The ISME Journal (2015) 9, 1246–1259 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Culture and molecular-based profiles show shifts in bacterial communities of the upper respiratory tract that occur with age This article has been corrected since Advance Online Publication and an erratum is also printed in this issue Jennifer C Stearns1, Carla J Davidson2, Suzanne McKeon2, Fiona J Whelan3, Michelle E Fontes1, Anthony B Schryvers2, Dawn ME Bowdish4, James D Kellner2,5 and Michael G Surette1,2,3 1Department of Medicine, McMaster University, Hamilton, Ontario, Canada; 2Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada; 3Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada; 4Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada and 5Department of Paediatrics, University of Calgary, Calgary, Alberta, Canada The upper respiratory tract (URT) is a crucial site for host defense, as it is home to bacterial communities that both modulate host immune defense and serve as a reservoir of potential pathogens. Young children are at high risk of respiratory illness, yet the composition of their URT microbiota is not well understood. Microbial profiling of the respiratory tract has traditionally focused on culturing common respiratory pathogens, whereas recent culture-independent microbiome profiling can only report the relative abundance of bacterial populations. In the current study, we used both molecular profiling of the bacterial 16S rRNA gene and laboratory culture to examine the bacterial diversity from the oropharynx and nasopharynx of 51 healthy children with a median age of 1.1 years (range 1–4.5 years) along with 19 accompanying parents. The resulting profiles suggest that in young children the nasopharyngeal microbiota, much like the gastro- intestinal tract microbiome, changes from an immature state, where it is colonized by a few dominant taxa, to a more diverse state as it matures to resemble the adult microbiota. Importantly, this difference in bacterial diversity between adults and children accompanies a change in bacterial load of three orders of magnitude. This indicates that the bacterial communities in the nasopharynx of young children have a fundamentally different structure from those in adults and suggests that maturation of this community occurs sometime during the first few years of life, a period that includes ages at which children are at the highest risk for respiratory disease. The ISME Journal (2015) 9, 1246–1259; doi:10.1038/ismej.2014.250; published online 9 January 2015 Introduction In addition to its beneficial role, the commensal microbiota can also be a reservoir of respiratory As the point of entry to both the lower respiratory pathogens as well as antibiotic resistance and tract and the gastrointestinal tract, the upper virulence genes (Garcia-Rodriguez and Fresnadillo respiratory tract (URT) is continuously exposed to Martinez, 2002; Liu et al., 2013). For instance, the outside world. The microbiota is involved in healthy people can have long periods of asympto- resistance to colonization by incoming pathogens matic carriage of classic respiratory pathogens (Kelley et al., 2005; Margolis et al., 2010), education (Garcia-Rodriguez and Fresnadillo Martinez, 2002) of the immune system (Lathrop et al., 2011) and and invasive disease can often arise from normally regulation of host immunocompetence in the lung benign bacterial residents (Bakaletz, 2004). This in response to infection (Ichinohe et al., 2011). duality blurs the definition of a commensal member of the respiratory tract. Instead, it’s likely that this body site harbours an indigenous microbiota whose Correspondence: MG Surette, Department of Medicine, McMaster members behave differently, depending on factors University, 1200 Main St West, Hamilton, Ontario L8S 4L8, such as their location in the body (Blaser and Canada. E-mail: [email protected] Falkow, 2009), bacterial community disturbance Received 11 July 2014; revised 18 November 2014; accepted (Lynch, 2013), environmental pressures (Feldman 24 November 2014; published online 9 January 2015 and Anderson, 2013) and/or immune responses in Changes in the microbiota of the upper airways with age JC Stearns et al 1247 the host (Starkey et al., 2013). Unlike many acute order to describe the healthy child URT microbiome infectious diseases where a single microbe can be in the context of the adults with whom they have the targeted and eradicated, lung infections are often most contact. We found that the nasopharynx of polymicrobial (Bakaletz, 2004; Han et al., 2012; young children is dominated by a small number of Huang et al., 2012, 2014; Dickson et al., 2013) and bacterial groups that are present in high total the organisms recovered from respiratory and numbers, in contrast to that in adults, which had invasive infections are often a mixture of common much lower bacterial carriage and a more diverse URT microbes (Laupland et al., 2000, Sibley et al., bacteria community. The oropharynx communities 2008; 2012). Respiratory infections have a higher were dominated by streptococci in all subjects and impact on health worldwide than all other infec- with higher bacterial biomass than the nasopharynx. tious diseases combined (Mizgerd, 2006) and mor- This study provides the first comprehensive look at tality rates associated with lung infections have not the healthy URT microbiota of young children along significantly improved in over 50 years (Mizgerd, with those of their parents. 2008). Children, in particular, are very susceptible to respiratory illness (Liu et al., 2012), therefore, an Materials and methods understanding of the makeup of the URT microbiota in this population is important to our understanding Cohort of the origin and progression of infection. Interac- The Calgary Area Streptococcus pneumoniae Epi- tions between resident airway pathogens are known demiology Research (team has conducted 10-point to occur, for instance, competitive exclusion prevalence surveys of pneumococcal NP coloniza- between Streptococcus pneumoniae and Staphylo- tion in healthy children o5 years of age attending coccus aureus (Lijek and Weiser, 2012); however, Community Health Centres for routine immuniza- these interactions have not been studied in the tion visits in Calgary, Canada since 2003 (Kellner context of the complete URT microbiota. et al., 2008; Ricketson et al., 2014). The study was Until recently, the focus of most URT microbiol- approved by the University of Calgary Conjoint ogy in healthy individuals has been on carriage of a Health Research Ethics Board. Each survey was few important pathogens such as S. pneumoniae, conducted over approximately a 6-week period in Haemophilus influenzae, Moraxella catarrhalis and seven Community Health Centres. After obtaining S. aureus (Dunne et al., 2013), however, our knowl- written informed consent, study nurses obtained edge of colonization and succession of bacterial demographic information and conducted a health communities in the healthy human URT is lacking. survey. An average of 615 children were enrolled in A few microbiome studies have focused on describ- each survey. During the 2011 and 2012 surveys, ing the communities at these sites (Lemon et al., additional written informed consent was obtained 2010; Charlson et al., 2011; Faust et al., 2012), for a convenience subset of the study population to however, only Bogaert et al. (2011) looked in depth provide additional samples that were used in the at children under 2 years of age, the population at present study to describe the healthy child URT greatest risk of respiratory illness and invasive microbiome. NP swabs were taken nasally and OP pneumococcal disease. No reports, however, have swabs orally according to the standard WHO looked at the healthy URT microbiota from both the method (O’Brien and Nohynek, 2003) by the public perspective of molecular profiling and quantitative health nurse from 51 children and 19 adults culture for all bacteria, an approach that provides a (Supplementary Table S4). We used Copan eSwabs more complete picture of bacterial colonization. (Alere Canada, Ottawa, ON, Canada), a flocked swab Molecular profiling can provide a snapshot of with 1 ml of Amies transfer fluid. All swabs were community structure, however, this picture is stored at room temperature and processed within imperfect because of its inability to distinguish 6–8 h. For processing, the swabs in their transfer between living cells and dead cells or cell-free fluid were vortexed vigorously for 15–30 s, then a DNA. It also suffers from bias against some bacterial 100 ml subsample was taken for plating. The remain- lineages, as no primer pair is perfect, and a lack of der was frozen at À 20 1C for molecular analysis. taxonomic resolution, as relatively short DNA sequences are used. Bacterial DNA isolation and Illumina sequencing of For the study herein, a subset of the swabs bacterial tags collected during a large point prevalence study of DNA was extracted from NP and OP swab samples S. pneumoniae serotype carriage in the Calgary area with a custom DNA extraction protocol involving (Ricketson et al., 2014) were used. A total of 51 mechanical and enzymatic lysis followed by a phenol:- healthy children, with a median age of 1.1 years, chloroform extraction and a clean-up step. First, 300