Project Summary

Escherichia coli O157:H7 Indicator Organism: Evaluation of Bovine-Associated Bacteria

Principle Investigator(s):
Mohammad Koohmaraie, Ph.D.
USDA-ARS U.S. Meat Animal Research Center
Completion Date:
June 2004



Beef processors have a need for quick, easily administered, accurate and cost-effective tests for detection of E. coli O157:H7. Historically, enumeration of various bacterial populations (total aerobic bacteria, Enterobacteriaceae, generic E. coli, etc.) has been used to give an indication of trends in pathogen population. These methods show the presence of the pathogen but do not estimate total numbers. Conversely, tests evaluating pathogen populations directly, especially E. coli O157:H7, are more costly, labor intensive and take multiple days to generate results. There are a variety of methods to identify members of diverse bacterial populations. Biochemical assay-based methods have traditionally been used to classify individual strains. These methods are slow and the results are biased towards those strains that are amenable to cultivation in the lab.

Molecular-based methods are now being used more frequently and they don’t require culturing of the organism prior to identification. One such method is denaturing gradient gel electrophoresis (DGGE). DGGE classifies organisms based on the melting profile from amplified segments of target genes. For the purposes of this study, the 16S ribosomal RNA gene served as the target gene. The genus and species of several bacteria can be derived based on the unique sequence of the 16S rRNA gene. The constituents of the microbial populations will be determined without enrichment, thus reducing bias towards those strains amenable to in vitro culture.

The objective of this study was to determine if a bacterial strain from the bovine-associated bacterial population cans serve as an indicator organism E. coli O157:H7.


A total of 150 carcass samples and 87 fecal samples were collected from two large, commercial processing plants and assayed for the presence of E. coli O157:H7. Isolates of E. coli O157:H7 were obtained from 84 (56%) of the carcass samples and 16 (18%) of the fecal samples. Total genomic DNA was obtained prior to enrichment from all 237 samples and was stored at –20°C. From the total genomic DNA, a portion of the 16S rDNA gene was amplified with highly conserved “universal primers” using the method described by Zoetendal et al. The fragments from different bacterial species contain variations in the DNA sequence and therefore have different melting temperatures. PCR reactions using such primers have been shown to amplify the rDNA sequences from several different bacterial genera. These reactions were expected to generate a comprehensive view of the microbial population present in each sample. Cumulative profiles for 30 fecal samples (15 E. coli O157:H7-positive and 15 E. coli O157:H7-negative) were obtained. As expected, these profiles showed excellent diversity of the bacterial communities contained in each sample. However, this great diversity of species caused the DGGE patterns to be too crowded for effective comparisons to be made.

To reduce the complexity of DGGE banding patterns, two bacterial groups were targeted: Lactic acid bacteria and Bifidobacteria. Each has been associated with antimicrobial activity against E. coli O157:H7.


It was believed that the presence of various members of the lactic acid bacteria or Bifidobateria might have a negative correlation with the presence of E. coli O157:H7. Unfortunately, such a relationship was not identified.


From this study, no bacterial species could be identified that correlated (positively or negatively) with the presence of E. coli O157:H7. Further study is needed to screen various bacterial groups for identification of a relationship.