Ear, Nose & Throat Journal2023, Vol. 102(12) 780–786© The Author(s) 2022Article reuse guidelines:sagepub.com/journals-permissionsDOI: 10.1177/01455613221135647journals.sagepub.com/home/ear
Objective: We aimed to investigate the difference between the bacterial profiles of the nasal cavities and adenoid surfaces of children with chronic rhinosinusitis (CRS). We also intended to determine and analyze the potential correlation between the pre- and post-adenoidectomy differences in the nasal bacterial profile and clinical prognosis. Methods: The clinical information of pediatric patients was collected. All the children underwent adenoidectomy (with or without tonsillectomy), and swab samples were collected during the operation. Visual analog scales (VAS) were used at 3, 6, and 12 months postoperatively. At the 12-month follow-up examination, swab samples were collected again. PCR amplification was performed of the v3-v4 variable regions of 16S rRNA of the collected specimens, as well as high-throughput sequencing using the Illumina platform. The species information was obtained by OTUs clustering, species annotation, and α-diversity analysis. Results: Twenty-two male and eight female pediatric patients were included in the investigation The most abundant genus level bacterial representatives on the nasal surface before adenoidectomy were Moraxella catarrh, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus, whereas on the surface of adenoids, they were Streptococcus pneumonia, Haemophilus influenza, Nucleobacter, and Moraxella catarrhalis. One year postoperatively, the bacteria with the highest abundance on the nasal surface at the genus level were Moraxella catarrhalis, Streptococcus pneumonia, Staphylococcus aureus, and non-culturable Dolosigranulum. One year postoperatively, the bacterial richness in the nasal cavity was significantly higher than at baseline (P < .05). Furthermore, the subjective nasal score of all children significantly decreased at 3, 6, and 12 months postoperatively (P < .01). Conclusion: The preoperative bacterial abundance of the nasal cavity and the adenoid surfaces was similar, showing a clear correlation. No single specific bacterium was established to be a dominant species associated with the development of CRS in children. The post-adenoidectomy bacterial richness in the nasal cavity was significantly increased, which may be closely related to the relief of postoperative sinusitis symptoms.
Keywordschronic rhinosinusitis in children, bacterial profile, 16S rRNA, adenoidectomy
Chronic rhinosinusitis (CRS affects roughly 4% of children and is characterized by easy relapse.1 However, scarce research has been conducted to elucidate pediatric CRS pathogenesis, and its etiology remains unknown.
A large number of studies have shown that bacterial colonization in the adenoid tissue or chronic infection is associated with CRS in children, and adenoidectomy is indicated to minimize bacterial load.2-6 Besides, inflammatory factors induced by bacterial infection have long been thought to play a role in the pathophysiology of CRS in children. Still, little research attention has been focused on the differences in the pre- and postoperative microbiota in children with CRS. Therefore, urgent investigations are needed on the type of bacterial infection, the bacterial abundance, and the changes in the diversity and richness of nasal flora during disease development and progression. The goal of this study was to investigate the pre- and post-adenoidectomy differences in the diversity and richness of the nasal flora in children with CRS to provide novel insights into the selection of effective treatment regimens.
This study was approved by the Ethics Committee of Beijing Children’s Hospital, Capital Medical University, and all patients wrote informed consent. Thirty children with CRS and AH (with/without tonsillar hypertrophy), admitted to the Department of Otorhinolaryngology, Head and Neck Surgery, National Center for Children’s Health, Beijing Children’s Hospital from October 2017 to October 2019, were enrolled in this study. Of them, 22 were males and 8 were females, aged from 3.17 to 10.67 years, with a median age of 5.80 years. Adenoidectomy (with or without tonsillectomy) was performed in all patients under general anesthesia. All patients did not treat with any medication for two weeks before surgery. Mometasone furoate aqueous nasal spray and physiological seawater nasal washing were used for one month after the operation. A month later, only symptomatic medication and physiological seawater nasal washing were used for the treatment of upper respiratory infection, and there was no other medication used in the nasal. All the children were followed up in the outpatient clinic at 3, 6, and 12 months postoperatively to complete the symptom scale score. Fifteen of the children that were followed up for a year postoperatively were willing to undergo nasal secretion sampling.
The following inclusion criteria were applied: history of sinusitis in the enrolled children more than three months, and at least one of the below clinical symptoms was manifested: nasal obstruction and mucopurulent nasal discharge, with or without pain in the head and face, decreased or lost smell, and cough. Electronic nasopharyngoscopy revealed purulent discharge in the middle meatus and confirmed the diagnosis of CRS. AH was diagnosed when the adenoids in the nasopharynx obstructed the choanal by more than 50%.
The exclusion criteria used were as follows: preoperative treatment with antibiotics for two weeks; history of respiratory infection within two weeks; recurrent pneumonia, bronchitis, suspected ciliary dysfunction since childhood; immunodeficiency diseases, cystic fibrosis, and other systemic diseases; and other medical problems that prevented the child from receiving surgery and medication.
Further screening was conducted based on the inclusion and exclusion criteria. Signed informed consent was obtained from the legal guardians (children under the age of eight) and/or from the children themselves (children aged eight years and above) at the time of admission. Then, clinical information questionnaires were completed to formally identify the children for enrollment.
The secretions from the adenoid surface and the left lateral wall of the nasal cavity were removed with a swab during the nasal endoscopic adenoidectomy, which was performed under general anesthesia (if tonsillar hypertrophy was present, it was simultaneously removed). The changes in the nasal symptom scores and the number of upper respiratory tract infections were documented during the outpatient follow-up at 3, 6, and 12 months postoperatively. Visual analog scales (VAS) were implemented to rate nasal symptoms, such as nasal congestion, sneezing, nasal discharge, and nasal itching pre- and postoperatively. Secretions from the left lateral wall of the nasal cavity were obtained in the outpatient clinic 12 months after the surgery.
Specimens from the left lateral wall of the nasal cavity and adenoid surface secretions were collected using swabs under strict aseptic conditions in the operating room. After general anesthesia and before any surgical procedure, to collect a sample, a moist sterile cotton swab was inserted in the left lateral wall of the middle meatus and the adenoid surface under endoscopic guidance. Next, the swab was taken out and placed in a sterile test tube. Further, it was frozen at –80°C within one hour after sampling until DNA was extracted. Postoperative nasal swab sampling was performed in the ENT clinic. Before any intervention in the nasal cavity, sterile nasopharyngeal swabs were used to obtain specimens under sterile endoscopic guidance with the child’s cooperation. Then, the samples were immediately frozen at -80°C until analysis. Each container had a barcode label with a unique identification number.
Total bacterial DNA was extracted from pre- and postoperative swab specimens. The extraction of DNA was performed following the instructions of the E.Z.N.A.® soil DNA kit (Omega Bio-Tek, Norcross, GA, USA). PCR amplification was carried out using primers specific for the bacterial 16S rRNA V3-V4 region. NanoDrop2000 was utilized to determine the concentration and purity of DNA in a reaction system volume of 20 μL. A 2% agarose gel was used to recover PCR results from the same sample. The recovered product was purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), followed by detection by gel electrophoresis with 2% agarose and quantification with a QuantusTM Fluorometer (Promega). Finally, high-throughput sequencing was performed using the Illumina platform.
The bacterial sequencing results were subjected to bioinformatic analysis for the determination of their bacterial richness and diversity.
Statistical analysis was performed using SPSS 19.0. The measurement data of each group pre- and postoperatively were compared and analyzed by paired t-test, and independent samples t-test made the comparison between groups; the counting data were compared by χ2 tests, and P < .05 was considered to indicate a statistically significant difference.
Based on the inclusion and exclusion criteria, a total number of 30 children were included. Fifteen children were followed up to one year postoperatively, who cooperated with the provision of nasal secretion sampling.
The predominant bacterial species on the surface of the nasal cavity were Moraxella catarrhalis, Streptococcus pneumonia, Haemophilus influenza, and Staphylococcus aureus. The main bacteria on the surface of the adenoids are Streptococcus pneumonia, Haemophilus influenza, Fusobacterium, nonculturable Neisseria, and Moraxella catarrhalis Figure 1.
The abundance of Streptococcus pneumonia, Haemophilus influenza, and Staphylococcus aureus was high both on the surface of the nasal cavity and on the adenoid surfaces, but with no statistical difference (P > .05). The abundance of Moraxella catarrhalis, non-culturable Dolosigranulum, Corynebacterium, and Neisseria was significantly higher on the surface of the nasal cavity, with a statistically significant difference (P < .05). Meanwhile, the abundance of Fusobacterium, Prevotella melaninogenica, and Porphyromonas gingivalis was significantly higher on the adenoid surface with statistical differences (P < .05). Other bacterial species were also present with a lower abundance Figure 2.
High-Throughput Sequencing Data Analysis. A number of 3,898,489 high-quality sequences were obtained from 30 samples through optimization. The average length of the sequences was 424 bp, and the distribution of the sequence length was between 420 and 460 bp. The OTU representative sequences with a 97% similarity were taxonomically analyzed. The preoperative group contained 6 phyla, 9 classes, and 15 genera, whereas 5 phyla, 8 classes, and 12 genera were represented in the postoperative group.
Comparison of the Nasal Flora at the Genus Level Pre- and Post-Adenoidectomy in Children With CRS. The five most abundant pre-adenoidectomy genera of the nasal flora were Moraxella catarrhalis, Streptococcus pneumonia, Haemophilus influenza, Staphylococcus aureus, and unculturable Cocci guile, accounting for 21.31%, 17.61%, 16.48%, 12.42%, and 9.36% before adenoidectomy and 22.25%, 19.63%, .76%, 15.57%, 9.82%, after adenoidectomy, respectively. There were no significant postoperative changes in the abundance Figure 3.
Analysis of the Species Diversity. The pre- and postadenoidectomy α-diversity indices are presented in Table 1. The data of both groups were subjected to t-test. Comparative assessments between the two groups pre- and postoperatively revealed a statistically significant difference (P < .05) between the values of the sobs, ace, and Chao indexes in both groups, that is, there was a significant post-adenoidectomy increase in the richness of the nasal flora. However, the Shannon and Simpson index values were not statistically different (P > .05). Both sets of coverage indices reached 99.9%, indicating that the majority of the species in the samples were detected and sequenced to a sufficient depth to reflect the composition of the original bacterial community in the samples.
Preoperative nasal symptoms including nasal congestion, nasal discharge, nasal itching, and sneezing were investigated by questionnaire. The preoperative VAS scores were compared with those at 3, 6, and 12 months postoperatively. Statistically significant (P < .05) improvement of the nasal symptoms was established Table 2.
CRS in children is common in otorhinolaryngology department settings. It is estimated that approximately 6–7% of upper respiratory tract infections may develop into adenoiditis and acute sinusitis,7 with a certain percentage developing into CRS.8 The nasal cavity and its surrounding tissues (including the adenoids) are part of the upper respiratory tract, and their anatomical proximity determines their pathophysiological and microbiological relevance. Expectedly, AH is common in children with sinusitis.
Earlier research has suggested that the adenoids represent a bacterial reservoir. In this regard, Bernstein et al. discovered that the bacteria on the adenoids were the same as those on the lateral wall of the nasal cavity in children with CRS, implying that the adenoids may be bacterial reservoirs in CRS.9 Fluorescence in situ hybridization (FISH) was earlier introduced by the use of immunostaining combined with observations under a confocal laser scanning microscope, whose results also showed that the bacterial strains in the adenoids were identical to the pathogens commonly detected in patients with CRS.10 These studies do not, however, indicate whether the microbiological profile of the adenoidal pathogens influences the microbiological profile of the pathogens present in the nasal cavity. To provide a more direct response to this query, further research on the alterations in the microbiological profile of the pathogens in the nasal cavity preand post-adenoidectomy is needed.
In the past, culture techniques were widely used for bacteriological research on CRS. However, this method may not truly reflect the diversity of the microorganisms in the sample and can obscure the presence of the real bacterial community representatives. Next-generation sequencing (NGS) has provided novel perspectives and advances to genomic research by increasing sequencing throughput and eliminating the need for pre-cloning.11 In this study, high-throughput sequencing of bacterial profiles was performed with the Illumina MiSeq platform.
Consistently with previous reports, here, we found that Moraxella, Haemophilus, and Streptococcus were the most abundant taxa in children with chronic respiratory infections.12 In the study of Galli et al., Haemophilus influenza was the most common bacterium isolated from adenoid tissue.13 A previous study revealed that the percentage of Haemophilus influenza decreases with age,14 and thus changes in Haemophilus influenza abundance may be observed more frequently in children with sinusitis. In this investigation, the post-adenoidectomy abundance of Haemophilus influenza in the nasal cavity was considerably lower than the preoperative but with no statistical difference. We speculate that this result is related to the small sample size, which highlights the need for larger sample sizes of future studies.
The nasal cavity is colonized by a large number and variety of bacteria not only in the disease state of chronic sinusitis, but also in the non-disease state.15 Bacteria with an abundance of more than 50% were considered dominant species, but no single specific bacterium species was detected to be dominant and linked to the development of CRS in children.
The microbiota’s composition, distribution, and abundance affect mucosal health, pathogen growth, function, pathogen overgrowth, and the susceptibility to infection may increase when the microbiota is disrupted (dysbiosis). In theory, increased biodiversity could boost the competition in the nasal microbiota, limiting the number of opportunistic infections. Biswas et al.16 discovered that the diversity of bacterial communities in samples taken from CRS patients was much lower than in those collected from healthy people whose bacterial burden was unrelated to the disease.17-19 In this study, we found differences between the preoperative and postoperative bacterial diversity and richness of the nasal flora in children with CRS. The increase in richness (P < .05) was statistically significant, indicating a healthier postoperative nasal cavity, which facilitates the stability and recovery of the nasal microbial ecosystem and reduces the incidence of sinusitis.
Due to difficulties in the postoperative follow-up examinations of children and their swab sampling, it was not possible to improve the collection of nasal secretions in all three follow-up visits of the enrolled children and perform dynamic analysis, Hence, only the postoperative 12-month bacterial profile, which was available in all children, was compared to the preoperative one in this study. Only 15 children participated in the standard postoperative sampling at the outpatient clinic, resulting in a limited sample size for each group. In the future, the conditions for children’s follow-up and communication should be improved, striving to promote more effective collection of laboratory specimen materials and to increase the sample size, which would facilitate the collection of more convincing information. Additionally, we were unable to screen for individuals with changes in their natural microbiota as a result of the repeated use of antibiotics. Thus, only children who had taken antibiotics within the previous two weeks were removed from the present research. Bacterial contamination from other tissues (e.g., nasal vestibular skin) and the presence of viruses (e.g., phages) in the samples should also be considered while sampling since these factors can influence microbial counts and genes. In this regard, the extraction procedures should be improved to limit contamination and error.
Little research has been conducted on post-adenoidectomy nasal microbiota of children. Therefore, this investigation could serve as the basis for an important comparison of the pre- and post-adenoidectomy microbiota of children with CRS. Due to the small sample size of the present study, further research is needed to confirm our present findings and conclusions.
WTG conceived and designed the experiments. LXT, PPW, XJY, XX, and YH collected the samples. WZ performed the experiments and analyzed the data. WZ and XXC wrote the manuscript under the review, editing, and supervision of WTG. All authors read and approved the final manuscript.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by The Special Fund of The Pediatric Medical Coordinated Development Center of Beijing Municipal Administration of Hospitals [XTZD20180103]; Beijing Hospital Authority Ascent Plan [DFL20191201]; National Clinical Research Center for Respiratory Diseases [HXZX-20210501]; and Research and transformation application of capital clinical diagnosis and treatment technology [Z20110000520084].
This study was approved by the Ethics Committee of Beijing Children’s Hospital, Capital Medical University, and all patients provided their informed consent. All experiments were performed based on relevant guidelines and regulations.
Xiaoxu Chen https://orcid.org/0000-0002-6617-1692
Wentong Ge https://orcid.org/0000-0002-2615-2702
Wei Zhang https://orcid.org/0000-0002-3594-6862
1 Department of Otorhinolaryngology, Head and Neck Surgery, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China
2 Beijing Key Laboratory for Pediatric Diseases of Otolaryngology Head and neck surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, China
Corresponding Author:Wentong Ge, MD, Department of Otorhinolaryngology, Head and Neck Surgery, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, No.56 Nanlishi Road, Beijing 100045, China.Email: gwt@bch.com.cn
