Determining Sources of Fecal Bacteria in Waterways

The microbiological contamination of waterways by pathogenic microbes has been, and is still, a persistent public safety concern in the United States and in most countries of the world. As most enteric pathogens are transmitted through the fecal–oral route, fecal pollution is generally regarded as the major contributor of pathogens to waterways. Fecal pollution of waterways can originate from wastewater treatment facilities, septic tanks, domestic- and wild-animal feces, and pets. Because enteric pathogens are derived from human or animal sources, techniques capable of identifying and apportioning fecal sources have been intensively investigated for use in remediation efforts and to satisfy regulatory concerns. Pollution of human origin is of the most concern, since human feces is more likely to contain human-specific enteric pathogens. Fecal indicator bacteria have been used successfully as the primary tool for microbiologically based risk assessment. However measurement of fecal indicator bacteria does not define what pathogens are present, or define the sources of these bacteria. Microbial source tracking (MST) methods that have the ability to differentiate among sources of fecal pollution are currently under development. These methods will ultimately be useful for risk assessment purposes and to aid regulatory agencies in developing strategies to remediate microbiologically impaired waterways.     

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.     

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.     

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.     

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.     

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。     

Ultracentrifugation as a direct method to concentrate viruses in environmental waters: virus-like particle enumeration as a new approach to determine the efficiency of recovery

Some health important enteric viruses are considered to be emerging waterborne pathogens and so the improvement of detection of these viruses in the aquatic environment is one of the most important steps in dealing with these pathogens. Since these viruses may be present in low numbers in water, it is necessary to concentrate water samples before viral detection. Although there are several methods to concentrate viruses in environmental waters, all present some drawbacks and consequently the method should be chosen that, despite its limitations, is adequate to achieve the aim of each study. As the effectiveness of the concentration methods is evaluated by determining the efficiency of viral recovery after concentration, it is important to use a simple and effective approach to evaluate their recovery efficiency. In this work ultracentrifugation, usually used as a secondary step for virus concentration, was evaluated as the main method to concentrate directly viruses in environmental water samples, using the microscopic enumeration of virus-like particles (VLP) as a new approach to estimate the efficiency of recovery. As the flocculation method is currently employed to concentrate viruses in environmental waters, it was also used in this study to assess the efficiency of the ultracentrifugation as the main viral concentration method in environmental waters. The results of this study indicate that ultracentrifugation is an adequate approach to concentrate viruses directly from environmental waters (recovery percentages between 66 and 72% in wastewaters and between 66 and 76% in recreational waters) and that the determination of VLP by epifluorescence microscopy is a simple, fast and cheap alternative approach to determine the recovery efficiency of the viral concentration methods.     

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.     

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。     

Geostatistical Uncertainty Modelling for the Environmental Hazard Assessment During Single Erosive Rainstorm Events

This paper presents an environmental hazard assessment to account the impacts of single rainstorm variability on river-torrential landscape identified as potentially vulnerable mainly to erosional soil degradation processes. An algorithm for the characterisation of this impact, called Erosive Hazard Index (EHI), is developed with a less expensive methodology. In EHI modelling, we assume that the river-torrential system has adapted to the natural hydrological regime, and a sudden fluctuation in this regime, especially those exceeding thresholds for an acceptable range of flexibility, may have disastrous consequences for the mountain environment. The hazard analysis links key rainstorm energy variables expressed as a single-storm erosion index (EI_sto), with impact thresholds identified using an intensity pattern model. Afterwards, the conditional probabilities of exceeding these thresholds are spatially assessed using non-parametric geostatistical techinques, known as indicator kriging. The approach was applied to a test site in river-torrential landscape of the Southern Italy (Benevento province) for 13 November 1997 rainstorm event.     

A comparison of rapid and conventional measures of indicator bacteria as predictors of waterborne protozoan pathogen presence and density

E. coli and enterococci in recreational waters are monitored as indicators of fecal contamination, pathogen presence, and health risk. Quantitative polymerase chain reaction (qPCR) tests for fecal indicator bacteria can provide beach managers with same-day information about water quality, unlike culture methods which provide that information the following day. The abilities of qPCR measurements of indicator bacteria, as compared to culture measurements of indicator bacteria, as predictors of pathogen presence or density in surface waters are not well understood. The purpose of this study was to make such comparisons between water samples collected from Chicago area surface waters, including rivers, inland lakes, Lake Michigan, and the Chicago Area Waterways System, which is dominated by wastewater effluent. A total of 294 twenty-litre samples were collected and analyzed for Giardia and Cryptosporidium. qPCR and membrane filtration methods were used to quantify E. coli and enterococci. Correlation, logistic regression, and zero-inflated Poisson modeling were utilized to evaluate associations between indicators and parasites. qPCR and culture measures of the indicator bacteria were similar in their ability to predict parasite presence and density. Correlations between parasites and indicators were generally stronger at waters not dominated by effluent. Associations between indicator density and Giarida presence were observed more consistently than between indicator density and Cryptosporidium presence. Associations between enterococci and parasites were generally stronger than associations between E. coli and parasites. The use of qPCR monitoring in our setting would generate more timely results without compromising the ability to predict parasite presence or density.     

粪大肠菌群酶底物法在环境监测中的应用研究

粪大肠菌群作为水体粪便污染的指示菌,对地表水的水质监测评价具有重要作用。采取较为精确、快速的粪大肠菌群检测方法,对控制流行疾病的发生和传播有重要的科学意义。从粪大肠菌群的检测意义、监测方法、标准化应用情况等方面进行归纳分析,提出推广和标准化酶底物法监测粪大肠菌群的迫切性。     

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.     

Impact of changed trophic status on the zooplankton composition in six water bodies of Dharwad district, Karnataka state (South India)

Morgan Island, located within the ACE Basin National Estuarine Research Reserve in South Carolina, is home to the only free-ranging colony of rhesus monkeys (Macca mulatta) in the continental United States. The purpose of this study was to assess environmental impacts of the monkey colony on water quality in adjacent tidal creeks and on island vegetation. Three tidal creeks were sampled: Morgan Creek, adjacent to the monkey colony; Back Creek, on Morgan Island not adjacent to the colony; and Rock Creek, on a nearby island unoccupied by monkeys. Temperature, salinity, pH, dissolved oxygen, nutrients and fecal coliform bacteria were measured six times at three sites in each of these creeks, and vegetation change analysis was conducted in a geographic information system using satellite imagery. Results showed elevated fecal coliform concentrations in the Morgan Creek site immediately adjacent to the colony, though no samples exceeded the standard set for recreational water use. Ribotyping reconnaissance matched four Escherichia coli isolates from Morgan and Back Creeks to the monkeys, identifying the colony as one source of fecal coliform bacteria, though relative source loadings could not be quantified. Significant differences were not observed between ammonia or orthophosphate levels in Morgan Creek relative to the other creeks tested; and vegetation change analysis showed a 35% increase in canopy cover between 1979 and 1999. Overall, these results suggest that the rhesus colony’s environmental impacts are localized and minimal. Results from this study provide baseline data on Morgan Island and may be useful in management decisions regarding the future of the monkey colony.     

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.     

粪大肠菌群酶底物法在环境应急监测中的应用

结合国外粪大肠菌群的酶底物检测方法,针对某次突发环境污染事件,用酶底物法和标准方法多管发酵法同步检测受污染地表水中的粪大肠菌群,讨论酶底物法在应急监测中检测粪大肠菌群的适用性。结果表明,两种方法的测定数据显著相关,没有统计学差异（ P＞0．05）。相对于多管发酵法,酶底物法特异性强,检测时间短,二次污染少,符合应急监测的要求。     

基于生物导向的水生态健康监测及评估概述

环境监测是水生态健康监测与评估的重要环节,基于物理、化学监测的传统水质监测通常仅能提供独立的数据信息,不能全面、直观地反映水环境状况。基于生物等生命体导向的水生态监测通过生物对环境的响应,能够直接反应复杂水体状况,在水环境健康监测与评估中占据重要地位。基于病原微生物、指示生物介绍了生物监测中的常规生物指标,总结了包括藻类、无脊椎动物和鱼类在内的常见指示生物在不同类型污染水体中的环境指示作用。从生物毒性效应出发介绍了常用的毒性效应测试方法、分析了污染物在不同生物学水平的响应,从而指明生物毒性效应在水环境健康评估中的发展优势。再从生态完整性角度阐述了生态完整性评价的一般方法和新兴分子生物学技术在水生态健康评估中的应用。重点指出环境毒理学和分子生物学在水环境监测的优势,以期为更加科学精确地进行水生态健康监测预警提供支撑。     

水环境中致病菌分子生物学检测技术研究进展

总结了水环境中主要致病菌及其导致的疾病;介绍了检测水环境样品中致病菌的聚合酶链式反应（PCR）、实时定量PCR、等温扩增技术、生物传感器和高通量测序等分子生物学技术研究进展;分析了不同检测技术的优缺点和应用特点;提出了水环境中致病菌分子生物学检测技术的未来发展方向,为水环境中致病菌分析检测和风险控制提供研究思路和技术支撑。     

Using Multiple Antibiotic Resistance and land use characteristics to determine sources of fecal coliform bacterial pollution

Multiple Antibiotic Resistance (MAR) analysis and regression modeling techniques were used to identify surface water areas impacted by fecal pollution from human sources, and to determine the effects of land use on fecal pollution in Murrells Inlet, a small, urbanized, high-salinity estuary located between Myrtle Beach and Georgetown, South Carolina. MAR analysis was performed to identify areas in the estuary that are impacted by human-source fecal pollution. Additionally, regression analysis was performed to determine if an association exists between land use and fecal coliform densities over the ten-year period from 1989 to 1998. Land-use variables were derived using Geographic Information System (GIS) techniques and were used in the regression analysis.MAR analyses were conducted by comparing the frequency and patterns of antibiotic resistance found in Escherichia coli isolates derived from surface water samples and from sewage sources in the Murrells Inlet sewage collection system. The MAR results suggest that the majority of the fecal pollution detected in the Murrells Inlet estuary may be from non-human sources, including fecal coliforms isolated from areas in close proximity to high densities of active septic tanks.A MAR Index, which measures the frequency of antibiotic resistance, was calculated for each of twenty-three water samples and nine sewage samples. The antibiotic resistance pattern comparisons were performed using cluster analysis. Although the MAR indices indicated that several surface water sites had potential human-source contamination, the cluster analysis suggests that only one sampling site had MAR patterns that were similar to those found in the sewage samples. This site was in close proximity to several large pleasure boats as well as a sewage collection system lift station, but was not near areas with active septic tanks. The results of the regression analysis also suggest that sewage sources and rainfall runoff from urbanized areas may contribute to fecal pollution in the estuary.     

Non-radiological contaminants from uranium mining and milling at Ranger,Jabiru, Northern Territory,Australia

Protection from the hazards from radioactivity is of prime importance in the management of uranium mine and mill wastes. Such wastes also contain non-radiological contaminants (heavy metals, acids and neutralising agents) which give rise to potential long-term health and environmental hazards and short-term hazards to the aquatic ecosystem, e.g. as a result of release of waste water. This study seeks to identify non-radiological contaminants (elements) transferred to waste water at the Ranger uranium mine/mill complex at Jabiru, which are likely to hazardous to the aquatic environment. The two principal sources of contaminants are:

1. ore and waste rock mobilised from mining; and
2. process reagents used in the milling and mineral extraction process.

These substances may or may not already be present in the natural environment but may lead to deleterious effects on the aquatic environment if increased above threshold levels. Rhenium, derived from the ore body, was found to be significantly enriched in waste water from Ranger, indicating its suitability as an indicator element for water originating from the mining and milling process, but only uranium, likewise derived from the ore, and magnesium, manganese and sulfur (as sulfate) from the milling process were found to be significant environmental contaminants.