Transcriptome Analysis of Escherichia coli O157:H7 Exposed to Lysates of Lettuce Leaves

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...

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

“…Bagged romaine lettuce was obtained from a local grocery store. Lettuce leaf lysate was extracted as described previously, with some modification (32). Lettuce leaves were homoge-nized by hand, using a mortar and pestle within 5 min and immediately collected into 50-ml conical tubes and centrifuged at 9,000 ϫ g for 4 min.…”

Section: Methodsmentioning

confidence: 99%

Requirement of Siderophore Biosynthesis for Plant Colonization by Salmonella enterica

Hao

Willis

Andrews‐Polymenis

et al. 2012

ABSTRACTContaminated fresh produce has become the number one vector of nontyphoidal salmonellosis to humans. However,Salmonella entericagenes essential for the life cycle of the organism outside the mammalian host are for the most part unknown. Screening deletion mutants led to the discovery that anaroAmutant had a significant root colonization defect due to a failure to replicate. AroA is part of the chorismic acid biosynthesis pathway, a central metabolic node involved in aromatic amino acid and siderophore production. Addition of tryptophan or phenylalanine to alfalfa root exudates did not restorearoAmutant replication. However, addition of ferrous sulfate restored replication of thearoAmutant, as well as alfalfa colonization. Tryptophan and phenylalanine auxotrophs had minor plant colonization defects, suggesting that suboptimal concentrations of these amino acids in root exudates were not major limiting factors forSalmonellareplication. AnentBmutant defective in siderophore biosynthesis had colonization and growth defects similar to those of thearoAmutant, and the defective phenotype was complemented by the addition of ferrous sulfate. Biosynthetic genes of eachSalmonellasiderophore, enterobactin and salmochelin, were upregulated in alfalfa root exudates, yet only enterobactin was sufficient for plant survival and persistence. Similar results in lettuce leaves indicate that siderophore biosynthesis is a widespread or perhaps universal plant colonization fitness factor forSalmonella, unlike phytobacterial pathogens, such asPseudomonasandXanthomonas.

“…Genomic DNA (2 g) was labeled via dye incorporation of Cy3-dCTP (GE Healthcare, Piscataway, NJ) using Klenow fragment (New England BioLabs, Ipswich, MA). Total RNA (20 g) from each experimental condition and each biological replicate was labeled via incorporation of Cy5-dCTP (GE Healthcare) into a cDNA product with the Fairplay III microarray labeling kit (Agilent, Santa Clara, CA) as previously described (26). Hybridization of Cy5-cDNA and Cy3-gDNA was carried out overnight at 42°C.…”

Section: Methodsmentioning

confidence: 99%

“…Transcriptional profiling experiments employing genomic DNA as a common reference were based on methods described previously (17,26). The use of a common reference allows the direct comparison of multiple isolates.…”

Section: Methodsmentioning

confidence: 99%

Distinct Transcriptional Profiles and Phenotypes Exhibited by Escherichia coli O157:H7 Isolates Related to the 2006 Spinach-Associated Outbreak

Parker

Kyle

Huynh

et al. 2012

Self Cite

In 2006, a large outbreak of Escherichia coli O157:H7 was linked to the consumption of ready-to-eat bagged baby spinach in the United States. The likely sources of preharvest spinach contamination were soil and water that became contaminated via cattle or feral pigs in the proximity of the spinach fields. In this study, we compared the transcriptional profiles of 12 E. coli O157:H7 isolates that possess the same two-enzyme pulsed-field gel electrophoresis (PFGE) profile and are related temporally or geographically to the above outbreak. These E. coli O157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig. The three clinical isolates and two spinach bag isolates grown in cultures to stationary phase showed decreased expression of many S -regulated genes, including gadA, osmE, osmY, and katE, compared with the soil, water, cow, feral pig, and the other three spinach bag isolates. The decreased expression of these S -regulated genes was correlated with the decreased resistance of the isolates to acid stress, osmotic stress, and oxidative stress but increases in scavenging ability. We also observed that intraisolate variability was much more pronounced among the clinical and spinach isolates than among the environmental isolates. Together, the transcriptional and phenotypic differences of the spinach outbreak isolates of E. coli O157:H7 support the hypothesis that some variants within the spinach bag retained characteristics of the preharvest isolates, whereas other variants with altered gene expression and phenotypes infected the human host.