Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection

2013 ◽  
Vol 51 (1) ◽  
pp. 31-35 ◽  
Author(s):  
Aphidech Sangdee ◽  
Sitakan Natphosuk ◽  
Adunwit Srisathan ◽  
Kusavadee Sangdee
Keyword(s):  
Escherichia Coli ◽  
Repetitive Dna ◽  
Dna Fingerprint ◽  
Scar Primers

scholarly journals Sample Size, Library Composition, and Genotypic Diversity among Natural Populations of Escherichia coli from Different Animals Influence Accuracy of Determining Sources of Fecal Pollution

2004 ◽  
Vol 70 (8) ◽  
pp. 4478-4485 ◽  
Author(s):  
LeeAnn K. Johnson ◽  
Mary B. Brown ◽  
Ethan A. Carruthers ◽  
John A. Ferguson ◽  
Priscilla E. Dombek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Fingerprinting ◽  
Correlation Coefficient ◽  
Average Rate ◽  
Dna Fingerprint ◽  
Correct Classification ◽  
Dna Fingerprints ◽  
Rarefaction Analysis ◽  
E Coli ◽  
Jackknife Analysis

ABSTRACT A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.

scholarly journals Prevalence and Genetic Relationship of Predominant Escherichia coli Serotypes Isolated from Poultry, Wild Animals, and Environment in the Mekong Delta, Vietnam

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Lam Thanh Nguyen ◽  
Nguyen Khanh Thuan ◽  
Nguyen Thu Tam ◽  
Chau Thi Huyen Trang ◽  
Nguyen Phuc Khanh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Systemic Disease ◽  
Mekong Delta ◽  
Dna Fingerprint ◽  
Poultry Production ◽  
Wild Animals ◽  
Genetic Characteristics ◽  
E Coli ◽  
Clinical Consequences ◽  
Relationship Of

Avian pathogenic Escherichia coli (APEC) is the main causative agent of avian colibacillosis, which is an important systemic disease of profound economic and clinical consequences for the poultry industry worldwide. In this study, 975 E. coli strains were isolated from 2,169 samples collected from cloacal swabs of chickens, in-farm wild animals (ants, geckos, flies, and rats), and environment. The highest proportion of E. coli isolation was obtained from chicken cloacal swabs with 71.05% (95% confidence interval (CI) 66.69–75.05%) followed by the proportions of 38.15% (95% CI 35.41–40.97%) and 38.11% (95% CI 34.15–42.24%) from wild animals or environment, respectively. Distribution of O-antigen serotypes of the E. coli isolates, including O1, O2, O18, and O78, was determined by PCR. The most predominant serotype was O18 (10.56%) followed by O2 (9.44%), O1 (7.79%), and O78 (6.56%). Of note, serotype O18 was more likely distributed in the examined wild animals, especially in geckos. Polymorphic DNA fingerprints, generated by ERIC-PCR, of representative E. coli strains of each serotype revealed genetic heterogeneity of the examined E. coli, and O18 was more divergent with 63 clusters formed from 66 isolates. Furthermore, several E. coli strains from different sample sources shared high DNA fingerprint relatedness, suggesting that there exists complex transmission of E. coli from chickens to wild animals and environment and vice versa in poultry husbandry settings. Although pathotypes of the examined E. coli were not determined in this study, our results provided important findings of epidemiological and genetic characteristics of E. coli in the Mekong Delta and highlighted the prerequisite of stricter biocontainment to reduce the prevalence and consequences of APEC in poultry production.

scholarly journals Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources

2007 ◽  
Vol 73 (18) ◽  
pp. 5703-5710 ◽  
Author(s):  
Satoshi Ishii ◽  
Katriya P. Meyer ◽  
Michael J. Sadowsky
Keyword(s):  
Escherichia Coli ◽  
Human Health ◽  
Animal Species ◽  
Virulence Gene ◽  
Health Hazards ◽  
Dna Fingerprint ◽  
Phylogenetic Groups ◽  
E Coli ◽  
Animal Hosts ◽  
Potential Human Health

ABSTRACT Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx 1c and/or stx 2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.

scholarly journals Presence and Growth of Naturalized Escherichia coli in Temperate Soils from Lake Superior Watersheds

2006 ◽  
Vol 72 (1) ◽  
pp. 612-621 ◽  
Author(s):  
Satoshi Ishii ◽  
Winfried B. Ksoll ◽  
Randall E. Hicks ◽  
Michael J. Sadowsky
Keyword(s):  
Escherichia Coli ◽  
Lake Superior ◽  
Fecal Contamination ◽  
Frozen Soil ◽  
Dna Fingerprint ◽  
E Coli ◽  
Soil Microbial ◽  
Soil Temperatures ◽  
Temperate Soils ◽  
Cell Densities

ABSTRACT The presence of Escherichia coli in water is used as an indicator of fecal contamination, but recent reports indicate that soil populations can also be detected in tropical, subtropical, and some temperate environments. In this study, we report that viable E. coli populations were repeatedly isolated from northern temperate soils in three Lake Superior watersheds from October 2003 to October 2004. Seasonal variation in the population density of soilborne E. coli was observed; the greatest cell densities, up to 3 × 103 CFU/g soil, were found in the summer to fall (June to October), and the lowest numbers, ≤1 CFU/g soil, occurred during the winter to spring months (February to May). Horizontal, fluorophore-enhanced repetitive extragenic palindromic PCR (HFERP) DNA fingerprint analyses indicated that identical soilborne E. coli genotypes, those with ≥92% similarity values, overwintered in frozen soil and were present over time. Soilborne E. coli strains had HFERP DNA fingerprints that were unique to specific soils and locations, suggesting that these E. coli strains became naturalized, autochthonous members of the soil microbial community. In laboratory studies, naturalized E. coli strains had the ability to grow and replicate to high cell densities, up to 4.2 × 105 CFU/g soil, in nonsterile soils when incubated at 30 or 37°C and survived longer than 1 month when soil temperatures were ≤25°C. To our knowledge, this is the first report of the growth of naturalized E. coli in nonsterile, nonamended soils. The presence of significant populations of naturalized populations of E. coli in temperate soils may confound the use of this bacterium as an indicator of fecal contamination.

scholarly journals Cladophora (Chlorophyta) spp. Harbor Human Bacterial Pathogens in Nearshore Water of Lake Michigan

2006 ◽  
Vol 72 (7) ◽  
pp. 4545-4553 ◽  
Author(s):  
Satoshi Ishii ◽  
Tao Yan ◽  
Dawn A. Shively ◽  
Muruleedhara N. Byappanahalli ◽  
Richard L. Whitman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Lake Michigan ◽  
Bacterial Pathogens ◽  
Dna Fingerprint ◽  
Most Probable Number ◽  
Cladophora Glomerata ◽  
Human Pathogens ◽  
Potential Health ◽  
Probable Number

ABSTRACT Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 × 103 cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 × 102 Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.

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