Clostridium tyrobutyricum is well known as one of the main causative agents of severe cheese spoilage. The metabolism of this anaerobic bacterium during ripening leads to textural and sensory defects in cheese and consequential loss of product value. The potential to induce cheese spoilage, however, may vary among different strains of the same species. Therefore, a better understanding of the intra-species diversity of C. tyrobutyricum may be of practical relevance for the dairy industry. In the present study, we compared the ability of three typing techniques to differentiate 95 C. tyrobutyricum strains on the subspecies level: (1) repetitive element palindromic PCR (rep-PCR) fingerprinting combined with conventional agarose gel electrophoresis, (2) hexaplex-PCR followed by an automated capillary electrophoresis and (3) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing. MALDI-TOF MS fingerprinting provided only moderate reproducibility and low discriminatory power. Both PCR-based methods were highly reproducible and discriminative, with hexaplex-PCR fingerprinting being slightly more discriminative than rep-PCR typing. Overall, a high intra-species diversity was observed among the tested strains, indicating that further investigations on the strain level may be of interest.

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6-8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

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 1755^T, 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 1755^T 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 104389^T and to members of the Clostridium botulinum Group I, such as Clostridium sporogenes DSM 795^T. Average nucleotide identity (ANI) values with related species were below the 95% prokaryotic species threshold, with the highest similarity to C. tepidum DSM 104389^T (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 1755^T included phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine, accompanied by an unidentified aminophospholipid, several unidentified aminolipids, phospholipids and lipids. Major cellular fatty acids were C_18 : 1 CIS 9, C_16 : 0, C_14 : 0, and C_18 : 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 1755^T (=DSM 117478^T=LMG 33232^T=CCOS 2102^T).

High incidences of the foodborne pathogen Listeria monocytogenes have been reported on smear cheeses, and despite increased hygiene efforts, this incidence has remained stable in recent years. Applying antilisterial strains may increase the safety of smear cheeses. To find and test antilisterial strains, we inoculated fresh soft cheeses from nine dairies with the surrogate species Listeria innocua and assessed its growth under standardized ripening conditions. Acetic acid at day 23 (r = −0.66), lactose in fresh cheese (r = −0.63), and glucose at day 10 (r = −0.62), as well as seven amplicon sequence variants (ASVs), were negatively correlated with L. innocua growth. Two of these ASVs were assigned to the genus Leuconostoc of Lactobacillaceae (r = −0.82 and −0.71). Isolates from this family, from Aerococcaceae, and Carnobacteriaceae were characterized according to their inhibitory properties, and those showing antilisterial properties were applied as protective cultures in challenge tests. The combined application of strains of Leuconostoc mesenteroides, Aerococcaceae, and Carnobacteriaceae successfully eliminated low levels of L. innocua in the final products. This is likely explained by antimicrobial compounds, including mesentericin Y105 and acetate, and competition for carbon sources and iron. This study shows a promising way to improve the safety of soft smear cheeses by applying defined protective cultures.