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United States Department of Agriculture

Agricultural Research Service

Research Project: CONTROL OF PATHOGENIC AND SPOILAGE BACTERIA ON RED MEAT

Location: Meat Safety & Quality Research

Title: Comparison of Colony Hybridization to Phenotype Screening on Washed Sheep's Blood Agar for the Isolation of Shiga toxin Producing Escherichia coli from Complex Matrixes

Authors
item BOSILEVAC, JOSEPH
item Koohmaraie, Mohammad - FORMER ARS EMPLOYEE

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 26, 2009
Publication Date: May 13, 2009
Citation: Bosilevac, J.M., Koohmaraie, M. 2009. Comparison of Colony Hybridization to Phenotype Screening on Washed Sheep's Blood Agar for the Isolation of Shiga toxin Producing Escherichia coli from Complex Matrixes [abstract]. 7th International Symposium on Shiga Toxin (Verocytotoxin)-Producing Escherichia coli Infections (VTEC2009) Paper No. P02.5.3. p. 106.

Interpretive Summary: No interpretive summary is required.

Technical Abstract: Purpose of study: Isolating Shiga toxin-producing Escherichia coli (STEC) from complex matrixes such as ground beef is a lengthy and laborious process. In our previous studies of STEC present in beef production, colony hybridization was used to identify suspect stx containing colonies for further characterization. Recently we have switched to using washed sheep’s blood agar containing mitomycin (WBAM) to phenotypically identify suspect STEC colonies. Differences between these two methods in the isolation rates and serogroup specificities of STEC have not been described. Methods: The success of isolating STEC using WBAM compared to stx colony hybridization was evaluated. Samples of beef (n = 186) that had screened positive for stx1 and(or) stx2 by PCR were used in each method. Isolates were serotyped using PCR and serological methods. The presence of virulence genes in each isolate was also determined using PCR. Results: Colony hybridization identified 80 STEC isolates in 67 samples, whereas WBAM identified 60 STEC isolates in 51 samples. Both methods isolated STEC from the same 44 samples, and 20 of these were of the same serotype, and presumably the same strain. In the other 24 cases, each method found a different serotype in the same sample. Colony hybridization found isolates in 23 samples WBAM did not, and WBAM found isolates in 7 samples colony hybridization did not. While colony hybridization found more isolates, the number was not quite statistically different (P=0.0953) from WBAM. WBAM isolated 24 different serogroups of STEC and colony hybridization isolated 23 different serogroups. When other virulence genes, such as intimin (eae), were considered, colony hybridization identified 3 eae positive isolates, while the WBAM method allowed isolation of 4 eae positive isolates. The use of WBAM also identified a serogroup O121, (a CDC top six human disease related non-O157 STEC) that was not found in the same samples using colony hybridization. The WBAM method allowed a single technician to process all 186 samples in a week, whereas three technicians required 3 weeks to process the 186 samples through the colony hybridization method. Conclusion: Even though fewer total isolates were recovered using WBAM instead of colony hybridization, the diversity of STEC recovered, and the number of STEC more commonly found in human disease were equal or greater using WBAM. This and the described increased throughput has made WBAM our preferred method for STEC isolation.

Last Modified: 7/25/2014