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

Date Published: March 22, 2019

Publisher: Public Library of Science

Author(s): Juliana M. Ansari, Christine Colasacco, Elli Emmanouil, Scott Kohlhepp, Olivia Harriott, Adriana Calderaro.


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.

Partial Text

A widespread awakening in the public and medical community’s interest in beneficial bacteria for promoting health is underway. Accelerated by the ease and affordability of rapid DNA sequencing technology, an avalanche of studies in animal models and humans has linked the microbiome (the microbial community inhabiting the human body) body to a wide range of diseases. This mounting knowledge of the human microbiome has stimulated interest in bacteria that confer a health benefit to the host (probiotics) or foods that selectively enhance growth of certain beneficial microbes (prebiotics). While fermented foods and beverages are common in the traditional diets around the world, market demand for probiotic foods is growing [1][2]. Probiotic strains are typically selected from lacto-fermented foods or the human digestive tract, then studied clinically for health-promoting effects. Two predominant groups of probiotic bacteria are the lactic acid bacteria (Lactobacillus and related genera) and bifidobacteria (Bifidobacterium spp). Certain Bacillus species and the yeast Saccharomyces boulardii are also sold in probiotic supplements and beverages. Candidate probiotic bacteria such as Akkermansia municiphila and Faecalibacterium prausnitzii, often termed “next generation probiotics,” have been identified from human microbiome studies but are not yet commercially available [3].

This study highlights that labeling of probiotic products with only the species name may not provide sufficient information about the strain-level diversity in these products. We found that on average, roughly half of the probiotics examined had the specific strain listed on the label, which varied considerably by store (Table 3). Adding strain information to labels would allow consumers and/or healthcare providers to more readily evaluate clinical studies of the probiotic’s effects for specific indications [15]. From survival in the GI tract (by tolerance to acidic pH and bile salts), to adhesion to intestinal cells, to competition with pathogens and production of bioactive compounds, the capacity and efficiency to perform these functions is often strain-dependent [33][53]. While it is well known that strain-level differences occur in the probiotic properties of microorganisms [6][14], our study showed that considerable variability in metabolism and environmental stress tolerance exists between these strains, with between 16–34% of the phenotypic assay wells yielding variable results. Microbial physiology is linked to “probiotic” phenotypes, because the food sources and molecular cues that cells encounter in their environment often directly regulate the expression of proteins and metabolites (or community-level behavior such as aggregation and biofilm formation) that confer the probiotic’s beneficial effect. While this study did not explicitly measure probiotic properties, testing probiotic characteristics would be a logical continuation of this research, and whole-genome sequencing could be used to identify the genetic basis of strain-specific differences.