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The U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex microbead-based suspension array used to screen colonies for 11 clinically relevant Shiga toxin-producing Escherichia coli (STEC) serogroups: 026, 045, 091, 0103, 0104, 0111, 0113, 0121, 0128, 0145, and 0157. We evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No falsepositive results were obtained. Of the 112 samples with a reported cycle threshold (CT) value, 101 undiluted, diluted, or extracted enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR CT values approached or were greater than 35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM Chapter 4a, the STEC tnicrobead-based suspension array can accurately screen food enrichment cultures. [ABSTRACT FROM AUTHOR]

The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce – bagged baby spinach, alfalfa sprouts, cilantro, and raw milk – we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:–, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices. [ABSTRACT FROM AUTHOR]

Abstract: The FDA Bacteriological Analytical Manual (BAM) method for the detection/isolation of Shiga toxin-producing Escherichia coli (STEC) involves enrichment of produce rinses, blended homogenates or stomached homogenates. However, the effectiveness of rinsing produce to remove attached bacteria is largely unknown. Moreover, PCR inhibitors can be released under physical treatment. The study objective was to determine the relative effectiveness of recovery methods for STEC contaminated produce. Spinach, lettuce, and cilantro were contaminated with E. coli O157:H7 or a non-O157 STEC, subjected to both the BAM method and a soak method, and tested by real-time PCR and cultural methods. For O157:H7 and non-O157:H7 STECs, the soak method was significantly more productive than leafy green rinses. Of 320 test portions, PCR of recovered colonies confirmed 148 were positive by rinsing and 271 were positive by soaking (an 83% increase in sensitivity). For recovery of O157:H7 from cilantro, of 60 test portions, positives were 38 by soaking, 41 by stomaching, and 28 by blending. Soaking and stomaching were significantly more productive than blending, although soaking was only arithmetically superior to stomaching. Based upon these results, it is recommended that a soak method replace the current BAM procedures. [Copyright &y& Elsevier]