Analysis of 16S rRNA gene sequence of nasopharyngeal exudate from healthy donors reveals changes in key microbial communities associated with aging

Background Functional or compositional perturbations of the microbiome can occur at different sites of the body and this dysbiosis has been linked to various diseases. Changes in the nasopharyngeal microbiome are associated to patient’s susceptibility to multiple viral infections, including COVID-19, supporting the idea that the nasopharynx may be playing an important role in health and disease. Most studies on the nasopharyngeal microbiome have focused on a specific component in the lifespan, such as infanthood or the elderly, or have other limitations such as low sample sizes. Therefore, detailed studies analyzing the age- and sex-associated changes in the nasopharyngeal microbiome of healthy people across their whole life are essential to understand the relevance of the nasopharynx in the pathogenesis of multiple diseases, particularly viral infections such as COVID-19. Results 120 nasopharyngeal samples from healthy subjects of all ages and both sexes were analyzed by 16s rRNA sequencing. Nasopharyngeal bacterial alpha diversity did not vary in any case between age or sex groups. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the predominant phyla in all the age groups, with several sex-associated differences probably due to the different levels of sex hormones between both sexes. Acinetobacter, Brevundimonas, Dolosigranulum, Finegoldia, Haemophilus, Leptotrichia, Moraxella, Peptoniphilus, Pseudomonas, Rothia , and Staphylococcus were the only 11 bacterial genera that presented significant age-associated differences. Other bacterial genera such as Anaerococcus, Burkholderia, Campylobacter, Delftia, Prevotella, Neisseria, Propionibacterium, Streptococcus, Ralstonia, Sphingomonas , and Corynebacterium appeared in the population with a very high frequency, suggesting that their presence might be biologically relevant. Conclusions In contrast to other anatomical areas such as the gut, bacterial diversity in the nasopharynx of healthy subjects remains very stable and resistant to perturbations throughout the whole life and in both sexes. Age-associated changes in taxonomic composition were observed at phylum, family, and genus levels, as well as several sex-associated changes probably due to the different levels of sex hormones present in both sexes at certain ages. Our results provide a complete and valuable dataset that will be useful for future research aiming for studying the relationship between changes in the nasopharyngeal microbiome and susceptibility to or severity of multiple diseases, including COVID-19. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the grant 00006/COVI/20 to VM and MLC funded by Fundacion Seneca-Murcia, the Saavedra Fajardo contract to SC funded by Fundacion Seneca-Murcia, the Juan de la Cierva-Incorporacion contract to SDT funded by Ministerio de Ciencia y Tecnologia/AEI/FEDER. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All procedures in this work were carried out following the principles expressed in the Declaration of Helsinki, as well as in all the other applicable international, national, and/or institutional guidelines for the use of samples and data, and have been approved by the Comite de Etica de la Investigacion (CEIm) at Hospital Clinico Universitario Virgen de la Arrixaca (protocol number 2020-10-12-HCUVA - Effects of aging in the susceptibility to SARS-CoV-2). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Raw sequencing data of all 16S rRNA sequences, metadata, and abundance tables are available at the open access repository Figshare under the accession numbers 10.6084/m9.figshare.19785991, 10.6084/m9.figshare.19786147, and 10.6084/m9.figshare.19785991, respectively.