scholarly journals Comparison of enterovirus and adenovirus concentration and enumeration methods in seawater from Southern California, USA and Baja Malibu, Mexico

2012 ◽  
Vol 10 (3) ◽  
pp. 419-430 ◽  
Author(s):  
Lauren M. Sassoubre ◽  
David C. Love ◽  
Andrea I. Silverman ◽  
Kara L. Nelson ◽  
Alexandria B. Boehm
Keyword(s):  
Cell Culture ◽  
Water Samples ◽  
Membrane Filtration ◽  
Baja California ◽  
Quantitative Polymerase Chain Reaction ◽  
Skim Milk ◽  
Water Quality Monitoring ◽  
Recreational Waters ◽  
California Water ◽  
Etiological Agents

Despite being important etiological agents of waterborne illness, the sources, transport and decay of human viruses in recreational waters are not well understood. This study examines enterovirus and adenovirus concentrations in coastal water samples collected from four beaches impacted by microbial pollution: (1) Malibu Lagoon, Malibu; (2) Tijuana River, Imperial Beach; (3) Baja Malibu, Baja California; and (4) Punta Bandera, Baja California. Water samples were concentrated using a flocculation-based skim milk method and dead-end membrane filtration (MF). Viruses were enumerated using cell culture infectivity assays and reverse transcription quantitative polymerase chain reaction (RT-QPCR). Across concentration and quantification methods, enteroviruses were detected more often than adenoviruses. For both viruses, MF followed by (RT)QPCR yielded higher concentrations than skim milk flocculation followed by (RT)QPCR or cell culture assays. Samples concentrated by skim milk flocculation and enumerated by (RT)QPCR agreed more closely with concentrations enumerated by cell culture assays than MF followed by (RT)QPCR. The detection of viruses by MF and (RT)QPCR was positively correlated with the presence of infectious viruses. Further research is needed to determine if detection of viruses by rapid methods such as (RT)QPCR can be a useful water quality monitoring tool to assess health risks in recreational waters.

scholarly journals DNA-based real-time detection and quantification of aeromonads from fresh water beaches on Lake Ontario

2009 ◽  
Vol 7 (2) ◽  
pp. 312-323 ◽  
Author(s):  
Izhar U. H. Khan ◽  
Alyssa Loughborough ◽  
Thomas A. Edge
Keyword(s):  
Real Time ◽  
Fresh Water ◽  
Water Samples ◽  
Quantitative Polymerase Chain Reaction ◽  
Lake Ontario ◽  
Enteric Bacteria ◽  
Recreational Water ◽  
Recreational Waters ◽  
Standard Curve ◽  
High Concentrations

The present study was designed to develop a novel, rapid, direct DNA-based protocol to enumerate aeromonads in recreational waters. An Aeromonas genus-specific real-time quantitative polymerase chain reaction (Q-PCR) protocol was developed and optimized using newly designed genus-specific oligonucleotide primers derived from the gyrase B subunit (GyrB) gene. A standard curve was developed based on the PCR protocol with a minimum quantification limit of 10 cell equivalents ml−1 achieved using an autoclaved water sample from recreational water spiked with known quantities of an Aeromonas ATCC strain. The Q-PCR protocol was validated and applied to detect and quantify the total number of aeromonads in water samples collected from two fresh water beaches on Lake Ontario. The Q-PCR protocol revealed significantly higher numbers of aeromonads in all water samples than a culture-based assay at both beaches. Foreshore sand was found to serve as a reservoir of high concentrations of Aeromonas similar to this phenomenon noted for enteric bacteria like Eschershia coli. The new real-time Q-PCR protocol facilitated the rapid quantification of total numbers of Aeromonas cells present in recreational water samples in <3 hours without culturing.

scholarly journals Comparison of Membrane Filtration and Multiple-Tube Fermentation by the Colilert and Enterolert Methods for Detection of Waterborne Coliform Bacteria, Escherichia coli, and Enterococci Used in Drinking and Bathing Water Quality Monitoring in Southern Sweden

1998 ◽  
Vol 64 (8) ◽  
pp. 3079-3083 ◽  
Author(s):  
Karl F. Eckner
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Water Samples ◽  
Membrane Filtration ◽  
Water Quality Monitoring ◽  
Coliform Bacteria ◽  
Southern Sweden ◽  
Standard Methods ◽  
Bathing Water ◽  
Drinking Water Samples

ABSTRACT A total of 338 water samples, 261 drinking water samples and 77 bathing water samples, obtained for routine testing were analyzed in duplicate by Swedish standard methods using multiple-tube fermentation or membrane filtration and by the Colilert and/or Enterolert methods. Water samples came from a wide variety of sources in southern Sweden (Skåne). The Colilert method was found to be more sensitive than Swedish standard methods for detecting coliform bacteria and of equal sensitivity for detecting Escherichia coli when all drinking water samples were grouped together. Based on these results, Swedac, the Swedish laboratory accreditation body, approved for the first time in Sweden use of the Colilert method at this laboratory for the analysis of all water sources not falling under public water regulations (A-krav). The coliform detection study of bathing water yielded anomalous results due to confirmation difficulties. E. coli detection in bathing water was similar by both the Colilert and Swedish standard methods as was fecal streptococcus and enterococcus detection by both the Enterolert and Swedish standard methods.

scholarly journals Rapid qPCR-Based Water Quality Monitoring in New York State Recreational Waters

2021 ◽  
Vol 3 ◽  
Author(s):  
Cristina P. Fernández-Baca ◽  
Catherine M. Spirito ◽  
Justin S. Bae ◽  
Zsofia M. Szegletes ◽  
Nathan Barott ◽  
...  
Keyword(s):  
New York ◽  
Water Samples ◽  
New York State ◽  
Water Quality Monitoring ◽  
Handheld Devices ◽  
Portable Devices ◽  
Recreational Waters ◽  
Fecal Indicator ◽  
York State ◽  
E Coli

Public swimming beaches often rely on culture-based methods to determine if fecal indicator bacteria (FIB) levels are greater than health risk-based beach action values (BAV). The slow turnaround time of culture-based assays can prevent effective beach closure and reopening decisions. Faster testing methods that can be completed on-site are needed. Additionally, beach closures are currently based on high FIB levels, but at-present there are no tools to examine the health risks to bathers from myriad pathogens (e.g., bacteria, viruses, protozoa) that may be present in recreational waters. Twelve New York State beaches (n = 9 freshwater and n = 3 marine) were monitored over the course of summer 2018, and two of the freshwater beaches were monitored in fall 2017 as part of a preliminary study. A rapid, in-field workflow for detecting fecal enterococci in water samples was tested using four assays on two Biomeme handheld devices. All Biomeme-based workflows involved in-field DNA extractions and qPCR using portable devices. Beach water samples were also analyzed using EPA-approved or EPA-based qPCR methods: two culture-based methods, Enterolert (targeting enterococci at freshwater and marine beaches) and Colilert (targeting E. coli at freshwater beaches); and one qPCR method based on EPA 1611.1. For low abundance pathogen quantification, nanoscale-qPCR was conducted in 2018 using the Pathogen Panel which targeted 12 viral, bacterial, and protozoal pathogens. In fall 2017, the qPCR-based methods performed similarly to Enterolert (r2 from 0.537 to 0.687) and correctly classified 62.5–75.0% of water samples for a BAV of 104 MPN per 100 ml. In summer 2018, the correlation between Enterococcus levels based on Biomeme qPCR and Enterolert varied substantially between the 12 beaches. Inclusion of diverse regions and beach types may have confounded the Biomeme qPCR results. The EPA 1611.1-based method showed a weak, significant correlation (r2 = 0.317, p = 0.00012) with Enterolert. Nanoscale-qPCR showed low-levels of pathogens present at all beach sites; but only three showed up with any substantial frequency, E. coli eae (25% of samples), norovirus (31.4%), and Giardia lamblia (11.4%). Preliminary studies to establish beach-specific correlation curves between rapid qPCR and Enterolert methods are needed before any qPCR assay is used to inform beach decisions.

A Most Probable Number Method for the Enumeration of Legionella Bacteria in Water

1991 ◽  
Vol 24 (2) ◽  
pp. 143-147 ◽  
Author(s):  
N. A. Grabow ◽  
R. Kfir ◽  
W. O. K. Grabow
Keyword(s):  
Water Samples ◽  
Membrane Filtration ◽  
Quantitative Method ◽  
Fluorescent Antibody ◽  
Most Probable Number ◽  
Probable Number ◽  
Comparative Tests ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Yeast Extract Agar

A new quantitative method for the enumeration of Legionella bacteria in water is described. Appropriate tenfold serial dilutions of water samples concentrated by membrane filtration are plated in triplicate on buffered charcoal yeast extract agar. After incubation for 3 days representative smears from individual plates are tested for the presence of Legionella by direct fluorescent antibody staining. The number of positive plates in each dilution is used to calculate the Legionella count by means of conventional most probable number statistics. In comparative tests on a variety of water samples this method yielded significantly higher counts than previously used procedures.

Pesticide Contamination of Groundwater in Virginia: BMP Impact Assessment

1993 ◽  
Vol 28 (3-5) ◽  
pp. 379-387 ◽  
Author(s):  
S. Mostaghimi ◽  
P. W. McClellan ◽  
R. A. Cooke
Keyword(s):  
Water Quality ◽  
Land Use ◽  
Best Management Practices ◽  
Water Samples ◽  
Management Practices ◽  
Water Quality Monitoring ◽  
Pesticide Contamination ◽  
Cropping Patterns ◽  
Creek Watershed ◽  
Surface And Groundwater

The Nomini Creek Watershed/Water Quality monitoring project was initiated in 1985, as part of the Chesapeake Bay Agreement of 1983, to quantify the impacts of agricultural best management practices (BMPs) on improving water quality. The watershed monitoring system was designed to provide a comprehensive assessment of the quality of surface and groundwater as influenced by changes in land use, agronomic, and cultural practices in the watershed over the duration of the project. The primary chemical characteristics monitored include both soluble and sediment-bound nutrients and pesticides in surface and groundwater. Water samples from 8 monitoring wells located in agricultural areas in the watershed were analyzed for 22 pesticides. A total of 20 pesticides have been detected in water samples collected. Atrazine is the most frequently detected pesticide. Detected concentrations of atrazine ranged from 0.03 - 25.56 ppb and occurred in about 26 percent of the samples. Other pesticides were detected at frequencies ranging from 1.6 to 14.2 percent of all samples collected and concentrations between 0.01 and 41.89 ppb. The observed concentrations and spatial distributions of pesticide contamination of groundwater are compared to land use and cropping patterns. Results indicate that BMPs are quite effective in reducing pesticide concentrations in groundwater.

Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 311-314 ◽  
Author(s):  
Aaron B. Margolin ◽  
Charles P. Gerba ◽  
Kenneth J. Richardson ◽  
Jaime E. Naranjo
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Water Samples ◽  
Cdna Probe ◽  
Culture Method ◽  
Tidal River ◽  
Nucleic Acid Hybridization ◽  
Treated Wastewater ◽  
Gene Probe ◽  
Naturally Occurring

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

scholarly journals Are the defined substrate-based methods adequate to determine the microbiological quality of natural recreational waters?

2009 ◽  
Vol 8 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Marta Sofia Valente ◽  
Paulo Pedro ◽  
M. Carmen Alonso ◽  
Juan J. Borrego ◽  
Lídia Dionísio
Keyword(s):  
Standard Method ◽  
Membrane Filtration ◽  
Microbiological Quality ◽  
Recreational Waters ◽  
Total Coliforms ◽  
E Coli ◽  
Quality Of Water ◽  
Routine Monitoring ◽  
Rapid Tests

Monitoring the microbiological quality of water used for recreational activities is very important to human public health. Although the sanitary quality of recreational marine waters could be evaluated by standard methods, they are time-consuming and need confirmation. For these reasons, faster and more sensitive methods, such as the defined substrate-based technology, have been developed. In the present work, we have compared the standard method of membrane filtration using Tergitol-TTC agar for total coliforms and Escherichia coli, and Slanetz and Bartley agar for enterococci, and the IDEXX defined substrate technology for these faecal pollution indicators to determine the microbiological quality of natural recreational waters. ISO 17994:2004 standard was used to compare these methods. The IDEXX for total coliforms and E. coli, Colilert®, showed higher values than those obtained by the standard method. Enterolert® test, for the enumeration of enterococci, showed lower values when compared with the standard method. It may be concluded that more studies to evaluate the precision and accuracy of the rapid tests are required in order to apply them for routine monitoring of marine and freshwater recreational bathing areas. The main advantages of these methods are that they are more specific, feasible and simpler than the standard methodology.

Flood and rainfall mobilisation of E. coli and faecal source tracking markers from decomposing cowpats 

2021 ◽  
Author(s):  
Megan Devane ◽  
Brent Gilpin ◽  
Jennifer Webster-Brown ◽  
Louise Weaver ◽  
Pierre Dupont ◽  
...  
Keyword(s):  
Water Quality ◽  
Overland Flow ◽  
Quantitative Polymerase Chain Reaction ◽  
Linear Regression Analysis ◽  
Water Quality Monitoring ◽  
Quality Monitoring ◽  
Source Tracking ◽  
Faecal Contamination ◽  
E Coli ◽  
Faecal Indicators

<p>The intensification of dairy farming on the agricultural landscape in New Zealand has raised concerns about pollution sources from dairy faecal runoff into waterways. The transport of faecal pollution from farms into waterways is facilitated by overland flow, which can result from rain and flood events, poorly designed irrigation practices and the washing down of milking sheds.</p><p>An important step for mitigation of pollution is the identification of the source(s) of faecal contamination. When elevated levels of faecal indicator bacteria (FIB) such as <em>Escherichia coli </em>are identified in a waterway, faecal source tracking (FST) tools such as microbial source tracking (MST) using quantitative polymerase chain reaction (qPCR), and faecal steroids (for example, cholesterol) provide information about the sources of faecal contamination. The understanding of the fate (degradation/persistence) and transport of these FST markers in the environment is recognised as an important requirement for the interpretation of water quality monitoring in aquatic environments.</p><p>This study investigated the effects of faecal decomposition on bovine faecal indicators (<em>E. coli </em>and FST markers: bovine-associated qPCR markers and ten faecal steroids) by monitoring the effect of flood and rainfall events on simulated cowpats over a five and a half month period under field conditions. Two separate spring/summer trials were conducted to evaluate: Trial 1) the mobilisation under simulated flood conditions of the faecal indicators from irrigated versus non-irrigated cowpats, Trial 2) the mobilisation of faecal indicators from non-irrigated cowpat flood runoff versus runoff after simulated rainfall onto non-irrigated cowpats.</p><p>The microbial community changes within the decomposing cowpat (as illustrated by amplicon-based metagenomic analysis) were expected to impact on the survival/persistence of the bacterial targets of the MST markers, and also alter the ratio between faecal sterols and their biodegradation products, the stanols. It was hypothesised, therefore, that there would be:</p><ul><li>Changes over time in the concentration of<em> E. coli </em>and the bovine-associated MST markers mobilised into the cowpat runoff</li> <li>Alterations in the FST ratio signature of the ten measured faecal steroids, resulting in a change from a bovine faecal steroid signature in fresh cowpat runoff to other animal faecal signatures in the runoff from decomposing cowpats</li> <li>A difference in the mobilisation decline rates of all FST and microbial markers within a treatment regime and between treatments.</li> </ul><p>Linear regression analysis was undertaken to establish mobilisation decline rates for each of the analytes in the mobilisable phase from the cowpat runoff treatments, with calculation of the time taken in days for reduction in 90% of the concentration (T<sub>90</sub>), and statistical comparison of the regression coefficients (slopes) of all analytes. The results will include a discussion of the impacts of the study’s observations on the interpretation of faecal indicator assessments for water quality monitoring in waterways influenced by sources of faecal contamination.</p>

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