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To preserve bovine hides until they are processed into leather, they are often cured in large raceways of saturated brine. This process prevents the growth of most microorganisms, but under some conditions these hides are still susceptible to decomposition by halophilic archaebacteria. Low amounts of porcine bile added to the brine used for curing, have exhibited control of growth of these primitive microorganisms. Reversed‐phase high pressure liquid chromatography (RPHPLC) has been applied to monitor bile concentration in saturated brine mixtures containing contaminants associated with animal hides (bacteria, blood, dirt, fats, hair, manure, urine, etc.). With minimal sample preparation, this method can be used by either the hide curing, packing, or tanning industry to assess the need to charge brine solutions with additional porcine bile during the continuous process of hide addition and removal. Calibration curves of porcine bile added to highly contaminated raceway brine have demonstrated lin...

In this manuscript we present a modified kinetic model for pathogen capture efficiency (E) variation with mixing time (Tmix) which is applicable for most combinations of immuno-magnetic bead (IMB)-target organism concentrations (e.g., [IMB] ˜ 8 × 106 IMB mL-1; Salmonella enterica serovar Enteritidis, [SE] ˜ 102-108 CFU mL-1). We found that as the ratio of [IMB] to [SE] was decreased from more than 105 to ca. 10-1, the average number of cells captured per IMB SE complex increased from ca. 1 to 4 ± 1. Concurrently, using a modified kinetic model for fitting E variation with Tmix, the maximum possible level of capture (§) was observed to decline from ˜ 100% (§˜ 1; [IMB] > > [SE]) to 23% (§˜ 0.23; [IMB]:[SE] ˜ 0.1). Using a purely kinetic approach we also found that an empirical mass transport term (yobs= (3.1 ± 1.2) × 10-9 mL IMB-1 min 1; averaged across all [SE] tested: [IMB]:[SE] ˜ 200000, 200, 3, and 0.1) did not differ much from yobs reported in earlier work (3.35 ± 0.2 × 10-9 mL min-1 IMB-1) at extremely low [SE] (e.g., ± 100 CFU mL-1). Upon inserting the classically-derived equation for γ into the kinetic model and solving for the effective IMB radius (rIMB), we found that the rIMB was 2.3 ± 0.16 μ (§ S; averaged across all [IMB]:[SE]) which was only 0.5–0.9 μ greater than the rIMB reported by the manufacturer (1.4 ± 0.2 μ). These results support previous work which showed a similar small rIMB deviation possibly due to the hydro-dynamic behavior of the IMBs in relatively dilute buffer suspensions ([IMB ˜ 8 × 106 IMB mL-1) and argues that IMB-based cell capture is almost exclusively a function of mass transport.

Background The disruption of the bacterial cell wall plays an important part in achieving quantitative extraction of DNA from Eubacteria essential for accurate analyses of genetic material recovered from environmental samples. Results In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70 × 106 (±9.05%), 4.77 × 108 (±31.0%), and 5.93 × 108 (±4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt; Gram-positive), Shigella sonnei (Ss; Gram-negative), and Escherichia coli O79 (Ec; Gram-negative). We have utilized real-time PCR (qPCR) quantification with two specific sets of primers associated with the 16S rRNA “gene” to determine the number of copies CFU-1 by comparing the unknown target DNA qPCR results with standards for each primer set. Based upon statistical analyses of our results, we determined that the Agencourt Genfind v2, High Pure PCR Template Prep Kit, and Omnilyse methods consistently provided the best yield of genomic DNA ranging from 141 to 934, 8 to 21, and 16 to 27 16S rDNA copies CFU-1 for Bt, Ss, and Ec. If one assumes 6–7 copies of the 16S rRNA gene per genome, between 1 and 3 genomes per actively dividing cell and ≥ 100 cells CFU-1 for Bt (found to be a reasonable assumption using an optical method expounded upon herein) or between 1 and 2 cells CFU-1 for either Ss or Ec, then the Omnilyse procedure provided nearly quantitative extraction of genomic DNA from these isolates (934 ± 19.9 copies CFU-1 for Bt; 20.8 ± 2.68 copies CFU-1 for Ss; 26.9 ± 3.39 copies CFU-1 for Ec). The Agencourt, High Pure, and Omnilyse technologies were subsequently assessed using 5 additional Gram-positive and 10 Gram-negative foodborne isolates (n = 3) using a set of “universal” 16S rDNA primers. Conclusion Overall, the most notable DNA extraction method was found to be the Omnilyse procedure which is a “bead blender” technology involving high frequency agitation in the presence of zirconium silicate beads. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0326-z) contains supplementary material, which is available to authorized users.

Campylobacter jejuni and Campylobacter coli are the two important species responsible for most of the Campylobacter infections in humans. Reliable isolation and detection of Campylobacter spp. from food samples are challenging due to the interferences from complex food substances and the fastidious growth requirements of this organism. In this study, a novel biosensor-based detection called BARDOT (BActerial Rapid Detection using Optical scattering Technology) was developed for high-throughput screening of Campylobacter colonies grown on an agar plate without disrupting the intact colonies. Image pattern characterization and principal component analysis (PCA) of 6909 bacterial colonies showed that the light scatter patterns of C. jejuni and C. coli were strikingly different from those of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. Examination of a mixed culture of these microorganisms revealed 85% (34/40) accuracy in differentiating Campylobacter from the other three major foodborne pathogens based on the similarity to the scatter patterns in an established library. The application of BARDOT in real food has been addressed through the analysis of Campylobacter spiked ground chicken and naturally contaminated fresh chicken pieces. Combined with real-time PCR verification, BARDOT was able to identify Campylobacter isolates from retail chicken. Moreover, applying passive filtration to food samples facilitated the isolation of pure Campylobacter colonies and therefore overcame the interference of the food matrix on BARDOT analysis.

A modified bacterial ice nucleation detection (BIND®) assay was used for rapid and sensitive detection of several Salmonella species. For the BIND assay, Salmonella cells are infected with bacteriophage genetically modified to contain DNA encoding an ice nucleation protein (INP). After infection, de novo protein synthesis occurs and INPs are incorporated into the outer membrane of the organism. After supercooling (−9.3°C), only buffer solutions containing transfected salmonellae freeze, causing a phase-sensitive dye to change color. This technique, and a probability-based protocol modification, provided quantitative detection with a minimum detectable level (MDL) of 2.0 ± 0.3 S. enteritidis cells/mL in buffer (about 3 h). The MDLs for S. typhimurium DT104 and S. abaetetuba were 4.2 ± 0.2 and 11.1 ± 0.4 cells/mL, respectively. Using salmonellae-specific immunomagnetic bead separation technology in conjunction with the modified BIND protocol, we achieved an MDL of about 4.5 S. enteritidis cells/mL with an apparent capture efficiency of 56%.

Direct isothermal titration calorimetric measurement of Δ H and Δ S for the inclusion of chlorogenic acid (CA) by epichlorohydrin-polymerized cyclomaltoheptaose (β-cyclodextrin, β-CD n ), β-CD, and hydroxypropyl-β-CD (HP-β-CD) was performed at various temperatures ( T =10, 25, 40, and 55 °C) in 0.1 M Na phosphate buffer (pH 6.7). β-CD n and HP-β-CD, but not soluble β-CD, binding reactions with CA exhibited a significant variation in Δ H as a function of T (Δ C p =−228±51, −145±32, and −10±38 J mol −1 deg −1 , respectively). These CD–CA systems exhibited normal enthalpy–entropy compensation ( T Δ S versus Δ H ) behavior with a slope close to unity ( α =1.05±0.1) and small intercept ( T Δ S 0 =18 kJ mol −1 ). Thus, as Δ G was fairly uniform (−17±2 kJ mol −1 ) across T , mutually compensating adjustments in solvent-associated perturbations in both Δ S and Δ H for binding occurred. Relatively large unfavorable changes in Δ S (e.g., Δ S solv decreased with T ) mainly occurred in the substituted β-CD–CA systems and were compensated for by commensurate changes in Δ H . This thermodynamically favorable enthalpic deficit (negative values increasing with T ) was negatively correlated with T -dependent alterations in the number of water molecules bound by the β-CD n –CA complexes. This interpretation agrees with previous work [ Carbohydr. Res. , 282 (1996) 65–79], whereupon the convex curvature in the partitioning of CA by β-CD n as a function of T , implying a non-vanishing Δ C p , was eliminated with low water activity.

A modified approach using a light addressable Potentiometric sensor (LAPS) method to detect Escherichia coli O157.H7 in solutions is described. The bacterial cells were captured by the specific antibody coated on magnetic beads. The immuno paramagnetic beads (IMB) were then concentrated by a magnetic separator. Fluorescein-labeled anti E. coli antibody and urease-conjugated anti-fluorescein antibody were then used to label the cells captured by the beads. After labeling, the mixtures were rapidly filtered through 0.45 or 1.0 μm nitrocellulose or polycarbonate filters. The production of NH3 from urea by the conjugated urease associated with the captured bacteria was then analyzed. The results indicate that IMB approach can be used to increase the specificity of the LAPS method for pathogenic bacteria detection. With this procedure, the presence of about one E. coli O157:H7 CFU per g of hamburger meat can be detected after a 6 h incubation.

We demonstrate substitution of the custom-synthesized alkaline phosphatase (AP) substrate, p -aminophenyl phosphate (pAPP), with the commercially available 1-naphthyl phosphate (1-NP) as applied in the enzyme-linked immunomagnetic electrochemical (ELIME) detection of the pathogenic bacterium, Escherichia coli O157:H7. ELIME entails ‘sandwiching’ bacterial analyte between antibody-coated magnetic beads and an AP-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multi-well plate format. Enzyme substrate (pAPP or 1-NP) was added and conversion to an electroactive product was measured using Osteryoung square wave voltammetry. Using this technique, quantitative detection of E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ≤4.7×10 3 cells ml −1 in buffer or porcine carcass wash water within ca. 80 min.

Live cells of E. coli O157:H7 were treated with 4′,6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for observation by epifluorescent microscopy. The treated bacteria, which exhibited minimal growth activity and partially retained respiratory function, were captured by goat anti-E. coli O157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB). The beads with captured bacteria were then concentrated by magnetic separators. The efficiency of this magnetic concentration step was less than that of using high speed centrifugation. The antibody-captured and IMB-immobilized bacteria were then applied on HF-treated, bovine serum albumin (BSA)-coated microscope slides mounted on an automated stage, and magnetically aligned before fluorescence distribution was measured by a cooled CCD attached to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bacteria. This approach could reduce the enumeration time for and increase the efficiency of bacteria counting by manual efforts. The developed procedure of capturing, concentrating and enumerating E. coli O157:H7 exhibited a similar applicability in buffer, pork carcass wash solution and apple juice as described in this study. The presence of about 100 cells/mL of the bacteria could be detected in 30 min with developed procedure.

The BIAcore biosensor was used to detect binding of Escherichia coli O157:H7 with specific antibodies. Immobilized Protein A or Protein G captured antibodies which in turn bound to the bacteria. Alternatively, immobilized antibody captured the E. coli O157:H7 and the bacteria were further probed by a second antibody which enhanced the signal. The regenerated sensor surfaces were used for at least 50 separate analyses. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. © Rapid Science Ltd. 1998

Immunoelectrochemical sensors in which the sensor surface functions as both analyte capture phase and electrochemical detector have recently been developed for bacteria analysis. The speed and sensitivity of these devices make them very attractive for applications such as the detection of pathogenic microorganisms in food and water. However, the development and optimization of assays utilizing these sensors can be complicated by undesired interactions between the capture and detection functions. Modification of the sensor to achieve improvements in one function can have deleterious effects on the other function, and such effects can be difficult to diagnose and correct. In the course of investigations on immunoelectrochemical detection of Salmonella, we developed a rapid, nondestructive epifluorescence microscopy method to determine bacteria capture efficiency. This method enabled us to study capture and detection functions independently and efficiently identify performance-limiting factors. Rapid-scan electrochemical methods were used to optimize detection sensitivity and to provide diagnostic information on detection performance.

There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures. We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry. In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody. With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format. Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection. The electrochemical response was directly proportional to the number of captured bacteria. Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca. 80 min.

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.

For antibody generation, pathogenic bacteria are often heat-treated prior to inoculation into host animals in order to prevent infection and subsequently, premature death of the host. Inoculation of host rabbits with gamma radiationkilled pathogenic bacteria was employed with the hopes of generating antibodies that would have higher affinity, relative to antibodies raised against thermally denatured microorganisms, to live pathogens. The two antibody sets, raised against either heat-killed or irradiated bacterial cells, were compared for immunological response with live, heattreated, chemically-treated (i.e., bleached), and irradiated Escherichia coli O157:H7 and Salmonella bacteria. With the exception of the chemically-treated cell immunological response, both antibody sets yielded similar responses— low for irradiated cells, moderate for live cells, and high for heat-treated cells. The results suggested that thermal or chemical treatment of live pathogen containing food samples will elicit higher immunological responses with either antibody system, indicating potential application for detection of the presence of live bacteria in non-irradiated food systems. In addition, these findings also indicated that an immunoassay analysis of irradiated foods may lead to low signals that might be interpreted as indicating the presence of live cells (i.e., may lead to a false negative result).

Background We have evaluated the antimicrobial properties of Ag-based nanoparticles (Nps) using two solid phase bioassays and found that 10-20 μL of 0.3-3 μM keratin-stabilized Nps (depending on the starting bacterial concentration = CI) completely inhibited the growth of an equivalent volume of ca. 103 to 104 colony forming units per mL (CFU mL-1) Staphylococcus aureus, Salmonella Typhimurium, or Escherichia coli O157:H7 on solid surfaces. Even after one week at 37°C on solid media, no growth was observed. At lower Np concentrations (= [Np]s), visible colonies were observed but they eventually ceased growing. Results To further study the physiology of this growth inhibition, we repeated these experiments in liquid phase by observing microbial growth via optical density at 590 nm (OD) at 37°C in the presence of a [Np] = 0 to 10-6 M. To extract various growth parameters we fit all OD[t] data to a common sigmoidal function which provides measures of the beginning and final OD values, a first-order rate constant (k), as well as the time to calculated 1/2-maximal OD (tm) which is a function of CI, k, as well as the microbiological lag time (T). Performing such experiments using a 96-well microtitre plate reader, we found that growth always occurred in solution but tm varied between 7 (controls; CI = 8 × 103 CFU mL-1) and > 20 hrs using either the citrate-([Np] ~ 3 × 10-7 M) or keratin-based ([Np] ~ 10-6 M) Nps and observed that {∂tm/∂ [Np]}citrate ~ 5 × 107 and {∂tm/∂ [Np]}keratin ~ 107 hr·L mol-1. We also found that there was little effect of Nps on S. aureus growth rates which varied only between k = 1.0 and 1.2 hr-1 (1.1 ± 0.075 hr-1). To test the idea that the Nps were changing the initial concentration (CI) of bacteria (i.e., cell death), we performed probabilistic calculations assuming that the perturbations in tm were due to CI alone. We found that such large perturbations in tm could only come about at a CI where the probability of any growth at all was small. This result indicates that much of the Np-induced change in tm was due to a greatly increased T (e.g., from ca. 1 to 15-20 hrs). For the solid phase assays we hypothesize that the bacteria eventually became non-culturable since they were inhibited from undergoing further cell division (T > many days). Conclusion We propose that the difference between the solid and liquid system relates to the obvious difference in the exposure, or residence, time of the Nps with respect to the bacterial cell membrane inasmuch as when small, Np-inhibited colonies were selected and streaked on fresh (i.e., no Nps present) media, growth proceeded normally: e.g., a small, growth-inhibited colony resulted in a plateful of typical S. aureus colonies when streaked on fresh, solid media.

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 x 10(-4) ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.

An immuno sandwich method was evaluated for the detection of Salmonella in liquid eggs. Liquid eggs spiked with different out-break strains of Salmonella were mixed with proper enrichment media and incubated at 37 C for 4 to 20 h. After enrichment, immunomagnetic beads (IMB) coated with anti Salmonella antibodies were used to capture the bacteria. Samarium (Sm) labeled anti Salmonella antibodies were then used to form sandwiched complexes with IMB captured bacteria. Sandwiched Salmonella were then treated with Sm-chelator to allow the measurement of the released Sm by time-resolved fluorescence (TRF). The processes ranging from IMB capture to Sm chelation were performed using an automated KingFisher apparatus. With this approach, the presence of ~ 1 CFU of outbreak strains of Salmonella Enteritidis per egg (~50 g of liquid eggs) could be detected after enrichment for 20 h at 37 C. For higher levels of Salmonella Enteritidis contamination, e.g., 10 CFU per 50 g of liquid eggs, the enrichment time could be reduced to 5 h at 37 C. The results demonstrated that a combination of IMB capture and TRF measurement could be a rapid and sensitive method for Salmonella Enteritidis detection in liquid eggs.

A sandwiched immunoassay consisting of toxin capture by immunomagnetic beads (IMB) and toxin detection by horseradish peroxidase (HRP) linked chemiluminescence was used to follow the production of Shiga-like toxins (SLT) by E. coli O157:H7. The intensity of luminescence generated by the oxidation of luminol-liked compounds was used to represent the concentration of toxins produced. The time-course of SLT production by E. coli O157:H7 under different conditions was investigated. In pure culture, optimal generation of SLT showed a significant delay than the steady state of cell growth. In mixed cultures of SLT producing E. coli O157:H7 and non-SLT producing E. coli K-12 strain, the production of toxins was substantially decreased. However, the growth of E. coli O157:H7 was not affected by the presence of K-12 strain. This decrease in SLT production was also observed in radiation-sterile ground beef. In regular ground beef that might contain numerous other bacteria, the growth of E. coli O157:H7 in EC media was not significantly affected but the lowered production of SLT was observed. The results showed that mechanism of inducing SLT production was complex with both the growth time and growth environment could influence SLT production. The addition of homo-serine lactone to the growth media enhanced the production of SLT. Thus, possibly cell-cell communication may have a role in SLT production by E. coli O157:H7.

A new fluorescent sandwich method for the detection of Escherichia coli O157:H7 was developed. Strepavidin coated magnetic beads and fluorescence beads reacted with biotinylated anti E. coli O157 antibodies to form the immuno magnetic beads (IMB) and immuno fluorescence beads (IFB), respectively. The E. coli bacteria captured by IMB were further labeled with IFB to form IMB M- ( E. coli O157:H7) N- IFB O sandwich complexes where the subscripts M, N and O were integral numbers. Using broth cultured E. coli O157:H7, the sandwich method was able to detect the bacteria at the level of ~ 10 3 to 10 4 CFU/mL. Known quantity of freshly cultured E. coli O157:H7 cells were added to ground beef obtained from local markets. The bacteria in inoculated beef patties were enriched in EC broth containing novobiocin. After enriched for 4 h at 40 °C, the developed IMB-IFB method was applied to detect the presence of E. coli O157:H7. The results demonstrated that the developed method could detect the presence of 1 CFU of E. coli O157:H7 per gram of ground beef.

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli O157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 x 10(3) cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.

Outbreaks of E. coli O157:H7 by the consumption of contaminated cantaloupes fruits have been documented. Pathogens harbored in the networked but porous veins in khaki colored skin are difficult to remove. Thus, sensitive and efficient methods are needed to detect the presence of E. coli O157:H7 in cantaloupes. In this work, known quantities of the E. coli were inoculated on cantaloupe skins or flesh at room temperature for 1 h. The contaminated samples were incubated in growth media at 37°C for 3.3h. The bacteria captured by magnetic beads coated with anti E. coli O157 antibodies were further sandwiched by second anti E. coli O157 antibodies containing peroxidase for chemiluminescent measurements of captured bacteria. Alternatively, the captured bacteria were treated with electron-shuttering reagent to detect the cellular level of NAD(P)H via bioluminescence. The detected enzyme activity (peroxidase) and the NAD(P)H were used to measure the presence of the pathogen. The results indicated both the chemiluminescence and the fluorescence methods, in 96 well microplate format, could be applied to detect the E. coli contamination of cantaloupes.

Commercially available alfalfa seeds were inoculated with low levels (~ 4 CFU/g) of pathogenic bacteria. The inoculated seeds were then allowed to sprout in sterile tap water at 22°C. After 48 hours, the irrigation water and sprouts were separately transferred to bovine heart infusion (BHI) media. The microbes in the BHI samples were allowed to grow for 4 hours at 37°C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E.coli O157 and/or Salmonella in the growth media. Separation and concentration of IMB-captured pathogens were achieved using magnetic separators. The captured E. coli O157:H7 and Salmonella spp were further tagged with europium (Eu) labeled anti-E. coli O157 antibodies and samarium (Sm) labeled anti-Salmonella antibodies, respectively. After washing, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent chelates. The specific time-resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and Salmonella enterica from the seeds inoculated with ~ 4 CFU/g of the pathogens. Non-targeted bacteria, e.g., Aeromonas and Citrobacter exhibited essentially no cross reactivity. Since the pathogen detection from the sprouts was achieved within 6 hours, the developed methodology could be use as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella enterica in this popular salad food.

Live cells of E. coliO157:H7 were captured by goat anti-E. coliO157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB). The captured bacteria were labeled by 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for observation by epifluorescent microscopy. The beads with captured bacteria were then concentrated by magnetic separators. The efficiency of this magnetic concentration step was less than that of using high speed centrifugation. The antibody-captured and IMB-immobilized bacteria were then applied on HF-treated, bovine serum albumin (BSA)-coated microscope slides mounted on an automated stage, and magnetically aligned before fluorescence distribution was measured by a cooled CCD attached to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bacteria. We applied this approach to determine the bacterial counts in spiked beef hamburger patties. The results showed that after a 6-hour enrichment, sufficient number of the bacteria could be detected from the samples spiked with 1 CFU of E. coliO157:H7 per gram of the hamburger.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

We describe the development of an immunoligand assay (ILA) in conjunction with a light-addressable potentiometric sensor (LAPS) for the rapid detection of Escherichia coli O157:H7 cells in buffered saline. The ILA protocol consists of "sandwiching" bacterial analyte between biotinylated and fluoresceinated antibodies, indirect enzyme labeling of the bacteria with urease-labeled anti-fluorescein antibody, and active capture of the immune complex at a biotinylated bovine serum albumin-blocked nitrocellulose filter membrane with streptavidin. Using live E. coli O157:H7, the efficiency of the ILA was compared using various ratios of the biotinylated and fluoresceinated antibodies. Simultaneous addition of equimolar biotinylated and fluoresceinated antibodies effected optimal urease labeling and subsequent active capture of the bacteria in the ILA. Equimolar concentrations of the antibodies were varied to achieve optimal LAPS detection response for the live bacteria. Using ILA with LAPS, a minimum detectable level of ca. 7.1 x 10(2) cells/ml of heat-killed or ca. 2.5 x 10(4) cells/ml of live E. coli O157:H7 bacteria was achieved in Tris-buffered saline in an assay time of ca. 45 or ca. 30 min, respectively.

Testing of foods for the presence of pathogenic organisms requires methods different than those employed in the clinical laboratory. In foods there could be very low levels of pathogenic bacteria and the organisms are often in a poor physiological state because of exposure to stressful conditions in the food environment or to stresses encountered during food processing and storage. The organism must be identified in the presence of a large population of competing flora. Therefore, a period of enrichment culturing of the sample in liquid growth medium is usually required in order to allow the organism to recover from injury and to allow for selective growth of low numbers of the target bacteria to detectable levels. Traditional methods for detection and identification of pathogenic bacteria in foods and other samples have relied on the use of specific microbiological media to isolate and enumerate viable bacterial cells followed by a series of biochemical and serological tests for confirmation.