Isolation and phenotypic and genomic characterization of Tetragenococcus spp. from two Spanish traditional blue-veined cheeses made of raw milk

High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties.

Automated summary

High throughput sequencing has revealed the presence of Tetragenococcus-related DNA sequences in dairy environments. In this study, a selective medium was developed to isolate Tetragenococcus spp. strains from Spanish blue-veined cheese made from raw milk. Phenotypic and genetic tests were conducted, and genome sequencing was performed on selected strains. The strains showed diverse genetic and phenotypic characteristics and exhibited poor growth in milk but utilized lactose and fermented milk citrate. Genome analysis identified lactose/galactose-related genes and genes encoding citrate metabolic enzymes. The strains were resistant to certain antibiotics but did not possess known antibiotic resistance genes or pathogenicity factors. No decarboxylase-encoding genes involved in biogenic amine production were found. Genome comparison revealed the presence of genes coding for proteolytic and lipolytic systems. These findings suggest that Tetragenococcus strains with desired traits could be used as cultures to enhance the flavor of traditional blue-veined cheeses.