Abstract
Lactic acid bacteria (LAB) are important for human health. However, the relative abundance of LAB in complex samples, such as fecal samples, is low and their presence and diversity (at the species level) is understudied. Therefore, we designed LAB-specific primer pairs based on 16S rRNA gene consensus sequences from 443 species of LAB from seven genera. The LAB strains selected were genetically similar and known to play a role in human health. Prior to primer design, we obtained consistent sequences for the primer-binding sites by comparing the 16S rRNA gene sequences, manually identifying single-stranded primers and modifying these primers using degenerate bases. We assembled primer pairs with product sizes of >400 bp. Optimal LAB-specific primers were screened using three methods: PCR amplification, agarose gel electrophoresis and single-molecule real-time (SMRT) sequencing analysis. During the SMRT analysis procedure, we focused on sequence reads and diversity at the species level of target LAB in three fecal samples, using the universal bacterium primer 27f/1492r as a reference control. We created a phylogenetic tree to confirm the ability of the best candidate primer pair to differentiate amongst species. The results revealed that LAB-specific primer L5, with a product size of 750 bp, could generate 3222, 2552, and 3405 sequence reads from fecal Samples 1, 2, and 3. This represented 14, 13 and 10% of all target LAB sequence reads, respectively, compared with 2, 0.8, and 0.8% using the 27f/1492r primer. In addition, L5 detected LAB that were in low abundance and could not be detected using the 27f/1492r primer. The phylogenetic tree based on the alignments between the forward and reverse primer of L5 showed that species within the seven target LAB genera could be distinguished from each other, confirming L5 is a powerful tool for inferring phylogenetic relationships amongst LAB species. In conclusion, L5 is a LAB-specific primer that can be used for high-throughput sequencing and identification of taxa to the species level, especially in complex samples with relatively low LAB content. This enables further research on LAB population diversity in complex ecosystem, and on relationships between LAB and their hosts.
Highlights
Lactic acid bacteria (LAB) are a group of non-spore-forming, gram-positive bacteria that produce lactic acid as the main product of sugar fermentation (Zhang and Zhihong, 2015)
We created a heatmap according to the relative abundance of the target LAB extracted from the total sequence at the species level (Figure 4B)
Some target LAB species with low relative abundance were detected in the L5 amplicons but not in the 27f/1492r and L6 amplicons, indicating that primer L5 was more efficient in detecting target LAB species with low relative abundances in complex samples
Summary
Lactic acid bacteria (LAB) are a group of non-spore-forming, gram-positive bacteria that produce lactic acid as the main product of sugar fermentation (Zhang and Zhihong, 2015). LAB are the most common probiotics found in the human gut microbiota and in a range of fermented foods. LAB can prevent the adhesion and reproduction of pathogens in intestinal mucosal tissues, relieve the symptoms of intestinal problems, and release various enzymes into the gut that aid host digestion (Perez et al, 2014). LAB helps maintain the stability of intestinal microbial communities by producing extracellular polysaccharides that can be used by other intestinal microorganisms (Hidalgo-Cantabrana et al, 2014). Since different types of LAB can affect the human intestinal microenvironment in different ways, it is important to identify which microorganisms are present in a microbial ecosystem, and which species are most likely to have beneficial effects. Accurate identification of these bacteria at the species level is challenging
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