In silico, in vitro and in vivo safety evaluation of Limosilactobacillus reuteri strains ATCC PTA-126787 & ATCC PTA-126788 for potential probiotic applications

Abstract

The last two decades have witnessed a tremendous growth in probiotics and in the numbers of publications on their potential health benefits. Owing to their distinguishing beneficial effects and long history of safe use, species belonging to the Lactobacillus genus are among the most widely used probiotic species in human food and dietary supplements and are finding increased use in animal feed. Here, we isolated, identified, and evaluated the safety of two novel Limosilactobacillus reuteri (L. reuteri) isolates, ATCC PTA-126787 & ATCC PTA-126788. More specifically, we sequenced the genomes of these two L. reuteri strains using the PacBio sequencing platform. Using a combination of biochemical and genetic methods, we identified the two strains as belonging to L. reuteri species. Detailed in silico analyses showed that the two strains do not encode for any known genetic sequences of concern for human or animal health. In vitro assays confirmed that the strains are susceptible to clinically relevant antibiotics and do not produce potentially harmful by-products such as biogenic amines. In vitro bile and acid tolerance studies demonstrated that the two strains have similar survival profiles as the commercial L. reuteri probiotic strain DSM 17938. Most importantly, daily administration of the two probiotic strains to broiler chickens in drinking water for 26 days did not induce any adverse effect, clinical disease, or histopathological lesions, supporting the safety of the strains in an in vivo avian model. All together, these data provide in silico, in vitro and in vivo evidence of the safety of the two novel candidates for potential probiotic applications in humans as well as animals.

Fig 1. Identification of L. reuteri strains by 16S rRNA amplicon sequencing.

L. reuteri strains were identified by PCR amplification and sequencing of the 16S rRNA variable region. A. Agarose gel electrophoresis of the 16S rRNA PCR product. B. Phylogenetic analysis of the 16S rRNA sequence along with other L. reuteri sequences. Streptococcus pyogenes was included as an outgroup.

Fig 2. Growth profiles of L. reuteri strains in MRS broth.

Growth profiles were assessed by growing the strains in MRS broth and determining the CFU counts at different time points. The data shown is representative of 3 independent experiments.

Fig 3. Chromosomal map of L. reuteri strains PTA-126787 and PTA-126788.

The concentric circles show, reading outwards: GC skew, GC content, AT skew, AT content, COG classification of proteins, CDS on reverse strand, ORFs on three frames in reverse strand, ORFs on three frames in forward strand, CDS on forward strand and COG classification of proteins on forward strand.

Fig 4. Phylogenetic relationship of L. reuteri strains PTA-126788 and PTA-126787 to other known human L. reuteri strains using 92 core genes.

The phylogenetic relationship was explored using UBCG v3.0 and a maximum likelihood tree was inferred using GTR+CAT model. Streptococcus thermophilus and Enterococcus faecalis were used as outgroups.

Fig 5

Distribution of predicted IS family within the genomes of Lactobacillus reuteri strains PTA-126787 (A) and PTA-126788 (B) using ISEScan.

Fig 6. Production of D- and L-lactic acid by L. reuteri strains.

L- and D-lactic acids were quantified using D-/L-lactic acid (D-L-lactate) Rapid Assay Kit (Megazyme). The data represent the mean ± SD from 3 independent experiments.

Fig 7. Ability of L. reuteri strains to undergo autoaggregation.

A. Ability to undergo autoaggregation was determined by growing the strains overnight in MRS broth and observing for aggregate formation. B. Autoaggregation was quantified by measuring the OD₆₀₀ in PBS after incubation for 5 hours and calculating the autoaggregation % as described in the methods section. The data represents the mean ± SE of 3 independent experiments.

Fig 8. Ability of L. reuteri strains to produce hydrogen peroxide.

Hydrogen peroxide production was assessed by growing the strains on MRS agar supplemented with 0.25mg/ml of tetramethylbenzidine and 0.01mg/ml of horseradish peroxidase and observing for color change. Dark blue coloration indicates high production of hydrogen peroxide. The data are representative of 3 independent experiments.

Fig 9. Tolerance of L. reuteri strains to 0.3% bile.

The ability of L. reuteri strains to tolerate bile salts was assessed by growing the strains in the presence of 0.3% bile salts for 4 hours and determining the CFU counts at 0 hours and 4 hours after incubation with bile salts. The data represent the mean ± SD from 3 independent experiments.

Fig 10. Tolerance of L. reuteri strains to acidic pH.

The ability of L. reuteri strains to tolerate acidic pH was assessed by growing the strains at pH 2.5 for 3 hours and determining the CFU counts at 0 hours and 3 hours after incubation. The data represent the mean ± SD from 3 independent experiments.