Origins of Botulinum Toxin Types | List Labs

July 16, 2018


By: T.J. Smith

Gram Strain of U10146, BoNT A type strain, ATCC 25763

Gram strain of C. botulinum

Origins of Botulinum Toxin Types – relationships between toxins and the bacteria that produce them

Soon after the discovery that botulism was caused by a toxin, multiple toxin types were identified. Initial characterizations were based mainly on serological differences, however other anomalies were noted, such as differences in toxicity, sensitivity or resistance of different animal species to intoxication, cultural or morphological characteristics, and, finally, genetic differences.


Historical Differentiation of Bacterial Organisms

In the late 1800s and early 1900s, differentiation of bacterial organisms was mostly a matter of observations related to colony and cell characteristics, growth characteristics, and biochemical usage. Organisms were often partly identified according to their Gram stain characteristics, either Gram stain positive (purple) or Gram negative (red), often with cocci (balls) or rod (rectangular) shapes. Variations in shape, size, or coloration served to further delineate certain genera, such as with the paired kidney bean shapes of Neisseria or the comma-shaped Vibrios. The presence of Gram positive organisms with subterminal oval spores served to further identify the anaerobic bacteria as Clostridium.

Additional delineations have come from biochemical reactions of the bacteria. These may involve the ability to break down and utilize different proteins in the environment (proteolysis) or to utilize various carbohydrates through sugar fermentation. Examples of these assessments include liquification of gelatin or color changes triggered by a lowered pH due to fermentation of lactose or sucrose. With toxin-producing clostridial species, the ability to break down fats using lipases (positive lipase reaction) coupled with an inability to break down lecithin (negative lecithinase reaction) were hallmarks of the presence of Clostridium botulinum. Differences in optimal growth temperatures and resistance of spores to heat treatment have also been used as tools for differentiation.


Different Bacterial Variants Found to Produce Both Same and Different Toxins

Differences in these characteristics were noted from the beginning, when the Ellezelles strain characterized by Dr. E. Van Ermengem was found to be a nonproteolytic organism that favored a moderate optimal growth temperature of 25-30° C, while the Darmstadt strain identified by Dr. G. Landmann was definitely proteolytic with a higher optimal growth temperature of approximately 37° C (Van Ermengem 1897, Leuchs 1910). The Darmstadt strain produced type A toxin, while the Ellezelles strain produced type B toxin. The fact that the two strains produced different toxin serotypes initially linked these toxin differences with the bacterial differences. However, it was quickly discovered that different bacterial variants could produce the same toxin and the same bacteria could produce different toxins. C. botulinum strains that produced type A toxin were identified from the west coast of the United States, while virtually identical strains from the east coast were identified as type B (Burke 1919). However, the bacteria producing type B toxins in Europe differed from those in the U.S. in that the European strains were nonproteolytic, while the U.S. strains were uniformly proteolytic. This provided clear evidence that the bacterial types and the toxins they produced were not linked.

In 1922, the literature began to reflect these bacterial differences by identifying proteolytic organisms that produce botulinum neurotoxin as “Clostridium parabotulinum” and nonproteolytic organisms remained “Clostridium botulinum”. Dr. H. R. Seddon first used the term C. parabotulinum to distinguish his type C strains, isolated from cattle in Australia, from those of Dr. Ida Bengtson, isolated from fly larvae in the U.S. (Bengtson 1922, Seddon 1922). In addition to difficulties encountered when neutralizing her toxins with his antisera, the strains themselves appeared to differ in proteolytic tendencies. For the next 30 years proteolytic type A and B strains and Seddon type C strains were labeled C. parabotulinum while the nonproteolytic European type B strains and U.S. type C strains were designated C. botulinum.

When type E-producing bacteria were characterized, they were found to be uniformly closely related to the nonproteolytic type B strains (Hazen 1937), and on rare occasions both proteolytic and nonproteolytic bacterial strains that produced type F toxin were isolated (Moller and Scheibel 1960, Eklund, Poysky et al. 1967).


Bacterial Variants of Botulinum Toxins

Despite these obvious bacterial strain differences, it was proposed in 1953 and decided over the following decade to designate all botulinum neurotoxin-producing organisms as “Clostridium botulinum” on the basis of that single overriding characteristic. This was problematic as bacterial strains were already known that had produced botulinum toxin in the past but were no longer toxin producers. A major surprise came with the identification of an entirely different clostridial species, C. baratii, that produced type F toxin (Hall, McCroskey et al. 1985). Shortly after this came the identification of a C. butyricum strain that produced type E toxin (Aureli, Fenicia et al. 1986). In addition, the characterization of the bacteria that produced type G toxin revealed that it was a distinct species, prompting its designation as C. argentinense (Gimenez and Ciccarelli 1970).

Based on phenotypic characteristics, at least six different bacterial variants that could produce one (or more) botulinum neurotoxins have been identified. The question of whether these variants are a single entity or represent separate species was later answered using technological advances in genetic analyses.


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About the Author

Theresa Smith has studied botulinum neurotoxins for over 25 years, specializing in toxin countermeasure research, and is considered a leading expert regarding diversity in botulinum neurotoxins as well as the organisms that produce these toxins.



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Bengtson, I. (1922). “Preliminary note on a toxin-producing anaerobe isolated from the larvae of Lucilia caesar.” Pub Health Repts 37: 164-170.

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Gimenez, D. F. and A. S. Ciccarelli (1970). “Another type of Clostridium botulinum.” Zentralbl Bakteriol Parasitenk Infektionskr Hyg Abt 215: 221-224.

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Moller, V. and I. Scheibel (1960). “Preliminary report of an apparently new type of Cl. botulinum ” Acta Path Microbiol Scand 48: 80.

Seddon, H. R. (1922). “Bulbar paralysis in cattle due to the action of a toxicogenic bacillus, with a discussion on the relationship of the condition to forage poisoning (botulism).” J Comp Path Ther 35: 147-190.

Van Ermengem, E. (1897). “A new anaerobic bacillus and its relation to botulism (originally published as “Ueber einen neuen anaeroben Bacillus und seine beziehungen zum botulismus” in Zeitschrift fur Hygiene und Infektionskrankheiten, 26:1-56) ” Clin Infect Dis 4: 701-719.


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