HIPPOKRATIA 2018, 22, 1: 29-36 RESEARCH ARTICLE 16S rRNA deep sequencing for the characterization of healthy human pharyngeal microbiome Gioula G, Melidou A, Siasios P, Minti F, Malisiovas N Microbiology Department, School of Medicine, Faculty of Health Science, Aristotle University of Thessaloniki, Thes- saloniki, Greece Abstract Background: The recent advent of high-throughput sequencing methods enabled the study of the composition of the upper respiratory tract (URT) microbial ecosystem and its relationship with health and disease in immense detail. The aim of the present study was the characterization of the human pharyngeal microbiome of healthy individuals in Greece. Materials and Methods: We obtained ten pharyngeal specimens from healthy volunteers, Greek resident, with Greek nationality, who were eligible to the selection criteria. The construction of DNA libraries was performed by using two primer sets that amplify selectively the corresponding hypervariable regions of the 16s region in bacteria (V2-V9). The Ion Torrent PGM platform was used for the performance of next-generation sequencing. Results: In the study samples, twelve phyla were identified. The most abundant ones were Firmicutes, Proteobacteria, Bacteroidetes, followed by Actinobacteria and Fusobacteria. Seventy-nine families, 79 genera and 137 species were identified and characterized. Moreover, 17 unique differentially abundant families, 30 unique differentially abundant genera and 24 unique differentially abundant species were identified among healthy subgroups with adjusted p-values <0.05. At the genus level, Moraxella (Proteobacteria) and Gemella (Firmicutes) were detected with a statistical signifi- cance in non-smokers, while Bifidobacterium (Actinobacteria), Alloscardovia (Actinobacteria), Dialister (Firmicutes) and Filifactor (Firmicutes) were present mostly in smokers. Conclusions: The URT is colonized by a variety of protective and potentially pathogenic bacteria. This microbiome sys- tem is highly diverse and varies significantly between individuals. Geographic location and ethnicity are considered to be a strong determinants and factors affecting the diversity and abundance of the URT microbiome. Although some of the most abundant families are common irrespective of these factors, the dominance patterns are usually different between the study subjects and between the studies from other geographic locations. Unique differentially abundant families, genera and species were identified, and further studies are needed to elucidate their role. Further studies should focus on the investigation of the URT microbiome dynamics and the interaction with the host in health and disease. HIPPOKRATIA 2018, 22(1): 29-36. Keywords: Pharyngeal microbiome, 16srRNA, healthy, sequencing, Greece Corresponding author: Georgia Gioula, Associate Professor of Medical Microbiology, Laboratory Department, School of Medicine, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece, tel: +302310999121, e-mail: [email protected] Introduction bacterial acquisition during the first years of life and host The upper respiratory tract (URT) is the natural niche factors profoundly influence the microbial colonization for respiratory bacterial and viral microorganisms and the succession. Unraveling the relationships between mi- cause of respiratory tract infections (RTIs). The URT is an crobes of the URT during perturbations is anticipated to interesting area from an ecological perspective: the con- provide insights into the pathogenesis of respiratory in- tinuous exposure to the external environment and inter- fections1. Discovery of the microbiome of both the upper nal host mechanisms, like respiration and gastrointestinal and the lower respiratory tracts created the opportunity processes, form different habitats for a wide variety of for understanding the disease onset, exacerbation, and microorganisms. Potentially pathogenic bacteria are em- progression of chronic respiratory diseases which may bedded in a community of commensals, forming together be directly associated with dysbiosis in the microbiome2. the nasopharyngeal microbiome. Many pathogenic spe- The recent advent of high-throughput sequencing cies, including Streptococcus pneumoniae, Haemophilus methods enabled the study of these communities and their influenza, Moraxella catarrahlis, Staphylococcus aureus, relationship with health and disease in immense detail. Ex- and Neisseria meningitidis, exist in the nasopharynx of tensive research has focused on the gut microbiome and apparently healthy individuals. Environmental factors, its role in metabolism, immune maturation, mucosal bar- 30 GIOULA G rier functions, and colonization resistance. Although much at any time of the day. Samples were immediately placed less extensively studied, the URT microbiome is equally at -80oC until DNA extraction and were only subjected to important and represents a strong determinant of respira- a single free-thaw cycle. tory health3. Deep sequencing methods have enabled the evaluation of the composition of the whole URT microbial DNA extraction ecosystem, giving the opportunity to explore its diversity For each subject, pharyngeal aspirates were collect- and relative abundance down to the species level and to ed, and total genomic DNA was extracted from 200 μl determine its relationship to human health and disease4. of sample according to the manufacturer’s recommend- Recent studies have focused on the differences in ed procedure using the Qiagen DNA mini kit (Qiagen, the microbiome composition between healthy individu- Hilden, Germany). The DNA was eluted in 100 μl of AE als from different races, ethnicity and geographic loca- elution buffer (Qiagen, Hilden, Germany) and stored at tion that are important determinants of the microbiome -20 °C until further processing. The total DNA concen- composition. Various factors are responsible for the geo- tration of each DNA extract was measured using Qubit graphical differences: host genetics, innate/adaptive im- fluorometer (Life Technologies Corporation, Carlsbad, munity, environmental factors, social and behavioral fea- CA, USA) with the Qubit dsDNA HS assay (Life Tech- tures (hygiene, diet, parasitic load)5. Fewer studies have nologies Corporation, Carlsbad, CA, USA). focused on such differences in the composition of the URT microbiome and generally, it is considered to be a 16S rRNA amplicon preparation, sequencing, and analysis less diverse system, compared to the gut, skin or oral mi- Library preparation crobiome. The URT microbiome has been characterized DNA libraries for next-generation sequencing were in geographically diverse populations from South Korea constructed using two primer sets that amplify selectively and USA, the Netherlands and Canada6-8. the corresponding hypervariable regions of the 16s region The present study aimed to characterize the relative in bacteria (V2, 3, 4, 6, 7, 8, 9). We used for amplification abundance of microbial communities of the pharynx at primers that contain adaptors for Ion Torrent sequencing the phylum, family, genus and species levels, by deep se- and Ion Xpress barcodes so that the polymerase chain quencing the 16S rRNA gene of bacteria in pharyngeal reaction (PCR) products could be pooled and sequenced swab samples of healthy individuals from Greece. This directly. For the library preparation, the end repair was study will form the basis for further studies in our geo- followed by purification of pooled amplicons with AM- graphical area that will focus on the relationship of the Pure beads, ligation of Ion Xpress barcode adapters and URT microbiome with disease. nick-repair, using Ion Plus Fragment Library kit (Life Technologies Corporation, Carlsbad, CA, USA). Purifi- Materials and Methods cation steps were performed using AMPure beads (Agen- Ten volunteers were eligible according to the selec- court), eluted in low Tris- ethylenediaminetetraacetic tion criteria and enrolled in this prospective study. Ethical acid (TE) buffer, and quantified by using a Qubit dsDNA approval was obtained from the Bioethics Committee of HS (high sensitivity) kit (Life Technologies Corporation, the Medical Faculty of Aristotle University and all study Carlsbad, CA, USA). The library concentration was de- subjects consented for their participation in the study. termined using the Ion Universal Library Quantification Their mean age was 32.3 years (range 5-61 years), while kit (Life Technologies Corporation, Carlsbad, CA, USA) 80 % of these volunteers were female. All individuals had in an ABI7500 real-time PCR system. The two resulting Greek nationality and were residents of areas of north- PCR libraries were equimolarly pooled after DNA puri- ern Greece at the time of sample collection (January to fication. March 2017). None of them were vaccinated against in- fluenza virus or suffered any known respiratory infection Template preparation and sequencing at least during the preceding year prior to sample collec- Before emulsion PCR, the library concentration was tion or required antibiotic treatment for the preceding six adjusted to 26 pM. Template preparation was performed months before sample collection. Their medical history in the Ion 400 Template One-Touch 2 following enrich- is shown in Table 1. For the calculation of the sample ment of the amplified Ion Sphere Particles using Dynal- size, G * Power 3.1.9.2v was used, and a priori power beads MyOne Streptavidin C1 beads (Life Technologies analysis was calculated to achieve a statistical power