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

Abstract

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 °C), 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 1 or 2 min, and soaking for 1 h or longer at 4 or 21 °C, respectively, resulted in maximum release of E. coli O157:H7 from seeds. Soaking seeds at 37 °C 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 °C, whereas at 37 °C, 0.1% or 0.5% Tween 80 was more effective. Detection of E. coli O157:H7 on seeds stored at 37 °C 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 °C and in sprouts produced from the seeds. The pathogen was not detected on seeds stored for 13 weeks at 37 °C 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 1 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 °C for up to 8 weeks as effectively as in sprouts produced from the seeds.