Detection of pathogen Escherichia coli O157:H7 using self-excited PZT-glass microcantilevers

Composite self-excited PZT-glass cantilevers (5 and 3 mm in length, 1.8 and 2.0 mm wide) were fabricated and their resonance characteristics were determined in air and at 1 mm liquid immersion. In air, resonance occurred at 65.8 and 63.4 kHz for the two cantilevers used in this paper. Monoclonal antibody (MAb) specific to the pathogen Escherichia coli (E. coli) O157:H7 was immobilized at the cantilever glass tip, and then exposed to pathogen in the concentration range of 7x10(2) to 7x10(7)bacteria/mL. Resonance of the second mode decreased due to pathogen attachment in accordance with a proposed kinetic model. The specific attachment rate constant was found to be 3x10(-9) to 5x10(-9) min-1 (cell/mL)-1. Exposure to a mixed population containing both a pathogenic and non-pathogenic strain showed that the antibody-immobilized cantilever is highly selective, thus demonstrating its usefulness for detecting water-borne pathogens.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

Detection of Escherichia coli O157:H7 with langasite pure shear horizontal surface acoustic wave sensors

The toxigenic Escherichia coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure and death. On average, O157:H7 causes 73,000 illnesses, 2100 hospitalizations and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0°, 22°, 90°) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a non-toxigenic test strain. To quantify the effect of non-specific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14° were measured for the E. coli detection, as compared to 2° due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

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.     

Oxygen sensitivity of heated cells of Escherichia coli O157:H7

Following defined heat treatments (55 °C for 100 min, 59 °C for 5 min, 61 °C for 1 min), a 6 decimal (6-D) reduction was obtained when cells of Escherichia coli O157:H7 were enumerated in aerobic growth medium. Part of this reduction (3-D) was due to thermal inactivation (as determined when cells were enumerated in anaerobic growth medium), and part (3-D) was due to the inability of sub-lethally heat-injured cells of E. coli O157:H7 to grow in the presence of oxygen. When held anaerobically, the injured cells regained their ability to grow in the presence of oxygen. Following heating at 59 °C for 5 min, repair took 4 h at 30 °C, 48 h at 20 °C, 95 h at 10 °C, but did not occur in 816 h at 5 °C. Recovery from sub-lethal heat injury was not influenced by heat shock. These findings are relevant to the safety of minimally-heated foods.     

An Aptamer-Based Biosensor for Colorimetric Detection of Escherichia coli O157:H7

Background

An aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of Escherichia coli (E. coli) O157:H7.

Methodology/Principal Findings

The aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS)-binding aptamer on the surface of nanoscale polydiacetylene (PDA) vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM) was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 10⁴∼ 10⁸ colony-forming units (CFU)/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor.

Conclusions

The new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Rapid Detection of Escherichia coli O157:H7 with Multiplex Real-Time PCR Assays

A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of stx1 and/or stx2 of Escherichia coli O157:H7. A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively. The two products were distinguished by melting point curve analysis.     

Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system

Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia coli O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughter-house in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2.     

Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4–6 h at 37°C. Journal of Industrial Microbiology & Biotechnology (2001) 26, 345–349. Received 30 August 2000/ Accepted in revised form 17 April 2001     

Detection of Verotoxin-Producing Escherichia coli O157:H7 by Multiplex Polymerase Chain Reaction

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin, Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Derivation of Escherichia coli O157:H7 from Its O55:H7 Precursor

There are 29 E. coli genome sequences available, mostly related to studies of species diversity or mode of pathogenicity, including two genomes of the well-known O157:H7 clone. However, there have been no genome studies of closely related clones aimed at exposing the details of evolutionary change. Here we sequenced the genome of an O55:H7 strain, closely related to the major pathogenic O157:H7 clone, with published genome sequences, and undertook comparative genomic and proteomic analysis. We were able to allocate most differences between the genomes to individual mutations, recombination events, or lateral gene transfer events, in specific lineages. Major differences include a type II secretion system present only in the O55:H7 chromosome, fewer type III secretion system effectors in O55:H7, and 19 phage genomes or phagelike elements in O55:H7 compared to 23 in O157:H7, with only three common to both. Many other changes were found in both O55:H7 and O157:H7 lineages, but in general there has been more change in the O157:H7 lineages. For example, we found 50% more synonymous mutational substitutions in O157:H7 compared to O55:H7. The two strains also diverged at the proteomic level. Mutational synonymous SNPs were used to estimate a divergence time of 400 years using a new clock rate, in contrast to 14,000 to 70,000 years using the traditional clock rates. The same approaches were applied to three closely related extraintestinal pathogenic E. coli genomes, and similar levels of mutation and recombination were found. This study revealed for the first time the full range of events involved in the evolution of the O157:H7 clone from its O55:H7 ancestor, and suggested that O157:H7 arose quite recently. Our findings also suggest that E. coli has a much lower frequency of recombination relative to mutation than was observed in a comparable study of a Vibrio cholerae lineage.     

Detection of Escherichia coli O157:H7 in the Beef Marketed in Malaysia

Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.