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Evaluation of techniques for enrichment and isolation of Escherichia coli
O157:H7 from artificially contaminated sprouts.
Int J Food Microbiol 2001 Dec 4;71(1):87-92
Weagant SD, Bound AJ.
Pacific Regional Laboratory Northwest, US Food and Drug Administration, Bothell,
WA 98021, USA. sweagant@ora.fda.gov
Because sprouted seed products are kept wet during and after production, have
high levels of nutrients, and a neutral pH, they are subject to the outgrowth of
pathogens such as Escherichia coli O157:H7. For these same reasons, these
products also contain high levels of heterotrophic organisms and in particular
coliform bacteria. Recent outbreaks have focused attention on the need to
improve methodology for isolating this pathogen from sprouts. When 40 E. coli
O157:H7 strains were grown in pure culture in enterohemorrhagic E. coli
enrichment broth (EEB) as prescribed in the U.S. FDA-Bacteriological Analytical
Manual (FDA-BAM) and in EEB modified by varying the cefixime concentration,
outgrowth for all strains in EEB was inhibited at 0.05 mg/l but for only 2 of 40
strains when the cefixime level was adjusted to 0.0125 mg/l. These two
enrichment formulae were compared to modified E. coli broth (mEC), modified
Tryptic Soy Broth with 20 mg/l novobiocin (mTSB + N), modified Buffered Peptone
Water (mBPW), and mBPW with added 10 mg/l acriflavin, 10 mg/l cefsulodin, and 8
mg/l vancomycin (mBPW + ACV) for isolation of E. coli O157:H7 from sprouts.
These comparisons were performed using low-level (0.12 to 0.42 cfu/g)
artificially contaminated alfalfa and mixed salad sprouts. After enrichment, two
isolation methods were compared for recovery; direct plating to
Tellurite-Cefixime Sorbitol MacConkey agar (TCSMAC) and immunomagnetic
separation (IMS) (Dynabeads anti-E. coli O157, Dynal, Oslo, Norway) followed by
plating to TCSMAC. In addition, an immunoprecipitin detection kit, VIP (BioControl,
Bellevue, WA), was evaluated for detection after enrichment. We found that five
of the six enrichments were equivalent for detection or recovery while one
enrichment (mTSB + N without agitation) was less productive. Incubation for 24 h
was more effective in recovering E. coli O157:H7 from sprouts than 6 h for all
enrichment broths. Plating after IMS was more productive than direct plating at
these low levels of contamination, yielding recovery in 70 of 90 trials compared
to 37 of 90 trials without IMS for six enrichments. The sensitivity of VIP for
detection of E. coli O157:H7 varied depending on the enrichment broth. Because
of the rapid rate of growth of E. coli O157:H7 in mBPW, the high productivity of
mBPW + ACV after 24-h enrichment and its compatibility with both IMS and
detection with immunoprecipitin tests, mBPW + ACV at 42 degrees C with agitation
was found to be the most promising enrichment protocol for testing sprouts.
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