Several Clostridium species are responsible for significant losses in the food industry. Here, a novel obligately anaerobic, spore-forming, motile bacterium, designated Clostridium caseinilyticum sp. nov., was isolated from hard and extra-hard cheeses with a defect consisting of a degradation of the cheese matrix. The type strain, FAM 1755T, was obtained from Swiss Emmentaler cheese in 1983, and additional strains CREA 4990 and FAM 27665 were isolated from Italian Grana Padano in 2022 and from Swiss silage in 2012, respectively. The rod-shaped, Gram-positive cells of FAM 1755T measured 3–4.5 µm in length and 0.7–0.9 µm in width. Phylogenetic analyses of whole-genome and 16S rRNA gene sequences placed the new taxon close to Clostridium tepidum DSM 104389T and to members of the Clostridium botulinum Group I, such as Clostridium sporogenes DSM 795T. Average nucleotide identity (ANI) values with related species were below the 95% prokaryotic species threshold, with the highest similarity to C. tepidum DSM 104389T (91.4% ANI). The new species grew at a slightly lower temperature range (20–40 °C) than C. tepidum and C. sporogenes and was more halotolerant [0–5% (w/v) NaCl] than C. tepidum. Although genetically closer to C. tepidum, its growth preferences were more similar to those of C. sporogenes. It was catalase-, oxidase- and urease-negative, fermented glucose, and hydrolyzed gelatin but not aesculin. The polar lipid profile of FAM 1755T included phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine, accompanied by an unidentified aminophospholipid, several unidentified aminolipids, phospholipids and lipids. Major cellular fatty acids were C18 : 1 CIS 9, C16 : 0, C14 : 0, and C18 : 1 DMA. The genomic DNA G+C content was 27.4 mol%. Unlike C. tepidum, the new species consistently hydrolyzed milk proteins, a feature implicated in cheese spoilage. Consequently, we propose the name C. caseinilyticum sp. nov., with the type strain FAM 1755T (=DSM 117478T=LMG 33232T=CCOS 2102T).
