By: T.J. Smith

The answer as to whether the botulinum neurotoxin (BoNT)-producing bacteria comprised six separate species required a complete revolution in microbial classification.  Up to the turn of the last century, bacterial differentiations were based on morphological characteristic and biochemical activities, known collectively as phenotypic characteristics.  However, the discovery of DNA as the ultimate code of life led to technological methodologies enabling the sequencing and comparisons of individual genes and, ultimately, entire bacterial genomes.  Initial studies used DNA-DNA hybridization (DDH) techniques which, due to the cumbersome nature of the assays, was followed quickly by comparative analyses of 16s rRNA gene, which is a highly conserved gene that is present among all bacterial species (Rossello-Mora and Amann 2001).  The results of these studies were remarkably similar, providing confidence in the predictability of both assays for bacterial speciation.

Setting the Stage for Current Classifications

16s rRNA analyses of various clostridial species verified earlier thoughts about their relationships (Collins 1998).   The proteolytic BoNT type A, B, and F-producing C. botulinum bacteria were found to cluster with closely related C. sporogenes, while nonproteolytic BoNT type B, E, and F-producing C. botulinum were determined to be a distinct species cluster.  Type C and D-producing bacteria were closely related to non-neurotoxigenic C. novyi strains.  Type G-producing bacteria, along with nontoxic C. subterminale, were deemed a distinct species, designated C. argentinense.  Type F-producing C. baratii and type E-producing C. butryricum were both found to be indistinguishable from their nontoxic counterparts using these techniques.  Thus, in addition to the neurotoxin-producing bacteria that had reverted to nontoxicity, additional connections between toxic and nontoxic organisms were seen.  This completely contradicted the theory that any botulinum neurotoxin-producing bacteria should be named “C. botulinum” and set the stage for current classifications based on whole genome analysis for differentiation of bacterial strains.

Seven Distinct Clostridial Species Produce Botulinum Neurotoxins

Currently, this analysis can be done at a very fine level, as each of the approximately 4 million nucleotide residues that reside within an average clostridial genome can be identified and compared.  Individual nucleotide differences among core, or shared, genes within a genome are analyzed using numerical computations that help determine species/species interfaces (Richter and Rossello-Mora 2009).  This technique is known as average nucleotide identity, or ANI.  It is known that the same bacterial isolate can mutate over time in the laboratory, so that sequencing of the same isolate over time should show a few minimal differences.  However, larger scale differences are seen in different strains within the same species and further numbers of differences separate distinct species.  These relationships are strengthened through analysis of large numbers of genomes, and this has helped to support an avalanche of bacterial genome sequencing studies.  To date over 200 Clostridium botulinum strains plus over 60 closely related strains have been sequenced and subjected to comparative analysis (https://www.ncbi.nlm.nih.gov/pubmed).  The results confirm that seven distinct clostridial species are capable of producing botulinum neurotoxins (Williamson, Sahl et al 2016).  These include three groups and four species. The first, Group I, proteolytic C. botulinum, had a name change, to C. parabotulinum and then changed back to C. botulinum Group I (Smith, Williamson et al 2018); Group II includes the nonproteolytic C. botulinum type B, E, and F toxin producers, and Group III, type C and D toxin-producing C. botulinum, a group name which has had a suggested change to C. novyi sensu lato (Skarin, Hafstrom et al 2011). In addition to these groups, four genetically distinct species which may produce botulinum toxin are C. argentinense; C. baratii; C. butyricum; and C. sporogenes.

Different species may produce the same toxin and different toxins may be produced by the same bacterial species.  In addition, there are documented non-neurotoxigenic members represented in each species.  A listing of BoNT-producing bacteria and their characteristics is shown in Table 1 (Hatheway 1988, Collins 1998).

Table 1.  An abbreviated table showing some major characteristics of various clostridia, that produce botulinum toxin.

Species/group	Toxins produced	Lipase	Lecithinase	Proteolytic
C. botulinum Group I	A, B, F, Ab, Ba, Af, HA	+	–	yes
C. botulinum Group II	B, E, F	+	–	no
C. botulinum Group III	C, D	+	variable	variable
C. argentinense	G	–	–	yes
C.baratii	F	–	+	no
C. butyricum	E	–	–	no
C. sporogenes	B	+	–	yes

It has been determined that there is a great deal of diversity among the bacteria that produce botulinum toxins, as well as among the toxins themselves.  The seven toxin serotypes differ to such a large extent that the antisera to one type cannot neutralize the toxin of a different type.  However, genetic analysis of these toxins has revealed yet another level of diversity.  The identification and study of BoNT subtypes has been the subject of increasing interest in the past three decades, leading to a whole new understanding of the complexity of these proteins.

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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.

References

Collins, M. D. (1998). “Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins.” J Appl Microbiol 84: 5-17. PMID: 15244052

Hatheway, C. L. (1988). Botulism. In A. Balows, W. H. Hausler, J. Ohashi and A. Turano (ed) Laboratory Diagnosis of Infectious Diseases New York, Springer-Verlag: 111-133.

Richter, M. and R. Rossello-Mora (2009). “Shifting the genomic gold standard for the prokaryotic species definition.” Proc Natl Acad Sci U S A 106(45): 19126-19131. PMID: 19855009

Rossello-Mora, R. and R. Amann (2001). “The species concept for prokaryotes.” FEMS Microbiol Rev 25(1): 39-67. PMID: 11152940

Skarin, H., T. Hafstrom, J. Westerberg and B. Segerman (2011). “Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements.” BMC Genomics 12(185): 1-13. PMID: 21486474

Smith, T. J., C. H. Williamson, K. Hill, J. W. Sahl and P. Keim (2018). “Botulinum neurotoxin-producing bacteria – isn’t it time we called a species a species?” MBio in press.

Williamson, C. H., J. W. Sahl, T. J. Smith, G. Xie, B. T. Foley, L. A. Smith, R. A. Fernandez, M. Lindstrom, H. Korkeala, P. Keim, J. Foster and K. Hill (2016). “Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.” BMC Genomics 17: 180. PMID: 26939550