No abstract

Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been actively conducted, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) is still a considerable technique in detecting and quantifying bacteria associated with the human oral and nasal cavities, due to the analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis for the identification and quantification of five bacterial genera (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, and Roseburia) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primers, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying the qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries.

Functional cosmetics industries using skin microbiome screening and beneficial materials isolated from key microorganisms are receiving increasing attention. Since Epidermidibacterium keratini EPI-7T was first discovered in human skin, previous studies have confirmed that it can produce a new pyrimidine compound, 1,1′-biuracil, having anti-aging effects on human skin. Therefore, we conducted genomic analyses to judge the use value of E. keratini EPI-7T and provide up-to-date information. Whole-genome sequencing analysis of E. keratini EPI-7T was performed to generate new complete genome and annotation information. E. keratini EPI-7T genome was subjected to comparative genomic analysis with a group of closely-related strains and skin flora strains through bioinformatic analysis. Furthermore, based on annotation information, we explored metabolic pathways for valuable substances that can be used in functional cosmetics. In this study, the whole-genome sequencing (WGS) and annotation results of E. keratini EPI-7T were improved, and through comparative analysis, it was confirmed that the E. keratini EPI-7T has more metabolite-related genes than comparison strains. In addition, we annotated the vital genes for biosynthesis of 20 amino acids, orotic acid, riboflavin (B2) and chorismate. In particular, we were able to prospect that orotic acid could accumulate inside E. keratini EPI-7T under uracil-enriched conditions. Therefore, through a genomics approach, this study aims to provide genetic information for the hidden potential of E. keratini EPI-7T and the strain development and biotechnology utilization to be conducted in further studies.

A non-motile, novel actinobacterial strain, designated Kera-3T, was isolated on 1/10 diluted R2A agar from human keratinocytes. Strain Kera-3T was Gram-stain-positive, aerobic and rod-shaped. The whole-cell hydrolysis of amino acids contained meso-DAP, alanine, and glutamic acids. The main menaquinone was MK-9 (H8). The major fatty acids were C16:0 and C18:1 ω9c. The major phospholipids were diphosphatidylglycerol and aminophospholipid, along with an unidentified phosphoglycolipid and an unidentified aminophosphoglycolipid. The G + C content of the genomic DNA was 70.5 % based on the complete genome sequence. Phylogenetic analyses of the 16S rRNA gene sequence and core-gene based on amino acid sequence clearly showed that strain Kera-3T formed a new lineage in the family Iamiaceae, being the closest neighbour Rhabdothermincola sediminis SYSU G02662T with a 91.19% 16S rRNA gene sequence identity. Phylogenetic data was supported with a comparative genomic study of the predicted general metabolism and carbohydrate active enzymes. Based on physiological and biochemical characteristics data analysis. Strain Kera-3T can be distinguished from known genera in the family Iamiaceae and represents a novel genus and novel species. Therefore, the name Dermatobacter hominis gen. nov., sp. nov. is proposed with the type strain being Kera-3T (= KACC 22415T = LMG 32493T).

Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been activated, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) are still of considerable technique in detecting and quantifying bacteria associated with the human mouth, nasal cavity, and pharynx due to analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis of identifying for five bacterial genera proportions (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, and Roseburia) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primer, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria ‘Porphyromonas gingivalis’, and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)–based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis–specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.