A time‐resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin‐labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)‐tagged anti‐E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu‐labels were then released from the antibodies and chelated to nitrilo‐triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu‐(2‐NTA)3(TOPO)2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K‐12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.
The use of an oxygen-reducing membrane fraction to stimulate growth of some anaerobic bacteria has been reported. In this study, stimulated recovery of Escherichia coli 0157:H7. Salmonella typhimurium, Streptococcus faecalis, and heat-injured Listeria monocytogenes was achieved by using broth media supplemented with Oxyrase "' (membrane-bound enzyme system derived from E. coli). The Oxyrase" enzyme allowed rapid growth of these facultatively anaerobic organisms in a nonselective broth. The activity of Oxyrase"' in four different selective enrichment broths to support growth of L. monocytogenes also was evaluated. Among tested broths, FDA-approved Listeria Enrichment Broth containing Oxyrase'" was found to be substantially superior for facilitating the growth of L. monocytogenes. Oxyrase"' promoted a signijicantly greater number of L. monocytogenes than commonly used but nonspecific reducing agents, such as L-cysteine HCl and sodium thioglycolate.Thesejndings indicated that all organisms tested can be grown well in liquid media using the oxygen-consuming membrane fragments to achieve and maintain oxygen-limited conditions. The Oxyrase "' enzyme system has potential applications in some selective media and other diagnostic tests specified for rapid detection of these facultatively anaerobic pathogens.
A rapid and simple method using a U‐shaped glass apparatus (Fung‐Yu tube) for early determination of the presence of Listeria monocytogenes and Listeria species in mixed cultures and inoculated meat samples has been developed. This system utilizes unique biochemical and physical properties of Listeria for selective enrichment. Fraser broth was used as a selective enrichment broth especially for observation of esculin hydrolysis (blackening of broth), and semisolid Modified Oxford agar was used for selective detection of motility of Listeria. When Fung‐Yu tubes containing 0.1 unit/mL of OxyraseTM (membrane fractions of Escherichia coli) were inoculated with L. monocytogenes, an enhanced early growth of L. monocytogenes occurred. A presumptive positive result for low numbers of L. monocytogenes (1–100 CFU/g) in the presence of large numbers of competitive microflora in pre‐enriched (24 h) ground beef samples using the Fung‐Yu tube method with the aid of OxyraseTMwas obtainable within 10 h. Using this system, isolation of Listeria in the presence of mixed bacterial flora (44 species), such as Bacillus, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Staphylococcus, and Streptococcus, and in inoculated ground beef was successful in 24–48 h. The Fung‐Yu tube procedure is a highly sensitive, selective, and easy‐to‐use method to separate and isolate L. monocytogenes and other Listeria spp. from other contaminating microorganisms in meats.
A modified procedure for magnetic capture of antibody‐conjugated bacteria for light addressable potentiometric sensor (LAPS) detection using the Threshold System was developed. Streptavidin coated magnetic beads, partially labeled with biotinylated anti Escherichia coli O157 antibodies, were used to capture Escherichia coli O157:H7. Captured bacteria were further labeled with fluorescein‐conjugated anti ‐E. coli O157:H7 antibodies and urease‐labeled. anti‐fluorescein antibody. Magnetically concentrated bacteria‐containing complexes were then immobilized through streptavidin‐biotin interactions on 0.45 μ biotinylated nitro‐cellulose membranes assembled as sample sticks for the Threshold instrument. The rate of pH change associated with the production of NH3 by the urease in urea‐containing solution was measured by a LAPS incorporated in the Threshold instrument. This approach allowed us to detect 103 to 104 CPU of cultured E. coli O157:H7 in PBS solutions. Furthermore, detectable LAPS signals of the sample sticks remained relatively constant for at least 24 h at 4C. The developed approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6‐h enrichment at 37C, as low as 1 CFU/g of E. coli O157:H7 in beef hamburger could be detected.
An approach to rapidly detect the presence of viable Escherichia coli O157:H7 is described. Specific immunomagnetic beads were applied to capture the bacteria in different solutions. The captured bacteria were then lysed by commercially available reagents to release cellular ATP which was detected by firefly luciferin‐luciferase induced chemiluminescence. The bioenergetic status of the bacteria was adjusted by the addition of glucose, a carbon nutrient source, and carbonyl cyanide meta‐chlorophenyl hydrazone (CCCP), a membrane protonophore. The addition of glucose restored oxygen consumption and medium acidification activities and increased the ATP content of harvested bacteria after storage. On the other hand, CCCP enhanced the oxygen consumption and medium acidification but significantly decreased the ATP content. None of the glucose and CCCP effects could be detected with heat‐killed and γ‐ray irradiated E. coli O157:H7. Thus, immunomagnetic capture of the E. coli followed by testing the bioenergetic responses of captured bacteria would qualitatively determine the presence of viable E. coli O157:H7. When applied, the developed procedure could easily detect the presence of less than one CFU of the E. coli per gram of beef hamburg after a 6‐h enrichment at 37C.
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