Concentration of Escherichia coli O157:H7 from Experimentally Infected, Pre-packaged Spinach by InnovaPrep’s Concentrating Pipette

Introduction: Many food outbreaks due to Escherichia coli O157:H7 are associated with vegetables and fruits.  Because these are perishable food items, procedures enabling same-day detection of low levels of pathogens are needed.   In this study, InnovaPrep's Concentrating Pipette was investigated as a bridge to concentrate large sample sizes into volumes more appropriate for rapid detection, allowing for reduced pre-enrichment times.

Purpose: The purpose of this study was to determine if same-day detection of experimentally infected spinach for E. coli O157:H7 is achievable.

Methods: An average of 10 CFU of E. coli O157:H7 was inoculated into 25 grams of pre-packaged fresh spinach, which was homogenized by a stomacher (AES) in 225 ml pre-warmed tryptic soy broth in a stomacher bag containing a 250 micron pre-filter (Interscience).  After 5 ½ hours of incubation, 100 ml of the homogenate was concentrated with a single-use, 0.45µm hollow fiber Concentrating Pipette Tip.  The eluted samples where then subjected to qPCR using DuPont’s BAX system and additionally plated onto tryptic soy agar plates.  

Results: Each experimentally infected spinach sample that was subjected to qPCR resulted in a positive detection (n=9), whereas blank controls were negative (n=3).  For concentration, the average time to concentrate 100 ml of homogenized spinach samples was 3.79 minutes ± 2.89.  The average concentration elution volumes were 216.70 µl ± 34.17 µl, which resulted in an average concentration factor of 349.18X ± 186.87X. Average time-to-result was 8 hours and 20 minutes.

Significance: These data suggest that spinach testing for E. coli O157:H7 can be performed in nearly the same time as an 8-hour work shift.