**5. Conclusions**

Our study shows that 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 taxa abundance 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. We 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.

**Supplementary Materials:** The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms24044127/s1.

**Author Contributions:** The authors offer the following declarations about their contributions: Conceived and designed the experiments: S.C., S.D.T., M.L.C., V.M. Performed the experiments: S.C., F.P.-S. Analyzed the data: S.C., S.D.T., F.P.-S., Á.E., M.L.C., V.M. Provided essential samples: A.M.-D. Writing—original draft: S.C. Writing—review & editing;: S.C., V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the grant 00006/COVI/20 to VM and MLC funded by Fundación Séneca-Murcia, the Saavedra Fajardo contract 21118/SF/19 to SC funded by Fundación Séneca-Murcia, the Juan de la Cierva-Incorporación contract to SDT funded by Ministerio de Ciencia y Tecnología/AEI/FEDER. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Institutional Review Board Statement:** 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 Comité de Ética de la Investigación (CEIm) at Hospital Clínico Universitario Virgen de la Arrixaca (protocol number 2020-10-12-HCUVA—Effects of aging in the susceptibility to SARS-CoV-2).

**Informed Consent Statement:** Nasopharyngeal swabs were collected for diagnosis of SARS-CoV-2 infection before this study was conceived, without the need of any informed consent as the collection procedure was non-invasive and risk-free. However, when the COVID-19 pandemic spread out of control, samples were kept at the Microbiology Service instead of destroyed after diagnosis as it was considered that they might be extremely relevant for research. This, together with the facts that (i) the retrospective use of these samples did not affect donor health or treatment at all, (ii) all data has been treated anonymously, and (iii) movement was limited due to the exceptional circumstances of the pandemic meant that it was not possible to obtain informed consent for the use of these samples in research. Moreover, none of the subjects expressly objected to their samples being used for research.

**Data Availability Statement:** 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 and 10.6084/m9.figshare.19786147, respectively.

**Acknowledgments:** We thank I. Fuentes, P. Martinez for their excellent technical assistance, Anabel Antón for 16S rRNA sequencing, and the staff of the Microbiology Service of HCUVA for sample collection and processing.

**Conflicts of Interest:** The authors declare no competing interest.
