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.

In the fight against the spread of antibiotic resistance (ABR), authorities usually require that strains “intentionally added into the food chain” be tested for their antibiotic susceptibility. This applies to strains used in starter or adjunct cultures for the production of fermented foods, such as many strains of Pediococcus pentosaceus . The European Food Safety Authority (EFSA) recommends testing strains for their antibiotic susceptibility based on both genomic and phenotypic approaches. Furthermore, it proposes a set of antibiotics to assess, as well as a list of microbiological cutoffs (MCs) allowing classifying lactic acid bacteria as susceptible or resistant. Accurate MCs are essential, on the one hand, to avoid false negative strains, which may carry ABR genes and remain unnoticed, and on the other, to avoid false positive strains, which may be discarded while screening potential candidates for food-technology applications. Due to relatively scarce data, MCs have been defined for the whole Pediococcus genus, although differences between different species should be expected. In this study, we investigated the antibiotic susceptibility of thirty-five strains of P. pentosaceus isolated from various matrices in the last seventy years. Minimal inhibitory concentrations (MICs) were determined using a standard protocol, and MIC distributions were established. Phenotypic analyses were complemented with genome sequencing and by seeking known antibiotic resistance genes. The genomes of all the strains were free of known antibiotic resistance genes, but most displayed MICs above the currently defined MCs for chloramphenicol, and all showed excessive MICs for tetracycline. Based on the distributions, we calculated and proposed new MCs for chloramphenicol (16 instead of 4 mg/L) and tetracycline (256 instead of 8 mg/L).

Enumeration and isolation of Streptococcus salivarius subsp. thermophilus from cheese is challenging, due to the relatively high number of species it may host. We describe medium SPY9.3 for the cultivation of S. salivarius subsp. thermophilus from cheese. The medium and related incubation conditions (SPY) was compared with 2 other protocols, M17 and ST: sensitivity was assessed by parallel cultivation of 55 strains of S. salivarius subsp. thermophilus, and selectivity by (i) parallel cultivation of 60 strains belonging to 20 different non-target species and sub-species and (ii) isolating bacteria from 3 raw-milk cheeses. Colony counts were similar on SPY9.3 and M17 (mean difference 0.07 log(cfu/mL), p > 0.001) and significantly higher on ST than on M17 and SPY9.3 (mean differences 0.42 and 0.48 log(cfu/mL), respectively, p < 0.001). SPY was more specific than ST and M17, with respectively 20%, 40%, and 50% of the investigated non-target species able to grow. S. salivarius subsp. thermophilus, Enterococcus spp., and Staphylococcus aureus were indistinguishable using all 3 protocols. Only SPY avoided growth of Lactobacillus delbrueckii subsp. lactis. Finally, ST and SPY displayed higher recoveries of S. salivarius subsp. thermophilus colonies from cheese than M17 (5.6, 5.5, and 3.0 adjusted log(cfu/mL), respectively) and the lowest proportion of non-specific isolates. The protocol described here and based on SPY9.3 presents a promising alternative to existing protocols for the enumeration and isolation of S salivarius subsp. thermophilus from cheese or other complex fermented products.

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either ‘sensitive’ or ‘resistant’ based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains’ genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing.

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).

OpenGenomeBrowser is a self-hostable open-source platform that manages access to genomic data and drastically simplifies comparative genomics analyses. It enables users to interactively generate phylogenetic trees, compare gene loci, browse biochemical pathways, perform gene trait matching, create dot plots, execute BLAST searches, and access the data. It features a flexible user management system, and its modular folder structure enables the organization of genomic data and metadata, and to automate analyses. We tested OpenGenomeBrowser with bacterial, archaeal and yeast genomes. The largest instance currently contains over 1,400 bacterial genomes. Source code, documentation, tutorials and a demo server are available at opengenomebrowser.github.io .