scholarly journals Development of Goose- and Duck-Specific DNA Markers To Determine Sources of Escherichia coli in Waterways

2006 ◽  
Vol 72 (6) ◽  
pp. 4012-4019 ◽  
Author(s):  
Matthew J. Hamilton ◽  
Tao Yan ◽  
Michael J. Sadowsky
Keyword(s):  
Escherichia Coli ◽  
Dna Markers ◽  
Fecal Contamination ◽  
Cost Effective ◽  
Source Tracking ◽  
E Coli ◽  
Hybridization Probes ◽  
Animal Hosts ◽  
Maximum Daily Loads ◽  
Microarray Screening

ABSTRACT The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.

scholarly journals Tracking the relative concentration between Bacteroidales DNA markers and culturable Escherichia coli in fecally polluted subtropical seawater: potential use in differentiating fresh and aged pollution

2017 ◽  
Vol 63 (3) ◽  
pp. 252-259
Author(s):  
Rulong Liu ◽  
Leo T.C. Yeung ◽  
Pui-Hei Ho ◽  
Stanley C.K. Lau
Keyword(s):  
Escherichia Coli ◽  
Dna Markers ◽  
Early Stage ◽  
Relative Concentration ◽  
Water Quality Monitoring ◽  
Source Tracking ◽  
E Coli ◽  
Sample Maximum ◽  
Pollution Events

Routine water quality monitoring practices based on the enumeration of culturable Escherichia coli provides no information about the source or age of fecal pollution. An emerging strategy is to use culturable E. coli and the DNA markers of Bacteroidales complementarily for microbial source tracking. In this study, we consistently observed in seawater microcosms of 3 different conditions that culturable E. coli decayed faster (T99 = 1.14 – 4.29 days) than Bacteroidales DNA markers did (T99 = 1.81 – 200.23 days). Concomitantly, the relative concentration between Bacteroidales DNA markers and culturable E. coli increased over time in all treatments. Particularly, the increase during the early stage of the experiments (before T99 of E. coli was reached) was faster than during the later stage (after T99 of E. coli was attained). We propose that the tracking of the relative concentration between Bacteroidales DNA markers and culturable E. coli provides an opportunity to differentiate a pollution that is relatively fresh from one that has aged. This method, upon further investigation and validation, could be useful in episodic pollution events where the surge of E. coli concentration causes noncompliance to the single sample maximum criterion that mandates high frequency follow-up monitoring.

scholarly journals Prevalence of Diarrhea-Associated Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Animal Hosts

2013 ◽  
Vol 79 (23) ◽  
pp. 7371-7380 ◽  
Author(s):  
Abhirosh Chandran ◽  
Asit Mazumder
Keyword(s):  
Escherichia Coli ◽  
Genetic Diversity ◽  
Virulence Genes ◽  
Diversity Index ◽  
Source Tracking ◽  
Taqman Assay ◽  
Content Type ◽  
E Coli ◽  
Atypical Epec ◽  
Animal Hosts

ABSTRACTIn order to assess the health risk associated with a given source of fecal contamination using bacterial source tracking (BST), it is important to know the occurrence of potential pathogens as a function of host.Escherichia coliisolates (n= 593) from the feces of diverse animals were screened for various virulence genes:stx1andstx2(Shiga toxin-producingE. coli[STEC]),eaeand EAF (enteropathogenicE. coli[EPEC]), STh, STp, and LT (enterotoxigenicE. coli[ETEC]), andipaH(enteroinvasiveE. coli[EIEC]). Eleven hosts were positive for only theeae(10.11%) gene, representing atypical EPEC, while two hosts were positive for botheaeand EAF (1.3%), representing typical EPEC.stx1,stx2, or bothstx1andstx2were present in 1 (0.1%,) 10 (5.56%), and 2 (1.51%) hosts, respectively, and confirmed as non-O157 by using aE. coliO157rfb(rfbO157) TaqMan assay. STh and STp were carried by 2 hosts (2.33%) and 1 host (0.33%), respectively, while none of the hosts were positive for LT andipaH. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 221 unique fingerprints with a Shannon diversity index of 2.67. Multivariate analysis of variance revealed that majority of the isolates clustered according to the year of sampling. The higher prevalence of atypical EPEC and non-O157 STEC observed in different animal hosts indicates that they can be a reservoir of these pathogens with the potential to contaminate surface water and impact human health. Therefore, we suggest thatE. colifrom these sources must be included while constructing known source fingerprint libraries for tracking purposes. However, the observed genetic diversity and temporal variation need to be considered since these factors can influence the accuracy of BST results.

The use of antibiotic resistance profiling as a means of tracing sources of fecal contamination in source waters

2010 ◽  
Vol 10 (2) ◽  
pp. 209-215
Author(s):  
M. S. Mthembu ◽  
P. T. Biyela ◽  
T. G. Djarova ◽  
A. K. Basson
Keyword(s):  
Antibiotic Resistance ◽  
Municipal Wastewater ◽  
Fecal Contamination ◽  
Source Tracking ◽  
Human Consumption ◽  
Biochemical Tests ◽  
Isolation And Identification ◽  
E Coli ◽  
Intestinal Pathogens ◽  
Source Waters

Fecal contamination of source waters and its associated intestinal pathogens continues to pose risks to public health although the extent and effect of microbial contamination of source waters gets very little attention in designing treatment plants in most developing countries. Coliform counts give an indication of the overall bacterial contamination of water and thus its safety for human consumption. However, their presence fails to provide information about the source of fecal contamination which is vital to managing fecal contamination problems in surface waters. This study explored the use of multiple antibiotic resistance (MAR) indexing as means of differentiating E. coli isolates from different sources. A total of 322 E. coli isolates were obtained from municipal wastewater and from fecal samples from domestic and wild animals. Conventional culture methods and standard chemical and biochemical tests were used for isolation and identification of E. coli. Isolates were assayed against 10 antibiotics using the micro-dilution technique. The results obtained generated antibiotic resistance profiles which were used to statistically group the isolates into different subsets. Correct source classification was obtained for 60% of human-derived and 95% non-human-derived E. coli respectively. These results indicate the validity of the usefulness of MAR indexing as a method of bacterial source tracking.

scholarly journals Escherichia coli is not a suitable fecal indicator to assess water fecal contamination by otters

2017 ◽  
Vol 78 (1) ◽  
pp. 155-159 ◽  
Author(s):  
M. Oliveira ◽  
D. Freire ◽  
N. M. Pedroso
Keyword(s):  
Escherichia Coli ◽  
Fecal Contamination ◽  
Microbiological Quality ◽  
Aquatic Environments ◽  
Pathogenic Microorganisms ◽  
Fecal Indicator ◽  
Fecal Samples ◽  
E Coli ◽  
Paulo State

Abstract The detection of pathogenic microorganisms in aquatic environments is extremely relevant in terms of public health. As these laboratorial methodologies are usually difficult, expensive and time-consuming, they are frequently replaced by the assessment of fecal indicator bacteria, such as Escherichia coli. This study aimed to assess the presence of E. coli in fecal samples from Neotropical otters, to evaluate its potential as fecal indicator to be applied to the determination of water microbiological quality in areas where otters’ populations are high. Twenty-six otter fecal samples, collected in Alto Paranapanema river basin, São Paulo State, Brazil, were analyzed for the presence of E. coli, using conventional bacteriological methods. Only 8 scat samples (30%) were E. coli positive, indicating that this microorganism is not a suitable fecal indicator to assess water fecal contamination by Neotropical otters, and should not be used to infer the presence of otter related pathogens in waters.

Development of Digoxigenin-Labeled PCR Amplicon Probes for Use in the Detection and Identification of Enteropathogenic Yersinia and Shiga Toxin–Producing Escherichia coli from Foods

1999 ◽  
Vol 62 (5) ◽  
pp. 438-443 ◽  
Author(s):  
STEPHEN D. WEAGANT ◽  
JAMES A. JAGOW ◽  
KAREN C. JINNEMAN ◽  
CURTIS J. OMIECINSKI ◽  
CHARLES A. KAYSNER ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pcr Amplification ◽  
Health Concern ◽  
E Coli ◽  
Hybridization Probes ◽  
Detection And Identification ◽  
Contaminated Food ◽  
Enteropathogenic Yersinia ◽  
Labeled Probe ◽  
Shelf Stable

By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), amplification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathogenic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. This technology allows the synthesis of a variety of shelf-stable probe reagents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: virF and yadA in enteropathogenic Yersinia and stx1 and stx2 in Shiga-like toxin–producing Escherichia coli. Results of DNA hybridizations of dot blots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent with results of equivalent PCR analyses. DNA colony hybridization with nonisotopic virF probes of colonies arising on spread plates from artificially contaminated food homogenates was able to detect potentially pathogenic Y. enterocolitica. When compared with oligonucleotide probes, amplicon probes are much less sensitive to changes in hybridization and wash temperatures, allowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at −20°C for more than 8 months. This method conveniently generates probes that are safe, stable, inexpensive, reusable, and reliable.

scholarly journals Identifying Host Sources of Fecal Pollution: Diversity of Escherichia coli in Confined Dairy and Swine Production Systems

2005 ◽  
Vol 71 (10) ◽  
pp. 5992-5998 ◽  
Author(s):  
Zexun Lu ◽  
David Lapen ◽  
Andrew Scott ◽  
Angela Dang ◽  
Edward Topp
Keyword(s):  
Escherichia Coli ◽  
Dairy Farm ◽  
Sampling Strategy ◽  
Farming Systems ◽  
Microbial Source Tracking ◽  
Fecal Pollution ◽  
Source Tracking ◽  
E Coli ◽  
Repetitive Extragenic Palindromic Pcr ◽  
Repetitive Extragenic Palindromic

ABSTRACT Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.

Effect of Prechill Fecal Contamination on Numbers of Bacteria Recovered from Broiler Chicken Carcasses Before and After Immersion Chilling

2004 ◽  
Vol 67 (9) ◽  
pp. 1829-1833 ◽  
Author(s):  
J. A. CASON ◽  
M. E. BERRANG ◽  
R. J. BUHR ◽  
N. A. COX
Keyword(s):  
Escherichia Coli ◽  
Broiler Chickens ◽  
Broiler Chicken ◽  
Tap Water ◽  
Fecal Contamination ◽  
Bacterial Counts ◽  
E Coli ◽  
Before And After ◽  
Marked Area ◽  
Skin Samples

Paired carcass halves were used to test whether fecal contamination of skin during processing of broiler chickens can be detected by increased bacterial counts in samples taken before and after immersion chilling. In each of three trials, six freshly defeathered and eviscerated carcasses were cut in half, and a rectangle (3 by 5 cm) was marked with dots of ink on the breast skin of each half. One half of each pair was chosen randomly, and 0.1 g of freshly collected feces was spread over the rectangle with a spatula. After 10 min, both halves were sprayed with tap water for 10 to 15 s until feces could no longer be seen in the marked area. Both halves were sampled with a 1-min carcass rinse and were then put in a paddle chiller with other eviscerated carcasses for 45 min to simulate industrial immersion chilling. Immediately after chilling, each carcass half was subjected to another 1-min rinse, after which the skin within the rectangle was aseptically removed from the carcass halves and stomached. Rinses of fecally contaminated halves had significantly higher Enterobacteriaceae immediately before chilling, but there were no differences in coliform and Escherichia coli counts. After chilling, there were no differences in Enterobacteriaceae, coliform, and E. coli counts in rinse or skin samples from the paired carcass halves. Correlations were generally poor between counts in rinse and skin samples but were significant between prechill and postchill rinses for both control and fecally contaminated halves. Correlations were also significant between counts in rinses of control and contaminated halves of the same carcass after chilling. Bacterial counts in postchill carcass rinses did not indicate that fecal contamination occurred before chilling.

scholarly journals Rapid Methods To Enumerate Escherichia coli in Foods Using 4-Methylumbelliferyl-ß-D-Glucuronide

2001 ◽  
Vol 84 (2) ◽  
pp. 407-415
Author(s):  
Diane F Ekholm ◽  
Irvin N Hirshfield
Keyword(s):  
Escherichia Coli ◽  
Cost Effective ◽  
Good Choice ◽  
Biochemical Tests ◽  
Rapid Methods ◽  
E Coli ◽  
Tree Nuts ◽  
Cost Effective Method ◽  
History Of ◽  
Hard Cheese

Abstract Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-ß-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)–MUG medium. An efficacious and cost-effective method is needed that can detect E. Coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. Coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. Coli and other lactose-fermenting organisms, the proposed method using regular LST/EC–MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC–MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC–MUG medium. For a product with a history of being positive for E. Coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST–MUG is the method of choice. A presumptive positive in the LST–MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. Coli. For samples spiked with E. Coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. Coli.

scholarly journals Heterogeneity of uidA gene in environmental Escherichia coli populations

2005 ◽  
Vol 3 (3) ◽  
pp. 297-304 ◽  
Author(s):  
Clarivel Lasalde ◽  
Roberto Rodriguez ◽  
Gary A. Toranzos ◽  
Henry H. Smith
Keyword(s):  
Escherichia Coli ◽  
Genetic Heterogeneity ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Dry Weight ◽  
Source Tracking ◽  
Tropical Rain Forests ◽  
Rain Forests ◽  
E Coli ◽  
Gradient Gel Electrophoresis

Previous studies have shown that Escherichia coli can be isolated from non-polluted rivers and from bromeliad axilae in pristine areas of tropical rain forests. Finding E. coli in pristine environments is unusual because this bacterium is thought to only survive in the gut of warm-blooded animals and thus its presence should indicate recent fecal contamination. The aims of this study were 1) to determine if E. coli is part of the native soil microbiota in tropical rain forests and 2) to determine if genetic heterogeneity exists among E. coli populations. High concentrations of total coliforms (104–105 cells per 10 g of soil dry weight) and low concentrations of thermotolerant coliforms (101–102 cells per 10 g dry soil, the majority of these were found to be E. coli) were detected. PCR using uidA-specific primers was done on DNA purified from E. coli isolates and the resulting amplicons analysed by denaturing-gradient gel electrophoresis (DGGE). Out of several hundred isolates, mixtures of nine different amplicons were consistently observed. The different patterns of DGGE observed indicate that the E. coli populations in these pristine soils are genetically heterogeneous. Fecal and environmental E. coli isolates were also analysed by pulsed-field gel electrophoresis (PFGE) which showed high DNA sequence variation among the E. coli isolates. Because of these differences in the genomes, PFGE did not allow grouping of environmental versus human isolates of E. coli when compared side to side. The apparent genetic polymorphisms, as a result of genetic heterogeneity, observed in isolates from the same pristine site indicate that source tracking may be difficult to carry out using E. coli as the target organism.

Survival of Escherichia coli in agricultural soil and presence in tile drainage and shallow groundwater

2010 ◽  
Vol 90 (3) ◽  
pp. 495-505 ◽  
Author(s):  
A C VanderZaag ◽  
K J Campbell ◽  
R C Jamieson ◽  
A C Sinclair ◽  
L G Hynes
Keyword(s):  
Escherichia Coli ◽  
Fecal Contamination ◽  
Shallow Groundwater ◽  
Drainage Water ◽  
Tile Drainage ◽  
Subsurface Water ◽  
Bacterial Survival ◽  
Manure Application ◽  
Monitoring Wells ◽  
E Coli

Animal agriculture and the use of manure as a soil amendment can lead to enteric pathogens entering water used for drinking, irrigation, and recreation. The presence of Escherichia coli in water is commonly used as an indicator of recent fecal contamination; however, a few recent studies suggest some E. coli populations are able to survive for extended time periods in agricultural soils. This important finding needs to be further assessed with field-scale studies. To this end, we conducted a 1-yr study within a 9.6-ha field that had received fertilizer and semi-solid dairy cattle manure annually for the past decade. Escherichia coli concentrations were monitored throughout the year (before and after manure application) in the effluent from tile drains (at approximately 80 cm depth) and in 5- to 8-m-deep groundwater wells. Escherichia coli was detected in both groundwater and tile drain effluent at concentrations exceeding irrigation and recreational water-quality guidelines. Within two of the monitoring wells, concentrations of E. coli, and frequency of detections, were greatest several months after the manure application. In two monitoring wells and one tile drain the frequency of E. coli detections was higher before manure was applied than after. This suggests the presence and abundance of E. coli was not strongly related to the timing of manure application. A laboratory study using naladixic acid resistant E. coli showed the bacteria could survive at least two times longer in soil samples collected from the study field than in soil from the adjacent riparian area, which had not received manure applications. Together, field and lab results suggest that a consistent source of E. coli exists within the field, which may include “naturalized” strains of E. coli. Further studies are required to determine the specific source of E. coli detected in tile drainage water and shallow groundwater. If the E. coli recovered in subsurface water is primarily mobilized from naturalized populations residing within the soil profile, this indicator organism would have little value as an indicator of recent fecal contamination. Key words: Bacterial survival, naturalized Escherichia coli, groundwater, tile drainage

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