Denise B. Lynch
a, b· Ian B. Jeffery
a, b· Siobhan Cusack
a·
Eibhlis M. O’Connor
c· Paul W. O’Toole
a, ba School of Microbiology and b Alimentary Pharmabiotic Centre, University College Cork, Cork , and
c Department of Life Sciences, University of Limerick, Limerick , Ireland
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 mi-crobiota 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 di-verse range of diseases [reviewed in de Vos and de Vos 1 ]. There is a particularly com-pelling case for studying the microbiota in aging subjects. This phase of life is accom-panied 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 indi-viduals, countries and populations, detailed analysis of gut microbiota-health interac-tions in older people is particularly appropriate. This review summarizes the differ-ences 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.
Physiological and Clinical Issues That Can Impact on the Gut Microbiota in Elderly
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 ag-ing 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 phys-iological function, which can in turn alter the composition of the microbiota.
Physiological, motor and sensory functions change with age. For instance, a natu-ral 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 sen-sation and consequently increased susceptibility to gastrointestinal complications.
Other age-related, digestive system complications include dysphagia (difficulty swal-lowing), functional dyspepsia (painful, difficult or disturbed digestion), gastroesoph-ageal reflux, delayed intestinal transit time, diverticulosis, and increased rates of con-stipation, 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 im-mune system by the microbiota, which influences the composition of the microbiota itself. Microbiota disturbance has been linked with an increased susceptibility to dis-orders including allergies, cancer, digestive/intestinal disdis-orders, frailty, obesity/meta-bolic disorder and its related conditions. It can also affect regulatory systems such as hormone signalling, leading to changes in mood and behaviour. Host metabolic path-ways that facilitate connection between the intestine and the brain, can be affected [3] . Disruption to this bidirectional homeostatic pathway has been associated with in-flammation and alterations in the stress response, among other stress-related symp-toms such as anxiety, commonly experienced in older age. A healthy, more diverse microbiota composition encourages resistance to pathogens and increased interac-tion 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 (immunosenes-cence) 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 fur-ther influence the composition of the microbiota. Health throughout life, and par-ticularly in later years, is dependent on the maintenance of homeostasis, the presence of a stable physiological environment. The relative stability of the adult intestinal mi-crobiota at a species level is a key contributory factor to the promotion and mainte-nance 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 micro-biota is able to respond to exogenous influences throughout life.
Culture-Based Analyses of Intestinal Microbiota of Elderly
Culture-based methods were traditionally used to analyze the intestinal microbiota.
An example of some of the methods utilized can be seen from experiments conduct-ed in 1989 in Japan [5] . Culture-basconduct-ed methods were usconduct-ed to compare the microbi-ota 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 sub-jects from each of the two areas was collected. A number of experiments were per-formed 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 deter-mination 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 jects had a larger bifidobacteria contingent than was observed from the Tokyo sub-jects. However the Yuzurihara subjects had fewer total bacteria, anaerobes, bacilli, clostridia, Bacteroides species, and Eubacterium aerofaciens . Four genera, Megamo-nas , Mitsuokella , SelenomoMegamo-nas , and Acidaminococcus , were isolated from the Yuzuri-hara subjects but not the Tokyo subjects. Intestinal bifidobacteria counts are known to decrease with age, and some Enterobacteriaceae increase. That the Yuzurihara el-derly had more bifidobacteria than the Tokyo elel-derly, 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 resis-tance to C. difficile infection. With the widespread use of antibiotics and increasing number of elderly, CDAD has become a challenging problem. Hopkins and Macfar-lane [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 el-derly 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 Bacteroi-des in elderly compared with the young subjects.
The benefits of using such culture-based methods for analyzing intestinal micro-biota 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 disad-vantage 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
distin-guish 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 Technological Revolution
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 lev-el of a specific group of bacteria. However, when trying to assess and compare a num-ber 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 dif-ferent 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, micro-bial 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 poten-tial 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 denois-ing, can be performed. Chimeric sequences are sometimes formed during PCR am-plification 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 multina-tional scientific consortiums such as the The Human Microbiome Project [10] , Me-taHIT (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 pop-ulations 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-abun-dance 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 cat-egorize samples based on the dominant genera that represent microbial populations that have a substantial scope to modify the phenotype of the individual through pro-duction of metabolites and immunomodulatory effects.
Culture-Independent Microbiota of Older Persons
Microbial-Based Changes in the Elderly
Numerous studies from different geographical locations have attempted to character-ize the microbiota of general healthy populations, and many have compared these
Table 1. Comparing enterotypes with CAGs (co-abundance groups)
Enterotypes CAGs
Each individual is associated with one enterotype group
Each individual is associated with multiple co-abundance groups
Enterotypes are mostly defined by the most abundant genera. Normally Bacteroides, Prevotella and Ruminococcus (or another genera)
The definition of co-abundance groups is based on gradients of taxa and associations between taxa The Enterotypes definition is rigorous Stable associations defining co-abundance groups are
yet to be finalized
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 se-quences 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 Enterobacteria-ceae , 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 , En-terobacteriaceae , 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 coccoides cluster, 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] .
Cultural Microbial Differences Observed in Different Age Groups
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. rec-tale-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 coun-tries, 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 el-derly subjects than their corresponding adult cohorts; however, this was not signifi-cant. 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
decreas-ing Firmicutes from community to long-stay. Reduced abundances of Coprococcus and Roseburia were observed in long-stay subjects, while they had increases in Para-bacteroides , Eubacterium , Anaerotruncus , Lactonifactor and Coprobacillus . This rela-tively 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 re-curring associations with increased Proteobacteria and Bifidobactia. Biagi et al. [18]
attempted to address this with centenarians. The centenarians tended to cluster sepa-rately 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 microbi-ota, and remind us of some of the difficulties we will face when trying to increase lon-gevity and quality of life. Somewhat personalized or community-based approaches may need to be considered in the future.
Diet as a Driver of Microbiota Variation in Older People
Factors Influencing the Gut Microbiota in Older Persons
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-individ-ual variability of the gut microbiota; (2) inter-individinter-individ-ual variance in dietary intakes even among seemingly homogenous population groups, e.g. the elderly [12] ; (3) vari-able effects of dietary intervention, dependent on the baseline microbiota; (4) the use of medical therapeutics, especially antibiotics [19] ; (5) classification of an appropriate
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-individ-ual variability of the gut microbiota; (2) inter-individinter-individ-ual variance in dietary intakes even among seemingly homogenous population groups, e.g. the elderly [12] ; (3) vari-able effects of dietary intervention, dependent on the baseline microbiota; (4) the use of medical therapeutics, especially antibiotics [19] ; (5) classification of an appropriate