Clostridium is a large genus of obligate anaerobes belonging to the Firmicutes phylum of bacteria, most of which have a Gram-positive cell wall structure. The genus includes significant human and animal pathogens, causative of potentially deadly diseases such as tetanus and botulism. Despite their relevance and many studies suggesting that they are not a monophyletic group, the taxonomy of the group has largely been neglected. Currently, species belonging to the genus are placed in the unnatural order defined as Clostridiales, which includes the class Clostridia. Here, we used genomic data from 779 strains to study the taxonomy and evolution of the group. This analysis allowed us to 1) confirm that the group is composed of more than one genus, 2) detect major differences between pathogens classified as a single species within the group of authentic Clostridium spp. (sensu stricto), 3) identify inconsistencies between taxonomy and toxin evolution that reflect on the pervasive misclassification of strains, and 4) identify differential traits within central metabolism of members of what has been defined earlier and confirmed by us as cluster I. Our analysis shows that the current taxonomic classification of Clostridium species hinders the prediction of functions and traits, suggests a new classification for this fascinating class of bacteria, and highlights the importance of phylogenomics for taxonomic studies.

Clostridium is a broad genus of anaerobic, spore-forming, rod-shaped, Gram-positive bacteria that can be found in different environments all around the world. The genus includes human and animal pathogens that produce potent exotoxins that cause rapid and potentially fatal diseases responsible for countless human casualties and billion-dollar annual loss to the agricultural sector. Diseases include botulism, tetanus, enterotoxemia, gas gangrene, necrotic enteritis, pseudomembranous colitis, blackleg, and black disease, which are caused by pathogenic Clostridium. Due to their ability to sporulate, they cannot be eradicated from the environment. As such, immunization with toxoid or bacterin-toxoid vaccines is the only protective method against infection. Toxins recovered from Clostridium cultures are inactivated to form toxoids, which are then formulated into multivalent vaccines. This review discusses the toxins, diseases, and toxoid production processes of the most common pathogenic Clostridium species, including Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Clostridium chauvoei, Clostridium septicum, Clostridium novyi and Clostridium hemolyticum.

Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings.

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a “fermentation map”, which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.

words)Clostridium is a large genus of obligate anaerobes belonging to the Firmicutes phylum of bacteria, most of which have a Gram-positive cell wall structure. The genus includes significant human and animal pathogens, causative of potentially deadly diseases such as tetanus and botulism. Despite their relevance and many studies suggesting that they are not a monophyletic group, the taxonomy of the group has largely been neglected. Currently, species belonging to the genus are placed in the unnatural order defined as Clostridiales, which includes the class Clostridia. Here we used genomic data from 779 strains to study the taxonomy and evolution of the group. This analysis allowed us to;(i) confirm that the group is composed of more than one genus (ii), detect major differences between pathogens classified as a single species within the group of authentic Clostridium spp. (sensu stricto), (iii) identify inconsistencies between taxonomy and toxin evolution that reflect on the pervasive misclassification of strains and, (iv) identify differential traits within central metabolism of members of what has been defined earlier and confirmed by us as cluster I. Our analysis shows that the current taxonomic classification of Clostridium species hinders the prediction of functions and traits, suggests a new classification for this fascinating class of bacteria and highlights the importance of phylogenomics for taxonomic studies.