Genetic diversity of Leuconostoc mesenteroides isolates from traditional Montenegrin brine cheese

Members of the genus Leuconostoc are Gram-positive, non-spore-forming, non-motile, and heterofermentative Lactic Acid Bacteria (LAB). Leuconostoc spp. are usually combined as an adjunct culture with fast acid producing Lactococcus sp. in undefined mixed (DL) type starter cultures. In many traditional dairy products, Leuconostoc spp. is a natural part of non-starter lactic acid bacteria (NSLAB), which play an important role in flavor development and therefore accounting for the typicity of cheeses. Despite the proven influence of Leuconostoc spp. on the final quality and characteristics of the product, data on the genomic diversity of Leuconostoc spp. isolates obtained from cheese are still scarce. Therefore, the focus of this study was the genomic characterization of Leuconostoc spp. isolated from different Montenegrin brine cheeses, in order to explore their diversity and provide genetic information for the selection of strains suitable for utilization as adjunct cultures in a future industrial cheese production.In spring and summer of 2019, sixteen Leuconostoc spp. isolates were obtained from white brine cheeses from nine different producers from three municipalities in the northern region of Montenegro. All isolates were identified as Ln. mesenteroides by MALDI-TOF-MS, digital DNA-DNA hybridization, 16s rDNA sequence analysis and ribosomal multilocus sequence typing (rMLST). Type Strain Server analysis (TYGS) assigned ten isolates to subspecies Ln. mesenteroides, and two each to Ln. cremoris, Ln. dextranicum and Ln. jonggajibkimchii, respectively. MLST and core genome MLST (cgMLST) revealed a high diversity of the Montenegrin Ln. mesenteroides cheese isolates. All isolates carried genes of the bacteriocin biosynthetic gene clusters, eight out of 16 strains carried the citCDEFG operon, 14 carried butA, and all 16 isolates carried alsS and ilv, genes responsible for the production of aromatic compounds. Safety evaluation indicated that isolates carried no virulence factors; five carried plasmids, one strain carried a partial beta-lactamase gene, and one strain had a C656T transition in the 23S rRNA gene, conferring resistance to erythromycin and clindamycin.In conclusion, Ln. mesenteroides isolates from Montenegrin traditional cheeses, based on MLST and cgMLST displayed a high genetic diversity and, in addition, lacked close genetic relatedness to the isolates currently deposited in GenBank.