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P1-34 Comparison of Different Agars for the Recovery and Isolation of Non-O157 STECs from Baby Spinach and a Raw Milk Cheese

Monday, July 23, 2012
Exhibit Hall (Rhode Island Convention Center)
Julie Kase, U.S. Food and Drug Administration-CFSAN, College Park, MD
Anna Maounounen-Laasri, U.S. Food and Drug Administration, College Park, MD
Tina Lusk, U.S. Food and Drug Administration-CFSAN, College Park, MD
Insook Son, U.S. Food and Drug Administration-CFSAN, College Park, MD
Willis Fedio, New Mexico State University, Las Cruces, NM
Thomas Hammack, U.S. Food and Drug Administration, College Park, MD
Introduction: The FDA Bacteriological Analytical Manual (BAM) recommends Levine’s Eosin Methylene Blue (L-EMB) agar for isolating non-O157 Shiga toxin-producing E. coli (STEC). However, because this agar cannot distinguish non-pathogenic E. coli from STEC, it is necessary to screen large numbers of colonies.  Other agars that target E. coli O157:H7 have not been thoroughly tested for the recovery of non-O157:H7 STECs.

Purpose: Evaluate the efficacy of different agar formulations for selective recovery of non-O157 STECs from various food matrices.

Methods:   Four replicate 25 g test-portions of baby spinach were spiked at levels of approximately 0.02 CFU/g with virulent STEC strains.  BAM procedures were followed using 6 agars: Rainbow O157, modified Rainbow O157, R & F E. coli O157, CHROMagar, washed blood (SHIBAM), and L-EMB.  All agars were also evaluated using a raw-milk cheese purchased in New Mexico and subjected to BAM procedures for the recovery of E. coli. 

Results:   For the spiked studies, the results were strain conditional.  For example, all of the agars recovered an O26 strain, while plates from the same batch (Rainbow O157 (modified and unmodified), CHROMagar, and R & F) failed to support the growth and recovery of an O111 strain.  When a raw-milk cheese was used for agar evaluation, generic E. coli was recovered from all agars.  However, the appearance of E. coli on both Rainbow O157 agar types varied from pink to blue to purple and required multiple colony picks as compared to the more straightforward color differentiation or hemolysis (SHIBAM agar) offered by the other agars.

Significance:   Agar performance was highly strain-dependent and variable colony coloration made picking colonies, based on color, difficult.  Follow-up studies evaluating agar performance with additional panels of STEC strains is warranted.  Until then, the use of multiple agars (including one without supplements to decrease background microflora) is recommended.

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