Optimization of reverse transcriptase PCR to detect viable Shiga-toxin-producing Escherichia coli.

The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing Escherichia coli (STEC) was investigated. Four primer sets, each targeting a specific region in the slt-II operon, were evaluated for their stringency and specificity for slt-II mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of E. coli slt-II mRNA is useful for detection of viable STEC in ground beef.

Survival of Escherichia coli O157:H7 in buttermilk as affected by contamination point and storage temperature.

The effects of contamination point (during fermentation versus postfermentation) and storage temperature (5 and 12 degrees C) were determined for survival of Escherichia coli O157:H7 in fermented buttermilk. E. coli O157:H7 was recovered from buttermilk inoculated during fermentation for 22 days and in buttermilk inoculated postfermentation for 32 days. For storage temperatures of 5 and 12 degrees C, D-values were lower for E. coli O157:H7 inoculated during fermentation (2.5, 2.2 days) than postfermentation (5.6, 4.8 days) (P < 0.05). Developed acidity in inoculated buttermilks was not different from controls (P > 0.05). The extended recovery of viable enterohemorrhagic E. coli O157:H7 from both processing scenarios indicates that the presence of E. coli O157:H7 in buttermilk is not limited to postprocessing contamination.