141 - 154: Diet-Microbiota-Health Interactions in Older Subjects: Implications for Healthy Aging

The human gastrointestinal tract has multiple critical roles that are central to health and well-being. It serves as a way to process foods and eliminate waste. It helps control appetite, it houses the largest mucosal surface in the body housing a large component of the immune system, and it even contributes to mood, behaviour and general well-being. The most numerate cellular part of the gastrointestinal tract is not human but microbial. Most of these bacteria are called commensals, meaning they are normally resident and metabolizing human dietary components. The microbes in and on the human body have a combined number of genes a hundred times that of the human gene complement. The gastrointestinal tract is one of the most diverse environments on the planet. The intestinal microbiota is now recognized as a major environmental modifier of health risk. Independent of genetic and other lifestyle factors, the gut microbiota has a coding capacity and potential metabolic activity that has a major impact on human physiology. In infancy, the microbiota composition trends towards an adult pattern over the first 2-3 years, with low initial diversity increasing over this time period. Disruptions of this process may be associated with risk for allergic disease in later life. In the adult years, alterations in the microbiota are associated with a diverse range of diseases [reviewed in de Vos and de Vos [1]]. There is a particularly compelling case for studying the microbiota in aging subjects. This phase of life is accompanied by a range of physiological and lifestyle changes that can have a big effect on the physical environment of the intestine. It has been known for several decades that the gut microbiota of older persons, similar to the very young, is in a state of flux [2]. Coupled with a wide range of reported alterations in the composition of the intestinal microbiota in seniors, and different rates of age-related health loss in different individuals, countries and populations, detailed analysis of gut microbiota-health interactions in older people is particularly appropriate. This review summarizes the differences between the physiology of older subjects and young adults that are relevant for microbiota changes and details the major findings of culture-based studies, and then examines the health implications of recent culture-independent studies, including those from the largest study to date, the ELDERMET consortium.

The global proportion of older people is rapidly and continually increasing. This has resulted in an increased need for healthcare and societal supports for this cohort of our society, and has highlighted the importance of not just longevity but healthy aging that maximizes functional capacity and quality of life in older age. The diversity of the microbiota of an individual is shaped by a number of factors, both internal to the host, and external. Common, age-related, physiological changes can modify physiological function, which can in turn alter the composition of the microbiota.

Physiological, motor and sensory functions change with age. For instance, a natural reduction in dentition and deteriorating muscle mass in later life can impact on mastication ability. This can limit dietary choices, and changes in the diet can greatly impact the microbiota. Aging may be accompanied by impairment of intestinal sensation and consequently increased susceptibility to gastrointestinal complications. Other age-related, digestive system complications include dysphagia (difficulty swallowing), functional dyspepsia (painful, difficult or disturbed digestion), gastroesophageal reflux, delayed intestinal transit time, diverticulosis, and increased rates of constipation, faecal and gaseous incontinence, all of which can significantly impact on microbiota composition and host health. Importantly, the impairment of taste and thirst sensation, olfaction and digestion, coupled with malabsorption and an increase in the levels of satiation in older people, can lead to imbalances in nutrient intake, malnutrition and significant perturbation of the microbiota [3].

Bilateral interaction with the host facilitates functional conditioning of the immune system by the microbiota, which influences the composition of the microbiota itself. Microbiota disturbance has been linked with an increased susceptibility to disorders including allergies, cancer, digestive/intestinal disorders, frailty, obesity/metabolic disorder and its related conditions. It can also affect regulatory systems such as hormone signalling, leading to changes in mood and behaviour. Host metabolic pathways that facilitate connection between the intestine and the brain, can be affected [3]. Disruption to this bidirectional homeostatic pathway has been associated with inflammation and alterations in the stress response, among other stress-related symptoms such as anxiety, commonly experienced in older age. A healthy, more diverse microbiota composition encourages resistance to pathogens and increased interaction with the host immune system. Loss of diversity in old age is associated with less resistance to pathogens and a natural decline of immune function (immunosenescence) with the development of chronic, low-grade inflammation typical of older age (inflammaging). Both low-diversity microbiota and immunosenescence can lead to increased rates of gastrointestinal infection.

In older age, complex and dynamic exogenous factors, including diet and lifestyle modifications, medication use (particularly antibiotics), disease, injury and stress further influence the composition of the microbiota. Health throughout life, and particularly in later years, is dependent on the maintenance of homeostasis, the presence of a stable physiological environment. The relative stability of the adult intestinal microbiota at a species level is a key contributory factor to the promotion and maintenance of health. However, at abundance level the composition of the microbiota can fluctuate substantially over a short period of time [4]. This suggests that the microbiota is able to respond to exogenous influences throughout life.

Culture-based methods were traditionally used to analyze the intestinal microbiota. An example of some of the methods utilized can be seen from experiments conducted in 1989 in Japan [5]. Culture-based methods were used to compare the microbiota of elderly people in Tokyo, Japan, with elderly in Yuzurihara, an area of Japan where the elderly tend to live longer. The faecal microbiota of 15 healthy elderly subjects from each of the two areas was collected. A number of experiments were performed to determine the genus, and where possible, species, of isolates found in these samples. Serial dilutions in an anaerobic diluent were made, and the samples were subsequently spread onto 4 non-selective and 11 selective agar plates. Subculturing from anaerobic plates to other plates helped determine which microorganisms were strict anaerobes.

In order to identify the isolates, many biochemical tests were performed on broth cultures. These tests include detection of bacteria-derived metabolites and the determination of the effect of bile on bacterial growth. Benno et al. [5] reported that while most of the same genera were observed between the two groups, the Yuzurihara subjects had a larger bifidobacteria contingent than was observed from the Tokyo subjects. However the Yuzurihara subjects had fewer total bacteria, anaerobes, bacilli, clostridia, Bacteroides species, and Eubacterium aerofaciens. Four genera, Megamonas, Mitsuokella, Selenomonas, and Acidaminococcus, were isolated from the Yuzurihara subjects but not the Tokyo subjects. Intestinal bifidobacteria counts are known to decrease with age, and some Enterobacteriaceae increase. That the Yuzurihara elderly had more bifidobacteria than the Tokyo elderly, despite being older, suggests that the Yuzurihara subjects were not displaying the same age-related microbiota changes that we see in other parts of the world. Benno et al. [5] suggest that this is due to the high-fibre diet of the Yuzurihara subjects, and that this is why the Yuzurihara people tend to live longer.

In 2002, another culture-based study focused on elderly suffering from Clostridium difficile-associated diarrhoea (CDAD) [6] who had a history of antibiotic treatments resulting in disturbed microbiota. This altered microbiota provided a reduced resistance to C. difficile infection. With the widespread use of antibiotics and increasing number of elderly, CDAD has become a challenging problem. Hopkins and Macfarlane [6] aimed to characterize the microbiota of elderly subjects with CDAD. They classified isolates according to their cellular fatty acid profiles. Their results showed that CDAD patients had the lowest species diversity when compared with healthy elderly and young subjects, particularly of bifidobacteria, Bacteroides and Prevotella. Facultative species were higher in CDAD patients than in healthy subjects. Together, this shows that C. difficile is associated with a greatly altered microbiota. The same group completed further studies in 2004 [7], this time comparing healthy young and elderly, and hospitalized elderly. Again, they used fatty acids to identify bacteria. They reported reduced numbers and species diversity in both bifidobacteria and Bacteroides in elderly compared with the young subjects.

The benefits of using such culture-based methods for analyzing intestinal microbiota include the low cost, and ability to retain isolates for further analyses. However, there are many disadvantages to culture-based methods. It is labour intensive, and with current approaches it is still not possible to culture the majority of the estimated gut bacteria (estimated 50-90%). Of those species that do grow on current artificial media, certain species will outgrow others, leading to further biases. Another disadvantage of culture-based approaches is difficulties in phylogenetic classifications. For some microbial families, multiple methods must be used to classify genera and species of different families, such as those discussed above. Benno et al. [5] required a large number of methods for classification of isolates. This indicates how complex it can be to identify isolates using culture-based methods. It also shows how much culture of a given isolate is required to identify it. Some of these methods could often not distinguish between two species of a given genus, so biologically-relevant species-specific genes or functions could not be accounted for. Speedy, high-throughput, specific and reliable alternatives were required.

The last decade has seen the introduction of increasingly intense research techniques. Rather than attempting to culture all isolates from a given environment, the DNA can be directly extracted from samples. In theory, this approach provides an unbiased view of the isolates within a sample. The preferred locus used for identification is the 16S ribosomal DNA. As a housekeeping gene found in almost all bacteria, often in high-copy numbers, it is easily amplified. It contains a number of variable regions that differ between species and/or genera and so allows efficient identification.

Real-time quantitative PCR is often used to determine the proportion of certain bacteria in a sample. This approach is fast, cheap and useful for determining the level of a specific group of bacteria. However, when trying to assess and compare a number of different groups, qPCR becomes laborious. This directed approach, while very useful in many cases, does not provide an exhaustive view of the gut population, which is proving to be increasingly important. In 2009, a phylogenetic microarray was developed specifically for the human intestinal tract, known as the HITChip [8]. This chip consists of 4,809 probes, and further probes can be added when required. However, microarrays are a high-throughput targeted approach. While they cover more targets at once, they are still limited by the probes. Different probes have different hybridization abilities, so biases can be introduced based on the choice of probes.

The current, more commonly used technology is high-throughput sequencing. There are a number of different sequencing technologies available; however, microbial community analyses based on 16S ribosomal DNA studies tend to use 454 FLX Titanium pyrosequencing due to the longer reads that can be obtained [9]. Up to 1.6 million reads can be sequenced in one run. Many different samples can be loaded on one slide using barcoded adaptors.

With any new technology such as pyrosequencing, programs for analysis must be developed. The aims are to maximize the data obtained while minimizing the potential for error. Speed and accuracy are paramount, and as increasing amounts of data are obtained, programs that can handle ample quantities of reads are essential. When handling pyrosequencing data, many steps can be executed. Multiplexed libraries must be separated. Adaptor sequences must be removed. Error correction, or denoising, can be performed. Chimeric sequences are sometimes formed during PCR amplification steps. Programs are available to remove these. Clustering is performed to reduce the time and volume for further steps. Finally, sequences must be classified at different phylogenetic levels.

Interest in these techniques has been huge with the formation of large multinational scientific consortiums such as the The Human Microbiome Project [10], MetaHIT (Metagenome of Human Intestinal Tract) [11] and the smaller ELDERMET project [12], as well as numerous labs around the world. These consortia have taken advantage of the new high-throughput technologies and have for the first time fully characterized the human microbiome in the gut and from other body sites.

The use of these techniques has illustrated the heterogeneity of the microbial populations in our gastrointestinal tracts with large inter-individual differences in the presence and absence of the bacterial species. Although some species are present in the majority of the population, these are in the minority in terms of the number of species that can be found in our gut. These rarer species are no less important for the well-being of the host. Species tend to co-occur and may be clustered into co-abundance groups (CAGs) [12] due to habit preference as defined by diet and cross-feeding events and the presence of bacteriocins, a type of bacterially produced antibacterial agent that is specific for a limited number or range of species. An alternative to the idea of CAGs are enterotypes. The idea of enterotypes predates that of CAGs and is different in a number of characteristics (table 1) [13]. Enterotype groups are distinct from one another and are often described as being similar to blood groups. Despite being controversial, the idea has become popular and has allowed researchers to categorize samples based on the dominant genera that represent microbial populations that have a substantial scope to modify the phenotype of the individual through production of metabolites and immunomodulatory effects.

Numerous studies from different geographical locations have attempted to characterize the microbiota of general healthy populations, and many have compared these with individuals carrying diseases, elderly, and even extreme elderly - individuals over 100 years of age. In 2001, Hopkins et al. [14] analyzed bacterial 16S rDNA sequences from children, adults, elderly, and C. difficile-infected geriatric patients from the UK. They revealed an overall decrease in bifidobacteria in elderly compared with adults, and a slight decrease in lactobacilli. There was no change in the Bacteroides-Porphyromonas-Prevotella group, contrary to their culture-based study 3 years later [7], discussed above. This group published again in 2004 [15], using real-time PCR on rDNA to compare bacteria from healthy elderly, hospitalized elderly, and elderly treated with antibiotics. The Bacteroides-Prevotella species were significantly less abundant in the hospitalized patients than in healthy elderly, whereas Enterobacteriaceae, Clostridium butyricum and Enterococcus faecalis were increased. Antibiotic-treated patients showed an increased abundance of E. faecalis compared with healthy elderly, but decreased abundances of Bacteroides-Prevotella group, Desulfovibrio, Enterobacteriaceae, Faecalibacterium prausnitzii, C. butyricum and Ruminococcus albus. F. prausnitzii has an anti-inflammatory affect, so reduced levels in antibiotic-treated patients may be associated with inflammaging.

Other studies have focused on other aspects of aging, such as frailty. A study on long-term care subjects in one elderly centre in The Netherlands [16] showed that an increase in frailty correlated with an increase in Ruminococcus and Atopobium, and a decrease in the Bacteroides-Prevotella group, the Eubacterium rectale-Clostridium coccoidescluster, Lactobacillus and F. prausnitzii. This frail microbial signature was similar to that found in the hospitalized subjects discussed by Bartosch et al. [15] and Claesson et al. [12].

A European study of subjects from four different countries, France, Italy, Germany and Sweden, provides evidence of location-based differences [17]. No differences were observed between age groups from the French or Swedish cohorts. The E. rectale-C. coccoides group increased with age in the German population, but decreased with age in the Italian subjects, a decrease similar to the Dutch study by Bartosch et al. [15]. German adults had lower Bacteroides-Prevotella than adults from other countries, while Italian elderly had lower proportions than elderly from other countries. F. prausnitzii decreased with age in the Swedish and Italian subjects, but not the French or German subjects. The Atopobium cluster increased with age in German and Swedish populations, but not French and Italian. Bifidobacterium was lower in all elderly subjects than their corresponding adult cohorts; however, this was not significant. Italian subjects had significantly more bifidobacteria than other populations.

Claesson et al. [12] assessed the microbiota of a large cohort of Irish elderly, from four different residence locations - community, long-term care, rehabilitation and day-hospital. They reported an overall trend of increasing Bacteroidetes and decreasing Firmicutes from community to long-stay. Reduced abundances of Coprococcus and Roseburia were observed in long-stay subjects, while they had increases in Parabacteroides, Eubacterium, Anaerotruncus, Lactonifactor and Coprobacillus. This relatively large study was able to identify a number of microbial relationships between microbiota and frailty and other clinical factors while controlling for confounding factors such as diet, medication and even age.

There are few differences between young and healthy elderly subjects with the recurring associations with increased Proteobacteria and Bifidobactia. Biagi et al. [18] attempted to address this with centenarians. The centenarians tended to cluster separately from elderly and young, which did not differ. The mainly centenarian cluster had higher Proteobacteria and Bacilli, and lower Clostridium cluster XIVa, but no reduction in Bacteroidetes was observed.

These studies convey the large inter-individual differences in microbiota in a given population. They also provide us a view of some of the differences that can be seen between populations. These illuminate the issues with generalized views of microbiota, and remind us of some of the difficulties we will face when trying to increase longevity and quality of life. Somewhat personalized or community-based approaches may need to be considered in the future.

There are a number of challenges facing the study of the role of diet as a modulator of gut microbiota variation in older persons including: (1) compositional inter-individual variability of the gut microbiota; (2) inter-individual variance in dietary intakes even among seemingly homogenous population groups, e.g. the elderly [12]; (3) variable effects of dietary intervention, dependent on the baseline microbiota; (4) the use of medical therapeutics, especially antibiotics [19]; (5) classification of an appropriate timeframe for dietary intervention and how to quantify its total microbiome effects.

The bacterial residents of the human gastrointestinal tract are capable of synthesizing a number of micronutrients including a number of B-group vitamins, vitamin K₂ isomers collectively known as menaquinones, and can aid calcium absorption, and regulate appetite and insulin release (fig. 1). In fact, long-term dietary choices can greatly influence the gut microbiota population, while nutritional status has also been shown to impact the composition of the gut microbiota and its functionality [20]. Dietary choice and variety are also influenced by disease which can affect the composition, diversity and metabolic capacity of the gut microbiota and have important implications for therapies aimed at modulating the large intestinal microbiota. The ELDERMET study illustrated the negative clinical implications of reduced dietary and microbiota diversity [12]. Targeted dietary interventions using prebiotics, probiotics or combinations (i.e. synbiotics) may counteract negative age-related changes, and address the detrimental effects of long-term broad-spectrum antibiotic therapy on the gut. Limited feeding trials show promising results with these supplements.

Diet can have a marked impact on the gut environment, including transit time and pH. Changing the intakes of the three main macronutrients (carbohydrates, protein and fats) can significantly affect the composition of the microbiota. In a study of the effects of long-term diet on the microbiota composition, Wu et al. [21] found that high dietary intakes of animal products were associated with Bacteroides, while a greater intake of plant material promoted the Prevotella genus. Similarly, the ELDERMET study found an abundance of bacterial species from the Prevotella genus among healthy elderly individuals [12]. A dietary intervention trial investigating the effect of different non-digestible carbohydrate (resistant starch and non-starch polysaccharide) on overweight individuals, revealed the adaptive nature of the microbiota [22]. Plant polysaccharides, excluding starches, are key structural and biological components of the cell membrane and are collectively known as non-starch polysaccharides. These structural components cannot be hydrolyzed by the endogenous enzymes of humans. Instead, a complex mutual dependence has developed between the mammalian host and symbiotic gut microorganisms that possess the ability to access this additional energy source which can contribute up to 10% of the body's daily energy requirements. In addition, extensive cross-feeding occurs in the colon between the primary degraders of complex substrates and other bacterial species that depend on their fermentation and partial degradation products from dietary substrates.

The importance of diet in modifying the gut microbiota in elderly populations is becoming increasingly apparent. A review by Woodmansey et al. [23] suggests a reduced and enhanced complement of carbohydrate and protein metabolizing bacteria in old age, respectively. Interestingly, it is thought increased proteolytic activity in the gut, caused by increased Bacteroides and Clostridium species is responsible for putrefaction and production of harmful ammonia, amines, phenols and indoles, while high fat feeding has been associated with increased systemic endotoxin production and low-grade inflammation in animal models [24]. High-fat, high-protein, low-complex carbohydrate diets promote the production of colonic residues that promote microbial production of potentially carcinogenic byproducts [25]. It is thought that the microbiota mediates the effect of diet on colon cancer risk by regulating the generation of butyrate, folate, and biotin, molecules known to play a key role in the regulation of epithelial proliferation. However, unlike high protein and fat intakes, carbohydrate increases gut microbial diversity [21], which has been associated with several health benefits in a number of population groups, including older populations [12]. Long-term high dietary fibre consumption has been associated with increased bacterial genes for cellulose and xylan hydrolysis and increased production of short-chain fatty acids [26], an energy source for epithelial cells which have been shown to have anti-inflammatory properties and may be particularly beneficial to aging population groups.

The majority of dietary intervention data has focused on the bifidogenic role of prebiotics in human subjects, which are defined as non-digestible carbohydrates, assumed to infer benefits for the host health by stimulation of a protective intestinal microbiota. The majority of research in this area has focused on the modulatory effects of single carbohydrate fractions, predominantly long- and short-chain carbohydrates which escape digestion in the upper gastrointestinal tract. These include fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), resistant starches and prebiotics such as inulin and oligofructose. They are then fermented in the colon and selectively stimulate the growth of bifidobacteria, which are known to exist at markedly reduced levels in old age (table 2). Studies have shown that prebiotics affect the immune system as a direct or indirect result of changes in the gut microbiota profile or in the fermentation potential. In addition, GOS and FOS have been attributed to functional claims related to the bioavailability of minerals, lipid metabolism and regulation of bowel habits.

A limited number of intervention trials have been conducted to determine the microbiota-modulatory effects of certain prebiotic ingredients. Walton et al. [27] conducted a study with thirty-nine, 50- to 81-year-olds supplemented with 8 g GOS or placebo for 6 weeks and found significant increases in bifidobacteria and butyrate. GOS-induced bifidogenesis was also reported by Vulevic et al. [28], who focused on the immunomodulatory effects of a mixed GOS preparation over a 10-week period, resulting in increased phagocytosis and an anti-inflammatory cytokine profile. However, the inter-individual variation in gut microbiota suggests the response of microbial communities to prebiotic supplementation/dietary modulation may also vary. In fact, certain study volunteers have been reported as ‘non-responders', indicating the influence of the initial composition of the gut microbiota.

Human studies have also investigated the effects of arabinoxylan-oligosaccharide supplementation, which have been reported to impact the protein/carbohydrate fermentation balance in the large intestine and thus affect the generation of potentially toxic metabolites in the colon. Supplementation trials have been reported to increase total bacterial populations including Bifidobacterium, faecal butyrate concentrations [27] and fermentation activity. Further investigation into the positive modulatory effects of other promising functional ingredients on microbiota composition in elderly cohorts is warranted.

It is clear from the preceding text that diet is a major driver of microbiota variation in the elderly (and other age groups). The large differences in the trending microbiota composition changes in European subjects in different countries measured by the CROWNALIFE study [17] are almost certainly due to factors including diet. As well as differences in the methods employed in culture-dependent studies, the difficulty in identifying unifying trends in microbiota composition change upon aging, as reviewed by Woodmansey [23], is also probably due in large part to diet influences. It is therefore necessary to conduct large longitudinal microbiota measurements in well-phenotyped individuals who consume a carefully measured diet, as well as to preform controlled dietary interventions in subjects whose microbiome has been analyzed at baseline. The NuAge project, funded by the European Commission (www.nu-age.eu), will go some way to tackling the latter aspiration because one of the objectives is to conduct a dietary intervention in 1,250 subjects across 5 geographically spread-out European cities, whereby the participants will be switched to a Mediterranean diet. Extensive physical and clinical measurements will then seek to correlate any changes recorded with alterations in the microbiome, inflammasome and peripheral blood lymphocyte epigenome in the subjects.

Even in healthier subjects, consumption of medication, often multiple types, is common in the elderly. However, there have been no dedicated studies of how the gut microbiota affects or is affected by multiple medications. At least 30 drugs are already known to be modified by the microbiota [29], and there is good evidence that the activity of the microbiota can affect the activity or bioavailability of drugs [30]. Coupled with dietary effects, this could present a complicated environmental modifier with particular relevance in the elderly. To unravel the interactions, strongly powered studies will be required.

The term ‘dysbiosis' is commonly used to convey an altered microbiota, but the difficulty of defining a core microbiota makes it very challenging to describe when dysbiosis has occurred. Despite this, what has certainly become clear from recent studies is that a low-diversity microbiota is a common feature of subjects undergoing obesity, inflammatory bowel disease [31] and accelerated aging-related health loss [12]. As noted above, our studies of increased frailty and health loss in long-term residential care subjects were characterized by a lower diversity microbiota and lower gene counts in shot-gun metagenome data [12]. While dietary adjustment would be the simplest way to restore microbiota diversity, it may prove practically difficult to implement because of physiological or financial barriers. Furthermore, in older subjects, there is a risk that the missing elements of the microbiota cannot be restored by diet alone. Faecal microbiota transplants have proved effective for treating C. difficile in humans [32] and for restoring glucose sensitivity in patients with metabolic syndrome [33]. Transplantation of the entire microbiota between humans poses residual safety concerns, but development of artificial consortia based on defined cultured microbes offers the prospect of a clean reproducible alternative. In the case of older subjects, one can imagine there being individuals who have lost the entire community of microorganisms capable of hydrolyzing or metabolizing certain dietary ingredients, such as resistant starch or other complex polysaccharide. So-called ‘bacteriotherapy' may also be the only way of restoring desired microbiota elements to these individuals.