*Article* **Analysis of 16S rRNA Gene Sequence of Nasopharyngeal Exudate Reveals Changes in Key Microbial Communities Associated with Aging**

**Sergio Candel 1,2,3,\*,†, Sylwia D. Tyrkalska 1,2,3,†, Fernando Pérez-Sanz 2, Antonio Moreno-Docón 4,5, Ángel Esteban 2, María L. Cayuela 2,3 and Victoriano Mulero 1,2,3,\*,‡**


**Abstract:** 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, 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 period in the lifespan, such as infancy or the old age, or have other limitations such as low sample size. 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. One hundred twenty 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. *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. Therefore, in contrast to other anatomical areas such as the gut, bacterial diversity in the nasopharynx of healthy subjects remains stable and resistant to perturbations throughout the whole life and in both sexes. Age-associated abundance changes 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.

**Keywords:** nasopharyngeal microbiome; age differences; sex differences; aging; human microbiome; 16S rRNA sequencing

**Citation:** Candel, S.; Tyrkalska, S.D.; Pérez-Sanz, F.; Moreno-Docón, A.; Esteban, Á.; Cayuela, M.L.; Mulero, V. Analysis of 16S rRNA Gene Sequence of Nasopharyngeal Exudate Reveals Changes in Key Microbial Communities Associated with Aging. *Int. J. Mol. Sci.* **2023**, *24*, 4127. https://doi.org/10.3390/ ijms24044127

Academic Editor: Maria Teresa Mascellino

Received: 9 January 2023 Revised: 13 February 2023 Accepted: 14 February 2023 Published: 18 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
