Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds

Isolating Escherichia coli O157:H7 from batches of alfalfa seeds used to produce sprouts implicated in human illness has been difficult, perhaps due to nonhomogenous and very low-level contamination and inaccessibility of the pathogen entrapped in protected areas of the seed coat. We evaluated the effectiveness of various treatments in releasing E. coli O157:H7 from seeds. The influence of homogenization (blending or stomaching for 1 or 2 min), rinsing method (shaking for 5 min), soaking time (0, 1, 3, 6, or 15 h), soaking temperature (4 or 21 degreesC), and the addition of surfactants (0.1%, 0.5%, or 1.0% Tween 80 or Span 20) to rinse water was determined. Blending or stomaching for I or 2 min, and soaking for I h or longer at 4 or 21 degreesC, respectively, resulted in maximum release of E. coli 0 1 57:H7 from seeds. Soaking seeds at 37 degreesC for 15 h increased cell populations of E. coli O157:H7 by approximately 3.6 log(10) CFU/g, likely due to bacterial growth. The maximum number of cells released from seeds by rinse water containing 1.0% Span 20 was at 21 degreesC, whereas at 37 degreesC, 0.1% or 0.5% Tween 80 was more effective. Detection of E. coli O157:H7 on seeds stored at 37 degreesC for up to 13 weeks and on sprouts derived from these seeds was compared. E. coli O157:H7 inoculated on seeds at 2.0 log(10) CFU/g was detected after storage of seeds for up to 8 weeks at 37 degreesC and in sprouts produced from the seeds. The pathogen was not detected on seeds stored for 13 weeks at 37 degreesC and was not isolated from sprouts produced from these seeds. Identifying seed treatment methods that enhance removal of E. coli O157:H7 from alfalfa seeds can aid the isolation and enumeration of the pathogen on seeds. With a combination of optimal conditions for detecting the pathogen, i.e. soaking seeds for 1 h and pummeling seeds for I min, an enrichment step in modified tryptic soy broth (TSB), and the use of immunomagnetic beads for separation of E. coli O157:H7 cells, E. coli O157:H7 was detected in alfalfa seeds incubated at 37 degreesC for up to 8 weeks as effectively as in sprouts produced from the seeds. Published by Elsevier Science B.V.