PCR of 16S rDNA sequences combined with analysis by DGGE has provided an efficient means of screening bacterial communities for changes in composition in response to allochthonous factors (19
). Nevertheless, the method has some disadvantages, notably that it is semiquantitative, it is difficult to standardize and hence to make valid comparisons of staining intensity of DNA fragments, and only the predominant populations (comprising 90 to 99% of the total community) are monitored when universal bacterial primers are used (36
). The last difficulty has been overcome through the use of group- or species-specific PCR primers that allow even minority populations in the community to be detected (26
). We have devised two important technical advances to improve analysis of fecal microbiota with PCR-DGGE. First, we used an internal standard by which DGGE profiles can be standardized to enable accurate comparison of profiles. The use of the internal standard permitted quantitative measurements of fragment staining intensities to be made that could then be evaluated statistically. Second, we used S1 nuclease treatment of DNA eluted from gels in order to remove single-stranded DNA. This resulted in a much higher success rate in cloning the DNA fragment of choice. Both of these technical improvements enhance PCR-DGGE as an analytical method.
A striking feature of our PCR-DGGE observations was the marked difference in bacterial community profiles generated with RNA compared to DNA as the PCR template. Profiles generated from bacterial RNA showed intensely stained fragments clustered in the middle of the denaturing gradient and were markedly different from those generated from DNA. RNA extracted from bacterial cells is mostly rRNA and can be used as an indicator of metabolic activity because the ribosome-per-cell ratio is roughly proportional to the growth rate of the bacteria (9
). While DNA-based analytical procedures provide a phylogenetic picture of the community, they do not reflect metabolic activity because the DNA could originate from living active cells, living dormant cells, lysed cells, or dead cells. On the basis of our observations, it can be proposed that the human gut provides a habitat for a diversity of bacterial species, as seen in the DNA-DGGE profiles, the composite of which varies from one human to another. The “core” or “true” gut microflora may be comprised of relatively few populations that provide the major metabolic activities. This is clearly a topic that requires further investigation.
The diets of our subjects were supplemented with biscuits that contained oligosaccharides at concentrations that produced edible products of commercial quality. The consumption of three biscuits provided a dose of oligosaccharides that did not cause flatus or abdominal pain in the subjects. The daily dosage was lower than that used in most prebiotic studies reported in the literature (4
), yet changes to fecal microbiota profiles were observed. Alterations to the bacterial community profiles in response to dietary supplementation were only detected in profiles that were generated from bacterial RNA. Therefore, we detected changes in bacterial metabolic activity in response to the consumption of the biscuits, but not changes in the population structure of the community. This seems a reasonable outcome considering the highly regulated nature of bacterial communities in which the proportions of the constituent populations are strictly maintained (1
Although oligosaccharides added to the gut ecosystem are generally expected to increase the number of bifidobacterial cells in particular, we did not observe any changes in population sizes by selective culture or nucleic acid-based analysis (FISH). Increased metabolic activity without an increase in the number of bacterial cells is not contradictory, since it has been shown in studies with rumen bacteria that “energy spillage” occurs in bacterial communities (28
). The correlation between ATP and biomass formation is often poor, and while some of this variation in growth efficiency can be explained by maintenance energy expenditure, bacteria have other mechanisms of nongrowth energy dissipation (futile cycles) (28
). Such energy spilling may occur when growth is limited by nutrients other than energy sources, as has been described for a rumen species, Streptococcus bovis
DNA fragments representing B. adolescentis
and C. aerofaciens
were present in RNA-DGGE gel profiles, especially when biscuits had been consumed. It can be argued that these bacterial cells were in a metabolically quiescent state prior to dietary supplementation with the biscuits. When oligosaccharides and/or lactose supplemented the host diet, a substrate for metabolism became available for the bacteria in the large bowel, but because other nutrients were growth limiting in the highly regulated bacterial community, an increase in bifidobacterial and colinsellal cell numbers did not occur. Therefore, bifidobacterial and colinsellal populations remained constant in size and the composition of the bifidobacterial population at the level of species and strains did not differ, but RNA-DGGE provided evidence of increased metabolic activity. The significance of this increased metabolism to the bacterial community and to the human host, even if it occurs to a greater magnitude in the proximal large bowel (7
), is not obvious but could provide important topics for future research.
β-Galactosidase activity was markedly increased in the feces of some subjects when the diet was supplemented with biscuits. At least twofold increases in activity were recorded only in samples collected at the end of periods during which biscuits had been consumed. The increases did not match alterations in RNA-DGGE profiles (increased staining of bifidobacterial and colinsellal fragments) and must therefore be considered to be due to alterations in the collective metabolism, including enzyme induction, of the ecosystem as a result of GOS and/or lactose entering the ecosystem.
We were interested in comparing the effect of consuming GOS in biscuits relative to FOS because most emphasis in the prebiotic literature has been placed on the latter substrate. Eleven of the 15 subjects showed alterations to RNA-DGGE profiles in association with biscuit consumption. Most of these changes were in profiles related to GOS biscuits (10 of 15), but the increased intensity of staining of B. adolescentis and C. aerofaciens fragments was more marked in samples collected at the end of the FOS biscuit consumption period. Some subjects showed altered profiles when NONE biscuits had been eaten, demonstrating that lactose itself had a “prebiotic” effect. The variation among subjects with respect to DGGE profiles and NONE biscuits could relate to the degree of lactose utilization in the small bowel and proximal large bowel of each individual.
As noted in a previous study (23
), each subject harbored a unique collection of bifidobacterial strains in the feces. High-pressure liquid chromatography analysis of culture residues of representative strains showed that they varied greatly in their ability to use the Oligovite (GOS) components. This method could provide a basis of selection for bifidobacterial strains suitable for use in synbiotics, which are products containing a combination of living bacterial cells and a substrate that they can metabolize (27
). Interestingly, strains that used a major portion of the oligosaccharide components of Oligovite were only isolated from subjects whose RNA-DGGE profiles were altered following GOS biscuit consumption.
Our study has provided important knowledge about the microbial ecology of the gut. First, we developed technical innovations for DGGE analysis of the bacterial community in feces (S1 nuclease activity and an internal DNA standard). Second, we obtained a new perspective on the composition of the fecal community by the use of RNA as the template in PCR-DGGE, which, after further investigation, may reveal the identity of the core, metabolically active members of the distal gut ecosystem. The use of highly purified RNA is of course critical to this analysis; otherwise, contaminating DNA will give the same profile obtained with a DNA extract. The RNA extraction method that we have described satisfies this criterion. Third, we demonstrated that evidence of increased metabolic activity in response to the consumption of GOS or FOS does not necessarily equate to increased numbers of bifidobacteria in a complex community, an observation that may explain the very small changes to bifidobacterial population sizes reported in other oligosaccharide studies, even when relatively large doses of prebiotics were administered (2
Our observations suggest that a phylogenetic approach to analysis of the gut ecosystem may not always be optimal and that a more physiological (biochemical) methodology might be more informative. Overall, our study has provided new perspectives to the analysis of the fecal microbiota in relation to the consumption of oligosaccharides in a concentration that is relevant to the food industry.