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DNA Metabarcoding of Microbial Communities for Healthcare I Reviews ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32. N 1. P 3–8 doi: http://dx.doi.org/10.7124/bc.000906 UDC 577.25 + 579.61 DNA metabarcoding of microbial communities for healthcare I. Ye. Zaets1, O. V. Podolich1, O. N. Reva2, N. O. Kozyrovska1 1 Institute of Molecular Biology and Genetics, NAS of Ukraine, 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2 Department of Biochemistry, Bioinformatics and Computational Biology Unit, University of Pretoria Lynnwood road, Hillcrest, Pretoria, South Africa, 0002 [email protected], [email protected], [email protected] High-throughput sequencing allows obtaining DNA barcodes of multiple species of microorganisms from a single environmental sample. Next Generation Sequencing (NGS)-based profiling provides new opportunities to evaluate the human health effect of microbial community members affiliated to probiotics. DNA metabar- coding may serve as a quality control of microbial communities, comprising complex probiotics and other fermented foods. A detailed inventory of complex communities is a pre-requisite of understanding their func- tionality as whole entities that makes it possible to design more effective bio-products by precise replacement of one community member by others. The present paper illustrates how the NGS-based DNA metabarcoding allows profiling of both wild and hybrid multi-microbial communities with the example of a kombucha probi- otic beverage fermented by yeast-bacterial partners. Keywords: DNA metabarcoding, microbial communities, healthcare, probiotics Introduction cultured in laboratory conditions that made species profiling incomplete. The introduction of next genera- High-throughput sequencing allows obtaining tion sequencing (NGS) advanced significantly the ap- DNA barcodes of microbial communities plicability of the barcoding approach by making it faster, cheaper and capable of simultaneous identify- DNA barcode is a short DNA sequence, selected in ing species of different taxonomic groups, including the genome, that can be used to identify different spe- those, which cannot be cultured [5]. The DNA me- cies, analogously to the black stripes of the Universal tabarcoding is referred to the automated identification Product Code used to distinguish commercial prod- of multiple species from a single bulk sample, con- ucts [1]. The traditional barcoding is designed to iden- taining entire organisms, or from a single environ- tify species isolated from individual specimens using mental sample, containing degraded DNA [6]. Sanger DNA sequencing. For different groups of or- Compared to the standard approaches, metabarcoding ganisms the specific barcodes were proposed. For ex- by pyrosequencing provides taxonomically more ample, the Fungal Working Group has identified a ri- comprehensive data and allows faster performing of bosomal internal transcribed spacer (ITS) as the best large-scale biodiversity research and reducing the la- barcode region for fungi [2, 3]. The gene of 16S RNA bor consumption during sample processing as well as became a universal barcode for prokaryotic microor- the overall cost of analyzing the bulk samples [7, 8]. ganisms [4]. The standardized barcoding is limited in The DNA metabarcoding approach has a wide range the identification of only those species, which can be of applications in forensics, ecology and paleoecolo- © 2016 I. Ye. Zaets et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited 3 I. Ye. Zaets, O. V. Podolich, O. N. Reva, N. O. Kozyrovska I. Ye. Zaets, O. V. Podolich, O. N. Reva, N. O. Kozyrovska gy. In this review, we highlight the use of metabarcod- contrast to probiotics, are non-digestible food ingre- ing for a quality control of beverages and fermented dients like oligosaccharides, which fuel beneficial products and for designing new probiotic beverages. host-associated microbiota and enhance the positive action of probiotic microbial organisms. Health- Functional beverages and analysis of microbial promoting products, containing a combination of communities in fermented products prebiotic and probiotic, are termed synbiotics [11]. DNA metabarcoding of microbial communities as an One-third of cancer cases might be prevented by the instrument of community analysis is of great impor- nutritious healthy diet, which also maintains healthy tance for resolving many healthcare problems. The weight and physical activity by consuming synbiot- U.S. National Institute of Health has initiated the ics. Further, the significance of tailor-made prebiot- Human Microbiome Project to characterize the hu- ics, probiotics and synbiotics in cancer prophylaxis man microbiota and actually to design the personal due to the bio-antimutagenic and desmutagenic ac- DNA metabarcode of microbial communities (bacte- tivities has been reported [15]. Because of the lac- rial, archaeal and fungal) of humans. Metabarcoding tose maldigestion problem and allergy to milk pro- of the human gut microbiota showed that different teins, which are widespread in human population, people harbor remarkably dissimilar microbiota, the fermented non-dairy products seem to become which may be affected by external factors such as more preferable [18]. These products also are cho- lifestyle and dietary specificities [9]. Long-term lesterol free and rich in neuroactive amines tyramine studies in the United States demonstrated a nearly and histamine, as well as in antioxidants, which pro- 100 percent reduction in the rate of coronary heart tect consumers from free radicals. disease, diabetes in women and colon cancer in men Fermented probiotic products are produced by in patients accepting “healthy lifestyle” (reviewed in complex microbial communities and characterized [10]). The conception of healthy lifestyle assumes by rather unstable species composition, which consuming the fermented beverages as natural pro- strongly depends on the nutritional sources and pos- biotics, as a part of the healthy diet, e.g., yogurt, sible contaminations. Usually, there exists a core miso, tamari and kombucha. The food industry needs consortium of microorganisms, which establish mu- to assess carefully the safety and efficacy of all new tualistic relationships and get rid of any contaminat- species and strains of probiotics before incorporat- ing intruders due to a successful competition and the ing them into the food products. The nutraceutical production of antimicrobial metabolites. On the oth- fermented products are produced by complex micro- er hand, the accessory members of the brewing com- bial communities characterized by rather unstable munities are more important for the quality of the species composition. Using the DNA metabarcoded final product, and the need in a robust control of pyrosequencing approach, the profiling of complex these communities is widely recognized. Taking into yeast-bacterial/archaeal communities in diverse bev- account that the majority of microbial species are erage fermentations allows the control of fermenta- uncultivable and cannot be detected by any micro- tion processes. biological methods, the DNA metabarcoding re- Probiotics are viable microorganisms, the health mains the only known tool to examine and control effect of which is independent of the site of action the fermented food microbial communities. This ap- and the route of administration [11]. Living probiot- proach is suitable for the analyses of archaeal, bacte- ic microorganisms occupy a wide niche because of rial and yeast diversity in natural fermented foods keeping gut health [12], boosting immune system [19–25] and it is of great importance for a new pro- [13], memory and mental sharpness [14], preventing biotic/synbiotic design. cancer [15], allergic disorders [16], diabetes and di- In this paper, we illustrate how this approach aims abetes-induced impairments [17], etc. Prebiotics, in at the analysis of hybrid complex microbial commu- 4 DNA metabarcoding of microbial communities for healthcare nities using as example the DNA metabarcoding of a tured several times. Total DNA has been extracted modified kombucha microbial consortium. from cultural liquid and pellicle. The DNA metabar- coding has been done by using amplification of 16S The DNA metabarcoding of a modified kombu- rDNA and ITS regions and further pyrosequencing cha microbial consortium for identification of bacterial and yeast species, re- Kombucha beverage is becoming increasingly popu- spectively [33]. The resulted metagenomic data, lar around the world today as a prophylactic probi- which summarized the most frequent BLASTN hits, otic and healing agent [26], and both the kombucha are shown in the histogram in Figure. brewed drink and processed pellicles may be prom- In summary, the results of pyrosequencing showed ising probiotic/synbiotic formulations [27]. The that the hybrid kombucha culture is enriched by bac- presence of tea polyphenols, gluconic acid, gluc- terial and yeast operational taxonomic units, which uronic acid, lactic acid, vitamins, minerals in the were not present in the stock KMC [33]. For exam- brewed drink and cellulose pellicle was reported ple, Lactobacillus sp. has been revealed in hybrid [26]. The prophylactic and
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