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Bacterial Microbiota Analysis Present in the Nose and Pharynx of a Mexican Young Population

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Bacterial Microbiota Analysis Present in the Nose and Pharynx of a Mexican Young Population Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 223-235 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 6 (2016) pp. 223-235 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.506.026 Bacterial Microbiota Analysis Present in the Nose and Pharynx of a Mexican Young Population Ana Karina Rodríguez-Vicente1, Jaime Bustos-Martínez2, Dolores Reyes-Duarte3 and Teresita Sainz-Espuñes4* 1Doctorado en Ciencias Biológicas y de la Salud. Universidad Autónoma Metropolitana-Xochimilco. Mexico City, Mexico 2Departamento de Atención a la Salud. Universidad Autónoma Metropolitana-Xochimilco, Mexico City, Mexico 3Departamento de Procesos y Tecnología. Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City, Mexico 4Departamento de Sistemas Biológicos. Universidad Autónoma Metropolitana-Xochimilco, Mexico City, Mexico *Corresponding author email id: ABSTRACT Culture-independent microbiota is relatively unexplored in Mexican population. The aim of this study was to characterize the microbiota of Mexican young healthy adults by means of K eywo rd s traditional culture and metagenomic analysis in order to provide novel insights and have a better understanding about the healthy baselines from which to detect differences associated Bacterial with diseases. The bacterial microbiota of the nose and pharynx from 75 healthy Microbiota, nonsmoking Mexican young adults was examined by conventional cultures and culture- Nose and independent methods. The hypervariable region (V6-V8) of the16S rRNA gene was PCR Pharynx , amplified from isolated DNA and DGGE analyzed, bands excised were sequenced and metagenomic phylogenetic analys is was done. The study showed that the bacterial microbiota of analysis . the pharynx was richer than that of the nose. Using conventional culture methods results showed that gram positive Staphylococcus aureus and S. epidermidis were found in both Article Info niches and gram negative bacteria such as Escherichia coli, Klebsiella sp. and Moraxella sp. were the most abundant genera in the nose and Enterobacter sp. in the pharynx. Accepted: Firmicutes and Proteobacteria phyla accounted for the majority of the bacteria detected in 12 May 2016 both niches. To our knowledge this is the first report describing partially the composition of Available Online: and the variability within the nasopharyngeal microbiota of a Mexican 10 June 2016 young adult population and therefore we will be able to investigate other different cities and compared them to find out where are the highest microbial contaminated zones and take some hygienic measures in accordance to the normativity reports. Introduction the nasal cavity. Like skin, the nostrils contain sebaceous glands, sweat glands, and The nostrils or anterior nares are the hairs and are lined by a keratinized, outermost segment of the nose and is stratified squamous epithelium more similar considered a transition zone from the skin to to that of skin than to the mucus-producing, 223 Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 223-235 ciliated, columnar epithelium of the nasal The nostrils are known to harbor bacteria cavity (Lemon, 2010; Wilson, 2005). from the genera Corynebacterium, Propionibacterium, and Staphylococcus The nostrils help filter inhaled air, which (Firmicutes and Proteobacteria phyla), contains low numbers of extremely diverse including the important pathogen microbes (Lemon, 2010; Brodie et al., 2007; Staphylococcus aureus found by molecular Fierer et al., 2008). The pharynx is methods (Lemon, 2010; Wilson, 2005). The constantly exposed to both inhaled and nasal cavity appears dominated by resident ingested microbes. This microbiota is aerobic microbiota as Corynebacterium spp. influenced by different factors like age, and Staphylococcus spp. detected by immunological conditions, and the cultivation (Rasmussen et al., 2000). In the environment that in this particular case is oropharynx species from the genera highly polluted in Mexico City (Rosas et al., Streptococcus, Haemophilus, Neisseria are 2006; Secretaria de Medio Ambiente, 2012). present, and to a lesser extent The nostril and oropharynx are considered Staphylococcus and various anaerobic distinct habitats. While the pathogen bacteria (Lemon, 2010; Wilson, 2005). Staphylococcus aureus colonizes both sites Oropharynx is the site of carriage of many (Lemon, 2010, Widmer et al., 2008, Mertz, important human pathogens, including 2007, Wilson, 2005). Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, The knowledge on nose and pharynx Neisseria meningitidis, Moraxella microbiota has focused on pathogen carriage catarrhalis, and Staphylococcus aureus using traditional cultivation methods, (Bogaert et al., 2011; Lemon, 2010; Mertz et however currently molecular approaches al., 2007; Widmer et al., 2008; Wilson including DGGE 16S gene analysis, 2005). pyrosequencing, 16S rDNA PCR-RFLP, analysis of phylochip, High-Throughput The aim of this study was to investigate the Sequencing (Chakraborty et al., 2014; Yi et microbiota of Mexican young healthy adults al., 2014, Ling et al., 2013; Aguirre et al., by means of traditional culture and 2012; Brugger et al., 2012; Lemon et al., metagenomic analysis in order to have a 2010). These methods have most used to better understanding about the healthy study the relation between the dynamic baselines from which to detect differences equilibrium of human health and disease, associated with diseases. Our approach was being the pathogenic microorganisms the chosen because this population has been best studied. In a variety of environmental scarcely studied in regard with children and samples the study of the microbial diversity older adults. is accomplished by metagenomic DNA studies and also by traditional culture Material and Methods methods. The later poorly predicts resident microbiota (Hauser et al., 2014). Cloning Ethic Statement and Ethical Approval and sequencing of 16S genes amplified directly from different human sites such as Samples were collected in accordance with the oral cavity for example, demonstrated relevance guidelines for ethical research that microbial diversity is by far more design, confidentiality and protection human extensive compared to culture-based studies subjects. Protocol was reviewed and (Chakraborty et al., 2014). approved by the ethics and biosafety committees of the Universidad Autónoma 224 Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 223-235 Metropolitana-Xochimilco (Hamdan et al., blood agar, chocolate agar, mannitol salt 2013). All procedures performed in studies agar and Mac Conkey agar plates and then involving human participants were in were incubated at 37°C for 24 h; chocolate accordance with the ethical standards of the agar plates with a 5% CO2 atmosphere. institutional and/or national research Bacteria identification was based on colony committee and with the 1964 Helsinki morphology and bacteriological biochemical declaration and its later amendments or conventional tests (Holtz, 1993; Winn et al., comparable ethical standards (World 2006). Medical Association Declaration of Helsinki Ethical Principles for Medical Research, Nucleic Acid Isolation 1964). Genomic DNA was extracted from the Subjects and Sample Collection swabs collected in saline buffer solution and from the enrichment media from nose and As in previous studies (Hamdan et al., 2013) pharynx using a commercial kit Fast ID informed consent was obtained from all Genomic DNA Extraction (Genetic ID NA individual participants for screening Inc, USA) following the manufacturer’s enrollment and specimen collection. Great instructions including some modifications: care was taken to ensure that all people ten microliters of proteinase K solution understood that they would participate as (10µg/µl), 5 µL of lysostaphin solution (0.5 volunteers; no academic or economic µg/µl) and 15 µL of mutanolysin solution incentives were offered. (1U/µl) were added for cellular lyses. Seventy five (75) healthy adult volunteers, PCR Amplification whose ages between 18-25 years old were included between January and March of Hypervariable region V6-V8 of the 16S 2012. Criteria for participation were: no rRNA gene was amplified from the isolated clinical signs of illness, no antibiotic therapy genomic DNA using universal primers 968f within 3 months before entering the study, 5’-AAC GCG AAG AAC CTT ACC-3’ and 1401r 5’–GCG TGT GTA CAA GAC CC-3’ no pregnancy or breast feeding and nonsmokers. (Ramírez-Saad et al., 2000). The gc968f forward primer contains additional 40 Separate swabs were used to collect the nucleotide GC-rich sequence the GC clamp samples, one for the nostrils and the other for DGGE technic. Amplification was for the posterior wall of the pharynx of each performed with a Whatman Biometra participant and were placed into enrichment TProfessional Basic 96 gradient thermal media (tripticase soy broth) and non cycler (Goettingen, Germany). Each mixture enrichment media (saline buffer solution). (25 µl final volume) contained 1 µl DNA The pharynx posterior wall was swabbed template, 0.5 mM primer concentration, 200 without touching the tonsils, uvula, tongue, mM dNTPs, 1X PCR buffer and Q solution or other oral structures. and 2.5 U of Taq polymerase (Qiagen TaqPCR Core kit Cat. no. 201225, Qiagen Microbial Cultivation GmbH, D-407224 Hilden ). DNA template was denatured for 5 min at 94°C. To Nasal and pharynx swabs were first enriched increase the amplification specificity and
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