Identification and Monitoring of Lactobacillus delbrueckii Subspecies Using Pangenomic-Based Novel Genetic Markers

Abstract

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

Pangenome Analysis Reveals the Genetic Basis for Taxonomic Classification of the Lactobacillaceae Family

“…Prior pangenome studies of lactic acid bacteria were focused on a few of the selected species, such as L. plantarum (127 genomes) (Carpi et al, 2022), L.delbrueckii (41 genomes) (Kim et al, 2021), L. acidophilus (46 genomes) (Huang et al, 2021), L. paracaseii (34 genomes) (Smokvina et al, 2013) and Lactobacillus spp. (Inglin et al, 2018).…”

Genomic Characterization of Lactobacillus delbrueckii Strains with Probiotics Properties

“…This genomic variation can be related to the differences in the number of unique genes observed across the strains, in which the subspecies lactis had the highest number. The pangenome analysis of Lactobacillus delbrueckii species has already been carried out by Inglin et al (2018) and Kim et al (2021) . However, the above authors did not perform a functional analysis related to the probiosis of these strains.…”