Strain-level diversity of commercial probiotic isolates of Bacillus, Lactobacillus, and Saccharomyces species illustrated by molecular identification and phenotypic profiling

Abstract

Probiotic products are becoming more prevalent as awareness of the role of beneficial microbes in health increases. Ingredient labels of these products often omit identifications at the strain level, making it difficult to track down applicable published research. In this study, we investigated whether products labeled with the same species name contained different strains of those species. From 21 commercially available probiotic supplements and beverages, we cultured five main species: Bacillus coagulans, Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus, and the yeast Saccharomyces boulardii. To confirm the identity of each bacterial isolate, we applied standard molecular approaches: 16S rRNA gene sequencing and Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS). Phenotypic profiling and identification were performed with the Biolog Microbial Identification system. All of the bacterial isolates were correctly identified by at least one approach. Sequencing the 16S rRNA gene led to 82% of species identifications matching the product label, with 71% of isolates identified by MALDI-TOF MS and 60% identified correctly with the Biolog system. Analysis of the Biolog phenotypic profiles revealed different patterns of carbon source usage by each species, with sugars preferentially utilized by all except B. subtilis. To assess the strain-level differences, we compared strains of the same species and found variability in carbohydrate utilization and tolerance to environmental stressors (salt, acidity, antibiotics). By demonstrating that products listing the same species often contain strains with different 16S sequences and phenotypes, this study highlights that current labels of probiotic supplements do not sufficiently convey the strain diversity in these products.

Fig 1. Typical colony morphologies of probiotic isolates grown on agar media.

(A) Bacillus coagulans on MRS agar, (B) Bacillus subtilis on TSA, (C) Lactobacillus plantarum on MRS agar, (D) Lactobacillus rhamnosus on MRS agar, (E) Saccharomyces boulardii on SDA agar.

Fig 2. Phenotypic profiling of probiotic bacteria and yeast.

(A) Probiotic bacteria Biolog results: Utilization of carbon sources (sugars). (B) Probiotic bacteria Biolog results: Utilization of carbon sources (other). (C) Probiotic bacteria Biolog results: Tolerance to compounds (Antibiotics, other). (D) Probiotic bacteria: Antibiotic susceptibility testing. Key for Disc diffusion: S = sensitive, I = intermediate, R = resistant. (E) Probiotic yeast Biolog results: Utilization of carbon sources. Wells of the yeast (YT) plate in Fig 2E contain different carbon sources than the same numbered wells of the Biolog GenIII plate for bacteria. Key for Biolog wells: P = positive, h = half (borderline positive/negative), (-) = negative. Note: only 47 of the 71 carbon sources on the GenIII plate are displayed in Fig 2A and 2B; the wells G1-8, H1-8, and Column 9 were mostly negative and are not shown.

Fig 3. Representative biolog plates.

(A) Bacillus coagulans (no Biolog ID), (B) Bacillus subtilis, (C) Lactobacillus plantarum, (D) Lactobacillus rhamnosus.

Fig 4. Species in this study and abundance in products for sale by major online retailer.

(A) Left: Bacteria and yeast isolated from probiotic products in this study (total = 20). Right: Number of products listed on Amazon.com containing the selected species of probiotic microbes.