Some chromogenic agars used to isolate E. coli O157:H7 rely on the inability of the organism to ferment sorbitol (Sorbitol-MacConkey or SMAC agar). Others depend on the organism’s inability to metabolize 4-methylumbelliferryl-b-D- glucuronide (MUG) due to inactive b-glucaronidase. However, sorbitol-fermenting O157:H7 and O157:HNM isolates have been isolated from patients with severe disease7,8 and have emerged as a major health problem in Europe.9 Further complicating the situation is the fact that most of the sorbitol-fermenting O157:HNM isolates do produce a functional b-glucaronidase and are also sensitive to telluride.
Thus, the three common culture methods for identifying E. coli 0157:H7 that rely on biochemical reactions are at risk to miss variants.
Genetic Detection Problems
The phenotypic variations that elude indirect techniques result from the E. coli O157:H7 genome being in a state of flux. In order to avoid similar misses, direct techniques that examine DNA structure must identify consistent markers that will not be lost as the genome shifts.
Several test developers have targeted virulence factors, following the logic that if the virulence factor isn’t present, the sample would not be pathogenic. Because the virulence factors occur not only in E. coli O157:H7, but also in other bacteria commonly associated with cattle, these methods must use an antigen-antibody screening prior to PCR to avoid widespread cross-reaction.
Two common markers for virulence are the shiga-like toxin genes (stx1 and stx2). E. coli O157:H7 may have one, both or neither of these genes. Furthermore, the stx genes are on prophages that pop in and out of the genome with such frequency that the stx genes become unreliable as markers.10 This explains why isolates from some patients with the most severe illness do not have either stx gene11 while others contain both stx-negative and stx-positive genes.
Another commonly targeted virulence marker is the attachment and effacement gene (eae). This gene encodes intimin, a protein used by the bacteria to attach to the intestinal wall. However, at least one documented outbreak of HUS has been caused by an eae-negative strain of E. coli O157:H7,12 indicating that lack of the eae strain does not necessarily mean lack of virulence.
Genetic Detection – An Improved Approach
Since the virulence factors may not be sufficiently consistent on the E. coli O157:H7 genome to give reliable results, a different approach is needed.
With new understanding of the variants described above, researchers at DuPont developed a multiplex PCR assay for detecting E. coli O157:H7 that provides more genetic information for additional cross-checks and greater reliability.
First, the developers looked for some rare genetic sequences in the more stable regions of the genome, rather than virulence factors that are carried on mobile genetic elements. Next, they examined those areas against a large panel of E. coli O157:H7, including rough and HNM varieties, along with closely related organisms. Finally, they added several sequences that were common to all the E. coli O157:H7 in the panel to the revised assay. This approach aims at providing a very high confidence level that variant strains will not be missed, and it is cross-reactive with only one non-O157:H7 serotype (O55:H7, the nearest genetic neighbor). This confidence has resulted in the USDA FSIS adopting the DuPont developed E. coli O157:H7 MP assay as its regulatory screening test for raw ground beef and beef trim.
Will a new variant of E. coli O157:H7 that defeats even this multiplex assay be identified? Although no one can predict the future, past experience has shown that the dynamic nature of E. coli O157:H7 tends to find an elusive spot in every detection method used for it so far. The challenge for scientists is to stay one step ahead of this potentially deadly pathogen. The challenge for food companies is to stay informed, focus on prevention and rely on proven, science-based detection methods to verify their food safety systems.
- USDA FSIS press release Feb. 28, 2005.
- USDA FSIS press release Feb. 23, 2006.
- Feng et al. (1998). J. Clin. Micro. (36): 2339-2341i.
- Vila et al. (1997). J. Clin. Micro. (35): 2279-2282.
- Barcoky-Gallagher et al. (2003). J. Food Protection (67):993-998.
- Taylor et al. (2002). J. Bacteriology (184): 4690-4698.
- Gunzer, F., Bohm, H., Russman, H., Bitzan, M., Aleksic, S., Karch, H. (1992). J. Clin. Micro. (30):1807-10.
- Fratamico, P.M., Buchanan, R.L., Cooke, P.H. (1993). Appl. Environ. Microbiol. (59): 4245-52.
- Karch and Bielaszewska (2001). J. Clin. Micro. (39):2043-2049.
- Shiakh and Tarr (2003). J. Bacteriology (185):3596-3605.
- Schmidt et al. (1999). J. Clin. Micro. (37):3491-3496.
- Paton et al. (1999). J. Clin. Micro. (37):3357-3361.
Frank Burns, Ph.D., is a senior scientist for DuPont Qualicon (Wilmington, Del.). Reach him at 302-695-5300.