Intra-species Genomic and Physiological Variability Impact Stress Resistance in Strains of Probiotic Potential

Abstract

Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the characterization of bacterial strains is critical for the development of novel probiotics, as variability between strains can dramatically alter bacterial physiology and functionality.

Keywords: Lactobacillus rhamnosus; bacterial stress; bile salt hydrolase (BSH); intra-Species variability; probiotics.

Figure 1

Comparative genomic analysis of Lactobacillus rhamnosus isolates. (A) Genome alignment of L. rhamnosus strains. Strains AMC43, AMC010, Lc705, and HN001 were compared to LGG using BRIG genome alignment software. Relevant stress-response genes are annotated. Comparison of relevant stress-response genes in L. rhamnosus, (B) ABC-F ATPase, (C) arcD, (D) bsh were aligned and compared in Geneious 10.1.3 software. The bottom figure in each graph represents single nucleotide polymorphisms within the compared gene.

Figure 2

Transcriptomics analysis. mRNA sequencing data from each treatment (pH3, pH8, 0.5%, w/v Bile) was mapped to LGG genome and compared to untreated cells to calculate Log2 differential expression ratio and p-value. The 50 genes with the highest differential expression ratio (FDR corrected p ≤ 0.05) from each strain were categorized by function (cell division, DNA/protein repair, transcription regulation, and molecular transport) and plotted in OriginLab software. Highly differentially expressed hypothetical genes and genes with functions that did not fit into a defined category were also included. Green text highlights genes differentially regulated in both AMC010 and AMC143.

Figure 3

Growth and survival of L. rhamnosus under pH-stress conditions. (A) Growth rates were calculated for cells grown at pH 6.6 (MRS) pH4 (adjusted with HCl), or pH8 (adjusted with NaOH). (^*p < 0.01). (B,C) Bacterial survival was assayed by plating and enumerating cells exposed to simulated gastric juice (NaCl, HCl, Pepsin pH3) or simulated intestinal juice (NaCl, HCl, Pancreatin pH8) for 1 or 2 h.

Figure 4

Growth and survival of L. rhamnosus under bile-stress conditions. (A) Growth rates relative to 0% bile were calculated for cells grown in MRS containing 0.1, 0.3 0.5, and 1.0% w/v oxgall. (^*p > 0.05, ^**p > 0.01). (B) Bacterial survival was determined by plating and enumerating colonies post exposure to 0.5% w/v oxgall in MRS devoid of any carbohydrate source for 2 h. ^*Indicates significant difference between AMC143 and each other strain tested. (^*p > 0.01, ND = No detected growth).

Figure 5

Bile salt hydrolase (bsh) expression and sequence analysis. (A) RTqPCR targeting bsh was performed on AMC010, AMC143, and LGG with and without acute bile treatment, (^*p > 0.05). (B) Sequence alignment of bsh performed in CLUSTAL including annotations of coding regions and ribosome binding site.

Figure 6

Survival of AMC010::bsh under bile-stress. Survival of AMC010::bsh was compared to wild type AMC010 by plating and enumerating colonies post exposure to 0.5% w/v oxgall (^*p < 0.01).