Bacteria holding times for fecal coliform by mFC agar method and total coliform and Escherichia coli by Colilert®-18 Quanti-Tray® method

Bacteria holding-time experiments of up to 62 h were performed on five surface-water samples from four urban stream sites in the vicinity of Atlanta, GA, USA that had relatively high densities of coliform bacteria (Escherichia coli densities were all well above the US Environmental Protection Agency criterion of 126 colonies (100 ml)^???1 for recreational waters). Holding-time experiments were done for fecal coliform using the membrane filtration modified fecal coliform (mFC) agar method and for total coliform and E. coli using the Colilert^®-18 Quanti-Tray^® method. The precisions of these analytical methods were quantified. Precisions determined for fecal coliform indicated that the upper bound of the ideal range of counts could reasonably be extended upward and would improve precision. For the Colilert^®-18 method, analytical precisions were similar to the theoretical precisions for this method. Fecal and total coliform densities did not change significantly with holding times up to about 27 h. Limited information indicated that fecal coliform densities might be stable for holding times of up to 62 h, whereas total coliform densities might not be stable for holding times greater than about 27 h. E. coli densities were stable for holding times of up to 18 h—a shorter period than indicated from a previous studies. These results should be applicable to non-regulatory monitoring sampling designs for similar urban surface-water sample types.     

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。     

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.     

Comparative study of fluorogenic and chromogenic media for specific detection of environmental isolates of thermotolerant Escherichia coli

In a field study 78 water samples were analysed employingFluorocult Brilla Broth (BB) and its performance was comparedwith standard MPN procedure. Out of 78 water samples analysed 56(71.7%) samples yielded positive reactions in BB whereas, 50(64.1%) samples were positive by standard fecal coliform test.A comparative study of fluorogenic and chromogenic mediacontaining substrate -D glucuronide for specificdetection of environmental isolates of 313 thermotolerant E.coli has been undertaken. Five fluorogenic media wereused: Fluorocult MacConkey agar (MCA), Fluorocult ECD agar(ECD), Fluorocult VRB agar (VRB), Fluorocult E. coli0157:H7 agar (ECH7) and Fluorocult Brilla Broth (BB) andChromogenic Chromocult agar (CCA). BB and CCA were found to behighly specific and sensitive media to detect E. coli asall E. coli yielded positive reaction on them. On ECH7 andECD agar 67.5 and 64.9 of E. coli isolates gave positivereaction, respectively. Low sensitivity was observed in case ofMCA and VRB agar in detecting E. coli. The performance ofBB appears to be better when compared with standard MPNprocedure employing MacConkey broth/Brilliant green bile brothin detecting E. coli in drinking water.     

Characterization and statistical modeling of bacterial (Escherichia coli) outflows from watersheds that discharge into southern Lake Michigan

Two watersheds in northwestern Indiana were selected for detailed monitoring of bacterially contaminated discharges (Escherichia coli) into Lake Michigan. A large watershed that drains an urbanized area with treatment plants that release raw sewage during storms discharges into Lake Michigan at the outlet of Burns Ditch. A small watershed drains part of the Great Marsh, a wetland complex that has been disrupted by ditching and limited residential development, at the outlet of Derby Ditch. Monitoring at the outlet of Burns Ditch in 1999 and 2000 indicated that E. coli concentrations vary over two orders of magnitude during storms. During one storm, sewage overflows caused concentrations to increase to more than 10,000 cfu/100 mL for several hours. Monitoring at Derby Ditch from 1997 to 2000 also indicated that E. coli concentrations increase during storms with the highest concentrations generally occurring during rising streamflow. Multiple regression analysis indicated that 60% of the variability in measured outflows of E. coli from Derby Ditch (n = 88) could be accounted for by a model that utilizes continuously measured rainfall, stream discharge, soil temperature and depth to water table in the Great Marsh. A similar analysis indicated that 90% of the variability in measured E. coli concentrations at the outlet of Burns Ditch (n = 43) during storms could be accounted for by a combination of continuously measured water-quality variables including nitrate and ammonium. These models, which utilize data that can be collected on a real-time basis, could form part of an Early Warning System for predicting beach closures.     

Identifying fecal pollution sources using 3M™ Petrifilm™ count plates and antibiotic resistance analysis in the Horse Creek Watershed in Aiken County,SC (USA)

Sources of fecal coliform pollution in a small South Carolina (USA) watershed were identified using inexpensive methods and commonly available equipment. Samples from the upper reaches of the watershed were analyzed with 3M^? Petrifilm^? count plates. We were able to narrow down the study’s focus to one particular tributary, Sand River, that was the major contributor of the coliform pollution (both fecal and total) to a downstream reservoir that is heavily used for recreation purposes. Concentrations of total coliforms ranged from 2,400 to 120,333 cfu/100 mL, with sharp increases in coliform counts observed in samples taken after rain events. Positive correlations between turbidity and fecal coliform counts suggested a relationship between fecal pollution and stormwater runoff. Antibiotic resistance analysis (ARA) compared antibiotic resistance profiles of fecal coliform isolates from the stream to those of a watershed-specific fecal source library (equine, waterfowl, canines, and untreated sewage). Known fecal source isolates and unknown isolates from the stream were exposed to six antibiotics at three concentrations each. Discriminant analysis grouped known isolates with an overall average rate of correct classification (ARCC) of 84.3 %. A total of 401 isolates from the first stream location were classified as equine (45.9 %), sewage (39.4 %), waterfowl (6.2 %), and feline (8.5 %). A similar pattern was observed at the second sampling location, with 42.6 % equine, 45.2 % sewage, 2.8 % waterfowl, 0.6 % canine, and 8.8 % feline. While there were slight weather-dependent differences, the vast majority of the coliform pollution in this stream appeared to be from two sources, equine and sewage. This information will contribute to better land use decisions and further justify implementation of low-impact development practices within this urban watershed.     

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.     

Assessment of animal impacts on bacterial water quality in a South Carolina, USA tidal creek system

Fecal pollution may adversely impact water quality in coastal ecosystems. The goal of this study was to determine whether cattle were a source of fecal pollution in a South Carolina watershed. Surface water samples were collected in June 2002 and February through March 2003 in closed shellfish harvesting waters of Toogoodoo Creek in Charleston County, SC. Fecal coliform concentrations in 70 % of the water samples taken for this study exceeded shellfish harvesting water standards. Ribotyping was performed in order to identify animal sources contributing to elevated fecal coliform levels. Escherichia coli isolates (n?=?253) from surface water samples were ribotyped and compared to a ribotype library developed from known sources of fecal material. Ribotypes from water samples that matched library ribotypes with 90 % maximum similarity or better were assigned to that source. Less than half of the unknown isolates (38 %) matched with library isolates. About half (53 %) of the matched ribotypes were assigned to cattle isolates and 43 % to raccoon. Ribotyping almost exclusively identified animal sources. While these results indicate that runoff from cattle farms was a likely source of fecal pollution in the watershed, wildlife also contributed. Given the small size of the library, ribotyping was moderately useful for determining the impact of adjacent cattle farms on Toogoodoo Creek. Increasing the number and diversity of the wildlife sources from the area would likely increase the usefulness of the method.     

Characterization of Escherichia Coli Isolates from Different Fecal Sources by Means of Classification Tree Analysis of Fatty Acid Methyl Ester (Fame) Profiles

Microbial source tracking (MST) methods need to be rapid, inexpensive and accurate. Unfortunately, many MST methods provide a wealth of information that is difficult to interpret by the regulators who use this information to make decisions. This paper describes the use of classification tree analysis to interpret the results of a MST method based on fatty acid methyl ester (FAME) profiles of Escherichia coli isolates, and to present results in a format readily interpretable by water quality managers. Raw sewage E. coli isolates and animal E. coli isolates from cow, dog, gull, and horse were isolated and their FAME profiles collected. Correct classification rates determined with leaveone-out cross-validation resulted in an overall low correct classification rate of 61%. A higher overall correct classification rate of 85% was obtained when the animal isolates were pooled together and compared to the raw sewage isolates. Bootstrap aggregation or adaptive resampling and combining of the FAME profile data increased correct classification rates substantially. Other MST methods may be better suited to differentiate between different fecal sources but classification tree analysis has enabled us to distinguish raw sewage from animal E. coli isolates, which previously had not been possible with other multivariate methods such as principal component analysis and cluster analysis.     

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.     

Comparative impacts of stormwater runoff on water quality of an urban, a suburban, and a rural stream

Water quality data at 12 sites within an urban, a suburban, and a rural stream were collected contemporaneously during four wet and eight dry periods. The urban stream yielded the highest biochemical oxygen demand (BOD), orthophosphate, total suspended sediment (TSS), and surfactant concentrations, while the most rural stream yielded the highest total organic carbon concentrations. Percent watershed development and percent impervious surface coverage were strongly correlated with BOD (biochemical oxygen demand), orthophosphate, and surfactant concentrations but negatively with total organic carbon. Excessive fecal coliform abundance most frequently occurred in the most urbanized catchments. Fecal coliform bacteria, TSS, turbidity, orthophosphate, total phosphorus, and BOD were significantly higher during rain events compared to nonrain periods. Total rainfall preceding sampling was positively correlated with turbidity, TSS, BOD, total phosphorus, and fecal coliform bacteria concentrations. Turbidity and TSS were positively correlated with phosphorus, fecal coliform bacteria, BOD, and chlorophyll a, which argues for better sedimentation controls under all landscape types.     

Linking on-farm dairy management practices to storm-flow fecal coliform loading for California coastal watersheds

How and where to improve water quality within an agricultural watershed requires data at a spatial scale that corresponds with individual management decision units on an agricultural operation. This is particularly true in the context of water quality regulations, such as Total Maximum Daily Loads (TMDLs), that identify agriculture as one source of non-point source pollution through larger tributary watershed scale and above and below water quality investigations. We have conducted a systems approach study of 10 coastal dairies and ranches to document fecal coliform concentration and loading to surface waters at the management decision unit scale. Water quality samples were collected on a storm event basis from loading units that included: manure management systems; gutters; storm drains; pastures; and corrals and lots. In addition, in-stream samples were collected above and below the dairy facilities and from a control watershed, managed for light grazing and without a dairy facility or human residence and corresponding septic system. Samples were analyzed for fecal coliform concentration by membrane filtration. Instantaneous discharge was measured for each collected sample. Storm runoff was also calculated using the curve number method (SCS, 1985). Results for a representative dairy as well as the entire 10 dairy data set are presented. Fecal coliform concentrations demonstrate high variability both within and between loading units. Fecal coliform concentrations for pastures range from 206 to 2,288,888 cfu/100 ml and for lots from 1,933 to 166,105,000 cfu/100 ml. Mean concentrations for pastures and lots are 121,298 (SE=62,222) and 3,155,584 (SE=1,902,713) cfu/100 ml, respectively. Fecal coliform load from units of concentrated animals and manure are significantly more than units such as pastures while storm flow amounts were significantly less. Compared with results from earlier tributary scale studies in the watershed, this systems approach has generatedwater quality data that is beneficial for management decisions because of its scale and representation of current management activities. These results are facilitating on-farm changes through the cooperative efforts of dairy managers, regulatory agency staff, and sources of technical and financial assistance.     

Microbiological water quality of the Mfoundi River watershed at Yaoundé, Cameroon, as inferred from indicator bacteria of fecal contamination

Using the membrane filtration technique to count total coliform (TC), fecal coliform (FC) and fecal streptococci (FS), the microbiological water quality of the Mfoundi River and four of its representative tributaries at Yaoundé, Cameroon, was assessed for human use and contact. Sampling was conducted so as to examine the potential origin of fecal contamination and how rainfall affects the measured concentrations of indicators organisms. Our results revealed that waters were not safe for human use or primary contact according to the standards for water quality established by the Word Health Organization (WHO). Indeed, these waters exhibited high concentrations of TC (Mean ± SD=5.6 × 10⁸ ± 2.5 × 10⁶ CFU/100 ml), FC (Mean ± SD=6.8 × 10⁵ ± 2.4 × 10³ CFU/100 ml) and FS (Mean ± SD=7.3 × 10⁵ ± 2.1 × 10³ CFU/100 ml) that varied with the sampling sites and points. FC/FS ratio suggested that this contamination was more from warm-blooded animals than humans and correlation analysis points to the role of rainfall as a contributing factor, which enhanced the bacterial numbers detected. We conclude that there is a great potential risk of infection for users of waters from the Mfoundi River and its tributaries at Yaoundé.     

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.     

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。     

Serogroups of Escherichia coli from Drinking Water

Fifty seven isolates of thermotolerant E. coli were recovered from 188 drinking water sources, 45 (78.9%) were typable of which 15 (26.3%) were pathogenic serotypes. Pathogenic serogroup obtained were 04 (Uropathogenic E. coli, UPEC), 025 (Enterotoxigenic E. coli, ETEC), 086 (Enteropathogenic E. coli, EPEC), 0103 (Shiga-toxin producing E. coli, STEC), 0157 (Shiga-toxin producing E. coli, STEC), 08 (Enterotoxigenic E. coli, ETEC) and 0113 (Shiga-toxin producing E. coli, STEC). All the pathogenic serotypes showed resistance to bacitracin and multiple heavy metal ions. Resistance to streptomycin and co-trimazole was detected in two strains whereas resistance to cephaloridine, polymixin-B and ampicillin was detected in one strain each. Transfer of resistances to drugs and metallic ions was observed in 9 out of 12 strains studied. Resistances to bacitracin were transferred in all nine strains. Among heavy metals resistance to As³⁺ followed by Cr⁶⁺ were transferred more frequently.     

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.     

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.     

Estimating the Microbial Risk of E. coli in Reclaimed Wastewater Irrigation on Paddy Field

Microbial risk was quantified to assess human health risk as a result of exposure to E. coli in reclaimed wastewater irrigation. Monitoring data on E. coli were collected from pond water in paddy rice plots during the growing season. Five treatments were used and each was triplicated to evaluate the changes in E. coli concentrations in experiments performed in 2003 and 2004. The Beta-Poisson model was used to estimate the microbial risk of pathogen ingestion among farmers and neighboring children. A Monte Carlo simulation (10,000 trials) was conducted to estimate the risk associated with uncertainty. In this study, risk values ranged from 10⁻⁴ to 10⁻⁸. UV-disinfected irrigation water showed a lower risk value than others, and its level was within the range of the actual paddy rice field with surface water. Agricultural activity was thought to be safer after 1–2 days, when the paddy field was irrigated with reclaimed wastewater. Also, children were found to have a greater risk of infection with E. coli. This paper should be viewed as a first step in the application of quantitative microbial risk assessment of wastewater reuse in paddy rice culture.     

Antibiotic resistance analysis of fecal coliforms to determine fecal pollution sources in a mixed-use watershed

Antibiotic resistance analysis was performed on fecal coliform(FC) bacteria from a mixed-use watershed to determine thesource, human or nonhuman, of fecal coliform contamination. The study consisted of discriminant analysis of antibioticresistance patterns generated by exposure to fourconcentrations of six antibiotics (ampicillin, gentamicinsulfate, kanamycin, spectinomycin dihydrochloride,streptomycin sulfate, and tetracycline hydrochloride). Areference database was constructed from 1125 fecal coliformisolates from the following sources: humans, domestic animals(cats and dogs), agricultural animals (chickens, cattle, andhorses), and wild animals. Based on similar antibioticresistance patterns, cat and dog isolates were grouped asdomestic animals and horse and cattle isolates were grouped aslivestock. The resulting average rate of correctclassification (ARCC) for human and nonhuman isolates was94%. A total of 800 FC isolates taken from the watershedduring either a dry event or a wet event were classifiedaccording to source. Human sources contribute a majority(>50%) of the baseflow FC isolates found in the watershed inurbanized areas. Chicken and livestock sources areresponsible for the majority of the baseflow FC isolates foundin the rural reaches of the watershed. Stormwater introducesFC isolates from domestic (16%) and wild (21%) sourcesthroughout the watershed and varying amounts (up to 60%) fromchicken and livestock sources. These results suggest thatantibiotic resistance patterns of FC may be used to determinesources of fecal contamination and aid in the direction ofwater quality improvement.