Thursday July 31, 2008 - Vol. VII Issue 6[Download PDF for Printing]
Biotoxins: Part 4
On June 12, 2002,
President George W. Bush signed into law the Public Health and Safety Act of
2002 (PL 107-188) which requires that the Department of Health and Human
Services maintain a list of biological agents and toxins which pose a severe
treat to public safety. The list of
biotoxins, as it appears in the August 23, 2002 Federal Resister, (see also 42
CFR Part 72, Appendix A) is as follows:
perfringens epsilon toxin
All of these biotoxins with the exception of shiga toxin,
shiga-like toxins, and staphylococcal enterotoxins have been topics of earlier
PEAC Newsletter Articles. Shiga toxin,
shiga-like toxins, and staphylococcal enterotoxins are fairly high molecular
weight proteins that are produced by microorganisms and are responsible for
some kinds of food poisoning. These
proteins can also be aerosolized, or potentially be aerosolized and used as a
Shiga toxin and Shiga-like Toxins
As the name implies, we are not dealing with a unique toxic
molecule but a class of toxins produced by several kinds of
microorganisms. Shiga toxin is produced
by the bacterial species Shigella dysenteriae.
Shiga-like toxins are produced by other bacterial species,
mostly by some Escherichia coli
[“E. coli”] strains. The different toxic protein molecules called
“Shiga toxin” or “Shiga-like toxins” differ slightly in their amino acid
sequence, but are all characterized in that they inhibit cell protein
synthesis. The toxin requires specific
receptor sites on the host cell surface in order to enter the cell membrane. Many animal species including pigs and cattle
lack these receptor sites and therefore are unaffected by the toxin, but still
pass the toxin and the bacteria in their feces. Unfortunately, human cells have these receptor sites and can get
very sick if they ingest food products contaminated with these bacteria or
toxins. Examples of food products have
included undercooked beef, hamburger, pork, unpasteurized fruit juice,
contaminated water, spinach, lettuce, and alfalfa sprouts. Person-to-person spread via fecal or oral
transmission may occur in high-risk settings such as day care centers and
Symptoms of Shiga-type food poisoning include diarrhea,
which is often bloody, and abdominal cramps.
There may be abdominal pain, vomiting, and a low-grade fever. Complications can occur, including damage to
kidneys, lungs, and the nervous system.
Blood in the urine can occur (hemolytic uremic syndrome, or HUS). In the absence of complications, recovery
takes place after several days.
Depending upon the circumstances, the incubation period from the time of
ingestion to onset of symptoms may vary from hours to about eight days, with 3
to 5 days being typical.
Additional information on HUS including treatment is in a
peer-reviewed article available from the Internet, http://www.emedicine.com/EMERG/topic238.htm
written by William Shapiro, MD, Department of Urgent Care and Emergency
Medicine, Scripps Clinic and Research Foundation. Most cases of HUS in the United States occur with young children
(5 years or younger), and about 75% of these cases are associated with
Shiga-type toxin food poisoning produced by E. coli
infections. About 85% of children
recover if given supportive care. The
mortality rate is 5 to 15%. The
prognosis for recovery for adults is poor, but the incidence of HUS among
adults due to Shiga-type toxins is less common. With adults, HUS is more likely to be associated with other
causes (other infectious agents, AIDS, administration of chemotherapeutic
agents such as mitomycin C, malignancies, etc.)
Thrombocytopenic purpura (TTP) is a complication of exposure
to Shiga-type toxin. Cell injury occurs
throughout the body including the brain, skin, intestines, skeletal muscle,
pancreas, spleen, adrenals, and heart.
HUS is the result of cell injury to the kidneys, and is a manifestation
of the same overall disease condition.
Hemorrhagic colitis (HC) refers to cell damage to the intestinal track.
About 73,000 laboratory-conformed E. coli
O157:H7 infections occur annually in the United States (1999 data, from http://www.ncbi.nlm.nih.gov
). About 10% of these result in TTP and/or
HUS. The O157:H7 strain of E. coli
is the most common cause of Shiga-type toxin poisoning, but about 100 other
strains of E. coli
can produce toxins.
There may be many more cases of food poisoning that are undiagnosed.
At this time, there is no vaccine or antitoxin available for
human use. Treatment is basically
The finding of grossly bloody stools in children often with
the absence of fever is a strong indicator of E. coli
infection. The major risk factors are eating
insufficiently cooked hamburgers or contact with cattle or other animals that
harbor the E. coli
. With adults,
other conditions may result in bloody stools.
A postdiarrheal HUS or TTP condition is also a strong indicator of
Shiga-type toxin poisoning.
The U.S. Center for Disease Control (CDC) [see http://www.epi.state.nc.us/epi/gcdc/manual/casedefs/E.COLI%20INFECTION-SHIGA%20TOXIN%20PRODUCING_CD.pdf
has published procedures for diagnosis:
Isolation of E. coli strain O157 from a clinical
specimen (probable case)
Identification of an elevated antibody titer to a known
Shiga toxin (confirmed case)
A clinically compatible case that is epidemiologically
linked to a confirmed or probable case.
The stx EIA Shiga toxin enzyme immunoassay is the most
common procedure used to confirm cases.
Public health authorities in all states recommend that
clinical laboratories forward Stx EIA positive specimens of stool samples to
the state laboratory for isolation and identification of the Shiga toxin-
producing E. coli
and report to the CDC. A South Dakota laboratory reported (see http://www.usd.edu/med/path/research_/shigatoxin.cfm
that a toxin-producing strain of E. coli
was isolated in 89% of the Stx
EIA positive specimens, and of these E
, 54% were strain
O157 and 46% were other strains.
Figure 1: Escherichia
Left: E. coli
electron microscope photo. E. coli
are rod-shaped bacteria 2 micrometers long and 0.8 micrometers in
Source: AP wire photo used on KPIX-TV (San Francisco)
website featuring a story on food poisoning due to spinach contamination.
The Shigella dysenteriae photo is similar
The Shiga Toxin Molecule:
The total molecular weight of this protein molecule is about
68,000 Daltons. The toxic subpart (A
subunit) is about 32,000 molecular weight.
The rest consists of five sections (B subunit) each of about 7700
molecular weight, and is responsible for binding to a particular cell
type. Once the toxin enters the host
cell, the A subunit splits into two parts; the A1 component binds to the
ribosome of the host cell disrupting protein synthesis. This kills the cells causing breakdown of
the vascular endothelium lining and hemorrhage. The B subunit binds itself to specific glycolipids on the host
cell, in particular globotriaosylceramide (Gb3), which is present in greater
amounts in renal epithelia tissues (kidneys).
Verotoxin is an alternative name for Shiga toxin and
(mouse) value for Shiga toxin and
Shiga-like toxin is less than 20 micrograms per kilogram, by either intravenous
or intraperitonal injection. The LD50
for aerosol exposure is 3 mcg/kg. [mcg = millionth of a gram; per kilogram of body weight].
Shiga toxin produced by Shigella
differs by only one amino acid from Shiga-like toxin 1 (Stx-1)
produced by some E. coli
Shiga-like toxin (Stx-2), produced by some stains of E. coli
differs from Stx (from Shigella dysenteriae
) by a different amino acid
sequence, and is 400 times as toxic (mouse injection) than Stx-1.
Potential for Terrorist Use:
We have not found any
confirmed terrorist use, [see:
http://www.cbwinfo.com/Biological/Toxins/Verotox.html], but the toxic protein
is robust and easy to manufacture. Both
and E. coli
(strain O157:H7 and other
strains) can be easily cultured in the laboratory. The bacterial genes responsible for producing the toxin have been
cloned, and the toxin can be manufactured using research strains of E. coli
The toxin is readily
soluble in water and can be aerosolized.
The toxin does not act through the skin, but is toxic by inhalation. Presumably a full face mask fitted with a
charcoal filter should provide adequate protection against aerosolized toxin.
In 1999, a scientist in
Germany received a request from North Korea asking that his laboratory furnish
them with plasmids used to produce non-toxic fragments of shiga toxin. The scientist was a group leader at the
European Molecular Biology Laboratory, and had just published a paper with his
colleagues on protein export in cells using the shiga toxin. The scientist did not honor the North Korean
request, and a warning was subsequently posted in EMBO Reports
dangers of possible transfer of the means for development of toxic weapons, http://www.nature.com/embor/journal/v1/n4/full/embor555.html
particular, scientists must adhere to article III of the United Nations
Convention on the prohibition of the development, production, and stockpiling
of bacteriological and toxic weapons [see http://www.state.gov/www/global/arms/treaties/bwc1.html
which obligates the countries “not to transfer to any recipient whatsoever,
directly or indirectly, and not in any way to assist, encourage, or induce any
State, group of States or international organizations to manufacture or
otherwise acquire any agents, toxins, weapons, or means of delivery…”.
Staphyloccus is a name for a microorganism. Enterotoxin means that the toxin produced by
the microorganism exerts its effects on the intestines. If allowed to grow in foods, enough toxin
may be produced to result in food poisoning after the foods are ingested. The most common symptoms of staphylococcal
food poisoning, which usually begin 2 to 6 hours after the food is ingested,
include nausea, vomiting, acute prostration, diarrhea, and abdominal
cramps. Unlike Shiga-like toxin, the
diarrhea is not bloody (unless there is some other pathological condition
present), and the onset of symptoms is sooner.
Patients may be dehydrated depending upon the severity of
nausea, vomiting, and diarrhea. There
may be non-localized abdominal pain.
Most enterotoxins causing human food poisoning are produced
by Staphylococcus aureus
, but several other Staphylococcus
species have been demonstrated to produce enterotoxins, including S.
, S. hyicus
, and S. epidermidius
. About 14 different enterotoxins have been
identified to date as distinct entities (types A, B, C1, C2, C3, D, E, G, H, I,
J, K, L, M, N, and O and the list is growing), of which at least seven are
produced by S. aureus
. The toxin
TSST-1, or “toxic shock syndrome toxin-1”, also appears in the literature, and
may have originally been called type F toxin.
The different toxins are usually called “Staphyococcal Enterotoxin A”,
“Staphylococcal Enterotoxin B”, or SEA, SEB, SEC1, …, SEJ.
is present almost
everwhere. At least 50% of healthy
people harbor these microorganisms naturally in their nasal passages and on
their skin. Food handlers can easily
infect food products. Staphylococcus
readily grow in unrefrigerated meats, dairy, bakery products, fish, chicken,
canned mushrooms, potato, and foods containing eggs. It is one of the most common causes of food poisoning. The actual incidence Staph-caused food
poisoning is unknown because many cases are mild and people do not seek
treatment, or if the patient visits an emergency room, diagnoses are usually
presumptive, and many other conditions mimic the symptoms of Staph-caused food
The threshold amount of enterotoxin for causing food
poisoning symptoms in humans is not known.
There have been some food outbreak studies and human challenge tests
indicating that at least 100 nanograms of Enterotoxin A (the most common type
of Enterotoxin involved in food poisoning) is required to produce illness [from
citations in http://www.cfsan.fda.gov/~ebam/bam-13a.html
Bacteriological Analytical Manual Online, U.S. Food and Drug
Administration]. The amount and type of
entertoxin is determined by analysis of the toxin in the food (ELISA Assay,
Micro-slide double diffusion methods, see citations for procedures). Oral doses of 20 to 500 micrograms of
Enterotoxin B produce emersis (vomiting) in nonhuman primates.
The biological mechanism by which the Staphylococcal
enterotoxins act on the body is complex and not fully understood. The enterotoxins are very compact and stable
molecules that are not cleaved by enzymes in the intestinal tract. The enterotoxin molecules bind to and
crosslink certain cell receptors (HLA-DR or DQ and T-cell receptors) resulting
in the production of inflammatory cytokines; therefore the observed symptoms of
nausea, vomiting, and diarrhea. The
condition is rarely fatal, and patients almost always recover. Death from this type of food poisoning has
occurred with infants, the elderly, and severely debilitated persons.
Treatment is supportive.
Persons already in poor health or persons unable to maintain their own
hydration require hospitalization.
The enterotoxin does not penetrate human skin and can be
removed using soap and water (good hand washing).
Figure 2: Staphylococcus
The bacteria are spherical shaped, one micrometer in
diameter, and often are together in grape-like structures [photo from University of
Wisconsin, Dept. of Bacteriology]
Sputum smear showing bacteria [photo from Loyle University Chicago, Stritch
School of Medicine]
and survival of Staphylococcus and toxins in cooking and storage of
chicken, in Appl Environ Microbiol. 2006
7057–7062, available at
of Pasteurized Chilled Food. About
90% of Staphylococcus aureus is destroyed in food at temperatures
of 140oF (60oC) for 5 to 8 minutes, or essentially
100% at temperatures above 180oF, but the toxin itself is heat
resistant under normal cooking methods.
This article also gives safe holding times and temperatures for
previously pasteurized foods, and temperatures required to kill different
kinds of bacteria and to deactivate toxins. See International Journal of Food Microbiology, 22 January 2001, pages 99-107, available
If bacteria are allowed to grow and produce toxin in
previously prepared food, cooking or recooking may kill the bacteria but not
deactivate the toxin. The enterotoxin
is heat resistant under ordinary cooking methods, and the enterotoxin can remain
intact in food even after boiling for 30 minutes at 100o
C. Cooking at temperatures of 100o
F) for more than 2 hours is required to destroy the toxin. A University of Wisconsin test [see Journal
of Food Science
57(3) 1992 available at http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2621.1992.tb08076.x
where mushrooms were inoculated with 100 to 1000 nanograms of SEA per gram of
mushrooms and then heated to 121.1o
C for 10 minutes still showed
detectable amounts of SEA after the heating.
Human production of antibodies in response to Staphylococcus
enterotoxins (SEA, SEB, SEC, SED, TSST-1) has been implemented as a
cause of persistent allergic upper airway/nasal/sinus rhinitis and/or asthma
conditions [see http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ProduktNr=224161&Ausgabe=229964&ArtikelNr=76833
]. The bacteria are present in the airway or
nasal passages and produce toxins.
Staphylococcal food poisoning is usually diagnosed by
clinical symptoms. The toxin can be
detected by enzyme-linked immunosorbent assays (ELISA Assays) and
chemiluminescence (ECL). A reverse
passive latex agglutination test may identify the toxin rapidly in food. Polymerase chain reaction assays can
sometimes find Staphylococcus aureus
genes in environmental
samples. If the toxin is inhaled, the
toxin can be found in respiratory secretions or nasal swabs for a short period
of time, as well as in blood and urine samples.
The enterotoxin molecule:
The molecular weight of the staphylococcal entertoxins vary
from about 23000 to 29600 Daltons.
Entertoxins A, D, and E form one group of closely related toxins (SEA,
SED, SEE) and Entertoxins B, C1, C2, and C3 (SEB, SEC1, SEC2, and SEC3) form a
second group. The different toxins vary
in molecular weight and amino acid sequencing.
SEB, for example, has a molecular weight of 28000 Daltons (one reference
says 28,336) and consists of 239 amino acid residues. SEA has a molecular weight of 27,000 and has 233 amino acid
residues. Toxic shock syndrome toxin
(TSST-1) form a third group, although analysis indicates that the three
dimensional structure is similar to SEB.
All are compact molecules that are not cleaved by intestinal
enzymes. The differences between these
types depend how they bind to host cell receptors, with additional binding
mechanisms unique to each toxin, and resulting different degrees of toxicity.
The U.S. CDC estimates that SEA accounts for 53% of the
Staphylococcal enterotoxin food poisoning.
SEB seems to be the second most common type. SED is another common type, typically associated with S.
growing in dairy products, and probably accounts for 5% of food
poisoning. Also, more than one
enterotoxin type can be produced by a given bacterial culture.
The lethal dose (LD50
) for humans for inhalation
of Staphylococcal Enterotoxin B (SEB) is 0.0004 mcg/kg.
The effective dose (ED50
) for incapacitating 50%
of exposed human populations for SEB inhalation is 0.02 mcg/kg.
A “mcg” is one millionth of a gram, and 0.02 mcg/kg means
0.02 mcg per kilogram of body weight.
[information on dose from http://www.emedicine.com/EMERG/topic888.htm
Potential for Terrorist Use:
Staphylococcal Enterotoxin B (SEB) was weaponized as an
incapacitating agent by the United States during the 1960’s. President Nixon in 1969 ordered the CIA to
destroy the entire stock of biotoxins and biological warfare agents collected
over the years and not engage in additional covert research. (see http://www.aarclibrary.org/publib/church/reports/vol1/pdf/ChurchV1_8_Exhibits.pdf
Other enterotoxins can be weaponized, but SEB is the
entertoxin which has been studied.
When inhaled as a respirable aerosol, SEB causes severe
respiratory distress, fever, headache, and sometimes nausea and vomiting. The mechanism of incapacitation is due to a
massive release of irritating cytokines, caused by binding of the toxin to specific
cell receptors. The onset of symptoms
may occur in about 2 to 12 hours after exposure and last up to two weeks. At low inhalation doses, symptoms include
sudden onset of headache, fever (103o
lasts up to 5 days, if significant pulmonary involvement is involved), myalgia
(muscle pain), shortness of breath, nonproductive cough (which may last up to 4
weeks), and retrosternal pain. At high
inhalation doses, the illness may progress with increasing respiratory
distress, hypoxis (inability to supply oxygen to body tissues), and ultimately
respiratory failure due to inflammation and pulmonary edema (fluid buildup in
the lungs). Higher levels may lead to
septic shock, multi-organ failure, and death.
Treatment (from SEB exposure due to inhalation) is
supportive. The basic treatment
includes use of humidified oxygen. If
exposure is significant, intubation and assisted ventilation with high oxygen
concentration may be dictated.
Attention should be made to the elimination of hypotension and hypoxia,
and pain control as needed. Cough
suppressants and acetaminophen for fever will make the patient more comfortable
[from emedicine.com, in article by Danielle M. Pesce, DO, at the Carl R.
Darnell Army Medical Center]. The
administration of antibiotics could make the patient’s condition worse.
More detailed studies on inhalation of SEB including a case
history investigation of nine laboratory workers who accidentally inhaled SEB
is in a paper by Robert Ulrich, et. al , “Staphylococcal Enterotoxin B and
Related Pyrogenic Toxins”, and is available from the Internet at http://www.au.af.mil/au/awc/awcgate/medaspec/Ch-31electrv699.pdf
information is available at http://www.cdc.gov/ncidod/eid/vol10no9/04-0250.htm
The bacteria can be readily grown. SEB is stable and is water soluble. Freeze-dried toxin can be stored for over one year. The toxin is moderately resistant to temperature
fluctuations, and can withstand boiling at 100o
C for several minutes
[reference emedicine.com, cited earlier].
There is potential for a terrorist release of the toxin as
an aerosol or a mist or a dust.
A military chemical protective mask or a full piece face
mask fitted with carbon cartridges is effective against inhalation of the