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
IMBM-(E. coliO157:H7)N-IFBO 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 ~ 103to 104 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.
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.
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