Introduction

The airway microbiota play a crucial role in local immune function [22]. Microbiota dysbiosis in the upper respiratory tract (URT) has been reported to be involved in disease development. The nasal microbiota are diverse, and the presence of opportunistic pathogens in the nasal cavity is associated with allergic rhinitis, chronic rhinosinusitis, pneumonia, otitis media, severe obstructive sleep apnea (OSA), and even neurological diseases [2, 6, 9, 43]. The presence of pathogenic bacteria in the adenoids and tonsils contributes to the development of chronic or recurrent middle ear disease, recurrent tonsillitis, and obstructive sleep apnea [13, 37]. Fago-Olsen et al. hypothesized that the adenoids are the main reservoir for otopathogens (e.g., Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis) leading to otitis media [7].

The entire URT is interconnected, with the adenoids and tonsils located at the junction of nose and throat. The bacterial community in the upper airway is affected by many factors, such as age, temperature, and density [3, 16]. For example, before adulthood, the proportion of pathogenic bacteria declines while that of the commensal Staphylococcus epidermidis increases [19]. Furthermore, microbiota structure differs across various sites. That is, Firmicutes and Actinobacteria form the majority of the bacteria in the nostrils, while Firmicutes, Proteobacteria, and Bacteroidetes are more abundant in the oropharynx [18].

Here, we identified the basic microbiota structure in the upper airway in a pediatric population. Samples including the adenoid and palatine tonsils tissues as well as swabs of the nostrils and oropharynx were collected. The microbiota alpha-diversity index in the lymphoid tissues was much higher than that of the swabs. The microbiota compositions differed significantly in the URT. In the nostrils, Firmicutes and Actinobacteria were the most abundant, while Bacteroidetes and Fusobacteria were the least. Proteobacteria were much less present in the adenoids and tonsils. At the genus level, Streptococcus was similarly prolific at all sites. Fusobacteria, the primary genus in the adenoids, was more abundant in the lymphoid tissues and the oropharynx. Haemophilus and Neisseria were more plentiful in the tonsils and oropharynx, while Staphylococcus and Moraxella were the most abundant genus in the nostrils. Notably, Klebsiella, which is usually localized to the gut, was ample in the adenoids.

Materials and methods

Participant enrollment and sample collection

Twenty-five children with hyperplasia of adenoids and tonsils were recruited for the study from Department of Otolaryngology-Head and Neck Surgery, the Second Hospital of Anhui Medical Universityn from January 2020 to July 2020. These children were associated with either OSA (14 cases) or recurrent tonsillitis (11 cases). OSA patients were diagnosed with overnight polysomnography and an Apnea–Hypopnea Index (AHI) score were calculated [36]. Here, we focused on uncomplicated childhood OSA, associated with adenotonsillar hypertrophy (> 50%) [24]. All enrolled OSA patients were associated with adenotonsillar hypertrophy with OSA AHI score > 5. The exclusion criterion was the use of any antimicrobials within the last two weeks. Infants younger than 1 year of age, with central apnea or hypoventilation syndromes were also excluded. OSA patients associated with other medical disorders including Down syndrome, craniofacial anomalies, neuromuscular disease, chronic lung disease were all excluded in our study. All participants do not have surgeries previously. The participants were unrelated individuals from both sexes. Separate mucosal swabs were collected from nostril or oropharynx of each participant, and the swabs were immediately submerged in 1 ml normal saline. The adenoids and tonsils tissues were collected during adenotonsillectomy operation. All the samples were kept at −80 °C until further use. The study was approved by the ethics committee of the Second Hospital of Anhui Medical University (No.20200402), and informed consent was obtained from all participants.

DNA extraction and 16S rRNA gene amplification

All mucosal swabs were centrifuged at 10,000 g for 10 min after completely vortex. The samples were dissolved in buffer ALT with lysozyme (1.25 mg/ml) for 30 min at 37 °C. For the tissue samples, the tissue was excised and homogenized in buffer ALT. After centrifugation at 6000 g for 10 min, the supernatant was incubated with lysozyme (1.25 mg/ml) for 30 min at 37 °C. All the samples were used for the following DNA extraction according to the QIAamp DNA Mini Kit (Qiagen) protocol. Bacterial DNA was amplified using primers 341F (CCTACGGGNBGCASCAG) and 805R (GACTACHVGGGTATCTAATCC). The amplicon-specific V3 and V4 hypervariable regions of the bacterial 16S rRNA gene were amplified and sequenced using the Illumina Hiseq 2500 platform according to the standard protocols. The raw sequencing data were submitted to the GenBank database under accession number PRJNA689096.

Processing of bacterial 16S rRNA sequenced data

After de-multiplexing and quality trimming, the forward and reverse sequences were combined using Fast Length Adjustment of SHort reads (FLASH). Low-quality reads were filtered by fastq_quality_filter (−p 90 −q 25 −Q33) in FASTX Toolkit 0.0.14. Usearch 64 bit (v8.0) was applied for quality control and chimera filtering. The cleaned FASTA sequences were combined and clustered into OTUs using Uclust. After normalizing based on the smallest size of samples by random subtraction, the numbers of reads for each sample were selected to construct the OTU abundance table. The taxonomic classification from the phylum to the genus level for the same number of reads was performed based on the Silva 16S rRNA v128 database using the RDP Classifier. The estimated sampling saturation and rarefaction curves were generated. Finally, the alpha-diversity and beta-diversity indices were calculated in the quantitative insights into microbial ecology (QIIME) and calculated based on weighted and unweighted Unifrac distance matrices. We used the Metastats method to identify species that show statistically significant differential abundances among groups.

Results

Demographic data of all the subjects

To identify the microbiota composition of different parts from one population, we enrolled 25 children who need operation due to the hyperplasia of adenoids and tonsils. The tonsillar tissue and adenoids of 25 children undergoing adenotonsillectomy due to OSA (14 cases) or recurrent tonsillitis (11 cases) were collected during operation. The average ages for all the enrolled participants are 7.43 and 7.82 y, respectively. AHI scores were calculated for all the participants (Table 1).

Table 1 Demographics characteristics of all the participants
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16S ribosomal ribonucleic acid (rRNA) sequencing

The microbiota from different sites of the same individuals were determined. URT samples (adenoids, palatine tonsils, oropharynx swabs, and nasal swabs) were collected from 25 children and underwent16S rRNA sequencing. After quality control and chimera filtration, we finally got the following sample sizes 21 adenoids, 23 palatine tonsils, 25 oropharynx swabs, and 25 nasal swabs. The rarefaction curve for the number of sequences was generated (Supplementary Fig. 1), which confirmed that our data were large enough for subsequent analysis.

Bacterial diversity

We first analyzed microbiota diversity. In terms of alpha diversity, the operational taxonomic units (OTUs) and Chao index revealed that community richness in the adenoids and palatine tonsils was higher than in the oropharynx and nostrils (Fig. 1a, b). The Shannon diversity index, taking into account both community richness and evenness, was calculated to determine the nature and range of the temporal variability in microbial diversity across individuals. The Shannon index indicated a lower microbial diversity in the nostrils compared to the other sites (Fig. 1c).

Fig. 1
figure 1

Alpha-diversity indices of microbiotas across different sites. The operational taxonomic units (A) and Chao index (B) revealed a higher community richness in the adenoids and tonsils compared to the other two sites. C The Shannon index indicated that the microbial diversity was lower in the nostrils compared to the other three sites. **p < 0.01, ****p < 0.0001

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Multivariate analysis of the microbial community across different sites

Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarity distance was used to analyze differences in inter-sample diversity. Samples from the nostrils had bacterial community structures that significantly differed from those of the adenoids. The bacterial compositions between the tonsils and oropharynx overlapped. Altogether, microbiota significantly differed in the URT of one pediatric population (analysis of similarities, R = 0.455, p = 0.001, Fig. 2).

Fig. 2
figure 2

Principal coordinates analysis based on Bray–Curtis dissimilarity among the four groups. Statistical analysis is by Analysis of Similarities (ANOSIM) test

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The overall microbiota structure in the upper airway

The microbiota distribution down to the genus level was analyzed across the different sites. In total, 18 phyla, 37 classes, 56 orders, 89 families, and 214 genera were detected from all the samples.

The analysis of 10 major microbial phyla revealed that the URT microbiota were composed of five predominant phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Firmicutes (around 50%) were the most abundant phylum across all sites. Generally, the microbiota composition in the nostrils significantly differed from that of the other three sites (Fig. 3a). In the nostrils, Firmicutes and Actinobacteria were the most plentiful, while Bacteroidetes and Fusobacteria were the least (Fig. 3b). Notably, Proteobacteria was much less prolific in lymphoid tissues compared to the nostrils or oropharynx (Fig. 3b).

Fig. 3
figure 3

The overall microbiota structure at the phylum level of the upper airway of children. A Relative abundance of the ten major phyla in subjects divided by age. B Abundance of Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, and Fusobacteria across different sites in single individuals. *p < 0.05, ***p < 0.001

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Bacteria at the genus level were also analyzed across the different sites. The distributions of the top 16 bacterial genera for all areas are shown in Fig. 4a. Similar to the phyla analysis, the top 16 genera in the nostrils were completely different from the tonsils, adenoids, and oropharynx. More specifically, the top three genera Staphylococcus (phylum Firmicutes), Moraxella (phylum Proteobacteria), and Corynebacteria (phylum Actinobacteria) in the nostrils were absent from the top 10 genera of the other three sites (Fig. 4b). Although Firmicutes was much more prolific in the nostrils than in the other areas, Streptococcus (phylum Firmicutes) was similarly abundant across all sites.

Fig. 4
figure 4

The overall microbiota structure at the genus level of the upper airway of children. (A) Relative abundance of the 16 major genera in the subjects divided by age. (B) Abundance of the top 10 genera across the different sites in single individuals

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Moraxella, Klebsiella, Haemophilus, and Neisseria are all pathogenic genera belonging to the Proteobacteria phylum. Although the abundance of Proteobacteria was very low in the adenoids, Klebsiella was the third-most ample genus at that site. Haemophilus and Neisseria were the most prevalent genera in the tonsils and oropharynx, respectively, and Moraxella was the second-most abundant genus in the nostrils (Fig. 4b), indicating that the entire upper tract serves as a possible reservoir for pathogenic bacteria.

The composition of the Bacteroidetes phylum is diverse: the Bacteoides, Prevotella 7, and Porphyromonas were all in the top 10 genera present in the adenoids, tonsils, and oropharynx; however, their amounts were very low in the nostrils (Fig. 4). In addition, Fusobacterium, which belongs to the Fusobacteria phylum, was more abundant in the adenoids and tonsils (Fig. 4b).

Comparison of potential pathogenic bacteria abundances in the upper airway

We observed a significant difference in microbiota composition in the nostrils compared to the other three sites; accordingly, the top four genera were analyzed. Consistent with the increased presence of the Firmicutes and Actinobacteria phyla, Staphylococcus (phylum Firmicutes) was approximately 29.6, 17.4, and 14.0 times more plentiful in the nostrils, respectively, than in the adenoids, tonsils, and oropharynx. Similarly, Dolosigranulum (phylum Firmicutes) was approximately 252, 191, and 161 times more abundant than in the adenoids, tonsils, and oropharynx, respectively. Finally, Corynebacterium 1 (phylum Actinobacteria) was present in amounts approximately 305, 489, and 820 times higher, respectively, than in the adenoids, tonsils, and oropharynx. Altogether, these three genera displayed the highest abundance in the nostrils compared to the other three sites (Fig. 5). Although the abundance of Proteobacteria was the lowest in the nostrils, the abundance of Moraxella (phylum Proteobacteria) was the highest (approximately 16, 178, and 30 times higher than the adenoids, tonsils, and oropharynx, respectively).

Fig. 5
figure 5

Mean abundance of the promising pathogenic genera in the human upper respiratory tract microbiota across the four sites. *p < 0.05, **p < 0.01, ***p < 0.001

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The pathogens reportedly involved in URT diseases were further analyzed. Traditional culture-based studies have shown that S. pneumoniae, H. influenzae, and Neisseria spp. are predominately isolated in patients with persistent otitis media with effusion [8]. Here, we observed that Streptococcus was similarly abundant across all sites, while Haemophilus and Neisseria were much more plentiful in the tonsils and oropharynx (Fig. 5). Notably, we observed that Klebsiella and Fusobacteria, normally localized to the alimentary canal, were much more ample in the adenoids as well as in the tonsils and oropharynx (Fig. 5), indicating that their presence in the throat, which connects the oral cavity and gastrointestinal tract, affects the localization of bacteria in lymphoid tissues.

Discussion

The URT microbiota influence human health. Commensal bacteria, no doubt, promote the maturation of the local immune system and the microbiota dysbiosis causes many diseases [32]. The adenoids are even coined the “pathogen reservoir” in children [13]. However, most of the current studies were based on the comparison of the microbiota between lymphoid tissues and middle ear specimens in patients. The exact distributions of the promising pathogens in the whole URT in children are still unknown. In this study, we analyzed the microbiota of the URT in a group of children without infections. The hyperplasia of adenoids and tonsils in these children were associated with OSA or recurrent tonsillitis. We found that Staphylococcus, Corynebacterium, Dolosigranulum and Moraxella were mainly present in the nostrils, while Haemophilus and Neisseria were mostly found in the tonsils and oropharynx. Notably, Fusobacteria and Klebsiella, which are normally present in the alimentary canal, exhibited high abundance in the adenoids. We concluded that promising pathogenic bacteria localize in the entire URT, and that specific bacteria are prone to localize in specific sites.

The adenoids and tonsils are lymphoid tissues that play important roles in immune defense [31]. The adenoids surface is colonized by commensal miocrobiota, which serve as a gateway to the respiratory tract [42]. However, the potential pathogenic bacteria localized to the adenoids and tonsils are involved in the development of various diseases in the pediatric population [23]. In fact, the adenoids are coined the “pathogen reservoir” in children due to its specific role in middle ear infections [13]. This “pathogen reservoir hypothesis” is supported by Ren et al. By surveying 16S rRNA, they found that the common pathogens including Haemophilus, Streptococcus, Moraxella, and Staphylococcus are prevalent and abundant in the adenoids [34]. However, others have cast doubt on the adenoid “pathogen reservoir hypothesis” because they failed to find a correlation between adenoid and middle ear microenvironments [1, 4, 12, 29]. Here, we observed that several pathogenic bacteria, such as Streptococcus and Haemophilus, were indeed localized to the adenoids at high amounts, supporting the pathogen reservoir hypothesis. However, Staphylococcus aureus and M. catarrhalis, bacteria frequently cultured from children with otitis media [25, 40], were the most abundant in the nostrils (Fig. 4 and 5), indicating that the potential pathogenic bacteria may also originate from the nostrils.

In our study, subjects including OSA or recurrent tonsillitis with the hyperplasia of adenoids and tonsils were recruited. It is reported that tonsils of patients with OSA present a defective regulatory B cell (Breg) compartment. Importantly, bacterial species isolated from the tonsils harbor the ability to breach the epithelial barrier [35]. We cannot exclude the possibility that the disease status affects the microbiota composition. The limit for our study is the lack of samples collected from healthy pediatric population. This constrains the conclusions of our study to the children with the hyperplasia of lymphoid tissues.

Notably, the top three genera in the adenoids included Fusobacterium and Klebsiella (Fig. 4b). Fusobacteria commonly inhabit the oral cavity and are involved in the development of colorectal cancer [14]. Klebsiella is commonly in the gut and is one of the most common pathogens in nosocomial infections [20]. The prevalence of gastroesophageal reflux disease is associated with otitis media in children [26, 40]. We hypothesize that these bacteria, normally localized to the alimentary canal, are also present in the adenoids due to the gastroesophageal reflux and contribute to the progression of infectious diseases in children.

Jensen et al. reported that the tonsillar crypts microbiota of children (2–4 years) are as diverse as that of adults. Pathogenic bacteria including H. influenzae, Haemophilus haemolyticus, Neisseria spp., and S. pneumoniae were detected in the tonsillar crypts of children [11]. Here, we observed a similar microbiota pattern in the tonsils and oropharynx, where Haemophilus and Neisseria were the most abundant pathogenic genera (Fig. 4 and 5). We hypothesize that pathogens localized to the tonsillar crypts partially originate from the oropharynx.

Patients with rhinosinusitis display reduced Corynebacterium levels compared to healthy individuals [21, 38]. The existence of bacterial interference between Corynebacterium and the pathogenic S. aureus and Streptococcus has been reported. Corynebacterium implantation can help eradicate S. aureus from the nasal cavities [33, 39]. Indeed, various Corynebacterium spp. can inhibit the growth or the virulence of S. aureus and S. pneumoniae [16], suggesting that the higher amounts of Corynebacterium in the nostrils may maintain nostril microbiota balance. Here, we observed that Corynebacterium and Staphylococcus were most plentiful in the nostrils, while Streptococcus, which is reported to be a prevalent pathogen in children [28], displayed similarly high abundance across all sites (Fig. 4b). Corynebacterium may help maintain microbiota balance in the nostrils by eradicating the pathogenic bacteria there. Due to limitations in sequencing, we were unable to identify the exact Staphylococci spp. In children, the carriage rate of pathogenic S. aureus is very high [19]. The pathogen S. aureus, commonly detected in patients with chronic sinusitis, nasal polyposis, or otitis media [5, 41], may mainly originate from the nasal carriage.

Dolosigranulum and Moraxella were the most abundant in the nostrils. Dolosigranulum pigrum, an unusual Gram-positive bacterium, is involved in nosocomial pneumonia and septicemia [10, 17]. M. catarrhalis is a common cause of otitis media and chronic obstructive pulmonary disease [27]. Coagulase-negative staphylococci (CoNS) can defend against pathogenic bacteria, such as Dolosigranulum and Moraxella, by producing antimicrobial peptides [15, 30]. Notably, the nostril microbiota are constantly changing because the nostrils are open to the air [3]. We hypothesize that the main Staphylococcus species, CoNS, is crucial in maintaining microbiota balance in the nostrils. However, why certain bacteria are prone to localizing at specific sites remain unknown. Further studies should focus on how microbiota balance maintenance affects human health.

In conclusion, our data shed light on the characteristics of the bacterial microbiota in the URT by analyzing the samples collected from the different sites of the same population. Our data confirm that upper tract lymphoid tissues, normally considered immune organs, may also serve as reservoirs for pathogenic bacteria.