Accurate Identification of 15 Strains of Lactobacillus in Commercially Available Probiotics and Study on Virulence Genes and Drug Resistance Genes

OBJECTIVE  With the development of microbial identification technology and the change of species classification and nomenclature rules, resulting in confusion of bacterial names. In this study, 15 strains of Lactobacillus from 13 batches of probiotics were identified by various methods, and the results of different identification methods were compared, the aim of this study was to establish a rapid and accurate standard method for the identification of Lactobacillus. Meanwhile, Whole Genome Sequencing(WGS) and bioinformatics methods were used to further evaluate the safety of Lactobacillus.

METHODS  According to the method of "GB 4789.35-2023 National food safety standard-Food microbiological examination-Lactic acid bacteria test", 15 strains of Lactobacillus were isolated and purified from 13 batches of commercial probiotic products. The 15 strains of Lactobacillus were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS), 16S rRNA gene sequencing and WGS, respectively. At the same time, WGS and bioinformatics methods were used to sequence assembly, gene prediction and functional annotation. In-depth excavation of information such as virulence genes and drug resistance elements of Lactobacillus strains.

RESULTS  MALDI-TOF MS, 16S rRNA gene sequencing and WGS technology were used to identify 4, 7 and 5 strains of Lactobacillus in 15 strains, respectively. The identification results were inconsistent with the label name. A04 was identified as Lactobacillus acidophilus, and the identification results of three sequencing techniques were all Lactobacillus paracasei, suggesting that there was confusion in the names of the products. Venn diagram analysis showed that the identification results of 5 strains were inconsistent between MALDI-TOF MS and 16S rRNA gene sequencing technology. The identification results of 6 strains of bacteria by 16S rRNA gene sequencing technology and WGS technology were inconsistent. The identification results of 3 strains of bacteria by MALDI-TOF MS and WGS were inconsistent. Using WGS and bioinformatics methods, it was found that no drug resistance elements and virulence elements were detected in 15 strains of Lactobacillus.

CONCLUSION  In this study, three identification techniques, MALDI-TOF MS, 16S rRNA gene sequencing and WGS, were used to confirm that the actual bacteria names of 5 batches of commercially available probiotic products were inconsistent with the product labels. There are some differences in the identification results of the three identification technologies, but WGS can obtain complete genomic information to achieve accurate identification of Lactobacillus and accurate analysis of virulence genes and drug resistance genes. Therefore, it can be used as a standard method for Lactobacillus identification and genome safety analysis, and realize tracking evaluation and supervision of commercially available probiotic products.