These studies have shown clearly, both by the use of an in vitro model and in vivo, that the composition of surface associated flora can be influenced by substrate and hence by diet. Moreover, the dietary changes required are very small; only a few grams of sugar-like carbohydrates were added to subjects’ food each day. Equally important is the nature of the change in the flora. An increase in bifidobacteria and lactobacilli in the gut, either by the use of pro or prebiotics, is currently considered a healthy objective. There are no previously reported studies in humans of any dietary factor that can be shown to alter the composition of the MAF. In a recent paper, Kleessen and colleagues28
showed in human flora associated rats that feeding an oligofructose/inulin mixture increased counts of mucosa associated bifidobacteria in the colon and improved mucosal architecture and mucus secretion.
In the in vitro model, the overall composition of the flora was the same whether in the planktonic or surface associated phase (table 1), although bacterial densities were lower at 12 hours on the agar-mucin surface. A clear bifidogenic effect was seen on the mucus baits with no notable changes in other genera or in the planktonic bacteria. This absence of a selective effect on the planktonic bifidobacteria may reflect the fact that truly free living (that is, not surface associated) bacteria do not exist in the luminal phase of the large bowel (see below). Alternatively, the use of multiple carbohydrate substrates in the batch culture medium, in addition to oligofructose and inulin, may have reduced the importance of the prebiotics in determining the pattern of overall growth in the mixed culture.
Short chain fatty acid concentrations increased substantially with the additional prebiotic substrate but no change in molar ratios was seen and thus these carbohydrates cannot be seen as providing selective increases in butyrate over other short chain fatty acids, although overall they are a good source of butyrate. These findings are similar to those observed in human studies of prebiotic feeding and faecal composition.16
The increase in lactate with added prebiotic is probably a consequence of the presence of rapidly metabolisable substrate in excess.
In the in vivo study, MAF reflected very much the bacterial composition that has been reported for planktonic and faecal populations. This is perhaps not surprising because the notion that a true planktonic flora exists in the large intestine is probably incorrect. Most, if not all, of the flora in the hind gut that are not mucosa associated are attached to particulate matter.29,30
Macfarlane and colleagues (in 1995 and 1997) showed that the composition of the flora attached to particles in the large bowel is very similar to that which is “free living” but that metabolically they are different. Increased activities of polysaccharidases, glycosidases, proteases, and arylamidases, and mucus degrading enzymes are seen in the particle associated species, probably reflecting the nature of the underlying surface material which will largely comprise plant cell walls, entrapped in a viscoelastic gel of the many mucins that are secreted into the gut. End products of fermentation also differ, with surface associated communities producing more acetate than planktonic flora.31
Addition of only 15 g/day of inulin and oligofructose produced significant changes in the MAF in both the proximal and distal colon. Most importantly, counts of bifidobacteria increased by 1 log10
or more, the principal species being B angulatum
. The increase in this particular genus is the classic prebiotic effect demonstrated previously in vitro32–34
and in vivo in faecal flora.16–18
The essence of prebiosis, however, is the selectivity of the effect. In the present study, the absence of significant increases in bacteroides, clostridia, coliforms, and total aerobes and anaerobes is reassuring. Numbers of lactobacilli also increased significantly, but this genus, like bifidobacteria, is thought to be part of the beneficial or protective flora of the gut. We also observed significant changes in eubacteria, an observation not previously noted in studies of prebiosis. It is not clear why this should occur in MAF and not planktonic or faecal flora. However, it is known that bacteria living in biofilms on surfaces show altered substrate use and efficiency of metabolism as well as resistance to host defence mechanisms and antibiotics.35–37
Metabolic consortia build up and substrate use becomes efficient. It is possible therefore that eubacteria benefited from cross feeding of carbohydrate residues from the initial actions of bifidobacteria and lactobacilli on prebiotics. Many eubacteria are saccharolytic.
Inulin and oligofructose are fructans that are not hydrolysed by pancreatic enzymes and escape digestion in the small bowel.38–40
Bifidobacteria have relatively high amounts of β-fructosidase that is selective for β1–2 glycosidic bonds in fructans.41
Subsequent transport mechanisms and sites of hydrolysis may also be faster. After oligosaccharide hydrolysis, monomers then serve as an efficient growth substrate for the bifidus pathway of hexose fermentation.42
In addition, the inhibitory effects of bifidobacterial growth on other colonic organisms6
are likely to assist in the competitive influence that occurred with oligofructose and inulin. Lactobacilli can also ferment FOS,43
and human feeding studies have shown that faecal populations of these bacteria can be increased in some subjects when fed prebiotics.18,44
Data presented in this study show that lactobacilli appear to compete equally effectively for prebiotic substrates at the mucosal surface.
In any study of the gut microflora, methodology is always an important issue. Culture based methods are known to introduce bias towards organisms that are easier to culture and recently, studies using molecular techniques have improved our knowledge of bacteria that are more difficult to culture and which form a significant portion of the microbiota. Culture based methodologies will never account for the entire microbial population, are slow, require immediate sample handling and identification by a variety of morphological, metabolic, and biochemical techniques. Phenotypic methods of identification are unlikely to match the discriminatory power of some genotypic tools. However, culture based methods are well established, cost effective, sensitive, and allow direct comparison with the many similar studies reported in the literature. They give valuable information of the likely metabolic potential of the overall population. Molecular analyses are important in contributing to the gaps in our knowledge but no method is infallible and other factors need to be considered with these newer methods. They are also subject to bias—for example, bacteria that are more susceptible to lysis, probe permeability, and amplification will be more readily detected. There are still a very limited number of probes available at species level. FISH methods are almost impossible to quantify and molecular techniques in general have lower sensitivities. Techniques such as DGGE (molecular fingerprinting) are not quantitative, and the metabolic contribution of “molecular species” cannot be determined. Both methodologies continue to contribute to our understanding of the large bowel ecosystem but it is important to consider the limitations of each technique, particularly when comparing populations of absolute cell numbers to those given as a proportion of the total bacteria present. Similar population sizes have been found by both in situ hybridisations and bacterial cell culture for organisms such as bifidobacteria and eubacteria, but the size of the total microflora is underestimated by the latter method, which would alter the proportional value.45,46
However, it currently remains prudent to consider evidence using both methodologies and this study clearly shows a significant change in the culturable portion of the MAF.
Widely differing results for the composition of the MAF have been reported. This is likely to be partly because of the different, and changing, methods referred to above, now used in bacteriology. Swidsinski and colleagues9
found low total counts, by conventional culture techniques, of MAF in 40 control subjects although these bacteria were similar in composition to faecal (planktonic) flora. However, biopsy specimens were vigorously washed before culturing. Much higher MAF counts were found in patients with active ulcerative colitis in this study. Schultsz and colleagues7
also observed very low counts of MAF in nine controls undergoing colonoscopy. Specimens were not washed but were fixed in formalin embedded in paraffin and bacteria detected by non-radioactive detection of rRNA by in situ hybridisation. Again, high counts were found in ulcerative colitis patients. Poor staining in the controls was ascribed to the MAF being inactive or dead, poor antibody penetration, and the barrier effect of mucus. Others have shown a numerous and diverse MAF in colonic or rectal mucosal from either sudden death victims47
or from colonoscopic and rectal biopsies of healthy subjects.8,48–51
In these reports, however, standard culturing techniques were used and biopsies were either not washed or washed gently to remove visible adherent faecal material. Similarly, Zoetendal and colleagues52
took biopsies at colonoscopy from the colons of 10 individuals and used 16S RNA probes to analyse bacterial diversity on paraformaldehyde fixed samples. Bacterial counts up to 106
were found in seven of 10 subjects but showed fewer species diversity than samples of faecal flora. Whether these bacteria are largely in the mucus layer, rather than adherent to epithelial cells, is a likely possibility but their proximity to the mucosal and thus interaction with epithelial and immune cells is an important interface. In situations where mucus secretion is thought to be abnormal, epithelial adherent populations exist.7,9
Alterations in the MAF had no effect on cell cycle entry by mucosal epithelial cells at four different sites in the large bowel. Our observation that Mcm-2 was present in significantly greater numbers of epithelial cells than the traditionally used proliferation markers Ki67 and PCNA is consistent with data from other studies.25,53
MCM proteins are the most sensitive markers available of cell cycle entry, being abundant in nuclei throughout the cell cycle and lost rapidly following differentiation, as occurs in large bowel crypts.25
It should be noted that Mcm-23, Ki67, and PCNA all essentially provide information concerning the cell cycle state of the stained tissue and more subtle differences in rates of cycling would not be identified using our approach.
Does the composition of the MAF matter? Almost certainly yes. Gut flora clearly interact with the mucosal immune system11–15
which is important in the process of developing tolerance to commensal flora. In mucosal disorders such as ulcerative colitis, this tolerance may have broken down.54–56
Changing the composition towards a flora with increased bifidobacteria and lactobacilli is thought to be beneficial to the host.57
These genera contain few if any pathogenic species and are a major part of the large bowel defence against invading pathogenic organisms through their capacity to secrete peptides that inhibit pathogen growth.6,58,59
When given as probiotics, lactobacilli adhere to the colonic and rectal mucosa46,60
although probably do not permanently colonise the gut epithelium.61
Probiotic bacteria are clearly established as being a benefit in the prevention of antibiotic associated diarrhoea62,63
and reducing the duration of infectious diarrhoea in children.64
Animal models indicate that they may have a protective effect against the development of colorectal cancer65,66
an effect that can be strengthened by combination with a prebiotic.67
The health benefits of prebiotic carbohydrates have yet to be established. Apart from some benefits to calcium absorption, no clear value has been shown.68
However, research in this area is relatively recent while the benefits of probiotics have been known for 100 years.
Moving the composition of the MAF towards one with increased numbers and more diverse species of bifidobacteria and lactobacilli should be beneficial to colonic health. If this can be achieved by a small change in the carbohydrate composition of the diet, then new possibilities emerge for public health and for preventive strategies by the gastroenterologist.