Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef

Abstract

Artificially inoculated Escherichia coli 0157:H7 was directly enumerated in ground beef and beef exudate, without enrichment or selection, by the antibody-direct epifluorescent filter technique (Ab-DEFT). The total assay time of the Ab-DEFT was less than 1 h. The beef was homogenized, treated for 15 min with trypsin and Triton X-100, and passed through a 5-,Im-pore-size prefilter and then through a 0.2-,um-pore-size black polycarbonate filter. The final filter was stained directly with fluorescein-labeled anti-0157 polyclonal antibody, rinsed, and examined by epifluorescence microscopy. The sensitivity of the Ab-DEFT was compared with that of a standard enrichment culture technique. Both methods reliably determined the presence of the pathogen in beef at 16 CFU/g. The Ab-DEFT was also useful for quantifying the pathogen and monitoring its growth in beef.

Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting

“…Similar phenomena have been described in other related studies [9] and represent an obstacle to increased sensitivity with this technology. In contrast, the Ab-DEFT relies on operator recognition of bacterial morphology to distinguish such debris from the target organism, and although recognition problems occur to some extent, a lower limit of detection by two orders of magnitude is achieved [3]. This level of sensitivity is of vital importance in the realm of direct detection, however, acceptable enrichment procedures generally increase the concentration to well above the threshold for FC detection at which point issues of analysis time and sample through-put come into play.…”

“…The streptomycin-resistant strain DHS-1 [3] of E. coli O157:H7 was used for all experiments. This strain is a variant of ATCC strain 35150.…”

“…The procedure used for sample processing was a modi¢cation of a previously reported method [3]. Brie£y, 2 ml of enrichment mixture was incubated at 50³C for 10 min with 0.5 ml trypsin and 2 ml 0.5% Triton X-100 (Sigma) and ¢ltered through a 5.0 Wm ¢lter.…”

“…As a result, many rapid and sensitive detection methods have been developed with DNA and immunologically based approaches in order to reduce the time required for detection of spe-ci¢c pathogens such as E. coli O157:H7 by conven-tional methods [2]. One antibody-based approach which has been successfully applied to E. coli O157:H7 is the antibody-direct epi£uorescent ¢lter technique (Ab-DEFT) [3]. This method is sensitive, economical, and takes about 1 h to perform the processing steps prior to microscopic examination of the sample.…”

“…In order to test FC technology in immuno£uorescent detection of E. coli O157:H7, mixtures of pure cultured E. coli O157:H7 and non-EHEC or arti¢cially contaminated non-selective enrichment cultures from ground beef were processed for the Ab-DEFT procedure [3]. Immuno£uorescent enumeration of organisms was done in parallel by epi£uorescence microscopy and by FC.…”