Intraspecific microdiversity and ecological drivers of lactic acid bacteria in naturally fermented milk ecosystem

Traditional fermented milks are produced by inoculating technique, which selects well-adapted microorganisms that have been passed on through generations. Few reports have used naturally fermented milks as model ecosystems to investigate the mechanism of formation of intra-species microbial diversity. Here, we isolated and whole-genome-sequenced a total of 717 lactic acid bacterial isolates obtained from 12 independent naturally fermented milks collect from 12 regions across five countries. We further analyzed the within-sample intra-species phylogenies of 214 Lactobacillus helveticus isolates, 97 Lactococcus lactis subsp. lactis isolates, and 325 Lactobacillus delbrueckii subsp. bulgaricus isolates. We observed a high degree of intra-species genomic and functional gene diversity within-/between-sample(s). Single nucleotide polymorphism-based phylogenetic reconstruction revealed great within-sample intra-species heterogeneity, evolving from multiple lineages. Further phylogenetic reconstruction (presence-absence gene profile) revealed within-sample inter-clade functional diversity (based on carbohydrate-active enzyme- and peptidase-encoding genes) in all three investigated species/subspecies. By identifying and mapping clade-specific genes of intra-sample clades of the three species/subspecies to the respective fermented milk metagenome, we found extensive potential inter-/intra-species horizontal gene transfer events. Finally, the microbial composition of the samples is closely linked to the nucleotide diversity of the respective species/subspecies. Overall, our results contribute to the conservation of lactic acid bacteria resources, providing ecological insights into the microbial ecosystem of naturally fermented dairy products.