Botulism
Clostridium Botulinum

Martone Gianluca, Nicotera Agostino

History

The first case described of Botulism dates back to 1793, when the german doctor Justinius Kerner, called to inquire into an event of alimentary intoxication, inferred that the responsible were a substance contained in some rotten sausages eaten by guests.

A long time after, in 1895, the Belgian bacteriologist Emile Van Ermengem isolated the bacterium causing botulism as a result of some analysis performed during an alimentary epidemic in Ellezelles: here, too, the poisoned had eaten rotten meat (raw ham).

Seen the relation of the bacterium with gone bad meat (especially charcuterie) - and not with its form, as has been supposed by a few - the microorganism was called botulinum from the latin term 'botulus', which stands for sausage.

(Botulino: cenni storici - Albanesi)

Epidemiology

Botulism is a clinical condition important to identify insofar as characterized by an high case fatality ratio: DL50 is of 1 μg/kg, so being the most powerful poison known. It is a pathology that despite being endemic has however a low incidence in the world; in Italy between 20 and 50 cases take place in a year, while increase in some areas of the world depending above all on eating habits. By way of example, alimentary botulism has an high incidence in eastern European countries, where there is a large consumption of vegetables canned at home, and in USA (110 cases in a year) where honey is more frequently given to infants.

Clostridium Botulinum

C. Botulinum is a Gram-positive , rod-shaped (8 μm long), known being an obligate anaerobe, even if recently has been found in it superoxide dismutase which makes it capable of surviving in presence of minimum quantities of oxygen.

It is a bacterium which in anaerobic environment acquires the capacity for sporulation: spores are ovoid and localized in subterminal position, so becoming difficult to see them.

C. Botulinum produces several exotoxins during the phase of sporulation, among which the most important are neurotoxins ; depending on the kind of neurotoxin produced, we can distinguish different phenotype groups:

*=chromosome/plasmid

(Wikipedia: C. Botulinum)

An eight toxin, H type, has been recently discovered in 2013 by the researcher of California Department of Public Health, and results now the most powerful between Botulinum toxins (New botox super-toxin has its details censored, 2013).

As we see from the table above, the only toxins which interests men are A,B,E and F; moreover they have a typical geographic correlation:
° North America -> type A C. Botulinum predominates the soil samples from the western regions while type B is the major type found in eastern areas.
° Europe -> type E is prevalent in aquatic sediments in Norway, Sweden, Denamrk, the Netherlands and in the Baltic coasts of Russia and Poland; type B is typical of more dried regions (e.g. United Kingdom and Italy)
(Wikipedia, Clostridium Botulinum in different geographical locations)

Types of infection

C. Botulinum lives in the soil, and its spores are very difficult to kill, since they survive high temperatures (100°C for a long time); because of this, many foods are canned with a pressurized boil that achieves an even higher temperature, sufficient to kill all spores. Furthermore some food qualities can be a handicap to the growth of the bacterium: a low pH (green beans and corn more at risk of contamination than tomatoes), high sugar concentrations and, obviously, aerobic conditions.

Botulin toxin on the other hand, of a proteinic nature and gastric juice - resistant, is instead thermolabile: a normal process of cooking (100°C for ten minutes) leads to the complete inactivation of the toxin.

There are different types of infection:

1. Iatrogenic botulism , due to a wrong use of toxin in therapy or cosmetics
2. Traumatic wound botulism , it occurs after the growth of the microorganism and its toxin production inside deep wounds, where there are anaerobic conditions. Is a rare and not well known form, and this is why wound botulism can take several months before being diagnosed. (Traumatic wound botulism, 2014)
3. Food poisoning , due to the ingestion of preformed toxins:
   1. Alimentary botulism as such -> following the consumption of conserves homemade, especially in oil, or sausages and fishes not well conserved.
   2. Infant botulism -> botulism in infants. Honey is the only food associated to this form until now; this because it is a natural source of spores which bees pick up during their activity, and once arrived in the gut they are able to germinate and to multiply producing the toxin causing the pathology. In the adult man all that can't happens because of the presence of a developed microbiota which contains bacterium growth.
   3. Adult intestinal toxemia -> it occurs with the same way of infant botulism but interests adults affected by anatomical gut malformation or exposed to a long antibiotic therapy.

Pathogenesis

All the seven serotypes of toxins have the same chemical model : they are a 450 kDa complex formed by the so called neurotoxin (BNT) and by non-toxic proteins. The neurotoxin in turn consists of one heavy amino acid chain (HC, 100 kDa) and one light chain (LC, 50 kDa) joined together by a disulfide bond.

Toxin's crystalline structure

In case of ingestion the toxin passes through mucose intestinal and enters the bloodstream, through which diffuses and reaches neuronal terminations of neuromuscular synapses. From this moment on the pathogenetic process can be described in three phases:

1. The bond of the acceptor ; the light chain recognizes a specific acceptor on the presynaptic acetylcholine terminal and binds it. Each serotype recognizes a specific receptor, and doesn't inhibit the bond of other serotypes with their own acceptors.
2. Internalisation ; the whole neurotoxin enters terminals by endocytosis. After endocytic vacuole's acidification the heavy chain forms a channel through the membrane, the disulfide bond breaks after reduction and so the light chain is able to pass in cytosol where employs its enzymatic function.
3. Inhibition of releasing mechanism ; the target of their action is the SNARE complex ,which regulates the join and the fusion of acetylcholine vesicles with the cell membrane. There are two possible site of action:
   1. Type A, C1 and E lyse a component of Snare complex called SNAP25 .
   2. Type B,D,F and G lyse synaptobrevin also called vesicle-associated membrane protein (VAMP) which so is not more able to bind syntaxyn.

The final effect is the would-be vesicles releasing, which clinically appears as the block of muscolar contraction (flaccid paralysis).

(Tossina botulinica,2013)

Clinical aspects

Seen the very high lethality is important to be able of identifying botulism as quickly as possible. The first symptoms of intoxication appear 12-26 hours after the ingestion (even if is difficult to speak about latency period in case of wound and infant botulism because exposure times cannot be ascertained), and consist of weakness, dizziness, dry mouth and pharynx.

Shortly after start to be interested the cranial nerves, by the rule of 4 D :

1. Diplopia , caused by flaccid paralysis of extrinsic muscles of the eye.
2. Dysphonia and Dysarthria , due to the reduced activity of secretory glands and to flaccid paralysis of muscles of phonation.
3. Dysphagia , caused by paralysis of the muscles of deglutition.

As time goes by the paralysis involves face (with ptosis), up to interest all the muscles of the body in a generalized condition said dystonia.

The patient however remains responsive and apyretic, since sometimes there is talk of classic triad : flaccid descending paralysis , no fever and state of awareness . Death occurs by respiratory paralysis in 70% of non-treated (botulism A type) and in 30% of non treated in B type.

Is well to underline symptoms are the same in all the forms of botulism, even if in infant distinguishing signs at first are weeping, suction difficulties, apnea and flaccid paralysis.

(Botulism: signs and prognosis, Wikipedia)

Diagnosis and therapy

Diagnosis in an important stage since we need to find the toxin at first in the patient and then in the food to identify the source and to avoid an epidemic phenomenon. Usually toxin is searched in serum, stool or gastric content by inoculating them in laboratory animals to see if paralysis occurs.

With regard to therapy, there are two principal intervention:
° Administration of serum, which is a pool of antitoxins against the toxins more frequently involved (A, B and E). The medicine operates binding the toxin present in bloodstream, avoiding further neuronal damages; if administrated in time the serum is able to reverse any suffering present once.
° Serum is not used in infants since it can't eradicate bacteria sited in the gut; so is utilized a different medicine called Botulism Immune Globulin , human-derived anti-botulism toxin antibodies approved by the U.S. Food and Drug Administration for the treatment of infant botulism types A and B.

(Farmaci per intossicazione da botulino: Mypersonaltrainer)

However artificial respiration is often placed side by side, while to quicken the elimination of the toxin can be administrated emetic or lassative medicine. Often antibiotic therapy is recommended.

The chances of survival increase in proportion to immediacy with which is done appeal to the right medical treatments; from here, the importance of being able to identify it.