreased likelihood of pathogens being present as well. The kinetic of enteric bacteria survival in natural waters is affected by a large number of factors. One of them is the temperature. The aim of this contribution was the experimentalresearch of the survival kinetic of enteric bacteria applying a simple mathematical formula, which describes the survival kineticpredicting the decay phase at various temperatures. We aspire that the results will lead both to the solution of many engineering problems and to future research.     

Characterisation of Indicator Organisms and Pathogens in Domestic Greywater for Recycling

Greywater from baths, showers and washbasins was collected separately from all other domestic wastewater at a university block of flats with a dual reticulation system and analysed for a range of contaminants including indicator organisms and pathogens. Greywater flow and temperature were also monitored and a diurnal variation was observed. Physical and chemical water quality parameters were similar to previously published data, although measured COD and BOD levels appeared to be lower, possibly due to settlement or biodegradation in the storage tanks. Plate counts and indicator organism concentrations were consistently high suggesting a high level of human bacterial contamination necessitating biological treatment and disinfection if the water is to be used for recycling. However, these high levels of indicator organisms did not correlate to pathogen presence and should not be used as pathogen indicators in greywater. One positive count of Salmonella veltereden was observed as well as low levels of Giardia. Cryptosporidium, Escherichia coli O157:H7, enteroviruses and Legionella were not identified in any of the samples. The research also highlighted a number of problems with the complexity of this type of sampling programme, such as identifying the most likely time to isolate pathogens and analysing an ‘unusual’ water source.     

Molecular approaches to microbiological monitoring: fecal source detection

Molecular methods are useful both to monitor natural communities of bacteria, and to track specific bacterial markers in complex environments. Length-heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNAs discriminate among 16S rRNA genes based on length polymorphisms of their PCR products. With these methods, we developed an alternative indicator that distinguishes the source of fecal pollution in water. We amplify 16S rRNA gene fragments from the fecal anaerobic genus Bacteroides with specific primers. Because Bacteroides normally resides in gut habitats, its presence in water indicates fecal pollution. Molecular detection circumvents the complexities of growing anaerobic bacteria. We identified Bacteroides LH-PCR and T-RFLP ribosomal DNA markers unique to either ruminant or human feces. The same unique fecal markers were recovered from polluted natural waters. We cloned and sequenced the unique markers; marker sequences were used to design specific PCR primers that reliably distinguish human from ruminant sources of fecal contamination. Primers for more species are under development. This approach is more sensitive than fecal coliform assays, is comparable in complexity to standard food safety and public health diagnostic tests, and lends itself to automation and high-throughput. Thus molecular genetic markers for fecal anaerobic bacteria hold promise for monitoring bacterial pollution and water quality.     

Changes in the sorption and rate of 17β-estradiol biodegradation by dissolved organic matter collected from different water sources

The potential biodegradation and subsequent transformation of 17β-estradiol (E2) to estrone (E1) were examined in the presence of various dissolved organic matter (DOM) isolated from effluent, river and lake waters. In addition, estrogenicity was estimated in association with the removal of E2 via its sorption onto DOM and biodegradation. The more biodegradable lake-derived DOM promoted more extensive transformation of E2 into E1 than the effluent organic matter through a biodegradation process. Overall, under all conditions, biodegradation dominated the removal of E2 in water. The increased dissolved organic carbon (DOC) concentrations in river and lake-derived DOM (e.g. 6.5 mg C L(-1)) reduced the removal of E2 by decreasing its biodegradation due to the moderate sorption of E2 onto DOM. The effluent organic matter showed greater removal of E2 via biodegradation, as well as significantly high sorption. This was associated with a large amount of hydrophobic fulvic acid (FA)- and humic acid (HA)-like organic components, as shown by the small increase in the specific UV absorbance at 254 nm (SUVA(254)). An increase in the DOC concentration reduced the removal of E2, resulting in high estrogenicity. The present study suggests that both organic composition and DOC concentration influenced the removal of E2 and, therefore, should be fully considered when assessing estrogenicity and its impacts on the aquatic environment.     

Improving water quality through California’s Clean Beach Initiative: an assessment of 17 projects

California’s Clean Beach Initiative (CBI) funds projects to reduce loads of fecal indicator bacteria (FIB) impacting beaches, thus providing an opportunity to judge the effectiveness of various CBI water pollution control strategies. Seventeen initial projects were selected for assessment to determine their effectiveness on reducing FIB in the receiving waters along beaches nearest to the projects. Control strategies included low-flow diversions, sterilization facilities, sewer improvements, pier best management practices (BMPs), vegetative swales, and enclosed beach BMPs. Assessments were based on statistical changes in pre- and postproject mean densities of FIB at shoreline monitoring stations targeted by the projects. Most low-flow diversions and the wetland swale project were effective in removing all contaminated runoff from beaches. UV sterilization was effective when coupled with pretreatment filtration and where effluent was released within a few hundred meters of the beach to avoid FIB regrowth. Other BMPs were less effective because they treated only a portion of contaminant sources impacting their target beach. These findings should be useful to other coastal states and agencies faced with similar pollution control problems.     

Comparison of beach bacterial water quality indicator measurement methods

Three methods (membrane filtration, multiple tube fermentation, and chromogenic substrate technology kits manufactured by IDEXX Laboratories, Inc.) are routinely used to measure indicator bacteria for beach water quality. To assess comparability of these methods, quantify within-laboratory variability for each method, and place that variability into context of variability among laboratories using the same method, 22 southern California laboratories participated in a series of intercalibration exercises. Each laboratory processed three to five replicates from thirteen samples, with total coliforms, fecal coliforms or enterococci measured depending on the sample. Results were generally comparable among methods, though membrane filtration appeared to underestimate the other two methods for fecal coliforms, possibly due to clumping. Variability was greatest for the multiple tube fermentation method. For all three methods, within laboratory variability was greater than among laboratories variability.     

Multi-scale landscape factors influencing stream water quality in the state of Oregon

Enterococci bacteria are used to indicate the presence of human and/or animal fecal materials in surface water. In addition to human influences on the quality of surface water, a cattle grazing is a widespread and persistent ecological stressor in the Western United States. Cattle may affect surface water quality directly by depositing nutrients and bacteria, and indirectly by damaging stream banks or removing vegetation cover, which may lead to increased sediment loads. This study used the State of Oregon surface water data to determine the likelihood of animal pathogen presence using enterococci and analyzed the spatial distribution and relationship of biotic (enterococci) and abiotic (nitrogen and phosphorous) surface water constituents to landscape metrics and others (e.g. human use, percent riparian cover, natural covers, grazing, etc.). We used a grazing potential index (GPI) based on proximity to water, land ownership and forage availability. Mean and variability of GPI, forage availability, stream density and length, and landscape metrics were related to enterococci and many forms of nitrogen and phosphorous in standard and logistic regression models. The GPI did not have a significant role in the models, but forage related variables had significant contribution. Urban land use within stream reach was the main driving factor when exceeding the threshold (> or =35 cfu/100 ml), agriculture was the driving force in elevating enterococci in sites where enterococci concentration was <35 cfu/100 ml. Landscape metrics related to amount of agriculture, wetlands and urban all contributed to increasing nutrients in surface water but at different scales. The probability of having sites with concentrations of enterococci above the threshold was much lower in areas of natural land cover and much higher in areas with higher urban land use within 60 m of stream. A 1% increase in natural land cover was associated with a 12% decrease in the predicted odds of having a site exceeding the threshold. Opposite to natural land cover, a one unit change in each of manmade barren and urban land use led to an increase of the likelihood of exceeding the threshold by 73%, and 11%, respectively. Change in urban land use had a higher influence on the likelihood of a site exceeding the threshold than that of natural land cover.     

Monitoring E. coli and total coliforms in natural spring water as related to recreational mountain areas

Natural spring water has unique properties, as it is rich in minerals that are considered to be beneficial to human health. A survey of the microbiological quality of natural spring water was conducted to assess possible risks from the consumption of the water by visitors in recreational mountain areas located in Seoul, South Korea. The densities of total coliforms and Escherichia coli were measured during the spring and the summer of 2002 to investigate the presence of coliform bacteria in the drinking spring waters. Total coliforms were detected in all samples and the mean density of total coliforms was up to a maximum of 228 CFU/mL. Detectable E. coli was found in 78% of all samples and the mean densities of E. coli varied from a minimum of 0 CFU/mL to a maximum of 15 CFU/mL in all samples. Malfunctioning septic systems and wildlife population appear to be the main source of E. coli contamination. Presence of E. coli in natural spring water indicates potential adverse health effects for individuals or populations exposed to this water. The fecal contaminated spring water may present an unacceptable risk to humans if it is used as raw drinking water.