Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

Abstract

Aim: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. Methods and Results: Raw milk, pasteurized milk with 1AE5% fat content and pasteurized milk with 3AE5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37°C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFoss TM ). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml -1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml -1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37°C) and mTSBn (16 h, 37°C), successively. Conclusions: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml -1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. Significance and Impact of the Study: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.

Growth of pure cultures of Verocytotoxin-producingEscherichia coliin a range of enrichment media

“…For the isolation of VTEC O26 and O111 strains, including the recovery of stressed cells from a variety of foods, Drysdale et al (2004) found that incubation at 42°C in BPW alone and BPW supplemented with 8 mg l )1 vancomycin gave better results than mTSB + n . By comparison, Reinders et al (2002) showed that detection of injured and uninjured E. coli O157 in raw or pasteurized milk following direct enrichment in mTSB + n was superior to BPW, although both media were incubated at 37°C in their study so it is not possible to draw a direct comparison with findings reported by Drysdale et al (2004).…”

“…By comparison, Reinders et al (2002) showed that detection of injured and uninjured E. coli O157 in raw or pasteurized milk following direct enrichment in mTSB + n was superior to BPW, although both media were incubated at 37°C in their study so it is not possible to draw a direct comparison with findings reported by Drysdale et al (2004). The optimal growth temperature of 20 E. coli O157 and 12 non-O157 VTEC strains in Mueller-Hinton broth was reported by Gonthier et al (2001) to be 40AE2 and 41AE2°C, respectively.…”

Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization following Formazan Reduction

“…In this study, the detection limit of CTC-FISH is 10 4 cells/ml by the use of the frame spotting method (12). Combination with concentration techniques, for example, filtration or immunomagnetic separation, which has been described for the detection of bacteria with a high recovery rate (22,25), should improve the limit. In addition, epifluorescence microscopy is a major tool for the direct detection of bacteria and was also used in this study.…”

Chromogenic Agar Medium for Detection and Isolation of Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Fresh Beef and Cattle Feces

“…First, there are no commercial IMS beads available for serogroups 045 and 0121. IMS has been shown to be an A 32 32 21 30 23 14 20 B 32 32 18 32 29 18 32 C 32 30 26 22 27 20 32 Total 96 effective and important tool for the isolation of STEC bacteria, especially E. coli 0157:H7 strains, from complex sample matrices, including bovine feces (19), soils (16), milk (27), and ground beef (2,33). Second, the background flora and/or other serogroups competed with each other either in the enrichment medium or on the chromogenic agar plate.…”