You can’t B. cereus? A Literature Review of Current Spores Research

Many Gram-positive bacteria (mainly Firmicutes) have the ability to undergo sporulation to survive in adverse conditions. Spores are small spherical (0.2µm), metabolically dormant and highly resilient structures that are resistant to extreme conditions, including: high temperatures, desiccation, extreme pH, chemical disinfection, ultraviolet light and aerobiosis _[2,3]. Spores are perceived as a threat to human health due to their ability to cause food spoilage and foodborne diseases, their pathological capabilities (such as botulism) and bioterrorism threat - Anthrax _[1,6].

A vegetative cell is triggered to sporulate by harsh environmental conditions, beginning with the formation of a forespore, followed by engulfment by the mother cell to form the endospore. This process is activated and regulated by sporulation-specific sigma (σ) factors in Bacillales and Clostridiales but there are distinct differences between the pathways in these 2 orders _[3,6]. A spore has several components as part of its structure. The core centre contains DNA protected by small acid soluble proteins (SASPs) and has a low water content due to the accumulation of dipicolinic acid (DPA) and Ca2+ _[1]. The core is surrounded by a low permeability inner membrane, a germ cell wall, a peptidoglycan cortex, multiple protein coats, and in some species; an exosporium _[1]. The exosporium in Bacillus and Clostridium spp. is made up of proteins and lipids arranged in a crystalline basal layer, such as in Bacillus cereus, the exosporium is formed mainly by the two cysteine-rich proteins CotY and ExsY _[4]. The shape and thickness of the exosporium varies across bacterial species. In Bacillus anthracis, the exosporium is thin and loose, the exosporium of Clostridium sordellii presents a smooth, balloon-like structure and spores across both genus (and several others) possess external hairlike or pili structures _[3]. These endospore appendages (Enas) have been described on the B. cereus spores with 2 distinct morphologies, denoted S-Ena (longer and thicker fibres) and L-Ena appendages (shorter and thinner fibres) _[5]. The role of these structures is not fully characterized in every species, but adhesion to surfaces and host cells is an integral part of cell survival and the infection process. Recent studies have suggested that Enas could enhance cellular auto-aggregation and aid in biofilm formation or play a role in transmission into a new host by adhesion to foods or the environment _[3,5].

Spore germination is instigated by external stimuli and identifying these specific germinants, such as L-alanine, Glucose and dodecylamine _[1], has been heavily researched over the last 50 years. Germinants bind to germinant receptors (GR’s) located on the inner membrane and causing the rapid release of DPA and Ca2+ through inner membrane channels, cortex hydrolysis and the intake of water by the spore to achieve outgrowth into a metabolically active cell _[6]. 

A research team from the University of Nottingham _[7] have recently looked into how spores can be harnessed to combat pathogenic Clostridioides difficile within the human gut. They have developed and trialled an oral vaccine of non-toxigenic C. difficile spores using an animal model and found that the vaccine induced an antibody mediated immune response that significantly reduced the adhesion of toxigenic C. difficile to the gut epithelial lining _[7]. Clostridium strains for their experiments were grown in a Whitley Anaerobic Workstation.

For more on the study of Clostridium watch our most recent webinar 'Toxigenic Clostridia in Humans and the Environment' or read another of our article's 'Clostridium ssp. and Clostridioides difficile: targeting Virulence Factors'.

Written by DWS Microbiologist, Charlotte Austin

References

1. Delbrück A., Zhang Y., Heydenreich R., Mathys A. (2021) Bacillus spore germination at moderate high pressure: A review on underlying mechanisms, influencing factors, and its comparison with nutrient germination. Comprehensive reviews in food science and food safety
2. Osman Erkmen (2021) Laboratory practices in Microbiology. 1^st Ed Elsevier Science 
3.  Portinha I., Douillard F., Korkeala H, Lindström M. (2022) Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum. International Journal of Molecular Sciences
4. Lablaine A., Serrano M., Bressuire-Isoard C., Chamot S., Bornard I., Carlin F., Henriques A., Broussolle V. (2021) The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus. mSphere
5. Nilsson D., Jonsmoen U., Malyshev D., Oberg R., Wiklund K., Andersson M. (2022) Physico-chemical characterization of single bacteria and spores using optical tweezers. BioRxiv
6. Christie G., Setlow P. (2020) Bacillus spore germination: Knowns, unknowns and what we need to learn. Cellular signalling
7. Hughes J., Aston C., Kelly M., Griffin R. (2022) Towards Development of a Non-Toxigenic Clostridioides difficile Oral Spore Vaccine against Toxigenic C. difficile. Pharmaceutics