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.
Highlights
Intestinal commensals play an important role in host health via being involved in various aspects of host physiology, such as tissue development, metabolism, and immunomodulation [1,2]
B. longum is distributed broadly across subjects of various ages [10], and is among the limited number of bacterial species that can colonize the gut over years [11]
Anaerobes (Bifidobacterium, Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides strains and Clostridium butyricum) were maintained in anaerobic chamber (80% N2, 10% H2, 10% CO2 ) during cultivation. b These strains were retrieved from Culture Collection of Food Microorganisms, Jiangnan university. c These strains were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). d The strain was isolated from the commercial probiotic product. e The strains were kindly provided by Biodefense and Emerging Infections Research Resources Repository (BEI Resources). f The strain was purchased from National Center for Medical
Summary
Intestinal commensals play an important role in host health via being involved in various aspects of host physiology, such as tissue development, metabolism, and immunomodulation [1,2]. The abundances of various Bifidobacterium species in the gut vary widely among individuals according to differences in dietary patterns [5,6], age groups [7], and physiological statuses [8]. Among these species, B. longum stands out as a member of the core human microbiome [9] and the most dominant species within the Bifidobacterium genus in the gut, regardless of the host age [7].
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