Development of a High-Resolution Single-Nucleotide Polymorphism Strain-Typing Assay Using Whole Genome-Based Analyses for the Lactobacillus acidophilus Probiotic Strain

Abstract

Lactobacillus acidophilus is one of the most commonly used industrial products worldwide. Since its probiotic efficacy is strain-specific, the identification of probiotics at both the species and strain levels is necessary. However, neither phenotypic nor conventional genotypic methods have enabled the effective differentiation of L. acidophilus strains. In this study, a whole-genome sequence-based analysis was carried out to establish high-resolution strain typing of 41 L. acidophilus strains (including commercial isolates and reference strains) using the cano-wgMLST_BacCompare analytics platform; consequently, a strain-specific discrimination method for the probiotic strain LA1063 was developed. Using a core-genome multilocus sequence-typing (cgMLST) scheme based on 1390 highly conserved genes, 41 strains could be assigned to 34 sequence types. Subsequently, we screened a set of 92 loci with a discriminatory power equal to that of the 1390 loci cgMLST scheme. A strain-specific polymerase chain reaction combined with a multiplex minisequencing method was developed based on four (phoU, secY, tilS, and uvrA_1) out of 21 loci, which could be discriminated between LA1063 and other L. acidophilus strains using the cgMLST data. We confirmed that the strain-specific single-nucleotide polymorphisms method could be used to quickly and accurately identify the L. acidophilus probiotic strain LA1063 in commercial products.

Keywords: Lactobacillus acidophilus; Strain-Specific Identification; core genome MLST; monophyletic bacterium; whole genome sequences.

Figure 1

The allele-based neighbor-joining tree constructed with core-genome multilocus sequence-typing (cgMLST) profiles for the 41 Lactobacillus acidophilus strains on the basis of a comparison of 1390 differentiated core genes. The bold letters indicate commercial isolates. Bar, allele numbers.

Figure 2

The allele-based minimum spanning tree constructed with cgMLST profiles for the 41 Lactobacillus acidophilus strains on the basis of a comparison of 1390 differentiated core genes. Each circle represents a different sequence type. Branch values indicate the number of loci that differ between nodes.

Figure 3

The allele-based neighbor-joining tree and heatmap constructed with cgMLST profiles for the 41 Lactobacillus acidophilus strains on the basis of 92 differentiated core genes. The bold letters indicate commercial strains. Different alleles in the same column are indicated by different colors. Bar, allele numbers.

Figure 4

Development of the LA1063 strain-specific PCR-based identification method. Heatmap and neighbor-joining tree of the analyzed the 41 Lactobacillus acidophilus strains based on the presence or absence of genes. Arrows indicate strain-specific markers for Lactobacillus acidophilus LA1063 and BCRC 17481 strains.

Figure 5

Single nucleotide polymorphism (SNP) genotyping of four polymorphisms in the phoU, secY, tilS, and uvrA_1 distinguished genes on the Lactobacillus acidophilus LA1063 strain. (a) Electropherogram of a 2% agarose gel containing multiple PCR products derived from four DNA fragments. Lane M, 100-bp ladder DNA marker; lane 1, LA1063 probiotic strain; lanes 2‒4, LA1063 isolated from separate batches of its derived probiotic product; and lane N, negative control. (b) Electropherograms obtained from the LA1063 strains by four-plex SNaPshot minisequencing assay. The X-axis represents the size of the minisequencing products (nucleotides); the Y-axis represents relative fluorescence units (RFUs). STD: GS120 LIZ size standard.