An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli

Abstract

A colony enzyme-linked immunosorbent assay using the hydrophobic grid membrane filter format was developed for the isolation of verotoxigenic Escherichia coli from human and food samples. The method utilizes monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1-and/or 2-producing serotypes. E. coli that produced a minimum of 2 x 102 and 2 x 103 50% cytotoxic doses per ml of verotoxins 1 and 2, respectively, were detectable. In a method comparison using human stool specimens, this procedure isolated 29%o more E. coli 0157 than did the standard sorbitol-MacConkey agar procedure, with no false-positive reactions. When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. coli of a variety of serotypes including 0157:H7. Four false positives were noted. This procedure provides a sensitive means for the isolation of verotoxigenic E. coli and should facilitate recovery of those serotypes that are otherwise indistinguishable from nonpathogenic strains.

Prevalence and Antibiotic Resistance of <i>Escherichia coli</i> O157: H7 Serotype from Chicken Droppings Produced by Free - Ranged and Poultry Birds in Cross River, Nigeria

“…The Enzyme-linked immunosorbent assay (ELISA) technique was used for the qualitative detection of E. coli O157 antigens in all enriched samples [14]. Two drops of each sample was introduced into separate wells until all the required number of wells (excluding the control wells) were used depending on the number of enriched samples to be assayed.…”

“…All enriched samples with positive ELISA results were analyzed using the standard E. coli O157:H7 culture technique as recommended by [14]. All enriched, ELISA positive stool samples were serially diluted to 10 -3 using physiological saline (0.85% w/v NaCl).…”

Assessment of <i>Escherichia coli</i> O157:H7 Contamination in Soil and Water Sources Proximal to Abattoirs Within Cross River State, Nigeria

“…The diagnostic automation Enzyme-linked immunosorbent assay (ELISA) technique was used for the qualitative detection of E. coli O157 antigens in all enriched samples [18].…”

Detection of Escherichia coli O157:H7 and other verocytotoxin-producing E. coli (VTEC) in food

“…In addition, as the toxins show considerable variation in their antigenicity and binding properties (even within the VT2 class toxins), care must be taken in the choice of reagents if the aim is to detect all VT-producing strains from a clinical specimen (Smith and Scotland 1993). Milley and Sekla (1993) developed a colony enzyme-linked immunosorbent assay using a hydrophobic grid membrane filter for the isolation of VTEC from human and food samples. The method utilized monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1 and/or 2 producing serotypes.…”

“…When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. roli of a variety of serotypes including 0157 : H7. According to Milley and Sekla (1993), reasons for why not all VT-positive samples yield a VTEC isolate might include a low number of VTEC present or a low proportion of VTEC relative to other Gram-negative organisms. Intrinsic to this problem were the facts that the VTs are a family of highly potent biological toxins that will exhibit a profound effect on Vero cell cultures at extremely low concentration and there are no bacteriological media or temperatures that will select for VTEC over other Gram-negative organisms (including non-toxigenic E. roli).…”