Clostridium Botulinum Catherine Chung Colin Brinkman Pam Chao
Introduction to botulism
Neuroparalytic disease caused by neurotoxins (BoNTs) produced by bacteria Clostridium botulinum.
7 different BoNTs (A-G) produced by different strains of C. botulinum.
BoNTs A, B, E, and F outbreaks in humans
BoNT C in birds
BoNT D in cattle
BoNT G isolated from soils
BoNT A most common and most potent
BoNTs also produced by other members of Clostridia family.
A long history
Botulism originally known as “sausage poisoning” in late 18th century and throughout 19th century.
From Latin botulus = sausage
Bacterial etiology recognized at end of 19th century
Outbreak of botulism in Belgium 1895 revealed cause as neuroparalytic toxin produced by anaerobic bacterium
Probably Type B
Outbreak in Germany several years later
Bacterium isolated; different from that in Belgium
Probably Type A
The 20th Century and Beyond
1949 Burgen et al. determines botulinum toxin blocks neurotransmitter release
1979 Simpson proposes cellular mechanism in 3 steps: binding, internalization, and intraneuronal action.
1990 a.a. sequence of one BoNT determined in Niemann’s laboratory
21st century – 3D structure of BoNT A resolved.
The History Continues…
First biological toxin used for treatment of human disease
Manufactured for medical use in 1989 under name “Oculinum”
Licensed for treatment of strabismus and blepharospasm (eye conditions characterized by excessive muscle contraction)
Blepharospasm treated with Oculinum
Vangelova, Luba. “Botulinum Toxin: A Poison that Can Heal.” Available at:
http://www.fda.gov/fdac/features/095_bot.html.
Medical uses of BoNT
Now manufactured under the name “Botox”
Experimentally used for treating migraine headaches, chronic low back pain, stroke, cerebral palsy, and dystonias (neurologic diseases involving abnormal muscle posture and tension)
Frequent injections allows an individual to develop antibodies
Studies carried out to determine feasibility of other strains of BoNT
BoNT B manufactured for treatment of cervical dystonia in 2000 as “Myobloc”
Cosmetic Use of BoNT
1994 FDA denounced the promotion of BoNT use for wrinkles as "an egregious example of promoting a potentially toxic biologic for cosmetic purposes."
Botox approved for Cosmetic use in April, 2002
Myobloc not approved for cosmetic use, but is used experimentally in many cosmetic procedures anyway
BoNT A (Botox)
Botox injection patient 13 weeks after injection
BoNT B (Myobloc)
Myobloc injection patient 11 weeks after procedure
The Smart Stuff
Structure:
Translated as a single chain precursor (pretoxin)
Cleaved to generate fully active neurotoxin composed of a light chain (LC) and heavy chain (H)
Light and heavy chains linked by single disulfide bridge
Light chain acts as an endopeptidase
When bridge is intact, BoNT has no catalytic activity
More on Structure
3D Structure
BoNTs prevent neurotransmitter release
SNARE proteins form complex to allow synaptic vesicles to fuse with plasma membrane neurotransmitter is released
3 different types of SNARE proteins
VAMPs
SNAP-25 (cleaved by BoNT A)
Syntaxin
BoNTs prevent neurotransmitter release
BoNTs cleave SNAREs
BoNTs block Acetylcholine release in three steps
Binding
BoNT binds by Hc to receptor on cell
Internalization
Toxins internalized via receptor-mediated endocytosis
THE POINT OF NO RETURN: once endocytosed, the toxins can no longer be neutralized by antisera
BoNTs block Acetylcholine release in 3 steps
Translocation
Heavy and light chains separate; light chain enters the cytosol and cleaves SNAREs
BoNTs cleave SNARE proteins and prevent Ach release
Clinical Perspective of Clostridium botulinum
There are four types of botulism, characterized by the method of delivery of the toxin.
The toxin cannot pass through the skin, thus, transmission requires a break in the skin or direct absorption through mucus membranes in the lungs or GI tract.
Foodborne botulism is the result of the ingestion of food contaminated with Clostridium botulinum containing the preformed toxin. Note: Ingestion of the toxin makes a person ill, not Clostridium botulinum itself.
Wound botulism occurs when a break in the skin becomes infected with Clostridium botulinum which then multiply and release botulism toxin into the blood.
Inhalation botulism occurs when aerosolized botulism toxin enters the lungs.
Infant botulism is the result of the infestation of the digestive tract with Clostridium botulinum.
In infant botulism, illness results from infestation of the GI tract with Clostridium botulinum. Such infestation is generally not an issue in individuals older than one year due largely to the large number of competing microorganisms found in the mature GI tract.
Roughly 100 cases of botulism are reported in the U.S. each year.
Approximately 25% are foodborne, 72% are infant botulism, and the remaining 3% are wound botulism.
Inhalation cases do not occur naturally.
Wound botulism is on the rise due to an increase in the use of black tar heroin. The source of the botulism could be the drug itself, a cut in the drug, dirty injection equipment, or contamination during the preparation process.
The incubation period varies according to the mode of transmission, rate of absorption of the toxin, and the total amount and type of toxin.
Foodborne botulism usually takes 24-36 hours to manifest itself.
Wound botulism often takes 3 or more days to appear.
Inhalation botulism has occurred very rarely, but incubation times may range from several hours to perhaps days, again depending upon the type and amount of toxin inhaled.
All four types of botulism result in symmetric descending flaccid paralysis of motor and autonomic nerves always beginning with the cranial nerves. These symptoms are preceded by constipation in cases of infant botulism.
If left untreated symptoms may expand to include paralysis of respiratory muscles as well as the arms and legs.
Asphyxiation due to respiratory paralysis is the most common cause of death in botulism cases.
Botulism results in death in approximately 8% of documented cases. The key to survival is early diagnosis. For the period 1899-1949 the case fatality ratio was approximately 60%. For the Period 1950-1996 the case-fatality ratio was 15.5%.
This improvement is largely attributable to improvements in respiratory intensive care and availability and prompt administration of the antitoxin.
Treatment
Antitoxin can halt the progress of symptoms if administered early to victims of food and wound botulism.
Antitoxin is not given to victims of infant botulism because when this is diagnosed it is generally too late for the antitoxin to do any good.
Treatment
Wound botulism is treated surgically to remove the Clostridium colony.
Artificial respiration is required if paralysis reaches the lungs. Such respiratory assistance may be required for weeks to months.
The paralysis induced by the toxin slowly improves over the course of many weeks.
Many patients make close to a full recovery following weeks to months of intensive care, however, lingering effects such as fatigue and shortness of breath may linger for years.
Treatment
Attempts to develop an effective botulism vaccine date back to the 1940’s. One current effort (now moving into clinical trials) uses injection of a non-toxic carboxy-terminus segment of the botulism toxin to confer immunity to the toxin.
Diagnosis
The symptoms of botulism are similar to those of Guillain-Barré syndrome, stroke, and myasthenia gravis.
As a result, botulism is probably substantially under-diagnosed.
Serum electrolytes, renal and liver function tests, complete blood tests, urinalysis, and electrocardiograms will all be normal unless secondary complications occur.
Diagnosis
A brain scan, spinal fluid examination, electromyograph, or tensilon test may be required to positively identify botulism.
The most effective test comes from the identification of botulism toxin in serum or stool. The test is most often carried out by injecting samples into a mouse and observing whether symptoms of botulism develop.
Diagnosis
However, the false negative rate for this test can be as high is 60% for serum samples and near 80% for stool samples in individuals clinically diagnosed with botulism.
Collection of samples early in the progression of the illness may be helpful, however, large outbreaks have occurred in which none or a very low percentage of victims produced a positive test result.
Diagnosis
In vitro methods utilizing ELISA are under development but are not yet validated.
Isolation of Clostridium botulinum from the patient’s feces or gastric sample is a good confirmation of botulism as the organism is rarely found in humans in the absence of the botulism poisoning, however, poisoning can occur without ingestion of the microorganism at all.
If botulism poisoning is suspected clinicians are advised to contact local and state health authorities who should then contact the CDC
Prevention
Proper food preparation is one of the most effective ways to limit the risk of exposure to botulism toxin.
Boiling food or water for ten minutes can eliminate some strains of Clostridium botulinum as well as neutralize the toxin as well. However, this will not assure 100% elimination.
Limiting growth of Clostridium botulinum and the production of botulism toxin is an alternative to their outright destruction.
Temperature, pH, food preservatives, and competing microorganisms are among the factors that influence the rate and degree of Clostridium botulinum growth.
Growth of most strains of Clostridium botulinum will not occur below 10 or above 50 degrees Celsius.
Clostridium botulinum will not grow in media with pH values lower than about 5.
Food preservatives such as nitrite, sorbic acid, parabens, phenolic antioxidants, polyphosphates, and ascorbates inhibit the growth of the microorganism.
Lactic acid bacteria including Lactobacillus, Pediococcus, and Pactococcus can inhibit the growth of Clostridium botulinum by increasing the acidity of the medium.
While the cause of roughly 85% of infant botulism cases is unknown, in up to 15% of infant botulism cases the causes was ingestion of honey. Infants younger than one year old should not be fed honey.
Avoiding Exposure
Avoid home-processed foods if at all possible, especially those with a low salt and acid content.
Botulism toxin is destroyed at a temperature of 176 F, thus if you must eat home-processed foods, boil them for 10 minutes before eating if at all possible.
If canning vegetables, use a pressure cooker, as it will kill any spores because it can reach temperatures above boiling.
Weaponization of Botulinum Toxin
What does it mean to “weaponize” a biological agent?
The "weaponization" of a microbial pathogen or toxin involves:
rendering the agent resistant to standard antibiotic drugs
freeze-drying and milling the agent into an extremely fine powder, consisting of particles tiny enough to become readily airborne and inhaled into the victims' lungs to cause infection
stabilizing the agent so that it will remain infectious for a longer period after release
treating the powder with chemical additives that absorb moisture and reduce clumping, so as to facilitate aerosolization.
Answer provided by Jonathan B. Tucker, Ph.D.
an expert on chemical and biological weapons in the Washington, D.C and a biological weapons inspector in Iraq under the auspices of the United Nations Special Commission in 1995
Least effective thus not practical to use in bioterrorism
Extremely small scale
Requires direct contact between wound and spores of C. Botulinum
Very primitive (may involve using knives)
Jermann M, Hiersemenzel LP, Waespe W. Drug-dependent patient with multiple cutaneous abscesses and wound botulism. Schweiz Med Wochenschr 1999, 129:1467
Food Botulism
Carried out by deliberate contamination of food or water supply:
In commercial production facilities or restaurants or reservoirs.
Produces either large epidemic (area) outbreak or separated episodic (time) outbreaks.
Recognition of intentional outbreaks:
Victims all share common dietary exposure
Average Incubation Period:
Neurological symptoms occurs ~12-36 hrs after ingestion
Food Botulism
BoNT can stay in untreated water or uncooked food for up to days
It is colorless, odorless and tasteless
However, BoNT:
Can be inactivated by heat (>85c for 5 min)
Can be rapidly inactivated by standard potable water treatments (e.g. Chlorination, aeration)
Large capacity reservoirs requires large* quantities of BoNT in order to cause severe contamination
*difficult for terrorists to make and carry around
There are obstacles in order to produce an effective large scale food botulism poisoning
Inhalational Botulism
Man-made (does not occur naturally)
Utilizes aerosolized Botulinum toxin
May involve freeze-drying and milling the toxin into an extremely fine powder
Absorption of toxin through mucosal surface in the lung
Incubation Period:
Neurological symptoms usually occurs 24-72 hrs after aerosol exposure
Inhalational Botulism: Aerosols, Missiles and Bombs
Large scale
More efficient way of bio-terrorism
Can equip war-heads of missiles or bombs and grenades
- As white powder or liquid slurry
Can be sprayed as aerosols
Point-source aerosol release can incapacitate or kill 10% of persons within 500 meters downwind.
1 gram of crystalline toxin can kill >1 million people if dispersed and inhaled evenly
Inhalation Botulism
More deadly than food botulism (because smaller lethal dose) but technically and financially difficult to carry out:
Instability
Inactivated by sunlight within 1-3 hours
Detoxified in air within 12 hours
Technically difficult and complicated for the insufficiently funded terrorist to:
Make the powder form for efficient dispersal
obtain the accurate dispersal equipment
This is illustrated by the lack of actual cases
(However we can never underestimate the ability of terrorists..)
Potential danger: Botulinum Superbug?!
WHY?
BoNT is non-contagious (just a toxin)
If it can be mae contagious, it will be even more deadly
One known study:
Gene splicing experiments in Soviet Union during 1970s
May have involved:
Splicing the BoNT gene into other contagious bacteria (e.g. Ebola) to increase the transmission rate.
Genetic modifications that removes the effectiveness of possible vaccines or immune responses
Genetic engineering to produce new virulent strains or new toxic genes
Here’s an Interview of a former Soviet scientist who was in the bioweapon program:
