In general, the intestinal bacterial levels determined in this study are within the range of those previously reported by us and other authors for similar human groups of age [6
]. Although our study does not provide a complete microbiota characterization, such as that obtained by using Next-Generation Techniques (NGS) techniques, it does provide truly quantitative data instead of relative proportions, shedding additional light on the age group comparisons conducted. Moreover, our results confirm the ageing-related decline in the levels of some microorganisms such as the Bacteroides
group, Bifidobacterium, Faecalibacterium,
cluster XIVa previously reported [6
]. This holds true for the group of elderly between 66 and 80 years of age, which matched the age range of human populations participating in most of the studies cited formerly, but strikingly, it is not true for the oldest subjects (over 80 years of age). In fact, the oldest group in our study showed higher levels of all the microorganisms analyzed, with the exception of Faecalibacterium
, than the elders between 66 and 80 years, with these differences reaching statistical significance for Akkermansia
. This suggests, in accordance with other authors [7
], that very long-living individuals present a microbiota profile distinct from that of elderly subjects not living so long. The data reported by other authors, together with our observations, point out at the potential association of the presence of high levels of some of these microorganisms, such as Akkermansia
, with a longer survival [29
Moreover, our data on the comparison of the elderly (group aged 66–80 years) with those over 80 shed some light on the apparent lack of agreement between studies in elderly subjects. Apparently contradictory results have been previously reported for some of the fecal microbiota groups analyzed, such as Akkermansia
] or lactobacilli [10
], among others. To this regard the different methodologies used among the studies may have an effect. Nevertheless, on the view of our results, the age of the elderly groups participating in the different studies should be also considered as an important factor of variation, since differences among them are apparent. Moreover, although the different studies published, like this one, have defined inclusion criteria it is not always possible to ascertain the lack of effects due to treatments or conditions that were present in the life of the individual before running in the study.
Contrary to that expected from the microbiota data, the levels of SCFAs showed a continued age-associated reduction, with the oldest age group (subjects over 80 years of age) displaying SCFA levels that were less than half of those found in younger adults (<50 years old). This reduction in the amount of SCFAs found by us in elderly subjects confirms previous observations [10
]. Given the important role played by the intestinal SCFAs [32
] this reduction on the levels of these compounds during ageing may have profound consequences in terms of gut barrier maintenance and host physiological homeostasis. Interestingly, this differential trend of microbial levels and concentration of microbial metabolites in subjects over 80 years of age, suggests that, despite bacterial levels remaining high, there is a drop in the metabolic activity of the microbiota in these very old subjects. However, it is also important to underline that the SCFA fecal levels may also be influenced by other factors such as the the carbon sources available from diet or the absorption/excretion of these metabolites, which may also be affected in subjects of advanced age.
Our results also suggest potential associations between nutrient intake and the gut microbiota. In accordance with that previously reported [33
] fiber associated positively with SCFA levels, whereas the polyphenols, compounds known to promote an appropriate immune status [34
], correlated with the levels of some microorganisms. Some differences in fiber intake, including soluble fiber, are apparent among age groups; however, these are within a narrow range, which together with the lack of correlation observed between fiber and microbial groups in the correlation analyses suggest that the variation in fiber intake does not have a major role in the observed differences. In this regard, it is important to take into consideration that some dietary polyphenols cannot just be metabolized by the microbiota but they can affect the microbiota composition [35
], even modulating some bacterial groups with a non-negligible role in the host immune system. Therefore, another aspect that is of great relevance in this sort of study is the potential impact of nutritional differences in the observed effects. Our data, in agreement with previous reports [9
], show significant differences in dietary habits and, therefore, nutritional status among the analyzed age groups. In this sense it is also important to aknowledge that, although we do not think this has a significant effect in our results, a certain recall bias in relation to age, affecting the accuracy of the dietary information collected, may be present. Taking into account that distinct population groups, such as the elderly, may have different nutritional needs, the potential influence of such differences may not be neglected. Despite malnutrition in the elderly being linked with frailty and an increased risk of disease, this situation does not seem to be extrapolable to our sample, where the intake of macronutrients is similar across age groups and is sufficient to satisfy the nutritional requirements of each life stage [36
]. However, some micronutrients and bioactive compounds are crucial for the maintenance of immunocompetence [38
]. At this point, it is noteworthy that the group of subjects aged >80 years-old presented mineral intakes comparable to the younger groups (including iron, for which intake was above the recommended 8 mg/day for this age group), and even higher daily intakes of vitamins B12
and E as compared the rest of the groups. Nevertheless, the detection, in the group of people over 80 years of age, of a lower intake of some micronutrients with well documented roles in promoting immune function in the elderly, such as carotenoids, folic acid, and vitamins A and D, may be considered in future studies aiming to prevent/minimize immunosenescense by means of specific nutritional supplementation [4
]. Also, polyphenols could play an additional key role in the complex bidirectional relationship between diet and immunity by means of microbiota modulation throughout life. Therefore, nutritional assessment and monitoring represent a key aspect when studying the age-associated changes in gut microbiota composition, and also in other aspects such as immune senescence. Such multifactorial approach promises to allow the development of nutritional strategies to positively modulate the fecal microbiota and immune system and to reduce nutritional deficiencies in the elderly. It is important, however, to underline that cross-sectional studies, like this one, present some limitations for the monitoring the shifts in the fecal microbiota along the lifespan. They may carry over important cohort effects and, while useful for understanding some phenomena and drawing hypotheses, validation from longitudinal studies is often needed.