Unraveling the world of halophilic and halotolerant bacteria in cheese by combining cultural, genomic and metagenomic approaches

Halophilic/halotolerant bacteria are generally assumed to live in natural environments, although they may also be found in foods such as cheese and seafood. These salt-loving bacteria have been occasionally characterized in cheese, and studies on their ecological and technological functions are still scarce. We therefore selected 13 traditional cheeses to systematically characterize these microorganisms in their rinds via cultural, genomic and metagenomic methods. Using different salt-based media, we identified 35 strains with unique 16S rRNA and rpoB gene sequences, whose whole genome was sequenced. Twenty are Gram-positive species including notably Brevibacterium aurantiacum (6) and Staphylococcus equorum (3), which are also frequently added as starters. ANI and pan-genomic analyses confirm the high genetic diversity of B. aurantiacum and reveal the presence of two subspecies in S. equorum, as well as the genetic proximity of several cheese strains to bovine isolates. Additionally, we isolated 15 Gram-negative strains, potentially defining ten new species of halophilic/halotolerant cheese bacteria, in particular for the genera Halomonas and Psychrobacter. The use of all the genomes sequenced in this study as a reference to complement those existing in the databases allowed us to study the representativeness of 66 species of halophilic/halotolerant bacteria in 74 cheese rind metagenomes. While Gram-positive strains may flourish in the different types of technologies, Gram-negative species are particularly abundant in cheeses with high moisture, such as washed-rind cheeses. Finally, analyses of co-occurrences reveal assemblies, including the frequent coexistence of several species of the same genus, forming moderately complex ecosystems with functional redundancies that probably ensure stable cheese development.