Comparison of Compositions and Metabolic Activities of Fecal Microbiotas in Young Adults and in Antibiotic-Treated and Non-Antibiotic-Treated Elderly Subjects

Volunteer recruitment.Inclusion criteria for the study for the healthy young (aged 19 to 35 years) and the healthy elderly (aged 65 years or more) volunteers were as follows. The volunteers were recruited as free-living individuals from the local community and were in general good health. Volunteers were excluded if they had received antibiotics in the 8 weeks prior to the start of the study. Current treatment involving the use of steroids, immunosuppressants, acetylsalicylic acid, lactulose, or other stimulants or laxatives was not permitted. Individuals who were medically unstable, terminally ill, or suffering from dysphagia or severe dementia were also excluded. Criteria were the same for hospitalized patients as for the healthy elderly volunteers except for the fact that they were all receiving antibiotics. These subjects were inpatients at Royal Victoria Hospital, Dundee, United Kingdom. Ethical permission for these studies was obtained from the Tayside Medical Research Ethics Committee.

Fecal material.For bacteriology studies, fresh stools were obtained from 12 healthy young adults aged 19 to 35 years (three males and nine females), 6 healthy elderly people aged 67 to 75 years (six females), and 10 elderly hospitalized patients receiving antibiotics, aged 73 to 101 years (two females and eight males). All samples were weighed and were processed under anaerobic conditions within 1 h of defecation to minimize loss of cell viability. Fecal slurries (10% [wt/vol]) were homogenized in a stomacher bag by using anaerobic 0.1 M phosphate-buffered saline (pH 6.8) and then passed through a 500-μm mesh sieve to remove large particulate matter. For measurements of bacterial fermentation products, stools were obtained from 14 healthy young people, 70 healthy elderly volunteers, and 9 antibiotic-treated elderly people, a group which included the subjects used for bacteriological analysis. Fecal slurries were prepared as described above, and aliquots (5 ml) were frozen and stored at −20°C for analysis of fecal organic acids and ammonia concentrations.

Isolation and enumeration of bacteria from feces.One milliliter of fecal slurry was vortex mixed with sterile anaerobic half-strength peptone water (9 ml) to form a 1 in 10 dilution series (10⁻² to 10⁻⁹). Samples (0.1 ml) from the tubes were spread onto prereduced duplicate agar plates, as outlined below. Anaerobes were isolated by using Wilkins-Chalgren agar. To observe proteolytic and amylolytic activities of bacteria after isolation, the medium was supplemented with casein (10 g liter⁻¹) or starch (10 g liter⁻¹). Zones of casein hydrolysis were observed directly as areas of opacity on the plates or as cleared areas after treatment with dilute HCl. Starch hydrolysis was observed by the addition of iodine. Neutral red (8 mg liter⁻¹) was also added to some of the agar plates to enhance the differentiation of bacterial colonies. Wilkins-Chalgren agar with gram-negative antibiotic supplements was used to isolate Bacteroides spp., Prevotella spp., and other gram-negative anaerobes. Beerens agar (1) was used to isolate bifidobacteria, while MRS agar was employed in the isolation of lactobacilli and lactococci. Clostridia were isolated by using perfringens agar with the manufacturer's antibiotic supplements, reinforced clostridial agar with added novobiocin and colistin (0.008 g liter⁻¹ each) and azide blood base agar. These media were also supplemented with casein to visualize proteolysis. Plates were incubated at 37°C in an anaerobic chamber (Don Whitley Scientific, Shipley, Yorkshire, England) containing an atmosphere of CO₂, H₂, and N₂ (10:10:80) for 48 to 72 h. Facultative anaerobes were cultured by using nutrient agar (for total counts), azide blood agar base with casein (for enterococci), and MacConkey agar number 2 (for enterobacteria) and were incubated aerobically at 37°C for 48 h.

Identification of bacteria.Bacteria were identified based on their cellular fatty acid profiles. Fatty acid methyl esters (FAME) were extracted from bacterial pellets obtained from approximately 40 ml of culture in anaerobic peptone yeast extract broth (21) supplemented with glucose (10 g liter⁻¹) for anaerobic isolates or from 40 mg of bacteria grown on BBL trypticase soy broth agar (Becton Dickinson Ltd., Oxford, United Kingdom) for aerobic isolates, by saponification, methylation, and extraction, as described previously (38). The FAME were separated by using a model 5898A microbial identification system (Microbial ID, Inc., Newark, Del.), which consisted of a Hewlett-Packard model 6890 gas chromatograph fitted with a 5% phenyl-methyl silicone capillary column (0.2 mm by 25 m), a flame ionization detector, a Hewlett-Packard model 7637A automatic sampler, and a Hewlett-Packard Vectra XM computer (Palo Alto, Calif.). Gas chromatography parameters were as follows: carrier gas, ultra-high-purity hydrogen; column head pressure, 60 kPa; injection volume, 2 μl; column split ratio, 100:1; septum purge, 5 ml min⁻¹; column temperature, 170 to 270°C; injection port temperature, 300°C; and detector temperature, 300°C. Peaks were automatically integrated, and fatty acid names and percentages were calculated, with numerical analysis done by using the standard MIS Library Generation software (Microbial ID, Inc.). Bacterial isolates were identified by comparing FAME profiles to those of known cultures in the MIS aerobe and anaerobic standard libraries. The system was calibrated by using a standard Microbial Identification System FAME calibration mix before each run and validated by using the type strains Stenotrophomonas maltophilia ATCC 13637, Bacteroides fragilis ATCC 25285, and Clostridium perfringens ATCC 13124. Yeasts were identified by using a API 20 C AUX biochemical identification system (bioMérieux, Basingstoke, Hampshire, England).

Chemical analyses.Ammonia levels were determined by using the phenol-hypochlorite method (51). Standards containing known concentrations of ammonium chloride were prepared. All samples were tested in triplicate. Ammonia levels were analyzed by using a microplate reader (model 450; Bio-Rad, Hercules, Calif.) with single wavelength absorbance (630 nm). SCFA, lactate, and succinate from fecal supernatants were analyzed by using methods described previously (21, 30). SCFA were first acidified with 50 mM H₂SO₄ to convert them into free fatty acids, which were subsequently extracted into ether. SCFA were separated by gas chromatography by using an Agilent Technologies model 6890N gas chromatograph, following split injection (40:1) with an HP-INNOwax column (model 19091N-133; cross-linked polyethylene glycol; 30 m by 0.25 mm by 0.25 μm; Agilent Technologies). Injector and detector temperatures were 250 and 300°C, respectively. The initial column temperature (120°C) was held for 1 min, thereafter increasing in stages of 10°C per min until reaching 265°C, which was maintained for 2 min. Flow rates of the helium carrier gas, H₂, air, and N₂ (the makeup gas) were set at 1.8 ml min⁻¹, 40 ml min⁻¹, 450 ml min⁻¹, and 45 ml min⁻¹, respectively. Lactate and succinate were measured by using the same analytical system and operating conditions, after methylation of samples and extraction into chloroform (21). All samples were quantitated by comparisons of sample peak heights with those of authentic standards and internal standards by using Hewlett-Packard Integrated Chemstation software.

Chemicals.All bacteriological culture media and selective antibiotic supplements were obtained from Oxoid (Basingstoke, Hampshire, England). Unless otherwise stated, all chemicals were purchased from the Sigma Chemical Co. (Poole, Dorset, England).

Statistics.Statistical analyses were done by using the SPSS version 9.0 statistics package (SPSS, Chicago, Ill.). The independent-sample t test was applied to compare bacterial counts from the healthy young and healthy elderly groups and from the healthy elderly and antibiotic-treated elderly groups. The Mann-Whitney U test was used to compare fecal SCFA and ammonia levels from the healthy young and healthy elderly subjects and from the healthy elderly and antibiotic-treated elderly groups.

Stool weight.Mean fecal weights were 107 g (± 22.2), 82.7 g (± 17.4), and 70.3 g (± 11.5) for the healthy young subjects (HY), healthy elderly subjects (HE), and antibiotic-treated elderly subjects (AE), respectively.

Bacteriology of different subject groups.The results shown in Fig. 1A demonstrate that while total anaerobe numbers were similar for all three study groups, the range of counts was noticeably increased for AE. Total facultative anaerobes (Fig. 1B) were generally lower in HY than in HE. However, a significant increase (P < 0.05) in levels of facultative anaerobes was found for AE.

• Open in new tab
• Download powerpoint

FIG. 1.

Box plot showing mean fecal bacterial counts from individuals in different age groups: a total anaerobe counts, b total facultative anaerobe counts. Results are expressed as mean log₁₀ CFU per gram of feces ± interquartile range. Error bars represent standard errors of the mean. Healthy young (HY), n = 12; healthy elderly (HE), n = 6; antibiotic-treated elderly (AE), n = 10; *, significant difference (P < 0.05) between HE and AE.

Bacteroides and prevotellas.Table 1 shows counts of bacteroides and prevotellas for each subject group, including details of the different species isolated. Marked reductions in total bacteroides occurred in both HE and AE, in comparison to HY, with lowered species diversity in the elderly subjects. The predominant Bacteroides spp. in samples from HY were B. ovatus and B. thetaiotaomicron, which had the highest prevalence and were present in the greatest numbers. Other common species, such as B. vulgatus, B. distasonis, B. fragilis, B. uniformis, and B. caccae, were detected in similar numbers but in fewer volunteers. Reductions in total bacteroides in HE and AE were accompanied by decreased species diversity. The principal bacteroides in HE was Bacteroides sp. strain FO, whereas B. ovatus was the predominant species in AE. A similar pattern was observed with prevotellas. Prevotella tannerae was the dominant species in HY and HE, although the organism was found in lower numbers in the latter group and was supplanted by Prevotella loescheii in AE. A general reduction in levels of prevotella was observed for both elderly groups, especially the group receiving antibiotics.

Bifidobacteria and lactic acid bacteria.Although there was a decline in total bifidobacterial counts from HY to HE (Table 2), the reduction was not significant. Bifidobacterium angulatum was the predominant species in both groups. A small decrease in the numbers of bifidobacterial species was found for HE. In comparison, total counts for AE were significantly lower (P < 0.05) than those for HE, with many patients having no bifidobacteria at all. Consequently, overall species diversity was low in these individuals. Bifidobacterium catenulatum, Bifidobacterium boum, and Bifidobacterium infantis were isolated from feces samples from HY but were not detected for either elderly group, whereas Bifidobacterium coryneforme was only present in samples from AE.

Total lactobacilli numbers in samples from HE were lower than in those from HY; however, the highest counts occurred in samples from AE, with almost a 100-fold difference compared to counts for HE. Samples from AE also had greater species diversity, and many of these organisms were not found in HE or HY. Large variations in total lactobacilli counts were evident for all three groups, particularly HE and AE.

Clostridia, fusobacteria, and propionibacteria.Counts of these bacteria are shown in Table 3. Clostridial diversity was markedly higher in AE patients compared to the other two groups. Conversely, the lowest numbers were found in samples from HE, with an associated reduction in species diversity. A large number of different species were observed in samples within each cohort, particularly HY and AE, with no particular organism predominating in any group. Nevertheless, many of the clostridia, such as C. difficile, in samples from AE were not detected in samples from the other volunteers. A comparatively limited number of fusobacteria and propionibacteria were isolated in this study. Fusobacteria were not detected at all in stools of HY; however, they were present in low numbers in samples from AE and HE. The HE had the highest counts, but AE had slightly higher species diversity. Propionibacteria counts were low, particularly in HY and HE, where this genus was detected in one individual only. The increased frequency of isolation of propionibacteria in AE was accompanied by an increase in the total numbers of propionibacteria for that group relative to other groups.

Eubacteria and anaerobic cocci.Fecal eubacterial levels were similar for the different study groups, though greater numbers were present in AE and HE than in HY (Table 4). Although the HE had the highest total eubacterial counts, samples from those individuals had the lowest species diversity, with only two different organisms, Eubacterium rectale and Eubacterium cylindroides, being detected. Eubacterium aerofaciens and Eubacterium rectale were the most prevalent species in HY, whereas Eubacterium limosum predominated in AE. Anaerobic gram-positive and gram-negative cocci were present in low numbers in HY, HE, and AE.

Aerobes and facultative anaerobes.The most predominant member of the Enterobacteriaceae detected in all three groups of volunteers was Escherichia coli (Table 5). This organism was the only enterobacterium isolated from samples from HE and had the largest numbers and prevalence in that group; samples from HY and AE showed similar prevalences, but there were reduced counts in samples from HY. Various other enterobacteria, including Providencia spp. and Proteus spp., were found in HY and AE.

Proportionally high numbers of enterococci were observed in samples from AE, with seven different species being detected. The HY had lower total numbers and reduced prevalences of these bacteria, although for both HY and AE, the individual counts were spread over a large range. E. faecalis was the predominant species in both HY and AE; however, E. durans had the greatest range, with some individuals having extremely high numbers in their feces. Enterococci were not isolated from any samples from the HE.

Streptococci were relatively uncommon, especially for HY, although the numbers of species observed was higher in samples from HE. Levels of staphylococci were also high for both elderly groups, compared to HY, with only a small increase in species diversity and a similar range of individual counts between the groups. Candida albicans occurred mainly in samples from HE, where it was detected more frequently than in samples from the other groups. HY had the lowest numbers and prevalence of Candida albicans. Pseudomonas aeruginosa was isolated from one AE fecal donor and was not detected in samples from HY or HE.

Microbiological species diversity.A summary of the changes in species diversity for each bacterial group detected in feces samples from HY, HE, and AE is shown in Fig. 2. A marked reduction in the numbers of species in both elderly populations was observed for many anaerobic bacterial groups, such as bacteroides, prevotella, and bifidobacteria. Lactobacilli and clostridial species diversity were decreased for HE compared to levels for the other groups, with samples from AE demonstrating the greatest species diversity in both bacterial genera. Similar changes occurred in eubacteria and enterobacteria populations. The large increase in enterococci in samples from AE can be clearly seen in Fig. 2, with an increase in streptococci and yeasts being evident for HE.

• Open in new tab
• Download powerpoint

FIG. 2.

Summary of bacterial species diversity in feces from individuals in different age groups. Results are expressed as mean values ± standard errors of the mean. Healthy young (white), healthy elderly (grey), and antibiotic-treated elderly (black).

Proteolytic and amylolytic activities.The abilities of fecal isolates to hydrolyze casein were determined by observing hydrolysis zones around bacterial colonies. The abundances of proteolytic species were calculated as percentages of the total numbers of bacteria detected, and the results showed that 15.8, 4.6, and 19.4% of all isolates from HY, HE, and AE, respectively, were proteolytic. Amylolytic activities were visualized similarly on starch plates, where hydrolysis was observed for 14.3, 3.6, and 9.0% of isolates from HY, HE, and AE, respectively.

Bacterial fermentation products in feces.Median fecal ammonia levels were highest in samples from HY and lowest in those from AE. Fecal ammonia was significantly lower (P < 0.05) for AE than for HE. The amounts of fermentation acids in samples from HY and HE were generally similar (Table 6), with only small reductions in fecal acetate, butyrate, and valerate, and total SCFA for HE. However, the production levels of these fermentation products were altered for AE, with significant decreases in propionate, butyrate, isobutyrate, and valerate being observed in comparison to levels for HE. The reduction in total SCFA between HE and AE was found to be approaching significance. Concentrations of fecal lactate and succinate were also determined. The results demonstrated the presence of succinate in samples from all three subject groups, with the highest concentrations occurring for AE and the lowest for HE (Table 6). Lactate was not detected in any of the fecal samples.