*2.3. Age- and Sex-Associated Changes in Relative Abundance of Bacterial Taxa in the Nasopharynx of Healthy Individuals*

To determine differences in nasopharyngeal taxa abundance among age and sex classes, we compared the nasopharyngeal microbiome of healthy women and men in the six different age groups established for this study (Table S1). The first general analysis at the phylum level revealed that Firmicutes and Proteobacteria relative abundances showed opposite kinetics with aging (Figure 3a–f), while the relative abundance of Firmicutes, which is the majority phylum in the youngest individuals (50%) (Figure 3a), clearly decreased with aging reaching its lowest values in subjects in their 50s and 60s (21% and 27%, respectively) (Figure 3d,e). The relative abundance of Proteobacteria presented its lowest value in the youngest people (24%) (Figure 3a) and increased in older individuals, peaking in subjects who were in their 50s (53%) (Figure 3d). The relative abundance of other phyla, such as Actinobacteria, Bacteroidetes, Tenericutes, or Cyanobateria, remained

more stable throughout life (Figure 3a–f). Moreover, when we continued working at the phylum level and searched for any abundance differences between the nasopharyngeal microbiota of females and males, we determined that, interestingly, the results were almost identical for both sexes within the two age groups containing the youngest (1–20 years) (Figure S3a) and oldest (>70 years) (Figure S3f) individuals. However, several differences between females and males were observed in other age groups, notably a higher relative abundance of Actinobacteria in males in their 20s and 30s compared to females of the same age group (20% vs. 7%) (Figure S3b), a higher relative abundance of Proteobacteria in males in their 40s compared to females of the same age group (46% vs. 29%) (Figure S3c), a higher relative abundance of Proteobacteria in females in their 50s compared to males of the same age group (57% vs. 47%) (Figure S3d), and a higher relative abundance of Firmicutes and lower of Actinobacteria in females in their 60s compared to males of the same age group (37% vs. 20% and 10% vs. 22%, respectively) (Figure S3e). Therefore, the fact that there were differences in taxa abundance when comparing the nasopharyngeal microbiota of females and males in most of the age groups studied (individuals between 21 and 70 years of age) (Figure S3b–e), but not within the two age groups containing the youngest and oldest individuals (people between 1 and 20 and older than 70 years of age) (Figure S3a,f), suggests that the different levels of sex hormones present in both sexes at different life stages might be modulating the nasopharyngeal microbiome.

Next, we proceeded further in this study and moved to the family level, finding that 24 distinct bacterial families presented an average abundance of >1% in at least one of the age groups studied (Figure 3g). Our analyses revealed the dominant family in each of the age groups: Staphylococcaceae in A1, Carnobacteriaceae in A2, Staphylococcaceae in A3, Burkholderiaceae in A4, Streptococcaceae in A5, and Staphylococcaceae in A6 (Figure 3g). Thus, it is curious that although Proteobacteria was the majority taxa in all the age groups when analyzing the nasopharyngeal microbiota at the phylum level (Figure 3a–f), the dominant family in all the age groups belongs to the Firmicutes phylum, except in the case of age group A4 where the family Burkholderiaceae, which belongs to the Proteobacteria phylum, was the most abundant taxa at family level (Figure 3g). Differences in the relative abundance of some families were detected when compared between age groups, but without following any easily interpretable pattern (Figure 3g). Analysis of differential taxa abundance at the family level between age groups, but separately in females and males, did not show any relevant sex-associated differences between sexes regarding the dominant bacterial families in each age group compared to the results described above for both sexes together (Figure 3g), excepting that Burkholderiaceae was the most abundant family in males of age group A3 instead of Staphylococcaceae, and that Corynebacteriaceae is the dominant family in males of age group A5 instead of Streptococcaceae (Figure S4). As mentioned above when working with females and males together, differences in relative abundance were detected in some families when comparing between different age groups in both sexes, but without following any easily interpretable patterns (Figure S4). Visualization of taxa abundance at the family level in all the individuals included in this study showed that most of them had a very diverse microbiome, with a high number of families with relative abundance of >1% (Figure S5). However, a few individuals had one dominant family that represented the majority of their nasopharyngeal microbiomes; these families tended to be Burkholderiaceae, Carnobacteriaceae and Staphylococcaceae (Figure S5).

**Figure 3.** Taxonomic composition and age-associated metagenomic changes in the nasopharynx of healthy individuals. (**a**–**f**) Krona charts showing the bacterial community composition at the phylum level in the indicated age groups. Stacked bar charts showing the relative abundance (%) of bacterial phyla. (**g**) Stacked bar charts showing the relative abundance (%) of bacterial families in the indicated age groups. (**h**) Stacked bar charts showing the relative abundance (%) of bacterial genera in the indicated age groups. For clarity, only bacterial families (**g**) and genera (**h**) with average abundance >1% at each age group are shown.

Working at the genus level, we determined that 24 bacterial genera presented an average abundance of >1% in at least one of the age groups studied (Figure 3h). Moreover, our results showed that *Staphylococcus* was the dominant genus in age group A1, *Dolosigranulum* in A2, *Staphylococcus* in A3 and A6, *Ralstonia* in A4, and *Streptococcus* in A5 (Figure 3h). No relevant differences were detected when comparing taxa abundance in the nasopharynx of females and males at the genus level within the age groups studied (Figure S6). Similar to what was described above at the family level, visualization of taxa abundance at the genus level in all the individuals included in this study showed that most of them had a high number of genera with relative abundance of >1%, with a few individuals presenting one dominant genus (mostly *Dolosigranulum* or *Staphylococcus*) that represented the majority of their nasopharyngeal microbiomes (Figure S7). Next, we focused on those genera whose relative abundance were significantly different between the different age groups established in this study, as this could help us to identify changes in the nasopharyngeal microbiota that are characteristic of aging. Thus, our analyses revealed that there were statistically significant differences (adjusted *p*-value < 0.05) in relative abundance between the distinct age groups in 11 bacterial genera: *Acinetobacter*, *Brevundimonas*, *Dolosigranulum*, *Finegoldia*, *Haemophilus*, *Leptotrichia*, *Moraxella*, *Peptoniphilus*, *Pseudomonas*, *Rothia* and *Staphylococcus* (Figure 4a and Table S2). Interestingly, most of these statistically significant differences in relative abundance between the age groups for the 11 mentioned genera were between age groups A1 or A6, which include individuals between 1 and 20 years of age and over 70 years old, respectively, and the rest of age groups (18 out of 37 cases for the age group A1 and 16 out of 37 cases for the age group A6) (Table S2). Among these statistically significant changes detected, it should be noted that *Acinetobacter* was the only genus whose relative abundance in the nasopharynx clearly increased progressively throughout life, peaking in individuals older than 70 years of age (Figure 4a and Table S2). In the cases of *Dolosigranulum* and *Rothia*, their relative abundance drastically increased and decreased, respectively, in individuals over 70 years of age, compared to middle-aged people in their 50s and 60s (Figure 4a and Table S2). Changes among the different age groups of *Finegoldia*, *Leptotrichia* and *Haemophilus* were also interesting, as their relative abundance was markedly reduced in elderly people over 70 years of age, even though they were present at other ages throughout life, mainly during middle age (Figure 4a and Table S2). The case of *Haemophilus* was particularly intriguing, as while its relative abundance was at least 10% of the nasopharyngeal microbiota composition in age groups A1-A5 (if we consider only these 11 genera that present statistically significant differences between age groups), it dramatically decreased in the group of individuals over 70 years old (Figure 4a and Table S2). Furthermore, it is noteworthy that the bacterial genera *Brevundimonas*, *Finegoldia*, *Leptotrichia* and *Peptoniphilus* presented a very low relative abundance in the youngest individuals, who are between 1 and 20 years old, compared to other age groups (Figure 4a and Table S2). Finally, although *Moraxella* and *Staphylococcus* showed significant differences in relative abundance between the distinct age groups in several cases, these differences did not seem to follow any easily interpretable pattern relating relative abundance levels to a particular life stage (Figure 4a and Table S2). Next, we wondered whether the significant differences in relative abundance between age groups observed for these 11 bacterial genera were due to sex-associated differences. Visualization of taxa abundance in females and males separately, considering only these 11 genera, showed no notable differences between both sexes (Figure S8). The only exception was a higher relative abundance of *Dolosigranulum* in males in their 20s and 30s compared to females of the same age, because this genus was clearly dominant in five males from that age group while only in one female of the same age (Figure S9). Besides this observation regarding *Dolosigranulum*, analyzing the taxa abundance in all the individuals included in this study also revealed that in most people, 1 out of these 11 genera was dominant compared to the relative abundance of the other 10 genera (Figure S9). Interestingly, 8 out of the 11 genera, excepting *Bevundimonas*, *Finegoldia* and *Peptoniphilus*, were determined to be the dominant genus in at least one individual, demonstrating that nasopharyngeal taxonomic composition at this level can

be very variable between different individuals, even if they belong to the same age or sex groups (Figures S9 and S10).

**Figure 4.** Taxonomic composition and frequency of appearance of the 11 bacterial genera which show significant differences between age groups. (**a**) Stacked bar charts showing the relative abundance (%) of the 11 bacterial genera indicated in the age groups established for this study. (**b**) Percentage of individuals, of the total included in this study, in which the indicated genera are present in the indicated age groups.
