Comparison of sporulation and germination conditions for Clostridium perfringens strains

Clostridium perfringens is a spore forming, obligate anaerobe, Gram-positive bacterium that causes a range of diseases in humans and animals. The infectious form of C. perfringens is the spore, a structure that is derived from the vegetative cell under conditions of nutrient deprivation. The first step in C. perfringens pathogenesis is the differentiation of spores into replicating bacteria. Previous work in analyzing C. perfringens spore germination has produced contradictory, strain-specific results. Hence, we analyzed the requirements for spore formation and germination in seven different C. perfringens strains. Our data showed that C. perfringens sporulation conditions are strain specific, but germination responses are homogenous. C. perfringens spores can germinate using two distinct pathways. The first germination pathway (the amino acid-only pathway or AA) requires L-alanine, L-phenylalanine, and sodium ions (Na+) as co-germinants. L-arginine is not a required germinant but potentiates germination. The AA pathway is inhibited by aromatic amino acids and potassium ions (K+). Bicarbonate (HCO3-), on the other hand, bypasses potassium-mediated inhibition of C. perfringens spore germination through the AA pathway. The second germination pathway (the bile salt and amino acid pathway or BA) is more promiscuous and is activated by several bile salts and amino acids. In contrast to the AA pathway, the BA pathway is insensitive to Na+, but can be activated by either K+ or HCO3-. We hypothesize that C. perfringens may have evolved these two distinct germination pathways to ensure spore response to different host environments. Manuscript contribution to the field Clostridium perfringens is a ubiquitous bacterium that can infect a wide variety of animal hosts, including humans. C. perfringens counts with a veritable arsenal of toxins that are differentially expressed depending on the host infected. Besides toxin production, C. perfringens can also form dormant and resistant spores that serve as infective vehicles. C. perfringens spores need to germinate back into vegetative cells to cause disease. Befitting to its wide host range, C. perfringens spore germination seems to use strain specific. In this study, we tested the ability of seven C. perfringens strains to produce spores under different conditions. We also tested the requirements for spore germination. We found that while C. perfringens sporulation was highly varied, the germination response for all strains could be divided into two distinct pathways. Since C. perfringens spores need to germinate to cause infection, understanding the germination behavior could lead to approaches for the prevention of diseases in humans and veterinary animals. ### Competing Interest Statement The authors have declared no competing interest.