Identification of Escherichia coli O157:H7 Genomic Regions Conserved in Strains with a Genotype Associated with Human Infection

Marina Steele, Kim Ziebell, Yongxiang Zhang, Andrew Benson, Paulina Konczy, Roger Johnson, and Victor Gannon

Applied and Environmental Microbiology 73(1), Jan. 2007, 22-31.


I'll presume we all know that E. coli O157:H7 is code for the most common seriously pathogenic form of E. coli currently present in the food supply. The numbers and letters represent the surface 'antigens' that are used to do a preliminary identification of strains of bacteria - the antigens are related to the structures on a bacterial cell surface that are accessible, presumably, to the immune system - but also accessible to assays for testing cell type. Because they are accessible to the immune system, antibodies (particularly monoclonals) can be generated to them and then used in immunoassays, which are very rapid and sensitive. The now classic 'ELISA' (enzyme-linked immunosorbent assay) allows single cells to be identified by their antigens almost immediately. The O antigen is part of the "LPS" of the gram negative cell wall, it is made of sugars. The K antigen is part of the 'capsule' layer - a protective layer of polymerized sugars more loosely connected with the cell. H antigens are derived from the flagella, which are also important to the cell in colonizing a host and are exposed on the cell surface.

Anyway, these antigens can also be used as markers for strongly selected genetic markers - certain antigens are better under certain conditions; particularly, some are more useful for evading predation (diversifying selection acts in this case) and some are more useful for evading the immune system.
Whatever the case, there are hundreds of antigens (~200 O, ~100 K, ~55 H). This allows for tens of thousands of combinations, helping sort the E. coli into clonal monophyletic populations. Of course, it is not necessary that this be true for any given combination, that it is clonal or monophyletic.

Everyone cares about O157:H7 because it keeps showing up in the ill and recently deceased - and in their spinach, tacos, etc. It also shows up in the rectum of ruminants (cattle, sheep) where it doesn't seem to cause the animals any trouble. Certainly, these E. coli have learned some tricks, and modern agriculture encourages their spread. One argument goes that animals being fed grain, not hay, has changed the stomach conditions to permit these E. coli to spread more readily (Diez-Gonzalez et al, 1998; Russell et al, 2000; Russell et al, 2000). The idea is that the E. coli are becoming acid tolerant. Stomach acid is one of the main barriers for humans to prevent infection by bacteria; it protects the established symbionts against temporary invaders. Without going into too much discussion of this point, there is reason to doubt the conclusion that the E. coli are really being selected for acid resistance by cattle diets (Carolyn J. Hovde, Paula R. Austin, Karen A. Cloud, Christopher J. Williams, and Carl W. Hunt, Applied and Environmental Microbiology, 65(7), July 1999, 3233-3235; Grauke LJ, Wynia SA,Sheng HQ, Yoon JW, Williams CJ, Hunt CW, Hovde CJ. Vet Microbiol. 2003 Sep 1;95(3):211-25; Science, April 1999, Vol. 284. no. 5411, p. 49; reviewed, "E. coli O157:H7 in hay- or grain-fed cattle," Dale Hancock and Tom Besser, October 12, 2006, citation not obvious). The evidence for this appears to be more 'suggestive' than 'conclusive.' Anyway, simply lowering the infectious dose may make it easier to become ill, but there must be other things going on to cause O157:H7 to be a problem.

One may be that these E. coli have learned tricks to get into plant roots. There are lots of studies about this issue as well:
JV. Gagliardi and JS. Karns. Environmental Microbiology 2002, Volume 4(2) 89.
Warriner K, Ibrahim F, Dickinson M, Wright C, Waites WM. J Food Prot. 2003 Oct;66(10):1790-7.
The second is interesting in that it presages the spinach problem by showing that E. coli can get internal to spinach cells, so the bacteria could never be washed off. Thus, eating raw spinach fertilized 'organically' with pathogen contaminated manure might create a hazard that cannot be dealt with by surface sterilization.

Anyway, back to the topic of the paper at hand: the relationships among these 0157:H7 bacteria that are causing all this mess.
There are apparently two major families of these bacteria. While even the dominant human pathogens don't seem to cause disease in animals, it seems from most experiments I've seen, that they also don't colonize the animals particularly stably. This doesn't really surprise me - stable intestinal colonization of a conventional (previously naturally colonized by bacteria) host by a laboratory isolate is pretty difficult, in the literature and in my experience. This is true even with large doses of the inocula.

As an aside, infectious doses vary greatly, and it may depend on everything short of the phase of the moon - and even that, in women. One of the great stories of early microbiology was when Koch was trying to demonstrate the germ theory of disease, and he had isolated the cholera causative agent (Vibrio cholera). He had a huge flask of this nasty bug, and another science professor, doubting Koch, postulates and all, decided to demonstrate his scorn. He drank the entire flask of the supposed cause of misery and massive suffering, still one of the most serious diseases - and he didn't get sick. I guess he had taken his vitamins.

Anyway, there are the human-derived Lineage I strains and the bovine Lineage II strains of E. coli O157:H7. they both appear around the world, but one tends to be in the cattle, the other, in disease. They appear to have diverged prior to global dispersal. So, perhaps, all those O157:H7 in cattle that people are all stressed about aren't a big deal... The authors in this paper decided to figure out what genes were different among the lineages. They found a bunch of regions that were different, and then characterized them to look for virulence factors which may make Lineage I a problem and Lineage II a bunch of bullcrap, literally.

They used 30 strains for the secondary screen - 10 Lineage I and 20 Lineage II, to confirm the differences found in a smaller primary set of 4 strains. Then they did a tertiary screen with 119 additional strains.

They found a bunch of regions that were relevant. The big finding is that most of these were associated with potential viruses, plasmids, and transposons - mobile genetic elements that can thereby change their background and associations. This begins to explain the apparent recent origin of the pathogens. Some of the genes look like obvious problems - colonization elements, hemolysin genes, outer membrane molecules. Others are not obvious.

One thing that this says is that these factors might reassemble in different combinations in other backgrounds. O157:H7 might have just assembled all the cool bad genes, a sort of Ocean's 11 of E. coli, all at once.

Another interesting thing is that it looks like 'lineage II' is just lineage I that has lost elements - perhaps there is selection against virulence elements in the cattle. Perhaps there is general pressure to be less virulent, not more. this would be very good for us and our food supply.

This is a very cool article on molecular ecology and evolution in the context of pathogenesis and symbiosis. It makes for some cool findings and begins to suggest some interesting approaches for prevention and therapy. For instance, curing bacteria of phage would likely be enough to eliminate many virulence factors without killing the cells. Another interesting point - if we can keep bacteria from being infected with phage, they might not emerge to virulence, if that is a major source of virulence factors which are otherwise being purified by selection.