Quantitative Detection of Bifidobacterium longum Strains in Feces Using Strain-Specific Primers

Abstract

We adopted a bioinformatics-based technique to identify strain-specific markers, which were then used to quantify the abundances of three distinct B. longum sup. longum strains in fecal samples of humans and mice. A pangenome analysis of 205 B. longum sup. longum genomes revealed the accumulation of considerable strain-specific genes within this species; specifically, 28.7% of the total identified genes were strain-specific. We identified 32, 14, and 49 genes specific to B. longum sup. longum RG4-1, B. longum sup. longum M1-20-R01-3, and B. longum sup. longum FGSZY6M4, respectively. After performing an in silico validation of these strain-specific markers using a nucleotide BLAST against both the B. longum sup. longum genome database and an NR/NT database, RG4-1_01874 (1331 bp), M1-20-R01-3_00324 (1745 bp), and FGSZY6M4_01477 (1691 bp) were chosen as target genes for strain-specific quantification. The specificities of the qPCR primers were validated against 47 non-target microorganisms and fecal baseline microbiota to ensure that they produced no PCR amplification products. The performance of the qPCR primer-based analysis was further assessed using fecal samples. After oral administration, the target B. longum strains appeared to efficiently colonize both the human and mouse guts, with average population levels of >10⁸ CFU/g feces. The bioinformatics pipeline proposed here can be applied to the quantification of various bacterial species.

Keywords: B. longum sup. longum; Roary; bioinformatics; gut colonization; probiotics; strain-specific qualification.

Figure 1

Phylogenetic relationship and genomic diversity of B. longum. (A) Phylogenetic tree (neighbor-joining method) of 205 B. longum strains. The three target strains used for strain-specific detection are colored. (B) Distribution of pair-wise SNP distances between 205 strains. (C) Pair-wise SNP distances between each of the target strains and all the other strains in the dataset. (D) Pangenome curve depicting the number of total genes detected versus the number of conserved genes as the number of included genomes increases.

Figure 2

The analysis pipeline for strain-specific primer design corresponding to the three B. longum strains (A) and the pangenome readout (B).

Figure 3

Electrophoresis results of PCR products generated using each strain-specific qPCR primer pair against DNA from the respective target B. longum strains and non-target microorganisms. Each gel includes 25 lanes (including a lane for the gene ruler). Order of microorganisms were as follows: for RG4-1-A (from right to left), gene ruler, B. longum RG4-1, B. longum FGSZY6M4, B. longum M1-20-R01-3, B. longum 274, B. longum FSHHK13M1, B. longum FSDLZ57M1, B. longum NaTon 49-4, B. longum FJSWXJ11M1, B. longum HUB 36-17, B. longum 28-10, B. longum ZCC7, Bifidobacterium breve DSM 20213, Bifidobacterium bifidum DSM 20456, Bifidobacterium pseudocatenulatum FQHXN5M4, Bifidobacterium pseudolongum 56M2, Bifidobacterium animalis BB12, Bifidobacterium adolescentis L2-32, Lactobacillus salivarius DSM 20555, Lactobacillus gasseri DSM 20243, Lactobacillus casei DSM 20011, Lactobacillus acidophilus DSM 20079, Lactobacillus plantarum DSM 20174, Lactobacillus reuteri DSM 20016, and Lactobacillus rhamnosus LMS2-1; for M1-A (from right to left), gene ruler, B. longum M1-20-R01-3, B. longum RG4-1, B. longum FGSZY6M4, and the order of following strains was the same as that of RG4-1-A; for GS-A (from right to left), gene ruler, B. longum FGSZY6M4, B. longum M1-20-R01-3, B. longum RG4-1, and the order of following strains was the same as that of RG4-1-A; for RG4-1-B, M1-B and GS-B (from right to left), Escherichia coli CMCC44102, Akkermansia muciniphila FJLHD50M21, Faecalibacterium prausnitzii ATCC 27768, Enterococcus faecalis CCFM596, Bacteroides fragilis NCTC9343, Bacteroides thetaiotaomicron FNMHLBE9-K-7, Bacteroides eggerthii FSDTA-HCK-B-9, Bacteroides cellulosilyticus FSDTA-ELI-BHI-5, Bacteroides nordii FNMHLBE13K2, Bacteroides stercoris FJSWX62K34, Bacteroides uniformis FJSWX62K43, Bacteroides caccae FFJLY22K5, Parabacteroides distasonis FSDTA-HCM-XY-12, Bacteroides dorei FJSWX61E4, Bacteroides faecis FTJS2E2, Bacteroides intestinalis FBJ60K5, Bacteroides vulgatus FSDLZ51K1, Bacteroides finegoldii FNMHLBE11E1, Bacteroides ovatus FBJ10-K-10, Bacteroides clarus F-FJ-LY 22-K-22, Bacteroides salyersiae FSDTA-ELI-BHI-9, Bacteroides xylanisolvens FSDTAHCMXY17, Parabacteroides merdae FSDTAELIBHI4 and Clostridium butyricum FJSCZD1G10.

Figure 4

qPCR standard curves for the three B. longum strains.

Figure 5

Colonized biomasses of the target B. longum strains in fecal samples from humans and mice. Panel (A), the results collected from the human trail; Panel (B), the results collected from the animal experiments.