by Erick Vandamme & Paul De Vos
The discoverer of Clostridium botulinum and botulin-toxin
Emile Pierre-Marie van Ermengem, (°1851, Leuven; † 1832, Elsene, Belgium) obtained his degree of Doctor in Medicine at the Catholic University of Leuven, Belgium, on September 20, 1875. In the subsequent period – before his appointment as Professor in Hygiene and Bacteriology at the State University of Ghent in 1888 – he improved his knowledge and skills at various renowned laboratories and clinics in Paris, London, Edinburgh and Vienna. He also worked at the famous laboratory of Robert Koch in Berlin where he experienced the so-called Koch-postulates in practice. In brief: i) the pathogenic agent must be present in high amounts in sick animals/plants and not in healthy ones, ii) the pathogenic agent must be isolated and enumerated, iii) test animals/plants of the same species must be inoculated with the pathogenic agent and iv) the infected animals/plants must show the same symptoms as the original sick animals/plants.
Emile Van Ermengem. Head of the Laboratory for Bacteriology (1886-1914) and of the Laboratory of Forensic Medicine (1896-1910), Dean of the Faculty of Medicine (1889-1890 and 1905-1906) of the State University of Ghent, Belgium.
The Ellezelles food poisoning accident
There is no doubt that, E. Van Ermengem investigated the severe food poisoning accident at Ellezelles a rural village near Ronse (Renaix) by applying the Koch postulates! On December 14, 1895, the musicians of the local brass band (Fanfare Royale Les Amis Réunis) participated at a meal in ‘Le Rustic’ a local pub where raw ham was served after they had performed at the funeral of Antoine Creteur. The next day, most of the 34 musicians fell ill and 3 young members (aged between 15 and 21) died within 5 to 7 days. All victims that had eaten from the pickled and smoked ham showed impaired eye vision, uncontrolled action of the eye lids, weakness of muscles, inflammation of mucous membranes and speech disorder. Brass members who had not eaten from the ham but from the fat bacon did not show the symptoms. This led to the conclusions of the local medical doctors that symptoms were due to the eating of spoiled ham meat. This serious case of food poisoning led to an judicial inquiry, tracking the origin of the ham, chemical analysis of it and autopsies of victims.
The discovery of Clostridium botulinum
A very extensive investigation took place at the laboratory of Prof. Van Ermengem at the State University of Ghent, showing that i) in the milt of the victims bacteria were present, ii) experiments with a series of laboratory animals provoked the same symptoms, iii) Van Ermengem named the isolated bacteria Bacillus botulinus (later Clostridium botulinum) referring to the first report of similar food intoxication caused by eating contaminated sausage (botulus, sausage; botulinum, pertaining to blood-sausage) by Justinus Kerner in 1817 also known as Kerner’s disease. Van Ermengem published his observations in 1897, indicating that botulism is i) an intoxication, not an infection; ii) the toxin is produced by a bacterium in food; iii) the toxin is not produced in the food if salt concentration in the food is high; iv) after ingestion the toxin is not inactivated by the normal digestive process; v) the toxin is heat labile and vi) not all animal species are equally susceptible.
Further research triggered by the discovery of C. botulinum
The toxin – named botulinum neurotoxin (BoNT) – produced and excreted by ‘named’ Clostridium botulinum strains and causing the food poisoning effect called ‘botulism’ was thus for the first time ever investigated at the laboratory of Prof. E. Van Ermengem. Intensive subsequent worldwide scientific research has led to the discovery of (at the moment) seven (A-G) types of toxins consisting of a heat-labile neurotoxin and an auxiliary protein. Starting with the discovery of two serologically different toxins by Leuchs in Germany and later (1919) named type A and B by Burke at Stanford University, CA,US; type C was identified (1922) in the US by Bengston and in Australia by Seddon; type D in the US by Meyer and Gunninson (1928); E in Ukraine (1936) by Bier and finally type F and G in 1960 by Möller and Scheibel in Scandinavia and in 1970 by Gimenex and Ciccarelli in Argentina.
BoNT is one of the most poisonous natural toxins known with and LD50 dosis of 0.2-2.5 ng per kg weight depending on the toxin producing strain. The toxin migrates to the muscles and nerves where it irreversibly blocks the release of the neurotransmitter acetylcholine, resulting in the weakening of the muscles and dizziness, difficulty in breathing and eventually resulting in death. However, most experts acknowledge that this effect alone appears inadequate to explain the entirety of the neurotoxin’s apparent analgesic activity. Consequently, scientific and clinical evidence has emerged that suggests multiple antinociceptive mechanisms for botulinum toxins in a variety of painful disorders, including: chronic musculoskeletal, neurological, pelvic, perineal, osteoarticular, and some headache conditions (Wheeler and Smith, 2013). Though the toxin is heat sensitive, the endospore forming capacity of C. botulinum results after (inadequate) sterilization of food products into an outgrowth of the spore with dramatic production of the BoTN’s in e.g. canned food.
Potential use of BoTN as biological weapon was already recognized during World War I. A stable acid precipitate of the toxin was made in the 1920s in the US and during World War II intensively further studied amongst other potential biological weapons in the US at Camp Detrick in Maryland, US, by Lamanna and Duff. Consequently, the methodology was used by E. J. Schantz to produce the first batch as basis for later clinical products. Further development occurred at the University of Wisconsin, Madison, US, (Schantz and Johnson) and collaborative work with and ophthalmologic surgeon Allan Scott (San Francisco, CA,US) in testing the product as alternative for strabismus surgery. Nowadays, many clinical trials are underway to extend the therapeutic indications for botulin toxin and to improve its safety. Thus not only for aesthetic applications (BoTox) in order to diminish ageing wrinkling effect of the facial skin but also and more importantly raising the quality of live for people suffering from excessive muscle contraction and/or other muscular disorders.
Clostridium botulinum is now known as a heterogenic group of bacteria. Originally the non-BoNT producing strains were classified under C. sporogenes. However, it turned out that named C. botulinum strains belong to four phylogenetic groups (I to IV). Some members (but not all) of these groups are known to produce BoNTs. Group I strains are proteolytic and saccharolytic and produce BoNT types A, B and F. Group II strains are non-proteolytic and produce BoNT types B, E and F, while group III strains produce BoNT C and D. Group IV strains (also identified as C. argentinense) produce BoNT G. Whole genome studies indicate that BoNT genes are sensitive to horizontal gene transfer (Weigand et al., 2015) and hence natural organismal relationships show that classification of BoNT producing strains into a single species – Clostridium botulinum – is no longer relevant. Based on the phylogenetic distances a reclassification at the generic and even family level would be a logic consequence.
References and further reading :
- Toxins and Known LD50 Values. (http://www.uab.cat/doc/DL50_biotoxines)
- Kerner J. Vergiftung durch verdorbene Würste. (1817). Tübinger Blätter Für Naturwissenschaften und Arzneykunde 3:1-25.
- Van Ermengem EP (1897). Über einen neuen anaeroben Bacillus und seine Beziehung zum Botulismus. Z. Hyg. Infectionskrankh. 26:1-56.
- Weigand MR, A. P-Gonzalez, TB Shirey, RC Broeker, MK Ishaq, KT Konstantindis, BH Raphael. (2015). Implications of genome-based discrimination between Clostridium botulinum Group I and Clostridium sporogenes strains for bacterial taxonomy. Appl. Env. Microbiol. DOI: 10.1128/AEM.01159-15
- Wheeler A and HS Smith (2013). Botulinum toxins: mechanisms of action, antinociception and clinical applications. Toxicology 306: 124-146.