Determining the Fate of NON-O157 Shiga Toxin-Producing Escherichia Coli in Dairy Compost During Storage using the Optimized Detection Method

Shiga toxin-producing Escherichia coli (STEC), a member of Enterobacteriaceae family, has been recognized as emerging pathogens. Dairy compost is commonly applied to farmland as a soil amendment. Despite the agricultural benefit of manure-based soil amendment, the inadequately treated compost can contribute to fresh produce contamination on the farm. Moreover, the epidemiological results showed that the non-O157 STEC cases have surpassed those of E. coli O157. Therefore, it is critical to evaluate the behavior of non-O157 STEC strains in the dairy manure-based compost. The objectives of this study were to: 1) optimize a culturing method for detecting non-O157 STEC from dairy compost, 2) determine the growth potential of top six non-O157 STEC serovars in dairy compost, and 3) conduct a persistence study of non-O157 STEC in dairy compost being held at room temperature. First, we optimized a culturing method for detecting STEC during enrichment. Cefixime-tellurite Sorbitol MacConkey Agar supplemented with 5 mg/l novobiocin (CTN-SMAC) was chosen for enumerating non-O157 STEC cells before or after enrichment, as CTN-SMAC is more cost effective than Modified Rainbow Agar (mRBA) and both agar plates enumerated the same level of STEC. The single step selective enrichment recovered ca. 0.54 log CFU/g more cells as compared to the two-step enrichment. In addition, without enrichment step, the detection limit of individual STEC serovar ranged from 250 to 2,500 CFU/g in dairy compost. For STEC O26 and O145, the detection limit by IMS was 2,500 CFU/g, but for other STEC serotypes (O45, O103, O111, and O121), the detection limit was 250 CFU/g. Our results demonstrated that a low level of STEC (ca. 100 CFU/g) could be detected within one day from dairy compost by culturing method through optimized enrichment procedure followed by immunomagnetic beads separation (IMS). Next, we investigated the survival potential of non-O157 STEC in dairy compost during storage at room temperature. A mixture of six non-O157 STEC serovars was inoculated into commercially available dairy compost with 30% moisture content at a final concentration of ca. 5.5 log CFU/g. During storage at room temperature for up to 42 days, STEC counts and other factors such as indigenous microorganism population, moisture contents and pH were analyzed at selected sampling intervals. Both moisture contents and pH values in dairy compost remained unchanged (p\u3e0.05) during the entire duration of trials, and so did the background bacterial level. As for the STEC population, a growth of ca. 0.5 log CFU/g was recorded within the first day post inoculation, followed by a rapid decrease of ca. 1.5 log CFU/g during 14 days of storage. By the end of the experiment, the population level of non-O157 STEC reduced ca. 1.7 logs, and the survival curve displayed an extensive tailing. Randomly selected colonies from the last 3 sampling times were confirmed as STEC by PCR. Our results demonstrated that low-level of STEC could be detected within one day from the finished dairy compost by culturing method through optimized enrichment procedure followed by IMS, and non-O157 STEC persisted in dairy compost for at least 42 days, indicating the long-term survival of non-O157 STEC in the finished dairy compost. Therefore, proper handling and testing of the finished dairy compost as soil amendment is critical for ensuring the microbiological safety of fresh produce and the farm environment