**3. Discussion**

Comparing bacterial species richness in the nasopharynx of healthy individuals in the different age groups established for this study and between females and males revealed no age- or sex-associated significant differences in alpha diversity. These results, which were confirmed by using three of the most reliable and commonly used alpha diversity indexes, such as the Shannon's diversity index, the inverse Simpson's diversity index, and the Chao1 diversity index, indicate that the nasopharyngeal microbiome is highly stable

and robust to perturbations throughout life as well as between sexes within each of the different age groups in healthy subjects. Alpha diversity could be expected to be lower in the youngest individuals compared to older individuals in all the anatomical areas, as the fetus is sterile until the moment of birth, when the newborn begins to be progressively colonized by microorganisms until its definitive microbiota is established [26]. This is exactly what happens, for example, with the gut microbiome of infants, whose alpha diversity is consistently determined to be lower than in adults [27,28], probably due to the introduction of new diversity from food, which increases with the consumption of foods other than breast milk [29]. Detailed metagenomic studies, which should include samples collected at multiple time points during childhood and during adulthood, would be needed to determine whether something similar occurs in the nasopharynx. In the case of the present study, we would not be able to detect such differences in diversity between the youngest individuals and the rest of age groups, if they exist, for two reasons: (i) because our first age group, which includes the youngest individuals, is very broad and encompasses individuals up to 20 years old, so any differences from young children could be diluted; (ii) because as we were not interested specifically in children, but in broader age groups spanning all stages of life, we excluded children younger than 1 year of age from this study, as their highly changing microbiota in formation could bias the analyses we were interested in. Interestingly, although it is known that there are relevant sex-associated differences in diversity in other anatomical areas such as the gut, and that such differences are probably due to the different levels of sex hormones between both sexes [30], we did not observe any significant diversity differences between females and males, even in the age groups where sex hormones levels should be quite different. It is also curious that, contrary to what occurs in the gut where the alpha diversity of the microbiota decreases with aging [24], we did not observe a similar reduction in diversity in the nasopharynx of older individuals. This difference between both anatomical areas might be explained by the fact that decreased microbial diversity of the gut in older subjects is associated with chronological age, number of concomitant diseases, number of medications used, increasing coliform numbers, and changes in diet [31], and although chronological age affects both anatomical areas in a similar way, it seems probable that other of these factors might affect the gut microbiota in a more intense way compared to the nasopharyngeal microbiota. In summary, we can say that the diversity of the nasopharyngeal community is very stable throughout life and between sexes.

Our analyses of taxa abundance in the nasopharynx at the phylum level revealed the existence of sex-associated differences within the age groups including individuals between 21 and 70 years of age, but not in the youngest and oldest people. As previously mentioned for the alpha diversity results, multiple sex-associated differences in taxonomic composition have also been described for the human gut microbiome [30], and numerous studies have reported evidence to support the idea that levels of sex hormones, such as progesterone [32], androgen [33], and estrogen [34], regulate its composition [35,36]. The effects of sex hormones on other microbial niches, such as the human vaginal microbiota, have also recently been demonstrated [37]. Although it has been shown that estrogen stimulation (hormone/gender effect) in the upper respiratory tract mucosa could reduce virus virulence by improving both nasal clearance and local immune response [38], the relationship between sex hormone levels and the nasopharyngeal microbiota has so far not been directly observed in clinical studies. However, the fact that our results reveal differences between females and males at the phylum level in all the age groups, except for those where differences in sex hormones levels do not exist or are not so strong (>70 years and 1–20 years old), suggests that sex hormones might be modulating the taxonomic composition of the healthy human nasopharynx. Note that although differences in sex hormones could be expected to be relevant in the age group that includes individuals between 1 and 20 years of age since pubescents and adolescents are part of this group, we assumed that such differences should not be so significant in our case because 70% of individuals in this age group are prepubescent in our study.

We might be tempted to think that the microbiota of nearby anatomical sites that are closely related in terms of structure and function should be practically identical. However, the reality seems to be much more complex. A good example of this is that although nasopharynx and nose are adjacent, and previous metagenomic studies comparing the microbiome of both anatomical areas have revealed a clear continuity, there are important differences between the two sites and even niche-specific bacteria [39]. Furthermore, this study also reported an evident heterogeneity among participants, since the nasopharyngeal microbiome of half of them was dominated by *Moraxella*, *Streptococcus*, *Fusobacterium*, *Neisseria*, *Alloprevotella* or *Haemophilus*, while in the other half it contained an intermixed bacterial profile where *Staphylococcus*, *Corynebacterium*, and *Dolosigranulum* seemed to be important bacterial members with varying relative abundances [39]. Our taxa abundance analyses at the genus level only detected statistically significant relative abundance differences between the different age groups for 11 bacterial genera: *Acinetobacter*, *Brevundimonas*, *Dolosigranulum*, *Finegoldia*, *Haemophilus*, *Leptotrichia*, *Moraxella*, *Peptoniphilus*, *Pseudomonas*, *Rothia,* and *Staphylococcus*. Interestingly, most of the 37 statistically significant differences detected between the different age groups for these 11 genera appear when comparing age groups A1 and A6 with the rest of the age groups (18 out of 37 and 16 out of 37, respectively). Therefore, these results reveal that, in terms of relative abundance of bacterial genera, the nasopharyngeal microbiota of the youngest and oldest subjects is more different from that of the other age groups than that of any other age group. Among these age-associated changes, from a clinical perspective, it is particularly concerning that *Dolosigranulum*, which is an opportunistic pathogen that causes pneumonia in elderly patients [40], is overrepresented in the nasopharynx of individuals over 70 years of age compared to middle-aged subjects. This suggests that the relative abundance of *Dolosigranulum* may be higher in elderly people due to the process of immunosenescence that occurs in them [41,42], or that its higher abundance may be due to other unidentified age-related factors. Nevertheless, the relevance of *Dolosigranulum* in the nasopharynx deserved further investigation, since nasal administration *Dolosigranulum pigrum* 040417 to mice increased the resistance against respiratory syncytial virus (RSV) and *Streptococcus pneumoniae* [43,44]. However, other strains of the same species failed to protect mice against these pathogens [43,44]. Another interesting observation of our study is that *Haemophilus* that causes pneumonia mainly in elderly people [45] is underrepresented precisely in individuals over 70 years of age. This suggests that their lower relative abundance in elderly subjects is due to other unidentified age-related factors, and that the elderly are much more susceptible to opportunistic infections caused by this bacterium, probably due to the previously mentioned process of immunosenescence. Further research will be necessary to elucidate the precise reason for this. Something similar could be said for *Rothia*, as its relative abundance also decreases drastically in people over 70 years of age while it is known to cause pneumonia mostly in aged individuals [46]. It is worth noting that, regardless of their relative abundance or whether they show statistically significant differences between age or sex groups, those bacterial genera that are present in most individuals or whose frequency of appearance changes drastically throughout life could be relevant from a biomedical and ecological point of view. Based on this idea, we highlight *Anaerococcus*, *Burkholderia*, *Campylobacter*, *Delftia*, *Prevotella*, *Neisseria*, *Propionibacterium*, *Streptococcus*, *Ralstonia*, *Sphingomonas* and *Corynebacterium* as candidate bacterial genera that could be playing an important role as they are present in the nasopharynx of most healthy individuals. In addition, we propose *Faecalibacterium*, *Stenotrophomonas* and *Phascolarctobacterium* as candidate bacterial genera that could be playing a relevant role, as their frequency of appearance in the nasopharynx of healthy subjects increases progressively throughout life, and *Aggregatibacter*, *Gemella* and *Fusobacterium* because their frequency of appearance in the nasopharynx decreases drastically and progressively as healthy people age. Elucidating the biomedical relevance of all these bacterial genera which are part of the healthy nasopharyngeal microbiota and determining their potential involvement in health and disease at different stages of life is certainly an exciting topic for future work.

Our study has several limitations. This was an observational, retrospective study, and collection of data was not standardized in advance. The 16S rRNA gene sequencing approach to study the microbiota could introduce bias in the obtained data because this method does not allow the study of the whole microbiome, but only the genera amplified by PCR. The taxonomic assignment at the species level may not be fully accurate. Nevertheless, it is the most common technique to study microbiota in clinical samples. Moreover, it was not possible to obtain serial samples. Furthermore, the groups are small, particularly the sex groups within each age group, so the study may have been underpowered to detect certain associations. Finally, we could not access any sociodemographic, environmental, lifestyle, or medical information of subjects enrolled in this study, which would have been helpful to better understand the characteristics of the cohort.

Although multiple studies have analyzed the microorganisms present in the nasopharynx in different contexts before this work, the characteristics of the healthy and mature human nasopharyngeal microbiota was largely unknown since (i) most studies focused on children or elderly people, (ii) confounding factors such as external drivers that alter it are not well known to date, and (iii) focus is generally shifted to its variation in diseases. With this work, we fill this important gap in knowledge. However, further research will be necessary to elucidate the effects of the nasopharyngeal taxonomic composition as well as the age- and sex-associated changes described here on the susceptibility of certain individuals to infectious diseases. Studying the case of the elderly people in detail will be particularly interesting from a biomedical and clinical perspective, since their nasopharyngeal microbiota is significantly different from that of younger subjects, and they are known to be much more susceptible to multiple infectious diseases, most notably COVID-19 [47]. Therefore, we hypothesize that there may be some correlation between the taxonomic composition in the nasopharynx of the elderly and their increased susceptibility to COVID-19, but this will be a challenge for future metagenomic studies that should include different age groups, both sexes, and patients infected with SARS-CoV-2 who have developed the disease with different severity.
