Ueli von Ah

The history of cheesemaking is deeply intertwined with the evolution of microbial communities, from spontaneous fermentation to modern, standardized practices. Despite centuries of refinement, the most profound shifts in cheese production occurred in the last century, driven by advances in microbiology and production technologies. These changes have shaped the bacterial and viral communities within cheese, yet the specific impacts remain underexplored. Using shotgun metagenomics and 16S rRNA gene amplicon sequencing approaches, we examined microbial community changes in Raclette du Valais, a traditional Swiss cheese, using preserved cheese wheels from 1875 to 2017 from the same alpine dairy in Switzerland. Our results reveal that significant shifts in microbial community composition coincide with changes in production practices. Notably, the oldest cheese harbored a distinct bacterial community, dominated by Lactiplantibacillus paraplantarum , Streptococcus thermophilus , Pseudolactococcus laudensis , and taxa commonly associated with the gut environment, indicative of spontaneous fermentation and the use of calf stomach for milk coagulation. Functionally, we can also track the rise and fall of antibiotic resistance genes mirroring their use. Furthermore, we found that domestication of lactic acid bacteria predates the studied period, and that bacteriophage genera detected in 1875 are representatives of those commonly found in modern cheesemaking. These findings highlight how microbial communities have adapted to changing production methods and how human intervention, through practices like antibiotic use in animal husbandry, has influenced these ecosystems in remote alpine cheesemaking. Significance Statement: Cheesemaking relies on complex microbial ecosystems shaped by long-standing human practices, yet how these communities responded to the modernization of food production has remained largely unknown. By analyzing DNA preserved in historical cheese wheels from a single alpine dairy, we examine microbial community changes across a key technological transition. We show that modernization impacted bacterial composition and functional potential, that cheese microbiomes record the rise of agricultural antibiotic use, and that major cheese-associated phages and domesticated starter bacteria were already established over a century ago. These findings demonstrate that historical cheeses preserve long-term microbial records and offer a glimpse how changes in food production practices shape fermented-food microbiomes.

Propionibacterium freudenreichii ( P. freudenreichii ) has various biotechnological applications, notably in the ripening of Swiss-type cheese, where it utilizes the two enantiomers of lactate as the main carbon source, contributing to flavor development and eye formation. Here, we genotypically characterized two P. freudenreichii strains unable to catabolize L-lactate through whole-genome sequencing and variant calling, using P. freudenreichii FAM-14222 as the reference genome, which highlighted a mutation in the lutB gene in both strains. This gene is part of the lutABC operon, which has been previously linked to lactate utilization in other bacterial species. Subsequently, we successfully restored the strains’ ability to utilize L-lactate by following an adaptive laboratory evolution approach, which involved repeated subculturing in a medium containing L-lactate as the main carbon source. Sequencing of the lutB gene confirmed that isolates with a restored ability to utilize L-lactate had also reverted the mutation back to wild-type, supporting the involvement of the lutABC operon in L-lactate catabolism in P. freudenreichii . Moreover, the phenotype of the two L-lactate-negative strains was confirmed under cheesemaking conditions, highlighting the potential of the strains as cheese ripening cultures. IMPORTANCE: Lactate catabolism is of paramount importance in Propionibacterium freudenreichii , particularly for its industrial applications, such as Swiss-type cheese ripening. Nevertheless, the genetic background of this metabolic process is not fully understood. In our study, we developed an adaptive laboratory evolution-based approach for the elucidation of L-lactate catabolism, starting from two strains unable to utilize L-lactate. Our results delivered experimental evidence of the role of the lutABC operon in this process, as opposed to the widespread theory of L-lactate dehydrogenase-mediated oxidation. A deeper understanding of this metabolic pathway will be beneficial for a more efficient selection of industrial strains, as well as for metabolic engineering.

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.

Domestication – the artificial selection of wild species to obtain variants with traits of human interest – was integral to the rise of complex societies. The oversupply of food was probably associated with the formalization of food preservation strategies through microbial fermentation. While considerable literature exists on the antiquity of fermented food, only few eukaryotic microbes have been studied so far for signs of domestication, less is known for bacteria. Here, we tested if cheese starter cultures harbour typical hallmarks of domestication by characterising over 100 community samples and over 100 individual strains isolated from historical and modern traditional Swiss cheese starter cultures. We find that cheese starter cultures have low genetic diversity both at the species and strain-level and maintained stable phenotypic traits. Molecular clock dating further suggests that the evolutionary origin of the bacteria approximately coincided with the first archaeological records of cheese making. Finally, we find evidence for ongoing genome decay and pseudogenization via transposon insertion related to a reduction of their niche breadth. Future work documenting the prevalence of these hallmarks across diverse fermented food systems and geographic regions will be key to unveiling the joint history of humanity with fermented food microbes.

A novel, facultatively anaerobic, Gram-stain-positive, motile, endospore-forming bacterium of the genus Paenibacillus , designated strain 2.1^T, was isolated from a colony of Apis mellifera affected by European foulbrood disease in Switzerland. The rod-shaped cells of strain 2.1^T were 2.2–6.5 µm long and 0.7–1.1 µm wide. Colonies of strain 2.1^T were orange-pigmented under oxic growth conditions on solid basal medium at 35–37 °C. Strain 2.1^T showed catalase and cytochrome c oxidase activity. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and phospholipid. The only respiratory quinone was menaquinone 7, and the major cellular fatty acids were anteiso-C_15 : 0, anteiso-C_17 : 0, iso-C_15 : 0, iso-C_17 : 0 and palmitic acid (C_16 : 0), which is consistent with other members of the genus Paenibacillus . The G+C content of the genomic DNA of strain 2.1^T was 53.3 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence similarity showed that strain 2.1^T was closely related to Paenibacillus dendritiformis LMG 21716^T (99.7 % similarity) and Paenibacillus thiaminolyticus DSM 7262^T (98.8 %). The whole-genome average nucleotide identity between strain 2.1^T and the type strains of P. dendritiformis and P. thiaminolyticus was 92 and 91 %, respectively, and thus lower than the 95 % threshold value for delineation of genomic prokaryotic species. Based on the results of phylogenetic, genomic, phenotypic and chemotaxonomic analyses we propose the name Paenibacillus melissococcoides sp. nov. for this novel Paenibacillus species. The type strain is 2.1^T (=CCOS 2000^T=DSM 113619^T=LMG 32539^T).

Genomic screening of bacteria is common practice to select strains with desired properties. However, 40-60% of all bacterial genes are still unknown, making capturing the phenotype an important part of the selection process. While omics-technologies collect high-dimensional phenotypic data, it remains challenging to link this information to genomic data to elucidate the impact of specific genes on phenotype. To this end, we present Scoary2, an ultra-fast software for microbial genome-wide association studies (mGWAS), enabling integrative data exploration. As proof of concept, we explore the metabolome of 44 yogurts with different strains of Propionibacterium freudenreichii , discovering two genes affecting carnitine metabolism.

Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

Reformulating recipes of ready-to-eat meat products such as salami to reduce salt content can mitigate the negative health impacts of a high salt diet. We evaluated the potential of potassium lactate (KL) as a sodium chloride (NaCl) replacer during salami production. NaCl and KL stress tolerance comparisons showed that four food-derived Listeria innocua isolates were suitable as biologically safe Listeria monocytogenes surrogates. Effects of the high salt (4% NaCl) concentration applied in standard salami recipes and a low salt (2.8% NaCl) plus KL (1.6%) combination on product characteristics and growth of contaminating Listeria and starter culture were compared. Simulated salami-ripening conditions applied in meat simulation broth and beef showed that the low salt plus KL combination retained similar to superior anti-Listeria activity compared to the high salt concentration treatment. Salami challenge tests showed that the low NaCl plus KL combination had comparable anti-Listeria activity as the high NaCl concentration during ripening and storage. No significant differences were detected in starter culture growth profiles and product characteristics between the high NaCl and low NaCl plus KL combination treated salami. In conclusion, KL replacement enabled a 30% NaCl reduction without compromising the product quality and antimicrobial benefits of high NaCl concentration inclusion.