Detection of Viable but Nonculturable
            <i>Escherichia coli</i>
            O157:H7 Bacteria in Drinking Water and River Water

Liu, Yanming; Gilchrist, Ainslie; Zhang, Jing; Li, Xing‐Fang

doi:10.1128/aem.02125-07

Cited by 114 publications

(87 citation statements)

References 43 publications

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“…Similar finding was observed in a previous study, in which the biological collection efficiencies of an electrostatic sampler were significantly higher when the Bacillus subtilis (Bacillus atrophaeus) spores were collected onto a filter than into the water (Mainelis et al 1999). A study indicated that Escherichia coli cells suffered starvation when suspended into deionized water, thus losing culturability (Liu et al 2008). Therefore, in this study, 0.9% NaCl solution was used to better preserve the culturability of the microorganisms being sampled.…”

Section: Resultssupporting

confidence: 67%

Development of an Automated Electrostatic Sampler (AES) for Bioaerosol Detection

Tan

Shen

Yao

et al. 2011

Aerosol Science and Technology

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In this study, an automated bioaerosol collection and output system was developed using a new electrostatic sampling method. The electrostatic sampler was designed using a half-ball shape steel electrode (radius is 45 mm) with three aerosol inlets (radius is 3.5 mm) on the top and a copper plate electrode (6 and 16 mm in diameter) suited inside a circular plastic support. Above the plate electrode, a plastic cylindrical reservoir (14 mm in diameter and 1 mm in height) was built with liquid inlet and outlet made of copper (2 mm in diameter). These outlets are connected to a peristaltic pump for liquid delivery. Next to the reservoir, there are two aerosol outlets (radius is 2.5 mm) connected to a vacuum pump. The physical collection efficiencies of the system were investigated when collecting indoor particles using an optical particle counter under different experimental conditions. The system was also tested when sampling indoor and outdoor bacterial aerosols. Experimental data showed that the system could collect 70%-90% of indoor particles of 0.3-2 µm at a low sampling flow rate of 1.2 L/min when a 20 kV voltage and a particle charger (1.5 V) were applied. Increasing sampling flow rate was observed to lead to the decrease of the collection efficiency. When a particle charger was applied, use of larger plate electrode (16 mm in diameter) was shown to significantly improve the collection efficiency close to two times than that of 6 mm. When operated for sampling environmental bacterial aerosols, the system obtained a similar magnitude of bacterial aerosol concentration levels compared to a mixed cellulose ester (MCE) filter. The integration of the automated electrostatic sampler (AES) sampler developed here with a biosensor device could offer a promising platform for the automated bioaerosol sensing.

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Section: Resultssupporting

confidence: 67%

Development of an Automated Electrostatic Sampler (AES) for Bioaerosol Detection

Tan

Shen

Yao

et al. 2011

Aerosol Science and Technology

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“…Currently, most available real-time PCR methodologies target virulence genes, such as stx 1 , stx 2 , and eaeA (2, 21, 32), or commonly shared phenotypic genes, such as uidA (5,20), fimA (18), rfbE (O antigen), and fliC (H antigen) (21). Indeed, using virulence genes as genetic markers will render an advantage to the assay, i.e., it can provide tangible forensic information as well as the identities of the pathogens.…”

Section: Discussionmentioning

confidence: 99%

Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

Chen

2012

Appl Environ Microbiol

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The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n ‫؍‬ 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 ؋ 10 7 dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources. E scherichia coli O157:H7 is a major food-borne pathogen responsible for a number of outbreaks in animals, poultry, and humans worldwide (1,7,14). It is estimated that E. coli O157:H7 outbreaks infect over 73,500 people annually in the United States (24), and this pathogen has not only become an important food safety concern but also a serious medical and public health problem. In order to effectively handle future outbreaks in a timely manner, it is necessary to have ample availability of sensitive, specific, and reliable methodologies that can be used for the rapid and selective detection of E. coli O157:H7.To date, great efforts have been made to develop appropriate methodologies for the detection of E. coli O157:H7. The traditional culture methods use selective media or chromogenic agar to detect E. coli O157:H7 (23). However, major limitations of this method are that it takes days to obtain the culture while providing little accurate information about the nature of the strain or isolate itself (39). PCR has been widely used for the detection of E. coli O157:H7 from foods and environmental samples. More recently, real-time PCR is gaining popularity for its enhanced sensitivity and specificity and its speedy turnaround time. Numerous real-time PCR-based methods have been reported for rapid and sensitive detection of E. coli O157:H7 (4, 7, 9, 10). One of the major obstacles for the PCR-based methodologies for sensitive and specific detection...

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“…Occasionally, a species or strain cannot be definitely identified by the virulence gene(s), such as stx1and stx2 genes, as those genes are present in different species or strains 16 as well. Using rfbE and fliC for target genes, both genes are needed to be used for complete identification 17 . Using ORF Z3276 as a biomarker, this qPCR assay has been demonstrated to be sensitive and specific assay ( Table 1).…”

Section: Discussionmentioning

confidence: 99%

Detection of Live <em>Escherichia coli</em> O157:H7 Cells by PMA-qPCR

Elkins

2014

JoVE

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A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

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Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water and River Water

Cited by 114 publications

References 43 publications

Development of an Automated Electrostatic Sampler (AES) for Bioaerosol Detection

Development of an Automated Electrostatic Sampler (AES) for Bioaerosol Detection

Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

Detection of Live <em>Escherichia coli</em> O157:H7 Cells by PMA-qPCR

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