High-Throughput and Quantitative Procedure for Determining Sources of
            <i>Escherichia coli</i>
            in Waterways by Using Host-Specific DNA Marker Genes

Yan, Tao; Hamilton, Matthew J.; Sadowsky, Michael J.

doi:10.1128/aem.01395-06

Cited by 24 publications

(15 citation statements)

References 37 publications

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“…E. coli strains were isolated from filter washings as previously described (Yan et al ., 2007). One to three ml aliquots of filter washings were spread-plated onto the surface of modified membrane thermotolerant Escherichia coli (mTEC) agar medium in 22-by-22-cm Q-tray bioassay plates (Genetix Boston, MA).…”

Section: Methodsmentioning

confidence: 99%

“…Plates were incubated at 35° C for 2 h, and then at 44.5° C for 22 h. After incubation, plates were stored at 4° C overnight to facilitate development of blue pigment in colonies and to differentiate E. coli from other coliform and gram-negative bacteria. Well-isolated blue colonies were picked by hand or by using a Q-Bot robotic system (Genetix, Boston, MA) into 384 well microplates containing Hogness modified freezing medium as previously described (Yan et al ., 2007). Microplates were incubated at 37° C overnight, and stored at -80° C until used.…”

Section: Methodsmentioning

confidence: 99%

“…Automated arraying of E. coli isolates onto positively charged, 22-by-22-cm, Performa II nylon membranes (Genetix) was done by using a QBot robot system (Genetix) as previously described (Yan et al ., 2007). Each membrane was divided into 6 sections, each section contained 384 subunits, and each subunit consisted of 4 individual spots.…”

Section: Methodsmentioning

confidence: 99%

“…We previously reported the successful use of high-throughput, semi-automated, robotic technology to detect the presence of host source-specific genes in environmental E. coli isolates (Yan et al ., 2007). In the studies presented here, we adapted this technology to quickly screen large numbers of E. coli isolates from marine recreational water for the presence of virulence determinants and confirmed positive hybridization results by using PCR.…”

Section: Introductionmentioning

confidence: 99%

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Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

et al. 2010

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Contamination of recreational waters with E. coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the beta intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

et al. 2010

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“…Ahmed et al 1) and Ishii et al 50) surveyed E. coli strains isolated from water samples by using PCR targeting virulence factor genes. The use of colony hybridization using virulence genespecific probes is a promising alternate method since it is reliable and can be applied to high-throughput and largescale studies 86,117) . The use of robots to pick and array E. coli colonies allows for the simultaneous analysis of up to 20,736 strains, with minimal time and human input 117) .…”

Section: Future Directionsmentioning

confidence: 99%

Escherichia coli in the Environment: Implications for Water Quality and Human Health

2008

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Escherichia coli is naturally present in the intestinal tracts of warm-blooded animals. Since E. coli is released into the environment through deposition of fecal material, this bacterium is widely used as an indicator of fecal contamination of waterways. Recently, research efforts have been directed towards the identification of potential sources of fecal contamination impacting waterways and beaches. This is often referred to as microbial source tracking. However, recent studies have reported that E. coli can become "naturalized" to soil, sand, sediments, and algae in tropical, subtropical, and temperate environments. This phenomenon raises issues concerning the continued use of this bacterium as an indicator of fecal contamination. In this review, we discuss the relationship between E. coli and fecal pollution and the use of this bacterium as an indicator of fecal contamination in freshwater systems. We also discuss recent studies showing that E. coli can become an active member of natural microbial communities in the environment, and how this bacterium is being used for microbial source tracking. We also discuss the impact of environmentally-"naturalized" E. coli populations on water quality.

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Detection of Microbial Water Quality Indicators and Fecal Waterborne Pathogens in Environmental Waters: A Review of Methods, Applications, and Limitations

Domingo

Rice

2009

Encyclopedia of Analytical Chemistry

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Environmental waters are important reservoirs of pathogenic microorganisms, many of which are of fecal origin. In most cases, the presence of pathogens is determined using surrogate bacterial indicators. In other cases, direct detection of the pathogen in question is required. Microbiological methods to determine the presence of indicator organisms or pathogens in environmental samples vary depending on the target organism, sample type, need for quantification, speed of detection, sensitivity, and detection limits. Classical methods depend on cultivation steps while emerging techniques rely on detecting genetic signals from nucleic acid extracted from water samples. Regardless of the method, accurate detection is important to public health officials and monitoring agencies. In this article, we review some of the methods used to detect and quantify indicators of microbial water quality and human fecal pathogens as well as other issues relevant to water fecal pollution.

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High-Throughput and Quantitative Procedure for Determining Sources of Escherichia coli in Waterways by Using Host-Specific DNA Marker Genes

Cited by 24 publications

References 37 publications

Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

Escherichia coli in the Environment: Implications for Water Quality and Human Health

Detection of Microbial Water Quality Indicators and Fecal Waterborne Pathogens in Environmental Waters: A Review of Methods, Applications, and Limitations

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