Header graphic for print
Marler Blog Providing Commentary on Food Poisoning Outbreaks & Litigation

Where the Hell did Shiga Toxin E. coli come from? A Literature Review – Part 2

The Discovery of E. coli O157:H7 as a Cause of Human Disease

Dr R.jpgThe discovery of E. coli O157:H7 as a cause of human disease was a relatively humble, unpublicized event in comparison to the identification of the E. coli O104:H4 strain that exploded in international headlines during the summer of 2011 following an outbreak in Germany (see Part 1 of this series).

In contrast, the E. coli O157:H7 story emerged quietly in 1975, two years after the Centers for Disease Control and Prevention (CDC) implemented a serotyping service for E. coli isolates.  That year, the CDC reference laboratory received a single E. coli strain from a California patient with bloody diarrhea, which typed as “O” antigen 157 and “H” antigen 7 (27).  It is worth noting that the E. coli O157 serotype was also described previously in the veterinary literature in the early 1970’s, but not linked to human illness (8-9).

The CDC subsequently described four sporadic illnesses caused by E. coli O157:H7, a publication that received little fanfare in a 1982 issue of CDC’s Morbidity and Mortality Weekly Report (3).  The original write-up was later highlighted in a 1997 MMWR commemoration of the agency’s 50th anniversary (4). 

Looking back, the earliest recognized outbreak may have occurred in 1980, as described by Steele and colleagues (25).  The authors reported an outbreak of hemolytic uremic syndrome (HUS) in Toronto, Canada associated with ingestion of fresh apple juice.  The etiologic agent was not identified during the investigation, but it is likely the illnesses were due to E. coli O157:H7 infections (4).  Another early outbreak of HUS probably due to E. coli O157:H7 was reported in Sacramento, California in July and November 1982, but the agent was not isolated from patient specimens (24).

In February and March 1982, the obscure E. coli O157:H7 strain was “re-discovered” during the investigation of two outbreaks of hemorrhagic colitis in Oregon and Michigan, respectively (22).  Dr. Lee Riley (pictured above) and colleagues authored the first paper describing an outbreak of hemorrhagic colitis associated with E. coli O157:H7.  The outbreaks involved 47 people who became ill from consumption of undercooked hamburgers prepared by Restaurant A (McDonalds) restaurant chains in Oregon and Michigan.  E. coli O157:H7 was isolated from patient stool, and the “smoking gun” was found in a raw ground beef hamburger patty from one of the fast-food restaurants implicated during the outbreak investigation (22, 27). 

During the same year, Canadian health officials were investigating cases of hemorrhagic colitis and diagnosed E. coli O157:H7 as the cause of another outbreak (10).  At this point, it appeared E. coli O157:H7 was a rare emerging foodborne disease in North America.

Throughout the 1980’s additional reports of sporadic- and outbreak-associated E. coli O157:H7 were published with a predominance of illnesses among children and the elderly (17, 21, 26).  However, the bacterium did not become a “household name” until 1993, when it made national headlines following a highly publicized multistate outbreak due to consumption of undercooked hamburgers served at Jack in the Box fast-food restaurants in the western United States (2).  With approximately 700 illnesses and 4 deaths, this outbreak was the impetus for policy changes in the ground beef industry, and pushed health officials in 1995 to add E. coli O157:H7 to the list of nationally reportable diseases the United States.  Likewise, Health Canada classified E. coli O157:H7 as a nationally notifiable disease in 1990.  E. coli O157:H7 infections from a wide range of food vehicles, as well as person-to-person transmission and direct contact with animals, have been described worldwide since the pathogen’s discovery in North America.

Did E. coli O157:H7 Cause Human Illness Before the 1970s-1980s?

An intriguing question is whether E. coli O157:H7 was an unrecognized cause of HUS before its link to outbreaks of hemorrhagic colitis.  E. coli O157:H7 is now recognized as the leading cause of HUS in children.  Karmali et al (1985) published an early report describing the association between HUS and vero (shiga) toxin-producing E. coli (11).  Interestingly, the authors point out that HUS was first described in 1955, which was twenty-years before the identification of E. coli O157:H7 as a human pathogen and pre-dated development of diagnostic assays for the strain.  In 1963, the first outbreak of HUS was reported in Wales (15).  Notably, the cases were all among children (range 6 weeks to 8 years old), and 7 of 10 patients had gastrointestinal illness with or without bloody diarrhea preceding onset of HUS.  The cause of the outbreak was not determined, but the authors speculated that the syndrome was initiated by an exogenous agent.  Was the cause E. coli O157:H7? 

It is unknown how many early cases of E. coli O157:H7 were missed pre-1970’s due to lack of awareness, diagnostic tests, and reporting.  Diagnosticians realized early on the need for appropriate laboratory tests to detect E. coli O157:H7, as well as proper specimen collection (4).  Even today, diagnosis of E. coli O157:H7 and other shiga toxin-producing E. coli (STEC) strains may be challenging and is dependent on the use of specific protocols for diagnosis in clinical, food, and environmental specimens.

Given what we know today about evolutionary changes and pathogenesis, these early HUS reports may very well have been due to E. coli O157:H7 or another STEC.

Was E. coli O157:H7 Born in a Feedlot in 1982?  Not According to the Molecular Clock

The molecular revolution in bacterial genetics began to really take off in the 1990’s with technological advances allowing researchers to study the genome sequences of microorganisms and identify genes associated pathogenic strains.  The complete genome sequence of E. coli O157:H7 was published in Nature in 2001 (19).  The first sequenced strain, EDL933, was isolated from hamburger meat associated with the 1982 McDonald’s outbreak (22).

Molecular biologists use the term “molecular clock” to describe the theory that evolutionary changes in bacterial strains can be measured over time by studying mutations in specific DNA sequences or the proteins they encode.  The theory assumes that these spontaneous mutations occur at constant rates, which allows calculation of an estimate of how long ago two related organisms diverged from a common ancestor.

The molecular clock theory is still a hypothesis and time estimates vary widely between papers depending on the genes or proteins studied and the methodology (28).  In a 2000 paper from the Whittman Lab, seven housekeeping genes and phylogenetic analysis were used to trace the history of virulence gene acquisition in pathogenic E. coli (20).  The authors estimated that E. coli O157:H7 separated from a common E. coli ancestor as long as 4.5 million years ago.  However, in a more recent paper, Leopold and co-authors examined E. coli O157:H7 strains from three continents over three decades from humans, cattle, and food (13).  They estimated the dominant E. coli O157:H7 cluster evolved on the in last millennium.  Similarly, Zhou et al (2010) conducted an analysis of mutation frequency to estimate the divergence of E. coli O157:H7 from E. coli O55:H7, its closest genetic relative (32).  These authors estimated the divergence occurred as recently as 400 years ago, or from 14,000 to 70,000 years ago depending on the clock rate used. 

“Stepwise” Long-term Evolution of Shiga Toxin-Producing E. coli

Feng and colleagues described a “stepwise” model for sequential evolution of E. coli O157:H7 from an ancestor (7). The model shown in the figure below suggests an explanation for  gentotypic and phenotypic differences between strains including changes in acquisition of shiga toxin genes, gain/loss of motility, and the ability to ferment sorbitol.  These findings have been exploited in the development of diagnostic assays including protocols that detect E. coli O157 based on a point mutation at +92 in the uidA gene (30-31).

3031.png

Figure 1. Evolution model for Escherichia coli O157:H7. Figure modified and updated from (1) to include the sequence type (ST) data showing subclones within clonal complexes. Some strains, whose position on the model remains to be determined, are shown with dashed lines (7).

The Whittam Lab subsequently published a detailed genetic analysis of E. coli O157:H7 associated with disease outbreaks thereby providing a deeper understanding of the relationship between genetics and pathogenesis (14).  They discovered nine distinct clades with Clade 8 containing strains linked to spinach and lettuce outbreaks that caused unusually high rates of hospitalizations and HUS.  The 1982 outbreak strain reported by Riley et al (1983) belonged to Clade 3, while the 1993 outbreak strain linked to Jack in the Box hamburgers belonged to clade 2.

JITB.jpg

Figure 2.  The phylogenetic network applied to 48 parsimoniously informative (PI) sites using the Neighbor-net algorithm for 528 E. coli O157 strains. The colored ellipses mark clades supported in the minimum evolution phylogeny. The numbers at the nodes denote the SNP genotypes (SGs) 1–39, and the white circle nodes contain two SGs that match at the 48 PI sites. The seven SGs found among multiple continents are marked with squares (14).

The Relentless Short-term Evolution of Pathogenic E. coli

Regardless of whether the E. coli O157:H7 serotype evolved hundreds or millions of years ago, it certainly did not suddenly mutate in a U.S. feedlot 30 years ago then spread worldwide as suggested by Pollan and other foodists.  However, their broad statements are supported, in part, by the ability of pathogenic E. coli strains to rapidly gain or lose virulence determinants through genetic mutations and gene transfer (5, 12, 18, 31).  These genetic changes can impact the ecology, epidemiology and pathogenesis of E. coli O157:H7 in a relatively short time period.  Specifically, mobile genetic elements such as bacteriophages and plasmids play a major role in the genetics of pathogenic E. coli, and can change significantly the virulence of a population of E. coli O157:H7 in as short as a single generation.    

Dr. Roy M. Robins-Browne reminds us of this relentless evolution of E. coli in an editorial published in Clinical Infectious Diseases (23):

“…bacterial evolution is an ongoing process that undoubtedly will lead to the emergence of other successful pathogenic clones of E. coli in the future.”

Notably, early in the recognition of E. coli O157:H7 as a human pathogen, shiga toxins (also referred to as vero toxins or cytotoxins) were characterized as important virulence factors in the pathogenesis of the syndrome (16).  These toxins do not originate from the bacterium, but rather from two lambda-like bacteriophages that infect the E. coli O157:H7 cell then integrate into its chromosome (23).  Bacteriophages carry the stx1 and stx2 genes responsible for shiga toxin-related disease.  We now understand that these toxins act like the ricin causing damage to the vascular system and potentially leading to HUS.  Other accessory virulence determinants include “pathogenicity islands” such as the locus for enterocyte effacement (LEE), which contains genes encoding virulence factors that assist E. coli in host invasion and influence the manifestation of diarrhea and other gastrointestinal signs and symptoms (23).

As shown in the diagram below, the hallmark of a pathogenic strain of E. coli is the presence of these additional mobile elements or gene clusters that convert a harmless, commensal E. coli strain into a potentially dangerous human pathogen (1). 

genetics.jpg

Figure 3. Contribution of horizontal acquisition of mobile genetic elements to the evolution of Escherichia coli pathotypes.The uptake of mobile genetic elements (phages, virulence plasmids and pathogenicity islands), as well as the loss of chromosomal-DNA regions in different E. coli lineages, has enabled the evolution of separate clones, which belong to different E. coli pathotypes and are associated with specific disease symptoms. LEE, locus of enterocyte effacement; PAI, pathogenicity island; pEAF, enteropathogenic E. coli adhesion-factor plasmid; pENT, enterotoxin-encoding plasmids; Stx, Shiga-toxin-encoding bacteriophage (1).

Summary

In summary, an overview of the literature demonstrates the importance of differentiating between a newly recognized pathogen versus a newly evolved organism.  The E. coli O157:H7 serotype clearly is not “new,” although its remarkable ability to evolve and adapt to different environments, particularly through acquisition and loss of mobile genetic elements, may result in new mechanisms of transmission and strain-dependent variations in pathogenicity. 

In Part 3, the role of agriculture, food distribution systems, and human activities in the spread of E. coli O157:H7 will be explored. 

Did humans turn an obscure E. coli strain into a dangerous emerging infectious disease?

References

  1. Ahmed, N., U. Dobrindt, J. Hacker, and S. E. Hasnain. 2008. Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention. Nat Rev Microbiol 6:387-94.
  2. Bell, B. P., M. Goldoft, P. M. Griffin, M. A. Davis, D. C. Gordon, P. I. Tarr, C. A. Bartleson, J. H. Lewis, T. J. Barrett, J. G. Wells, and et al. 1994. A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. JAMA 272:1349-53.
  3. CDC. 1982. Isolation of E. coli O157:H7 from sporadic cases of hemorrhagic colitis – United States. MMWR Morb Mortal Wkly Rep 31:580, 585.
  4. CDC. 1997. Isolation of E. coli O157:H7 from sporadic cases of hemorrhagic colitis–United States. 1982. MMWR Morb Mortal Wkly Rep 46:700-4.
  5. Coombes, B. K., M. W. Gilmour, and C. D. Goodman. 2011. The evolution of virulence in non-O157 shiga toxin-producing Escherichia coli. Front Microbiol 2:90.
  6. Donnenberg, M. S., and T. S. Whittam. 2001. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J Clin Invest 107:539-48.
  7. Feng, P. C., S. R. Monday, D. W. Lacher, L. Allison, A. Siitonen, C. Keys, M. Eklund, H. Nagano, H. Karch, J. Keen, and T. S. Whittam. 2007. Genetic diversity among clonal lineages within Escherichia coli O157:H7 stepwise evolutionary model. Emerg Infect Dis 13:1701-6.
  8. Furowicz, A. J., and F. Orskov. 1972. Two new Escherichia coli O antigens, O150 and O157, and one new K antigen, K92, in strains isolated from veterinary diseases. Acta Pathol Microbiol Scand B Microbiol Immunol 80:441-4.
  9. Glantz, P. J., C. L. Gyles, J. Greenfield, and F. Otskov. 1973. Isolation of Escherichia coli O157 from pigs with colibacillosis in Canada and the United States. Can J Comp Med 37:200-2.
  10. Johnson, W. M., H. Lior, and G. S. Bezanson. 1983. Cytotoxic Escherichia coli O157:H7 associated with haemorrhagic colitis in Canada. Lancet 1:76.
  11. Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis 151:775-82.
  12. Kudva, I. T., P. S. Evans, N. T. Perna, T. J. Barrett, F. M. Ausubel, F. R. Blattner, and S. B. Calderwood. 2002. Strains of Escherichia coli O157:H7 differ primarily by insertions or deletions, not single-nucleotide polymorphisms. J Bacteriol 184:1873-9.
  13. Leopold, S. R., V. Magrini, N. J. Holt, N. Shaikh, E. R. Mardis, J. Cagno, Y. Ogura, A. Iguchi, T. Hayashi, A. Mellmann, H. Karch, T. E. Besser, S. A. Sawyer, T. S. Whittam, and P. I. Tarr. 2009. A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis. Proc Natl Acad Sci U S A 106:8713-8.
  14. Manning, S. D., A. S. Motiwala, A. C. Springman, W. Qi, D. W. Lacher, L. M. Ouellette, J. M. Mladonicky, P. Somsel, J. T. Rudrik, S. E. Dietrich, W. Zhang, B. Swaminathan, D. Alland, and T. S. Whittam. 2008. Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks. Proc Natl Acad Sci U S A 105:4868-73.
  15. McLean, M. M., C. H. Jones, and D. A. Sutherland. 1966. Haemolytic-uraemic syndrome. A report of an outbreak. Arch Dis Child 41:76-81.
  16. O’Brien, A. O., T. A. Lively, M. E. Chen, S. W. Rothman, and S. B. Formal. 1983. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (SHIGA) like cytotoxin. Lancet 1:702.
  17. Pai, C. H., R. Gordon, H. V. Sims, and L. E. Bryan. 1984. Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7. Clinical, epidemiologic, and bacteriologic features. Ann Intern Med 101:738-42.
  18. Perna, N. T., G. F. Mayhew, G. Posfai, S. Elliott, M. S. Donnenberg, J. B. Kaper, and F. R. Blattner. 1998. Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 66:3810-7.
  19. Perna, N. T., G. Plunkett, 3rd, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-33.
  20. Reid, S. D., C. J. Herbelin, A. C. Bumbaugh, R. K. Selander, and T. S. Whittam. 2000. Parallel evolution of virulence in pathogenic Escherichia coli. Nature 406:64-7.
  21. Remis, R. S., K. L. MacDonald, L. W. Riley, N. D. Puhr, J. G. Wells, B. R. Davis, P. A. Blake, and M. L. Cohen. 1984. Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7. Ann Intern Med 101:624-6.
  22. Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 308:681-5.
  23. Robins-Browne, R. M. 2005. The relentless evolution of pathogenic Escherichia coli. Clin Infect Dis 41:793-4.
  24. Rogers, M. F., L. D. Budnick, I. Kirson, E. S. Hurwitz, M. H. Hatch, C. A. Bopp, M. A. Karmali, G. W. Gary, R. Layne, and L. B. Schonberger. 1986. Hemolytic-uremic syndrome–an outbreak in Sacramento, California. West J Med 144:169-73.
  25. Steele, B. T., N. Murphy, G. S. Arbus, and C. P. Rance. 1982. An outbreak of hemolytic uremic syndrome associated with ingestion of fresh apple juice. J Pediatr 101:963-5.
  26. Symonds, J. 1988. Haemorrhagic colitis and Escherichia coli O157–a pathogen unmasked. Br Med J (Clin Res Ed) 296:875-6.
  27. Wells, J. G., B. R. Davis, I. K. Wachsmuth, L. W. Riley, R. S. Remis, R. Sokolow, and G. K. Morris. 1983. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J Clin Microbiol 18:512-20.
  28. Whittam, T. S., S. D. Reid, and R. K. Selander. 1998. Mutators and long-term molecular evolution of pathogenic Escherichia coli O157:H7. Emerg Infect Dis 4:615-7.
  29. Yoshitomi, K. J., K. C. Jinneman, and S. D. Weagant. 2006. Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR. Mol Cell Probes 20:31-41.
  30. Yoshitomi, K. J., K. C. Jinneman, and S. D. Weagant. 2003. Optimization of a 3′-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR. Mol Cell Probes 17:275-80.
  31. Zhang, Y., C. Laing, M. Steele, K. Ziebell, R. Johnson, A. K. Benson, E. Taboada, and V. P. Gannon. 2007. Genome evolution in major Escherichia coli O157:H7 lineages. BMC Genomics 8:121.
  32. Zhou, Z., X. Li, B. Liu, L. Beutin, J. Xu, Y. Ren, L. Feng, R. Lan, P. R. Reeves, and L. Wang. 2010. Derivation of Escherichia coli O157:H7 from its O55:H7 precursor. PLoS One 5:e8700.
  • http://www.thecompletepatient.com David Gumpert

    Great, highly readable historical analysis. Thanks for setting O157 in context:: …”it certainly did not suddenly mutate in a U.S. feedlot 30 years ago then spread worldwide as suggested by Pollan and other foodists. However, their broad statements are supported, in part, by the ability of pathogenic E. coli strains to rapidly gain or lose virulence determinants through genetic mutations and gene transfer.”

  • Dan Cohen, Maccabee Seed Co. Davis CA

    Bill,

    My view goes briefly as follows.

    The question is the weaponization of O157:H7 as an effective, virulent human pathogen and not the long term evolution of the O157:H7 serotype.

    I remember many O antigen serotypes were associated with cattle in particular in the 1960′s and it is not surprising that O157 also was noted in the veterinary literature. What changes were required for O157′s — in this case — to (first) transform into a human associated disease and (second) become a human disease with serious and sometimes devastating consequences, that is become “weaponized”?

    The molecular clock assumptions are dependent on knowing the rate of “spontaneous mutations in housekeeping genes” that otherwise remain stable; at best the clocks measure genetic distance (without time as an independent constant factor) and various assumptions are made to add time as a linear inference. It’s not bad for guessing at vertebrate species evolution, much more problematic with bacterial evolution where microenvironmental changes cause tremendous selection pressure, and the ability to genetically respond to such changes is itself highly evolved, and not dependent on an assumed-to-be-constant spontaneous mutation rate.

    In contrast virulence factors change during the course of a single infection, as noted in the same German studies mentioned in my FSN article “A Parallel Clinical Pattern”.

    When I look at changes in selection pressures that could have enabled new pathogen emergence, the following stand out for cattle and dairy: the routine use of antibiotics, either not for specific disease symptoms or to make up for poor routine handling; the refeeding of manures to cattle and dairy; the refeeding of cattle meat byproducts to cattle. The last included brains and nerve tissue which lead to the emergence of BSE (mad cow disease) not just in cattle but then in dairy associated cats (“mad cat”) and finally in humans (BSE).

    All of these were economist driven changes in feeding practices, recommendations made in an absence of considering biology but just “nutritional value” after the second world war. They pre-date feedlots and CAFO’s and can occur on ranch, but feedlot and CAFO conditions have been associated with all of them. A similar factor is the reusing of dried manure as dairy cow bedding.

    What they do is create a closed loop selection system which favors the emergence and spread of enteric pathogen sub-types that otherwise might be outcompeted in a more complex life-history, and in the absence of selection for antibiotic resistance.

    I’ll be interested to see your views in part three,

    Dan Cohen

  • Tim Hall

    Dan Cohen’s comments are excellent and consistent with observation which is still the best tool in the toolbox. The dairy and feedlot scenarios resulting in partially damaged bacteria equipped with repair mechanisms in search of spare parts can only lead to a reservoir which favors the emergence and spread of enteric pathogens. These selections made in the presence of antibiotics guarantees future variants with a range of unattractive characteristics that have yet to be envisioned.
    The routine use of prophylactic antibiotics in the cattle and dairy industry was discouraged many years ago by the nations’s scientiists. No creedence was given then and I doubt if we can expect a differenct outcome now. Given the economics involved I no longer believe that science has any potential to trump greed but I still maintaint that we have the ability to partially mitigate the damage.

  • http://organicpastures.com mark mcafee

    Bill,
    Great stuff…
    “Did humans turn an obscure E. coli strain into a dangerous emerging infectious disease?”
    I always thought that GMO researchers using ecoli as their target bacteria when bombarding DNA could have played a part in the change of a simple bacteria into a virilent strain. The antibiotics fed in feedlots place massive pressure on bacteria to evolve and survive as well. This stress, and or the random bombardment in a lab results in all sorts of Genetic Sports that are unknown and result in weird evolutions that can be virilent…. mankind knows so very little about how its miraculous solutions to medical problems come back to haunt us. It is very much the “Faustian deal with the bacterial devil”.
    Perhaps it was this random engineering experiment in the late 1970s that created this virilent form of ecoli. We are all guessing.
    I like the work done that shows that simple mineral deficiencies may play an essential role in ecoli-HUS prevention.
    God knows….but we need to keep on looking.
    Perhaps the best medicine is less medicine and stronger immune systems through consumption of biodiverse whole food diets. Bacteria in the healthy GUT have a way of protecting us all very well.
    Mark McAfee
    Founder – CEO
    Organic Pastures Dairy Fresno CA

  • Anonymous

    While I personally suspect e. coli infections have plagued humanity long before the advent of modern “corporate” agriculture, I must share that when I was working on the Bovine Growth Hormone project at Monsanto during the mid-1980′s the recombinant bovine somatatropin was “cultivated” in and harvested from strains of e. coli on the bench top level and in the pilot plant. Probably just coincidental, but you never know…