Abstract
Two pairs of species-specific PCR primers targeting the housekeeping groEL gene, Spa146f-Spa525r and Spa93f-Spa525r, were designed to quantify human oral and fecal Streptococcus parasanguinis. Blast analysis against reference sequences of NCBI nucleotide collection database and the Chaperonin Sequence Database showed the forward primers Spa146f and Spa93f 100% matched only with S. parasanguinis, and the in silico Simulated PCR algorithm showed both primer pairs hit only S. parasanguinis groEL gene in Chaperonin Sequence Database. The two primer pairs were respectively used to perform PCR with saliva DNA of each of 6 human subjects, and the amplicons of individual PCR reactions were cloned. The phylogenetic analysis showed cloned sequences were all affiliated to S. parasanguinis, which further validates the specificity of two primer pairs, and that individual subjects harbored multiple genotypes of S. parasanguinis in saliva. By spiking S. parasanguinis into human fecal samples, we found the quantification limit of quantitative real-time PCR (qPCR) assays for both primer pairs was 5–6 log10 groEL copies/g feces. Human fecal S. parasanguinis amounts quantified with qPCR using each of the two primer pairs correlated well with those determined with metagenomic sequencing. qPCR with either primer pair showed periodontitis patients had significantly lower level of saliva S. parasanguinis than healthy people. In both feces and saliva, the S. parasanguinis abundances quantified with two primer pairs exhibited strong and significant correlation. Our results show that the two S. parasanguinis-specific primer pairs can be used to quantify and profile human saliva and fecal S. parasanguinis.
Highlights
Streptococcus parasanguinis is a common human commensal bacterial species colonizing multiple body sites
Primer Spa146f, Spa93f, and Spa525r showed 100% similarity with the groEL gene of all 7 S. parasanguinis strains deposited in Chaperonin Sequence Database6 (Table 2 and Supplementary Tables S1–S3)
In many previous studies that developed PCR primers specific for certain bacterial taxa, PCR assays were performed with the genomic DNA of dozens of bacterial strains belonging to targeted and non-targeted taxa as the templates, and the specificity of the primer pairs were validated by the results that only targeted bacteria produced amplicons of expected size (Junick and Blaut, 2012; Leigh et al, 2018)
Summary
Streptococcus parasanguinis is a common human commensal bacterial species colonizing multiple body sites. It is a prevalent bacteria in the oral cavity of both adults and infants (Franzosa et al, 2014; Dzidic et al, 2018), and plays an important role in dental plaque formation (Garnett et al, 2012) and significantly inhibits the growth the periodontopathogens by producing hydrogen. S. parasanguinis is one of the dominating pioneer colonizers of human infant intestine in first days of life (Songjinda et al, 2005), a predominant bacterial species in the small intestine of adults (van den Bogert et al, 2013b), and detected in the feces of children (Zhang et al, 2015) and adults (Franzosa et al, 2014). It is necessary to develop molecular methods to quantify and profile S. parasanguinis in human microbiome samples to understand its role in human health and diseases
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