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.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Immunoliposome sandwich assay for the detection of Escherichia coli O157:H7

We describe the development of a field-portable colorimetric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating dye as an analytical reagent. Antibodies (anti-E. coli O157:H7) thiolated by 2-iminothiolane were coupled to malemide-tagged liposomes encapsulating the marker dye, sulforhodamine B. Transmission electron microscopy showed that the immunoliposomes bound only to the serotype without any cross-reactivity with tested negative controls. A wicking reagent containing immunoliposomes and the test sample and a plastic-backed nitrocellulose strip with a measurement zone were used in a sandwich (noncompetitive) assay format. During the capillary migration of the wicking reagent, E. coli, with surface-bound immunoliposomes, was captured at the measurement zone on which antibodies to E. coli O157:H7 were immobilized. The color density of the measurement zone was directly proportional to the amount of E. coli O157:H7 in the sample. The detection limit of the current assay with pure cultures of the serotype was ca. 10(4) colony-forming units (CFU)/mL. The assay, which does not need washing and incubation steps, can be completed in 8 min. These results demonstrate the feasibility of using dye-encapsulating immunoliposomes in microporous membranes for the rapid detection of molecules with multivalent antigenic sites.     

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.     

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.     

Multiplex PCR for detection of Escherichia coli O157:H7 in foods

Escherichia coli O157:H7 is well known enterohemorrhagic pathogen responsible for infections among animals including a man. The main source of this bacterium is cattle, that is mostly asymptomatic and through that E. coli O157:H7 can simple transfer to food products. Therefore, there is a need for rapid, sensitive and specific detection method. The present work is focused on its detection by a heptaplex polymerase chain reaction, which targets genes from known virulent regions of E. coli O157:H7. According to obtained results this approach is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture and 6 and 8 CFU from milk and meat samples, respectively, independently of tested sample volume.     

Bile salts induce resistance to polymyxin in enterohemorrhagic Escherichia coli O157:H7

Many enteric bacteria use bile as an environmental cue to signal resistance and virulence gene expression. Microarray analysis of enterohemorrhagic Escherichia coli O157:H7 (EHEC) treated with bile salts revealed upregulation of genes for an efflux system (acrAB), a two-component signal transduction system (basRS/pmrAB), and lipid A modification (arnBCADTEF and ugd). Bile salt treatment of EHEC produced a basS- and arnT-dependent resistance to polymyxin.     

Tir细胞骨架偶联蛋白:大肠杆菌O157：H7的一种新的毒力因子

肠出血性大肠杆菌O157:H7是一种重要的传染性病原菌,可引起多种致死性疾病的爆发流行。Tir细胞骨架偶联蛋白(TccP)是近年来发现的O157:H7的一种新的重要的毒力因子,在O157:H7黏附宿主细胞造成黏附擦拭(A/E)损伤过程中起重要作用,TccP相关研究对阐明O157:H7致病机制具有重要意义。     

rpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7.

An rpoS mutant (rpoS::pRR10) of Escherichia coli O157:H7 ATCC 43895 was generated. Stationary-phase acid, heat, and salt tolerance was significantly reduced, and starvation-induced acid tolerance did not develop in the mutant. RpoS was also important for survival of E. coli O157:H7 in dry, fermented sausage.     

Starvation- and stationary-phase-induced acid tolerance in Escherichia coli O157:H7.

Stationary phase and the starvation of log-phase cells increased the acid tolerance of Escherichia coli O157:H7 strains. Although the degree of acid tolerance varied, the survival of most O157:H7 strains exceeded that of other, related, pathogens in a synthetic gastric fluid.     

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.     

Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars

A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10(10) and 10(4) cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10(6) to 10(9) CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as L-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of "nonfermentable" sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.     

Osmotic stress on dilution of acid injured Escherichia coli O157:H7

To determine surviving numbers of Escherichia coli from cultures or food systems, dilution with 0.1% peptone is regularly used. Higher numbers of survivors could be obtained from an acid-treated culture if 0.5 mol l-1 sucrose was added to the 0.1% peptone. Sorbitol, glucose or sodium chloride, but not glycerol, could be used in place of sucrose. Using electron microscopy distinct differences could be seen between acid-treated and untreated cells. The osmolarity of the diluents ranged from 5 to 500 mosmol kg-1 H2O for the 0.5 mol l-1 sugar or glycerol solutions, to about 1000 mosmol kg-1 H2O for the salt solution. Maximum recovery diluent has an osmolarity of about 300 mosmol kg-1 H2O and resulted in recovery of similar numbers of injured cells as a 0.5 mol l-1 solution of sugar in 0.1% peptone. Taking into account the observed damage to acid-treated cells and the differences in osmolarity of the diluents, it is likely that dilution in 0.1% peptone imposed additional stress on the acid-injured cells which caused further cell damage. Dilution in a more osmotically stable solution alleviated this osmotic stress.     

The strain-specific dynamics of Escherichia coli O157:H7 faecal shedding in cattle post inoculation

This study reports analysis of faecal shedding dynamics in cattle for three Escherichia coli O157:H7 (ECO157) strains (S1, S2 and S3) of different genotype and ecological history, using experimental inoculation data. The three strains were compared for their shedding frequency and level of ECO157 in faeces. A multistate Markov chain model was used to compare shedding patterns of S1 and S2. Strains S1 and S2 were detected seven to eight times more often and at 10⁴ larger levels than strain S3. Strains S1 and S2 had similar frequencies and levels of shedding. However, the total time spent in the shedding state during colonization was on average four times longer for S1 (15 days) compared to S2 (4 days). These results indicate that an ECO157 strain effect on the frequency, level, pattern and the duration of faecal shedding may need to be considered in control of ECO157 in the cattle reservoir.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network

A hand held device has been designed for the immunomagnetic detection and quantification of the pathogen Escherichia coli O157:H7 in food and clinical samples. In this work, a technology to manufacture a Lab on a Chip that integrates a 3D microfluidic network with a microfabricated biosensor has been developed. With this aim, the sensing film optimization, the design of the microfluidic circuitry, the development of the biological protocols involved in the measurements and, finally, the packaging needed to carry out the assays in a safe and straightforward way have been completed. The biosensor is designed to be capable to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody-antigen reaction. The giant magnetoresistive multilayer structure implemented as sensing film consists of 20[Cu(5.10nm)/Co(2.47 nm)] with a magnetoresistance of 3.20% at 235Oe and a sensitivity up to 0.06 Omega/Oe between 150Oe and 230Oe. Silicon nitride has been selected as optimum sensor surface coating due to its suitability for antibody immobilization. In order to guide the biological samples towards the sensing area, a microfluidic network made of SU-8 photoresist has been included. Finally, a novel packaging design has been fabricated employing 3D stereolithographic techniques. The microchannels are connected to the outside using standard tubing. Hence, this packaging allows an easy replacement of the used devices.     

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.