scholarly journals Application of denaturing gradient gel electrophoresis (DGGE) for assessing fecal pollution sources at a recreational beach

2014 ◽  
Vol 12 (4) ◽  
pp. 846-857 ◽  
Author(s):  
M. A. Esseili ◽  
I. I. Kassem ◽  
J. Lis ◽  
V. Sigler
Keyword(s):  
Gel Electrophoresis ◽  
Membrane Filtration ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Polymerase Chain Reaction Analysis ◽  
Fecal Pollution ◽  
Pollution Sources ◽  
E Coli ◽  
Recreational Beach ◽  
Gradient Gel Electrophoresis ◽  
Storm Sewer

We investigated the effectiveness of Escherichia coli community fingerprinting for identifying fecal pollution sources impacting a recreational beach. E. coli in water collected from the beach, nearby creek and storm sewer outfall were enumerated using membrane filtration, while E. coli communities were characterized following polymerase chain reaction analysis and denaturing gradient gel electrophoresis (DGGE) fingerprinting. Analysis of E. coli densities to determine the contributions of the creek and storm sewer during dry weather was inconclusive. However, DGGE fingerprinting indicated that the creek E. coli communities had a greater impact on the beach community composition (80–95% similarity), than on storm sewer communities (41–64%). Following rainfall events, E. coli communities in the creek were at least 93% similar to those at the beach, while the similarity of the outfall and beach communities varied from 72 to 96%. Furthermore, E. coli communities at the beach were more similar to creek communities than to storm sewer communities after the first 2 h and 48 h following the onset of rainfall, and of comparable similarity following 24 h of rainfall, suggesting transient contributions from the storm sewer. DGGE analysis of E. coli communities provided evidence that the creek was a consistent source of E. coli to the beach, while the storm sewer was a transient source.

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.

Source tracking ofEscherichia coliby 16S–23S intergenic spacer region denaturing gradient gel electrophoresis (DGGE) of therrnBribosomal operon

2007 ◽  
Vol 53 (10) ◽  
pp. 1174-1184 ◽  
Author(s):  
Thomas V. D’Elia ◽  
Chester R. Cooper ◽  
Carl G. Johnston
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Intergenic Spacer ◽  
Source Tracking ◽  
Combined Sewer Overflow ◽  
Intergenic Spacer Region ◽  
E Coli ◽  
Combined Sewer ◽  
Gradient Gel Electrophoresis

This research validates a novel approach for source tracking based on denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from Escherichia coli isolates. Escherichia coli from different animal sources and from river samples upstream from, at, and downstream of a combined sewer overflow were subjected to DGGE to determine sequence variations within the 16S–23S intergenic spacer region (ISR) of the rrnB ribosomal operon. The ISR was analyzed to determine if E. coli isolates from various animal sources could be differentiated from each other. DNA isolated from the E. coli animal sources was PCR amplified to isolate the rrnB operon. To prevent amplification of all 7 E. coli ribosomal operons by PCR amplification using universal primers, sequence-specific primers were utilized for the rrnB operon. Another primer set was then used to prepare samples of the 16S–23S ISR for DGGE. Comparison of PCR–DGGE results between human and animal sources revealed differences in the distribution and frequency of the DGGE bands produced. Human and Canada Goose isolates had the most unique distribution patterns and the highest percent of unique isolates and were grouped separately from all other animal sources. Method validation suggests that there are enough host specificity and genetic differences for use in the field. Field results at and around a combined sewer overflow indicate that this method can be used for microbial source tracking.

Allelopathic and Medicinal plant. 26. Millettia speciosa

2020 ◽  
Vol 51 (2) ◽  
pp. 125-146
Author(s):  
Nasiruddin Nasiruddin ◽  
Yu Zhangxin ◽  
Ting Zhao Chen Guangying ◽  
Minghui Ji
Keyword(s):  
Community Structure ◽  
Medicinal Plant ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Wiener Index ◽  
Pseudomonas Spp ◽  
Evenness Index ◽  
Gradient Gel Electrophoresis ◽  
Rhizosphere Pseudomonas

We grew cucumber in pots in greenhouse for 9-successive cropping cycles and analyzed the rhizosphere Pseudomonas spp. community structure and abundance by PCR-denaturing gradient gel electrophoresis and quantitative PCR. Results showed that continuous monocropping changed the cucumber rhizosphere Pseudomonas spp. community. The number of DGGE bands, Shannon-Wiener index and Evenness index decreased during the 3rd cropping and thereafter, increased up to the 7th cropping, however, however, afterwards they decreased again. The abundance of Pseudomonas spp. increased up to the 5th successive cropping and then decreased gradually. These findings indicated that the structure and abundance of Pseudomonas spp. community changed with long-term cucumber monocropping, which might be linked to soil sickness caused by its continuous monocropping.

scholarly journals Impact of Intensive Land-Based Fish Culture in Qingdao, China, on the Bacterial Communities in Surrounding Marine Waters and Sediments

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Qiufen Li ◽  
Yan Zhang ◽  
David Juck ◽  
Nathalie Fortin ◽  
Charles W. Greer
Keyword(s):  
Bacterial Communities ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Fish Culture ◽  
Fish Ponds ◽  
Marine Area ◽  
Gradient Gel Electrophoresis ◽  
Reducing Bacteria ◽  
The Impact ◽  
Nitrate Reducing Bacteria

The impact of intensive land-based fish culture in Qingdao, China, on the bacterial communities in surrounding marine environment was analyzed. Culture-based studies showed that the highest counts of heterotrophic, ammonium-oxidizing, nitrifying, and nitrate-reducing bacteria were found in fish ponds and the effluent channel, with lower counts in the adjacent marine area and the lowest counts in the samples taken from 500 m off the effluent channel. Denaturing gradient gel electrophoresis (DGGE) analysis was used to assess total bacterial diversity. Fewer bands were observed from the samples taken from near the effluent channel compared with more distant sediment samples, suggesting that excess nutrients from the aquaculture facility may be reducing the diversity of bacterial communities in nearby sediments. Phylogenetic analysis of the sequenced DGGE bands indicated that the bacteria community of fish-culture-associated environments was mainly composed of Flavobacteriaceae, gamma- and deltaproteobacteria, including generaGelidibacter, Psychroserpen, Lacinutrix,andCroceimarina.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

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