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Microbial Pathogenesis 143 (2020) 104115 Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath The characterization of bacterial communities of oropharynx microbiota in healthy children by combining culture techniques and sequencing of the 16S rRNA gene T Abbas Malekia, Maryam Zamirnastaa, Morovat Taherikalania, Iraj Pakzada, Jasem Mohammadib, Marcela Krutovac, Ebrahim Kouhsaria, Nourkhoda Sadeghifard (Professor)a,∗ a b c Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran Department Pediatrics, Ilam University of Medical Sciences, Ilam, Iran Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic A R T I C LE I N FO A B S T R A C T Keywords: Respiratory microbiome Oropharynx 16S rRNA Gram-negative bacteria S. aureus The high incidence of bacterial respiratory infections has led to a focus on evaluating the human respiratory microbiome. Studies based on culture-based and molecular methods have shown an increase in the bacterial community that includes the bacterial phyla Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria in the oropharynx of healthy individuals. Therefore, recognizing this microbial compound and subsequently identifying those carriers of specific pathogens can be of great help in predicting future infections and their control. In this prospective study, we sought to characterize the bacterial communities of the respiratory microbiome in healthy children aged between 3 and 6 years old by combining both cultural techniques and sequencing of the 16S rRNA gene. Seventy-seven oropharynx samples using Dacron swabs were collected from 77 healthy children in the kindergartens of Ilam, Iran. Bacterial identification was performed by phenotypic methods and in house developed PCR-based sequencing (the V1–V9 hypervariable region of the bacterial 16S ribosomal RNA gene). In total, 346 bacterial isolates were characterized based on phenotypic and sequencing-based molecular methods. The 3 most predominant phyla were Firmicutes (74%), Proteobacteria (22%), and Actinobacteria (4%). At the level of the genus, Staphylococci (coagulase-positive and coagulase-negative) and Streptococci were dominant. Also, the most commonly identified potentially pathogenic colonisers were S. aureus (75%), Enterobacteriaceae spp. (40.1%), and A. baumannii (15.6%). The present study identified 3 phyla and 9 family of bacteria in the oropharyngeal microbiome. Remarkably, the presence of potential pathogenic bacteria in the nasopharynx of healthy children can predispose them to infectious diseases, and also frequent exposure to human respiratory bacterial pathogens are further risk factors. 1. Introduction The WHO reported that respiratory tract infections are still among the leading cause of death in children and adults worldwide [1,2]. Furthermore, the low diversity of respiratory microbiota in early life is one of the major risk factors for the development of allergies [3]. Nasopharyngeal carriage plays a significant role in the spread of bacterial pathogens since excretions from the nasopharynx are a major source of potential pathogens. The colonization of the nasopharynx by specific pathogenic bacteria can lead to an invasion of the blood, tissues, cerebrospinal fluid. Nevertheless, the disease arises only in a small group of individuals who have been colonized [4–6]. Given this background, a study into the prevalence of bacteria in ∗ children's respiratory system, could not only provide a better understanding of the establishment of bacteria and their interactions, but also help to predict bacterial ecological changes and the design of control strategies. Predicting the spread of the infection in the community and designing solutions for its prevention and treatment – such as selecting the correct mathematical model and estimating parameters correctly may provide estimates on the effectiveness of interventions as well as the screening of individuals to determine if they have been infected or exposed to an infectious disease [7]. It is known that, some bacterial species are a common part of microbiota that have a positive effect on health but in some conditions, e.g. when they colonize a different environment such as the mucous membrane, they then may become pathogenic [8]. The fast and Corresponding author. Banganjab, Pazhouhesh Blvd, Ilam University of Medical Sciences, 6939177143, Ilam, Iran. Tel./fax: +98 841 2227101 E-mail addresses: Sadeghifard@gmail.com, Sadeghifard@gmail.com (N. Sadeghifard). https://doi.org/10.1016/j.micpath.2020.104115 Received 1 November 2019; Received in revised form 1 March 2020; Accepted 1 March 2020 Available online 03 March 2020 0882-4010/ © 2020 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 143 (2020) 104115 A. Maleki, et al. accurate detection of bacteria is difficult and a major facet of medicine [9]. Thus, investigating microbiota for further analysis could prevent many diseases. The technical challenges and variability in respiratory microbiome research, including the absence of uniform laboratory practices (sampling, processing biases, DNA isolation, selection of 16S rRNA variable region, sequencing, and bioinformatics analysis), resulted in a difficult and variable assessment of the microbial respiratory tract communities [10,11]. There are many limitations related to culture-based methods for the determination of human microbiome communities e.g. labor intensive, time-consuming (media preparation, dilution, plating, incubation, counting, isolation, and characterization) and the considerable cost of consumables [12,13]. In addition, using biochemical approaches to identify a particular bacterial strain can throw up false results. This may particularly be explained when the microbial species are so similar that phenotypic methods are not always helpful [12,13]. Currently, different methods are being developed to investigate the human respiratory microbiome such as combining cultural techniques, sequencing of the 16S rRNA gene. The 16S ribosomal RNA (rRNA) gene comprises conserved stretches of sequences that can be used to design universal primers to amplify the gene from the majority of known bacterial species. These regions are interspersed with variable sequence regions. These sequences can be used to assign the identity and phylogeny of the organisms in a microbial community [14–16]. Reports published previously have shown that Firmicutes, Bacteroidetes, and Proteobacteria are the most frequently identified bacteria at the phylum level in healthy individuals [17]. The purpose of this prospective study, was to characterize the microbiome of oropharynx (pharyngeal area) in children of Ilam aged between 3 and 6 years-old by combining culture and 16S rRNA gene sequencing methods. In order to assess the relationship between the oropharynx microbiome and risk factors for infection, epidemiological data were collected such as age, gender, attendance at kindergarten, size of family, type of birth, breastfeeding or formula feeding and the parents’ smoking habits. medium (Merck, Darmstadt, Germany). The criteria for inclusion in this study were: healthy children aged between 3 and 6 years old with no signs of respiratory diseases and who had not taken any antibiotics during the last three months. The swabs were cultured in the selected media under aerobic and anaerobic conditions. The media used were Brucella blood agar (HiMedia, India) (containing 5% sheep blood), chocolate agar (HiMedia, India) (containing 5% sheep blood), MacConkey agar (HiMedia, India), and mannitol salt agar (HiMedia, India). In the following, media were incubated for 24–48 h under both aerobic and anaerobic conditions at 37 °C. Then, the isolates were identified as standard phenotypical protocols including, biochemical and microbiological tests [18–20]. To identify the Gram-positive cocci (such as Staphylococcus spp., Streptococcus spp. and Enterococcus spp.) the following biochemical tests were performed after cultural growth on mannitol salt agar, the production of catalase, coagulase, DNase, indole, methylred, Voges-Proskauer reaction, citrate utilization, urease production, optochin sensitivity, bacitracin sensitivity, camp, bile esculin hydrolysis, nitrate broth, starch hydrolysis, growth in 6.5% NaCl, and motility agar. Finally, the biochemical identification of the species was confirmed by API Staph and API 20 Strep (bioMérieux, Marcy-l'Étoile, France). To identify the Gram-negative bacilli (such as Enterobacteriaceae, non-fermentative bacilli) using biochemical approaches included the following biochemical testes were applied: oxidase, indole production, methyl red, the voges-proskauer, citrate, urease, H2S production, motility, lysine decarboxylase, lactose and glucose fermentation, ONitrophenyl-β-D-galactopyranoside (ONPG), Oxidative/Fermentation (OF), pigmentation, growth at 42 °C, and gelatin liquefaction. Finally, the biochemical identification of the species was confirmed by API Rapid 20E API 20NE (bioMérieux, Marcy-l'Étoile, France). Mixed cultures cannot be applied in our study (may receive false or no results). Thus, stock isolates (pure culture) were kept in the TSB broth (Conda, Spain) containing 20% glycerol at −80 °C for further analysis. 2. Methods 2.3. DNA extraction 2.1. Study design and subjects The DNA of all isolates (pure culture) was extracted and PCR-based sequencing was performed to verify the identification result previously obtained for each strain using a biochemical approach. Purified genomic DNA was extracted from fresh overnight cultures of bacterial isolates and oropharynx swabs using QIAamp DNA mini kits (Qiagene, Hilden, Germany), according to the manufacturer's instructions. DNA purity, quality, and quantity was measured by absorbance spectrophotometry (Nanodrop-1000; NanoDrop Technologies, Wilmington, DE, USA) and agarose gel-electrophoresis. The extracted DNA was stored at −20 °C for further analysis. This prospective study was conducted from 2015 to 2016. Seventyseven samples were collected from children between the ages of 3–6 years from several healthcare centers and kindergartens of Ilam, Iran. A questionnaire determining the age, sex, history of hospitalization, history of respiratory diseases, size of family, history of smoking, childbirth (cesarean or natural delivery), and breast or formula feeding in the first two years of life and attendance at kindergarten. After obtaining verbal and written consent of the parents as well as the completed questionnaires, the criteria study for inclusion in the study were discussed. This study was approved by the ethics committees of the Ilam University of Medical Sciences (EC/94/H/263). 2.4. PCR amplification of 16S rRNA genes PCR was performed using primers covering the hypervariable region V1–V9 of the 16SrRNA gene (Table 1). The Master Mix contained 100 μM of each deoxynucleotide (dNTP), 1 U of DNA polymerase, 10 pM of each primer, and 10 μL of the DNA template in a final volume of 25 μL of 1 × PCR buffer containing 1.5 mM MgCl2. After initial denaturation at 94 °C for 3 min, PCR was performed using 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 2.2. Sample collection, processing, and culture of samples Seventy-seven oropharynx samples were collected by trained field workers using sterile Dacron‐tipped swabs (BD™, BBL, USA). After sampling of the posterior oropharynx, the swabs were immediately transported to Skim milk, Tryptone, Glucose, and Glycerin (STGG) Table 1 The primer sequences used for PCR assay. Target sequence Primer Sequence (5′-3′) GC% Amplicon size V1, 16S rRNA gene V9, 16S rRNA gene 27F 1492R AGAGTTTGATCATGGCTCAG CGGTTACCTTGTTACGACTT 45.00 45.00 1500 bp 2 Microbial Pathogenesis 143 (2020) 104115 A. Maleki, et al. aureus, and A. baumannii and attendance at kindergarten (P < 0.05). The carriage of S. aureus and A. baumannii is maybe related to the large family sizes, lower socio-economic development, crowded environments, increased multiple drug resistance followed by more infections caused by S. aureus and/or A. baumannii in children. The analysis also showed that there was no correlation in the age of the children in the first two years of life (Table 3). 60 s, and extension at 72 °C for 90 s, followed by a final extension step at 72 °C for 3 min (MyCycler Thermal Cycler, Bio-Rad, Munich, Germany). PCRs were run in duplicate and amplified products were pooled and purified using a Qiaquick PCR purification kit according to the manufacturer's instructions (Qiagene, Hilden, Germany) and the final obtained products were sent for Sanger sequencing (Macrogene, South Korea). The sequences were analyzed by Chromas 2.5 software (Technelysium, Tewantin, Australia; http://technelysium.com.au/wp/ chromaspro/), and final identification of sequences was performed via Blast algorithm in the NCBI database (http://www.ncbi.com). 4. Discussion We investigated the simultaneous characterization of bacterial communities of oropharynx samples in healthy children aged between 3 and 6 years old by combining culture techniques and sequencing of the 16S rRNA gene. The importance of acute respiratory infections in children, especially in developing countries is very evident and consequently, 30% of the annual mortality rate in children is associated with acute respiratory infections [21]. The oropharynx of children is colonized by invasive and non-invasive bacteria such as: Haemephilus influenzae, Neisseria meningitides, and Corynebacteria species [22,23]. Alpha-hemolytic and nonhemolytic Streptococci, commensal Neisseria spp., coagulase-negative Staphylococci, and diphtheroids are considered the normal flora of the oropharynx and nasopharynx [22]. Potential pathogens such as beta-hemolytic Streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis, and Prevotella melaninogenica are the bacteria that are commonly isolated from infections in the oropharynx and nasopharynx [23]. Variations in the microbial flora in the oropharynx may predispose children to infection. As expected the results of this study revealed the presence of highly diverse bacterial communities include 3 phyla Firmicutes, Proteobacteria, and Actinobacteria and 9 families Staphylococcaceae, Streptococcaceae, Micrococcaceae, Enterococcaceae, Bacillaceae, Moraxellaceae, Neisseriaceae, Corynebacteriaceae, and Enterobacteriacae in the oropharynx of healthy individuals. There was a moderate inter-individual variability in the composition of the microbiota up to phyla level, and in the relative abundance of the individual bacterial inhabitants. Similar to other human body habitats, we found a complex, diverse, and highly variable microbiota. Anaerobic bacteria are significant because they dominate the diagnose flora [24,25]. However, the recovery of anaerobes relies on prompt and proper management; thus, the isolation, identification, and, restoration of anaerobes can be complicated, challenging, time-consuming, labor-intensive, and expensive. Furthermore, their exact role is difficult to determine and they are often overlooked [26]. This study was carried out using bacterial culture and 16S rRNA gene and sequencing. Einarsson et al. [27], using the same techniques in human lung specimens, showed that bronchial lavages revealed a dominance of Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria. Additionally, at the genus level of Streptococcus, Prevotella, Actinomyces and Veillonella were dominant [27]. Similar results were also obtained from the study of Humpreys et al. investigating the bacterial colonization of tonsil and pharyngeal mucosal walls [25]. Our results support previous findings and showed that the highest amount of colonization in the childrens’ oropharynx was detected as Firmicutes (69.37%), Proteobacteria (16.19%), and Actinobacteria (4.33%). Also, in this research, 15 bacterial communities were detected and from them Streptococcaceae and Staphylococcaceae were the most common. In addition, it was also found that at the level of the genus, the most frequent microbiome present in the oropharynx of healthy children were the genera of Staphylococcus, Streptococcus, Pseudomonas, Klebsiella, Acinetobacter, Escherichia, Micrococcus, Neisseria, and Enterococcus. Some species are part of the regular oral flora and are not known to cause diseases, although some of these bacteria are pathogenic and if, in their carriers, change to the invasive form they can cause serious diseases [28]. The importance of this issue is increased when children who carry these bacteria are asymptomatic, and are, 2.5. Statistical analysis Data collected from the cultures, sequencing and questionnaires, were analyzed by SPSS software (19.0). A chi-square test was used to determine the correlation between variables. A P value of 0.05 was considered to be statistically significant. 3. Results 3.1. Study population The frequency of variables in the study is displayed in Table 2. 3.2. Frequency of bacterial isolates In total, 346 isolates were obtained from 77 oropharynx samples by phenotypic and PCR sequencing based on 16S rRNA gene region methods. At the phylum level, the oropharynx microbiota was dominated by Firmicutes (74%), Proteobacteria (22%), and Actinobacteria (4%). At the genus level, oropharynx microbiota mainly contained Staphylococci (coagulase-positive and coagulase-negative) and Streptococci, which were dominant. Also, a study of pathogenic species showed that S. aureus (75%), members of the Enterobacteriaceae (40.1%) and A. baumannii species (15.6%) were the most frequent species. At the family level, Staphylococcaceae, Streptococcaceae, and then, Enterobacteriacae were the most frequent. Also, S. aureus (75.3%; n = 58), S. viridans (54.5%; n = 42) and S. epidermidis (42.8%; n = 33) were the most frequent species. To differentiate the obtained sequences in each clade, the phylogenetic hierarchy was drawn phylogenetic maximum likelihood tree based on 16S rRNA gene sequence data was constructed using a database http://www.ebi.ac.uk/. (Fig. 1). 3.3. The correlation between pathogenic bacteria with the present study variables A statistical significance was observed between the frequency of S. Table 2 Demographic characteristics among of 77 individuals. Characteristics Number of subjects Male Female Age range (years) <5 5–6 Type of delivery born Vaginal delivery Caesarean section Fed in the first two years of life Breastfeeding Formula Combination of both Kindergarten Healthcare center 31 (40.3%) 46 (59.7%) 30 (39%) 47 (61%) 36 (46.8%) 41 (53.2%) 50 12 15 61 16 (64.9%) (15.6%) (19.5%) (79.2%) (20.8) 3 Microbial Pathogenesis 143 (2020) 104115 A. Maleki, et al. Fig. 1. The phylogenetic tree based on identities sequences with clades was drawn Phylogenetic maximum likelihood tree based on 16S rRNA gene sequence data was constructed using a database available at: http://www.ebi.ac.uk/. in different geographical regions and include: methodological procedure (quality of the sampling, number and frequency of specimens), genetic background variables, socioeconomic status, age, season, acute respiratory illness, diet (breast-feeding versus bottle-feeding), sleeping thus, a potential source of the disease in the community. Therefore, the timely identification and treatment of these pathogenic carriers can contribute to the health of the community. Many factors are known to affect nasopharyngeal colonization rates 4 Microbial Pathogenesis 143 (2020) 104115 A. Maleki, et al. knowledge of the morphological, physiological, and ecological characteristics of bacteria. On the other hand, new-generation sequencing (NGS) methods can be considered as a superior method for the determination of respiratory microbial populations [42–44]. NGS platforms are, in their present versions, too expensive, need special trained staff, unpractical, and not validated for use in clinical microbiology laboratories [43,45,46]. Despite the benefit of methods based on n 16S rRNA gene sequencing, especially in the identification of low abundant, fastidious or bacterial species with unknown culture conditions, the culture method still is important [47]. According to some reports, a number of bacterial species that were identified by culture, could not be traced using molecular-based methods [47]. In support of this claim, a study has proven that a combination of both used a culture-based and independent culture-based methods increases the sensitivity of detection in microbiome and is superior to using either technique in isolation [48]. On the other hand, it is difficult to assemble an optimum set of culture media to meet the demands of growing different bacterial species. In support of this, in a study conducted by Mahboubi et al. [47], who used a culture-based and high throughput culture-independent techniques to determine the microbial population, it was found that 30% of samples that were positive for Haemophilus were not identified in cultures and the probable reason for that may be related to the selected media. So, in the current study, the lack of isolation of some bacteria could be attributed to the non-use of specific culture media. However, there are a few points about the limitation of sequencing of the 16S rRNA gene such as high sequence similarity, sequencing errors, differences arising from the different regions chosen [49], and difficulties in assessing operational taxonomic units (OTUs) [50]. Combining culture techniques, and the sequence-based molecular methods, provides a broader perspective of bacteria in airways. This approach represents a new tool for the detection, identification, and understanding of bacterial interactions in disease processes as well as providing reliable epidemiological data for tracing the source of human infections [51–54]. Table 3 The correlation of bacterial pathogens with age of children. Bacterial pathogens Age of children P (value) Staphylococcus aureus <3 4–5 6 <3 4–5 6 <3 4–5 6 <3 4–5 6 <3 4–5 6 0.39 Acinetobacter baumannii Pseudomonas aeruginosa Escherichia coli Klebsiella pneumonia 0.12 0.59 0.051 0.52 position, housing, access to health care, poor hygiene, parents’ smoking habits, the number of family members, day-care contact and the number of siblings [4,29–31]. The parents’ smoking habits has been well-known as a risk factor related with increased carriage of respiratory pathogens, since parents who smoke harbor more potential pathogens, fewer interfering organisms [4,29–31]. In addition, smoke may damage and inflame nasopharyngeal mucosa, intensifying susceptibility to viral and bacterial colonization [4]. A prospective cohort study [29] of preschool healthy children (3–6 years old) showed that M. catarrhalis was more commonly isolated from children with smoking parents (p < O.O3, OR 1.4, 95% Cl 1.4–2.0). Although, a number of studies showed that passive smoking did not affect the carriage rate of respiratory pathogens [32–34]. Thus, the outcome of Parents’ smoking habits on colonization rate of children remains controversial. The important risk factors for respiratory infection also include overcrowding, i.e. due to attending day care centers or residing in orphanages, common viral infections, and an excessive use of antibiotics [4]. Kindergartens, a place with risk of infection transmission, are extremely controlled in many countries. Our findings showed a significance relationship between pathogenic bacteria (S. aureus, and A. baumannii) with the attendance at kindergarten (P < 0.05). Although, no significant relationship was observed between frequencies of pathogenic bacteria with others variables. A longitudinal analysis [35] was carried out in healthy kindergarten children (aged 3–7 years) and elementary-school healthy children (aged 7–10 years) was showed that the most common carriers of M. catarrhalis and S. pneumoniae were kindergarten children, the prevalence of carriers of S. aureus only was highest in the elementary-school children. The global kindergarten carriage rates of S. aureus (10–50%) [29,36–39] are lower to the corresponding rate as 75% in this study. This supports the notion that children can act as significant carriers of bacterial pathogens and, therefore, can increase the risk of respiratory diseases [4,40]. It has been suggested that, in addition to investigating carriage of specific pathogens in the respiratory tract, it may be better to measure the prevalence of the total bacterial population [41]. Thus, one of the most important aspects of determining the bacterial population in the upper respiratory tract in healthy people is the identification of pathogenic bacteria and also symbiotic populations. The carriage of specific pathogens in the nasopharynx can lead to an invasion of the blood, tissues, cerebrospinal fluid. However, the disease occurs only in a small group of individuals who have been colonized [4–6]. Developments in molecular biology have helped significantly with investigations into bacterial diversity and advanced notably our 5. Conclusion Our study into respiratory microbiomes, based on culture and molecular methods, showed that the oropharynx is a suitable habitat for the colonization of Firmicutes, Bacteroidetes, and Proteobacteria. This study is the first to report on the respiratory microbiome in the oropharynx region of healthy children in Iran. These findings can be used as a reference for the identification of the important components of microbiota in the oropharynx of healthy children as well as a means to identify the carriers of specific pathogenic agents and help to predict the potential risks of infections. We suggest that further studies are needed in order to identify potential risk factors, their relation to different populations, the identification of people at high-risk, early detection, and the mechanisms involved in the pathogenesis of potential pathogens of lower respiratory tract infections in children. Source(s) of support This research was financially supported by Ilam University of Medical Sciences, Ilam, Iran (941003/4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CRediT authorship contribution statement Abbas Maleki: Conceptualization, Investigation, Methodology. Maryam Zamirnasta: Conceptualization, Investigation, Methodology. Morovat Taherikalani: Supervision, Validation. Iraj Pakzad: Supervision, Validation. Jasem Mohammadi: Supervision, Validation. Marcela Krutova: Writing - original draft, Writing - review & editing. 5 Microbial Pathogenesis 143 (2020) 104115 A. Maleki, et al. Ebrahim Kouhsari: Writing - original draft, Writing - review & editing. Nourkhoda Sadeghifard: Writing - original draft, Writing - review & editing. [20] W.C. Winn, Koneman's Color Atlas and Textbook of Diagnostic Microbiology, Lippincott williams & wilkins, 2006. [21] M.R. Boloursaz, F. Lotfian, F. Aghahosseini, A. Cheraghvandi, S. Khalilzadeh, A. Farjah, et al., Epidemiology of lower respiratory tract infections in children, J. Compr. Pediatr. 4 (2013) 93–98. [22] E. Karaman, O. Enver, Y. Alimoglu, N. Gonullu, H. Bahar, M.M. Torun, et al., Oropharyngeal flora changes after tonsillectomy, Otolaryngology-Head Neck Surg. (Tokyo) 141 (2009) 609–613. [23] I. Brook, P. Yocum, E.M. Friedman, Aerobic and anaerobic bacteria in tonsils of children with recurrent tonsillitis, Ann. Otol. Rhinol. Laryngol. 90 (1981) 261–263. [24] T. Özen, A. Kilic, O. Bedir, Ö. Koru, K. Sorkun, M. Tanyuksel, et al., In vitro activity of Turkish propolis samples against anaerobic bacteria causing oral cavity infections, Kafkas Univ Vet Fak Derg 16 (2010) 293–298. [25] S. Finegold, Anaerobic Infections in Humans, Elsevier, 2012. [26] I. Brook, Anaerobic bacteria in upper respiratory tract and head and neck infections: microbiology and treatment, Anaerobe 18 (2012) 214–220. [27] G. Einarsson, D. Comer, L. McIlreavey, J. Parkhill, M. Ennis, M. Tunney, et al., Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers, Thorax 71 (2016) 795–803. [28] C.A.C. Gioia, A.P.S. de Lemos, M.C.O. Gorla, R.A. Mendoza-Sassi, T. Ballester, A. Von Groll, et al., Detection of Neisseria meningitidis in asymptomatic carriers in a university hospital from Brazil, Rev. Argent. Microbiol. 47 (2015) 322–327. [29] S. Jourdain, P. Smeesters, O. Denis, M. Dramaix, V. Sputael, X. Malaviolle, et al., Differences in nasopharyngeal bacterial carriage in preschool children from different socio-economic origins, Clin. Microbiol. Infect. 17 (2011) 907–914. [30] I. Brook, Effects of exposure to smoking on the microbial flora of children and their parents, Int. J. Pediatr. Otorhinolaryngol. 74 (2010) 447–450. [31] I. Brook, The impact of smoking on oral and nasopharyngeal bacterial flora, J. Dent. Res. 90 (2011) 704–710. [32] M.P. Fairchok, W.S. Ashton, G.W. Fischer, Carriage of penicillin-resistant pneumococci in a military population in Washington, DC: risk factors and correlation with clinical isolates, Clin. Infect. Dis. 22 (1996) 966–972. [33] N. Principi, P. Marchisio, G.C. Schito, S. Mannelli, Risk factors for carriage of respiratory pathogens in the nasopharynx of healthy children, Pediatr. Infect. Dis. J. 18 (1999) 517–523. [34] A.S. Neto, P. Lavado, P. Flores, R. Dias, M.A. Pessanha, E. Sousa, et al., Risk factors for the nasopharyngeal carriage of respiratory pathogens by Portuguese children: phenotype and antimicrobial susceptibility of Haemophilus influenzae and Streptococcus pneumoniae, Microb. Drug Resist. 9 (2003) 99–108. [35] S. Bae, J.-Y. Yu, K. Lee, S. Lee, B. Park, Y. Kang, Nasal colonization by four potential respiratory bacteria in healthy children attending kindergarten or elementary school in Seoul, Korea, J. Med. Microbiol. 61 (2012) 678–685. [36] X-m Luo, J-p Liang, S-z Gao, Z-q Li, R-d Zhou, Surveys on bacteria nasopharyngeal carriage prevalence in 186 children, Chin. J. Microecol. 18 (2006) 204–208. [37] H. Žemličková, P. Urbášková, V. Adamkova, J. Motlova, V. Lebedova, B. Prochazka, Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus isolated from the nasopharynx of healthy children attending day-care centres in the Czech Republic, Epidemiol. Infect. 134 (2006) 1179–1187. [38] L. Liu, W. Xie, C. Jing, Survey of Respiratory Tract Microbial Population in Children, (2007). [39] H. Pan, B. Cui, Y. Huang, J. Yang, W. Ba-Thein, Nasal carriage of common bacterial pathogens among healthy kindergarten children in Chaoshan region, southern China: a cross-sectional study, BMC Pediatr. 16 (2016) 161. [40] A. Sulikowska, P. Grzesiowski, E. Sadowy, J. Fiett, W. Hryniewicz, Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolated from the nasopharynges of asymptomatic children and molecular analysis of S. pneumoniae and H. influenzae strain replacement in the nasopharynx, J. Clin. Microbiol. 42 (2004) 3942–3949. [41] L.O. Bakaletz, Developing animal models for polymicrobial diseases, Nat. Rev. Microbiol. 2 (2004) 552. [42] V.T. Aho, P.A. Pereira, T. Haahtela, R. Pawankar, P. Auvinen, K. Koskinen, The microbiome of the human lower airways: a next generation sequencing perspective, World Allergy Organ. J. 8 (2015) 1. [43] G. Casey, D. Conti, R. Haile, D. Duggan, Next generation sequencing and a new era of medicine, Gut 62 (2013) 920–932. [44] B. Goldberg, H. Sichtig, C. Geyer, N. Ledeboer, G.M. Weinstock, Making the leap from research laboratory to clinic: challenges and opportunities for next-generation sequencing in infectious disease diagnostics, mBio 6 (2015) e01888-15. [45] J.D. Khoury, D.V. Catenacci, Next-generation companion diagnostics: promises, challenges, and solutions, Arch. Pathol. Lab Med. 139 (2014) 11–13. [46] S. Yohe, B. Thyagarajan, Review of clinical next-generation sequencing, Arch. Pathol. Lab Med. 141 (2017) 1544–1557. [47] M.A. Mahboubi, L.A. Carmody, B.K. Foster, L.M. Kalikin, D.R. VanDevanter, J.J. LiPuma, Culture-based and culture-independent bacteriologic analysis of cystic fibrosis respiratory specimens, J. Clin. Microbiol. 54 (2016) 613–619. [48] C.D. Sibley, M.E. Grinwis, T.R. Field, C.S. Eshaghurshan, M.M. Faria, S.E. Dowd, et al., Culture enriched molecular profiling of the cystic fibrosis airway microbiome, PloS One 6 (2011) e22702. [49] N. Youssef, C.S. Sheik, L.R. Krumholz, F.Z. Najar, B.A. Roe, M.S. Elshahed, Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys, Appl. Environ. Microbiol. 75 (2009) 5227–5236. [50] S.M. Huse, D.M. Welch, H.G. Morrison, M.L. Sogin, Ironing out the wrinkles in the rare biosphere through improved OTU clustering, Environ. Microbiol. 12 (2010) 1889–1898. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We would like to thank the Ilam University of Medical Sciences for their professional support. Ethical approval This project was approved by the Ilam University Human Ethics committee (EC/94/H/263). References [1] W.H. Organization, Pneumococcal conjugate vaccine for childhood immunization—WHO position paper, Wkly. Epidemiol. Rec.= Relevé épidémiologique hebdomadaire 82 (2007) 93–104. [2] D. Bogaert, B. Keijser, S. Huse, J. Rossen, R. Veenhoven, E. Van Gils, et al., Variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis, PloS One 6 (2011) e17035. [3] O. Salami, B.J. Marsland, Has the airway microbiome been overlooked in respiratory disease? Genome Med. 7 (2015) 62. [4] J.Á. García-Rodríguez, M.J. Fresnadillo Martínez, Dynamics of nasopharyngeal colonization by potential respiratory pathogens, J. Antimicrob. Chemother. 50 (2002) 59–74. [5] P.G. Peerbooms, M.N. Engelen, D.A. Stokman, B.H. van Benthem, M.-L. van Weert, S.M. Bruisten, et al., Nasopharyngeal carriage of potential bacterial pathogens related to day care attendance, with special reference to the molecular epidemiology of Haemophilus influenzae, J. Clin. Microbiol. 40 (2002) 2832–2836. [6] T. Tenenbaum, A. Franz, N. Neuhausen, R. Willems, J. Brade, S. Schweitzer-Krantz, et al., Clinical characteristics of children with lower respiratory tract infections are dependent on the carriage of specific pathogens in the nasopharynx, Eur. J. Clin. Microbiol. Infect. Dis. 31 (2012) 3173–3182. [7] R.H. Chisholm, P.T. Campbell, Y. Wu, S.Y. Tong, J. McVernon, N. Geard, Implications of asymptomatic carriers for infectious disease transmission and control, Roy. Soc. Open Sci. 5 (2018) 172341. [8] B. Wang, M. Yao, L. Lv, Z. Ling, L. Li, The human microbiota in health and disease, Engineering 3 (2017) 71–82. [9] R. Franco-Duarte, L. Černáková, S. Kadam, K.S. Kaushik, B. Salehi, A. Bevilacqua, et al., Advances in chemical and biological methods to identify microorganisms—from past to present, Microorganisms 7 (2019) 130. [10] W.H. Man, W.A. de Steenhuijsen Piters, D. Bogaert, The microbiota of the respiratory tract: gatekeeper to respiratory health, Nat. Rev. Microbiol. 15 (2017) 259. [11] M. Depner, M.J. Ege, M.J. Cox, S. Dwyer, A.W. Walker, L.T. Birzele, et al., Bacterial microbiota of the upper respiratory tract and childhood asthma, J. Allergy Clin. Immunol. 139 (2017) 826–834 e13. [12] on Earth D, National Academies of Sciences E, Medicine, Current Methods for Studying the Human Microbiome. Environmental Chemicals, the Human Microbiome, and Health Risk: A Research Strategy, National Academies Press (US), 2017. [13] C. Zapka, J. Leff, J. Henley, J. Tittl, E. De Nardo, M. Butler, et al., Comparison of standard culture-based method to culture-independent method for evaluation of hygiene effects on the hand microbiome, mBio 8 (2017) e00093-17. [14] M.J. Claesson, Q. Wang, O. O'Sullivan, R. Greene-Diniz, J.R. Cole, R.P. Ross, et al., Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions, Nucleic Acids Res. 38 (2010) e200-e. [15] J. Ghyselinck, S. Pfeiffer, K. Heylen, A. Sessitsch, P. De Vos, The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies, PloS One 8 (2013). [16] O. Mizrahi-Man, E.R. Davenport, Y. Gilad, Taxonomic classification of bacterial 16S rRNA genes using short sequencing reads: evaluation of effective study designs, PloS One 8 (2013). [17] R. Faner, O. Sibila, A. Agusti, E. Bernasconi, J.D. Chalmers, G.B. Huffnagle, et al., The microbiome in respiratory medicine: current challenges and future perspectives, Eur. Respir. J. 49 (2017) 1602086. [18] C.R. Mahon, D.C. Lehman, G. Manuselis, Textbook of Diagnostic Microbiology-EBook, Elsevier Health Sciences, 2018. [19] P. Tille, Bailey & Scott's Diagnostic Microbiology-E-Book, Elsevier Health Sciences, 2015. 6 Microbial Pathogenesis 143 (2020) 104115 A. Maleki, et al. of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients, FEMS Immunol. Med. Microbiol. 27 (2000) 51–57. [54] J.K. Harris, M.A. De Groote, S.D. Sagel, E.T. Zemanick, R. Kapsner, C. Penvari, et al., Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis, Proc. Natl. Acad. Sci. Unit. States Am. 104 (2007) 20529–20533. [51] G. Rogers, M. Carroll, D. Serisier, P. Hockey, G. Jones, K. Bruce, Characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16S ribosomal DNA terminal restriction fragment length polymorphism profiling, J. Clin. Microbiol. 42 (2004) 5176–5183. [52] G.B. Rogers, M.P. Carroll, D.J. Serisier, P.M. Hockey, V. Kehagia, G.R. Jones, et al., Bacterial activity in cystic fibrosis lung infections, Respir. Res. 6 (2005) 49. [53] A. ven Belkum, N.H. Renders, S. Smith, S.E. Overbeek, H.A. Verbrugh, Comparison 7