The impact of electrochemical disinfection on Escherichia coli and Legionella pneumophila in tap water

In order to reduce the risks of Legionnaires' disease, caused by the bacterium Legionella pneumophila, disinfection of tap water systems contaminated with this bacterium is a necessity. This study investigates if electrochemical disinfection is able to eliminate such contamination. Hereto, water spiked with bacteria (10(4)CFU Escherichia coli or L. pneumophila/ml) was passed through an electrolysis cell (direct effect) or bacteria were added to tap water after passage through such disinfection unit (residual effect). The spiked tap water was completely disinfected, during passage through the electrolysis cell, even when only a residual free oxidant concentration of 0.07 mg/l is left (L. pneumophila). The residual effect leads to a complete eradication of cultivable E. coli, if after reaction time at least a free oxidant concentration of 0.08 mg/l is still present. Similar conditions reduce substantially L. pneumophila, but a complete killing is not realised.     

Bacteria detection utilizing electrical conductivity

Real-time and specific detection of single bacterium remains a fundamental challenge and draws very much attention. Using test patterns composed of interdigitated Au-electrode arrays modified with antibody, the specific and quantitative detection of the electrical conductivity of a single Escherichia coli (E. coli, JM109) has been carried out in this work. The key is to ensure low background current of the antibody-modified test patterns before bacteria detection (<0.7pA in this case) and minimize the residual moisture or hydration after E. coli immobilization, such as via the use of 1-min bake at 50 degrees C prior to electrical measurement. This method holds great potential for future application in the real-time, specific, and quantitative bacterium detection down to a single bacterium cell.     

Escherichia coli and its application in a mediated amperometric glucose sensor

Escherichia coli cells, which contain apo-glucose dehydrogenase, were used in constructing a mediated amperometric glucose sensor. The E. coli modified glucose sensor, which was prepared by immobilizing E. coli cells behind a dialysis membrane on a carbon paste electrode containing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q(0)), produced a current for the electrocatalytic oxidation of glucose with Q(0) as an electron transfer mediator only after the addition of a trace amount of pyrroloquinoline quinone (PQQ), the cofactor of the enzyme. This allows a novel method of glucose measurements free from the interference of the redox active substances, if contained, in a sample solution. The glucose sensor was insensitive to dioxygen; the currents measured under anaerobic and aerobic conditions, and even under dioxygen saturated conditions were almost the same in magnitude at a given concentration of glucose over the range of 0.2-10 mM. Response time of the glucose sensor was 2 min to attain 90% level of the steady-state current. The E. coli modified glucose sensor was reusable when treated with ethylenediaminetetraacetic acid (EDTA). When E. coli cells were lyophilized, they could be stored at room temperature in a dry box for more than six months without loss of the catalytic activity.     

Evaluation of a microwire sensor functionalized to detect Escherichia coli bacterial cells

A functionalized microwire sensor based on dielectrophoresis (DEP) and antigen-antibody reaction was initially developed for sensitive and selective detection of E. coli O157:H7. The dynamics of gold-tungsten microwires were manipulated using an automated X-Y-Z stage and the sensing process included antibody immobilization and bacterial detection, and cell quantification. Antibodies were first immobilized on surface of the microwire to improve sensing specificity, and then coupled with DEP for capture of E. coli cells in a mixture of E. coli cells and non-conductive polystyrene beads. Afterward, fluorescein-conjugated secondary antibodies were applied to the wire for quantification of captured bacteria. Field Emission Scanning Electron Microscope (FESEM) figures and fluorescence intensities of bacteria on the wire validated the sensing mechanism. The entire immobilization and detection procedure could be completed within 30 min with simple operations. Performance of the microwire sensor was not significantly affected when conducted in orange juice. In addition, the detection limit of this sensor was about 5 bacterial cells per microwire in 1000 CFU/mL bacterial suspensions when the electric field generated at 3 MHz and 20 peak to peak voltage (V(pp)), and only targeted E. coli cells were concentrated and captured.     

A Love wave immunosensor for whole E. coli bacteria detection using an innovative two-step immobilisation approach

The efficiency of a monomolecular film of (3-glycidoxypropyl) trimethoxysilane (GPTS) on a shear horizontal guided (Love) acoustic wave immunosensor to detect whole Escherichia coli (E. coli) bacteria is demonstrated. Direct anti-E. coli antibodies grafting onto the sensor surface did not lead to a significant bacteria immobilisation, partially attributed to the SiO2 sensor surface roughness. An innovative method has been set up to get around this difficulty and to detect whole bacteria. It consists in grafting goat anti-mouse antibodies (GAM) onto the sensor surface in a first step and introducing E. coli bacteria mixed with anti-E. coli antibodies onto the sensor in a second step. We describe the characteristics of such a technique like sample preparation time (lower than 30 min) and temperature improvements. A 37 degrees C experimental temperature led to the fastest bacteria binding kinetic, reducing the total analysis time. This method enables to keep the specificity of the antibody/antigen interaction and provides significant results in less than 1h. This leads to a detection threshold of 10(6) bacteria/ml in a 500 microl chamber.     

Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.     

Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli

The surface display of heterologous proteins on live Escherichia coli using anchoring motifs from outer membranes proteins has impacted on many areas of biochemistry, molecular biology and biotechnology. The Anaplasma marginale major surface protein 1a (MSP1a) contains N-terminal surface-exposed repeated peptides (28-289 amino acids) that are involved in pathogen interaction with host cell receptors and is surface-displayed when the recombinant protein is expressed in E. coli. Therefore, it was predicted that MSP1a would surface display on E. coli peptides inserted in the N-terminal repeats region of the protein. The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that a recombinant protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region is displayed on the E. coli surface and is recognized by anti-BM86 and anti-MSP1a antibodies. This system provides a novel approach to the surface display of heterologous antigenic proteins on live E. coli and suggests the possibility to use the recombinant bacteria for immunization studies against cattle tick infestations.     

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.     

Quantitative detection of Escherichia coli O157 in surface waters by using immunomagnetic electrochemiluminescence.

A protocol for the quantitative detection of Escherichia coli O157 in raw and concentrated surface waters using immunomagnetic electrochemiluminescence (IM-ECL) was developed and optimized. Three antibody sandwich formats were tested: commercial anti-O157:H7 IM beads, IM beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-house with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. The monoclonal IM bead-polyclonal TAG format was chosen for optimization because it gave lower background levels and linear regression slopes of ca. 1.0, indicative of a constant ECL signal per cell. The dynamic range was ca. 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples. The monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 10(8) cells of a non-O157 strain of E. coli ml(-1). Background ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than raw water samples. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone. Successive cell capture incubations, termed sequential bead capture (SBC), were optimized for establishing baseline ECL values for individual water samples. The linear dynamic range with SBC was ca. 10(2) to 10(5) E. coli O157 cells ml of concentrated water(-1). To validate the protocol, 10-liter surface water samples were spiked with ca. 5,000 E. coli O157 (Odwalla) cells and concentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by IM-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statistically significant and were generally consistent with standard curves for these cell concentrations. Enrichments were conducted with aliquots of spiked raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E. coli O157 (>10,000 ECL units); MLB gave a somewhat higher detection rate with spiked raw water. The potential sensitivity of the IM-ECL assay is ca. 25 E. coli O157 cells ml of raw water(-1), 25 cells 100 ml of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) with concentration and enrichment. The IM-ECL assay appears suitable for routine analysis and screening of water samples.     

Development of QCM biosensor to detect a marine derived pathogenic bacteria Edwardsiella tarda using a novel immobilisation method

QCM technology offers a real time output, simplicity of use and cost effectiveness in addition to high sensitivity. Sensitivity of QCM immunosensor can be enhanced by improving the immobilisation procedure on the quartz surface. The immobilisation strategy should be able to control both the amount and the orientation of the antibody (immunoglobulin; IgG) on the transducer for high affinity to antigens. This study introduced a new methodology recruiting oxidised IgG to expose aldehyde group in Fc region to cross-link to hydrazide conformed on self assembled monolayer (SAM) and compared with three conventional methods. Consequently, it was proved that considerable amount of antibody was immobilised and the sensitivity of new methodology was higher than other methods while ability of new methodology to immobilise IgG was lower than the conventional methods. The frequency shifts following bacterial cell injection were positively related to the frequency shifts after the injection of IgG and the amounts of bacterial cells, revealing that the frequency shifts after bacterial cell injection fully represented the weight change by specific attachments of bacterial cells to the IgG cross-linked on the gold surface. Specificity was tested on different bacteria including E. coli, V. vulnificus and A. hydrophila and showed no significant non-specific affinity on the tested bacteria. It was also demonstrated that the prepared sensor chip was stable enough to withstand repeated surface regeneration. Indeed, polyclonal antibody was more effective to detect antigen than monoclonal antibody which binds to only one epitope of antigen. Conclusively, the new methodology is appeared to be more sensitive than conventional methods tested and reusable for 10 times.     

Direct detection by polymerase chain reaction (PCR) of Escherichia coli in water and soft cheese and identification of enterotoxigenic strains

PCR was used to develop a method to detect Escherichia coli in surface water and soft cheese, which does not require cultivation of bacteria. DNA sequences from the ma /B operon of E. coli were amplified to specifically detect this bacterium. Samples of surface water and soft cheese naturally contaminated with E. coli from less than 100 cells per g up to several times 10⁵cells per g were analysed by both the classical culture method and the PCR assay. Comparable results were obtained with both methods. Soft cheese samples artificially contaminated with various levels of enterotoxigenic E. coli were analysed with a second PCR test specific for the heat-labile enterotoxin type I (LTI) of E. coli. The detection limit was about 1000 bacteria per g of soft cheese. In addition, two soft cheese samples naturally contaminated with 2 times 10⁵and 6 times 10⁵ E. coli per g as determined by the culture method were analysed by LTI-PCR and found to contain low levels of enterotoxigenic E. coli.     

Novel immuno-FRET assay method for Bacillus spores and Escherichia coli O157:H7

Novel immunofluorescence resonance energy transfer (immuno-FRET) assays for both Bacillus cereus spores and Escherichia coli O157:H7 are reported. Both assays involve the use of dual (QSY-7 and Oregon Green 514-antibody)-labeled spores or vegetative bacteria, such that Oregon Green 514-labeled antibodies are quenched by proximal QSY-7 molecules that are covalently bound to the dual (Oregon Green 514 and QSY-7)-labeled cells. Upon introduction of unlabeled bacteria or spores, in the respective assays, an increase in fluorescence is observed in proportion to the numbers of unlabeled cells. This is due to migration of Oregon Green 514-labeled antibody from the dual-labeled cells to the unlabeled target cells as verified by fluorescence microscopy. Optimization of the QSY-7 surface density led to a B. cereus spore detection sensitivity of approximately 1.0 x 10(5) to 2.5 x 10(5) spores per milliliter and 3.5 x 10(5) cells per milliliter for E. coli using a conventional cuvette-based spectrofluorometer.     

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.     

Microbiological analysis of cryopegs from the Varandei Peninsula,Barents Sea

The paper deals with the microbiological characterization of water-saturated horizons in permafrost soils (cryopegs) found on the Varandei Peninsula (Barents Sea coast), 4–20 m deep. The total quantity of bacteria in the water of cryopegs was 3.5 × 10⁸ cells/ml. The population of cultivated aerobic heterotrophic bacteria was 3–4 × 10⁷ cells/ml and the number of anaerobic heterotrophic bacteria varied from 10² to 10⁵ cells/ml depending on cultivation temperature and salinity. Sulfate-reducing bacteria and methanogenic archaea were found as hundreds and tens of cells per ml of water, respectively. A pure culture of a sulfate-reducing strain B15 was isolated from borehole 21 and characterized. Phylogenetic analysis has shown that the new bacterium is a member of the genus Desulfovibrio with Desulfovibrio mexicanus as its closest relative (96.5% similarity). However, the significant phenotypic differences suggest that strain B15 is a new species of sulfate-reducing bacteria.     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献   

Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 10(2) to 10(7)CFU/ml could be detected. The detection limit was approximately 6CFU/strip in PBS buffer and 50CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.     

Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining.

Reclaimed water is an important resource for areas with inadequate water supplies. However, there have been few studies on the variety of microorganisms found in this type of water, since typically reclaimed water is examined only for the presence of coliform bacteria. Many microorganisms, including the legionellae, are known to be more resistant to chlorine than are coliform bacteria. Previously, we detected > 10(3) Legionella cells per ml in primary and secondary sewage effluents and observed no significant reduction in population numbers throughout the treatment process. In this study, we detected Legionella spp. in chlorinated effluent by using an EnviroAmp Legionella PCR kit and direct fluorescent antibody (DFA) staining. However, we were not able to isolate Legionella spp. from either natural or seeded reclaimed water samples. This suggests that the Legionella spp. detected by the PCR and DFA methods may be injured or viable but nonculturable after exposure to the high residual chlorine levels typically found in this type of water source. The numbers of coliform bacteria were low (< 2 cells per 100 ml) in most reclaimed water samples and were not correlated with the presence or absence of Legionella spp. We also collected air samples from above a secondary aeration basin and analyzed them by using the PCR, DFA, and plate culture methods. Legionella spp. were detected in the air obtained from above the secondary basin with all three methods. We concluded that the PCR was superior to the culture and DFA methods for detecting Legionella spp. in environmental water samples.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.