Comparative genomic analysis of the multispecies probiotic-marketed product VSL#3

Abstract

Several probiotic-marketed formulations available for the consumers contain live lactic acid bacteria and/or bifidobacteria. The multispecies product commercialized as VSL#3 has been used for treating various gastro-intestinal disorders. However, like many other products, the bacterial strains present in VSL#3 have only been characterized to a limited extent and their efficacy as well as their predicted mode of action remain unclear, preventing further applications or comparative studies. In this work, the genomes of all eight bacterial strains present in VSL#3 were sequenced and characterized, to advance insights into the possible mode of action of this product and also to serve as a basis for future work and trials. Phylogenetic and genomic data analysis allowed us to identify the 7 species present in the VSL#3 product as specified by the manufacturer. The 8 strains present belong to the species Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium breve and B. animalis subsp. lactis (two distinct strains). Comparative genomics revealed that the draft genomes of the S. thermophilus and L. helveticus strains were predicted to encode most of the defence systems such as restriction modification and CRISPR-Cas systems. Genes associated with a variety of potential probiotic functions were also identified. Thus, in the three Bifidobacterium spp., gene clusters were predicted to encode tight adherence pili, known to promote bacteria-host interaction and intestinal barrier integrity, and to impact host cell development. Various repertoires of putative signalling proteins were predicted to be encoded by the genomes of the Lactobacillus spp., i.e. surface layer proteins, LPXTG-containing proteins, or sortase-dependent pili that may interact with the intestinal mucosa and dendritic cells. Taken altogether, the individual genomic characterization of the strains present in the VSL#3 product confirmed the product specifications, determined its coding capacity as well as identified potential probiotic functions.

Development of omics‐based protocols for the microbiological characterization of multi‐strain formulations marketed as probiotics: the case of VSL#3

“…Using this sequence coverage, a strain with a load of one billion of CFU up to an overall load of 450 billions of CFU (declared in the product label), and therefore representing approximately 0.22% of the blend, should be theoretically covered by approximately 13 333 reads. The results obtained showed that 99.8% of the total reads matched with an identity of > 99% to the genomes of the 8 strains that had been previously described (Douillard et al, 2018). This confirmed the identity of the strains in this commercial VSL#3 product.…”

“…E). Since the urease activity was unique for S. thermophilus (Douillard et al , ) as confirmed by the metagenomic analysis that also showed S. thermophilus to have over 50 % abundance, we conclude that S. thermophilus in the VSL#3 blend is responsible for the urease activity.…”

“…The tetW gene detected is present in well‐characterized and commercialized probiotics (Kleerebezem et al , ; Garrigues et al , ) but has so far not found to be transferable (Gueimonde et al , ). The tetW gene was previously identified in VSL#3 strains BB02, BL03 and BI04, as was a potential aminoglycoside aminotransferase coding gene in strain BI04 (Douillard et al , ). Metagenomic data confirmed the absence of pathogenic islands or genes coding for toxic compounds, thus confirming previous data obtained from the genome analysis of the individual VSL#3 strains (Douillard et al , ).…”

“…The tetW gene was previously identified in VSL#3 strains BB02, BL03 and BI04, as was a potential aminoglycoside aminotransferase coding gene in strain BI04 (Douillard et al , ). Metagenomic data confirmed the absence of pathogenic islands or genes coding for toxic compounds, thus confirming previous data obtained from the genome analysis of the individual VSL#3 strains (Douillard et al , ).…”

“…Protein separation, excision of gel slices (four per loaded sample), reduction and alkylation, tryptic digestion and analysis by nLC (Proxeon EASY) connected to a LTQ‐Orbitrap XL (Thermo Electron) were performed as described previously (Oosterkamp et al , ; Buntin et al , ). Spectra were analysed using MaxQuant 1.5.2.8 and a database of common contaminants, next to protein databases of the reported genomes of the VSL#3 strains, which were used as input (Douillard et al , , 16.468 sequences) and those of Escherichia coli K12 and BL21‐DE3 as possible contaminants. The ‘Specific Trypsin/P’ digestion mode was used in MaxQuant as described previously (Cox et al , ) with maximally 2 missed cleavages and further default settings for the Andromeda search engine (including first search 20 ppm peptide tolerance, main search 4.5 ppm tolerance, IT‐MS‐MS fragment match tolerance of 0.5 Da, carbamidomethyl (C) set as a fixed modification, while variable modifications were set for protein N ‐terminal acetylation and M oxidation, which were completed by non‐default settings for de‐amidation of N and Q; the maximum number of modifications per peptide was 5).…”