Knowledge of the intestinal flora has developed simultaneously with the methods of inves-tigation with regard to both sampling technique and analysis of the flora.
A. Methods of Evaluation
Conventional stool collection allows only investigation of the terminal flora of the diges-tive tract. The dominant flora consists of highly anaerobic microorganisms, which are therefore difficult to isolate and keep alive. More elaborate sampling methods, such as biopsy of the intestinal mucosa and collection of luminal aspirate fluid using a Camus probe or weighted tube, have made it possible to carry out detailed exploration of all portions of the digestive tract. These new sampling methods and the design of more appro-priate culture media for the various species now make it possible to use a good direct
Hoang-Dung TRAN and friends approach to understanding the intestinal bacterial flora by identifying and counting the
species samples.
The importance and role of the microbial flora within the host can also be assessed indirectly by determining the bacterial metabolites produced using the following methods:
The breath test or measurement of the hydrogen expired by a subject after ingesting lactulose.
Measurement of the activities of various bacterial enzymes in intestinal samples.
Tests for fatty acids excreted in the stools by gas chromatography reflecting bacterial metabolism.
Counting of a given species of Bacteroides from a mixture of bacterial DNA isolated directly from the stools and exposed to a species-specific labeled DNA probe has been suggested.[212,213] However, this method would not make it possible to differentiate between viable and nonviable bacteria, and a confirmation of the percentage of live bacteria would still have to be carried out using conventional dish culture methods.
B. Composition
The intestinal flora of a human weighs between 1 and 2 kg, that is, roughly the same weight as organs such as liver, brain, or lungs. The digestive tract houses about 1014 bacteria, which means there are more living entities in the flora than there are cells in a normal body and the bacteria consist of 500 species. This flora can be divided into two categories.
1. The dominant population, the effects of which on the host were the first to be understood.
2. The subdominant population, which accounts for less than 1% of the total bacterial population but which, according to studies,[146]may play a nonnegli-gible role in the equilibrium of the intestinal ecosystem.
Numerous studies have been carried out to define the composition of the intestinal flora. The main results, shown inTable 12,show some discrepancies, which can easily be explained from the choice of culture methods, isolation media, and counting methods.
Generally, the most numerous population in adults is Bacteroides (about 1010.3/g of feces). Thereafter, in decreasing order, come Eubacterium, Bifidobacterium, and then the Peptococcaceae. Of the aero-anaerobes, there are the enterobacteria (108.2) followed by Streptococcus, aerobic Lactobacillus, and finally Staphylococcus (104.4).[5]
C. Factors Affecting the Flora
1. Factors Ensuring the Equilibrium of the Intestinal Flora
The diversity of bacterial species and their quantity at various levels within the digestive tract can be preserved only by means of physical, chemical, and biological regulatory mechanisms.
Intestinal peristaltism results in the elimination of many microorganisms.
The acidity of the stomach maintains a low concentration of bacteria in the upper part of the digestive tract and destroys some pathogens.
The interactions that exist between various bacterial species are also important in maintaining the equilibrium of the intestinal microflora. It is possible to observe
Hoang-Dung TRAN and friends
symbioses between species as a result of the production of vitamins or amino acids or other metabolites which can be assimilated by other species and also of antagonisms due to the release of antibiotics, bacteriocins, or factors such as the volatile fatty acids.
2. Location and Physiology
The composition of the intestinal flora varies depending on the rate of transit and luminal secretions as well as the intestinal segment.[214]Thus, a given well-defined resident flora corresponds to each portion of the tract. The various factors active along the digestive tract result in qualitative and quantitative differences in the digestive flora, as shown in Table 13.Thus, the flora present in the proximal small intestine (duodenum and jejunum) consists of aerobic gram-positive microorganisms (streptococci and staphylococci) and a few yeasts.
This aero-anaerobic flora is subsequently replaced, within the ileum, by a flora consisting of E. coli and anaerobes such as Clostridium, Fusobacterium, and Bac-teroides (106total bacteria per mL).[5]This switch from a dominant aerobic population within the stomach to a strictly anaerobic population within the colon can be explained if we accept that the aero-anaerobic bacteria use any oxygen present, creating the redox conditions for the implantation of anaerobic species in more distal portions.
Finally, two parts should be distinguished within the colon: (a) the ascending colon, which contains mainly gram-positive bacteria, which have the primary role of sugar fermentation, and (b) the descending colon, in which the flora, known as “putrefaction flora,” consists mainly of gram-negative bacteria but also some gram-positive bacteria (Clostridium, Bacteroides).
Table 12 Fecal Flora of Different Human Groups
Bacterian group
Total bacteriaa 10.1 10.5 10.8 10.8 10.5
Aerobic or facultative anaerobic
Enterobacteria 9.3 8.8 8.0 8.2 7.8
Streptococcus 8.5 8.1 7.8 7.7 8.2
Lactobacillus 6.4 7.3 7.0 6.7 8.0
Staphylococcus 6.2 6.8 4.0 4.4 4.3
Yeast 3.5 4.0 4.2 3.7 4.6
Anaerobic
Bacteroides 8.6 8.2 10.4 10.3 10.0
Eubacteria 0 9.7 9.9 9.9 9.5
Bifidobacterium 9.3 9.9 10.1 9.8 9.4
Peptococcus 0 9.0 8.2 8.9 7.7
Clostridium 5.9 6.9 5.7 4.8 6.6
perfringens
Veillonella 5.6 6.3 5.2 4.8 6.1
alog cfu.
Source: Ref. [153].
Hoang-Dung TRAN and friends
This conventional theory, associating a region of the colon with a bacterial function and consequently with particularly dominant species[214]has been challenged by the work of Croucher et al.[206]Their studies of human colon biopsies tend to demonstrate that there is no specific location for the various species within the colon.
D. Age and Diet
This change in the flora is closely linked with the maturation of the digestive system, once more highlighting the importance of the reciprocal host-bacteria relationship. Bacter-iological examination of the feces shows that diet has little or no effect on the consti-tution of the dominant intestinal flora.[215]
E. Role and Effect of the Intestinal Flora
“The gastrointestinal tract is a complex ecosystem with characteristics which depend at each moment on a dynamic equilibrium between the host and the native bacteria.”[214]
Exogenous bacteria also influence all the bacteria within the intestinal flora. Some may be probiotic and others simply commensal, whereas others may be pathogens. The overall effect of the microbial flora on the host is generally evaluated by comparing an axenic animal with a holoxenic animal in which the flora has developed normally. This tool has been found to be most useful in demonstrating the effects of a given species or small group of species on the host.
1. Effect on Physiology of the Intestinal Wall and the Immune Defense System
The intestinal flora modifies the morphology of the mucosa and the rate of turnover and differentiation of epithelial cells. It also follows enterocyte maturation and development Table 13 Human Gastrointestinal Flora
Stomach Jejunum Ileum Colon
Total microbial concentration 0 – 103a 0 – 105 103– 107 1011– 1012 Strict aerobic or facultative anaerobic
bacteria
Enterobacteria 0 – 102 0 – 103 102– 105 104– 1010
Streptococcus 0 – 103 0 – 104 102– 106 105– 1010
Staphylococcus 0 – 102 0 – 103 102– 105 104– 107
Lactobacillus 0 – 103 0 – 104 102– 105 106– 1010
Fongy 0 – 102 0 – 102 102– 103 102– 106
Anaerobic bacteria
Bacteroides Rare 0 – 102 103– 106 1010– 1012
Bifidobacterium Rare 0 – 103 103– 107 108– 1012
Peptococcus Rare 0 – 103 103– 104 108– 1012
Clostridium Rare Rare 103– 104 106– 1011
Fusobacterium Rare Rare Rare 109– 1010
Eubacteria Rare Rare 103– 105 109– 1012
Veillonellae Rare 0 – 102 103– 104 103– 104
aNumber per gram of intestinal contents.
Hoang-Dung TRAN and friends of the velocities in the neonate.[214,216,217]
In the axenic animal, Simon and Gorbach[215]
observed an increase in the activity of enterocytic enzymes, in particular alkaline phospha-tase, disaccharidase, andb-glucosidase. All these studies have demonstrated the import-ance of the role of the flora, since it determines the uptake of nutrients and permits the formation of the ecological site for other bacteria.
An important role played by the flora is its action in cell maturation observed in the normal development of Peyer’s patches. These observations, first made as a result of histological investigation of the intestinal wall, have subsequently been confirmed by numerous studies demonstrating that resistance to various pathogens is conditioned by the presence of a flora. The intestinal bacteria ensure the maintenance of the immune status by providing repeated antigen stimulation throughout the human life span.
2. Bacteria as Nutrient Sources
The bacterial mass of the intestine is itself an important source of nutrients: thiamine, ribo-flavin, folic acid, vitamin B12, pantothenic acid, short-chain fatty acids, amino acids, and proteins, which are partially absorbed and used by the host.[43,218,219]
3. Metabolic Effects
The bacterial flora produces a very large and varied quantity of enzymes, which are used by the flora itself but also by the host. All the aspects of the intestinal metabolism of the host are influenced by the enzymatic activity of the bacteria it shelters and more particularly the anaerobic bacteria. We will list here some examples of the effects of bacterial metabolism on the host.
Enzymatic Action. These bacteria are able to compensate for enzymatic. deficiencies of the host if they are introduced in a sufficiently large number into the digestive tract. This is the case of lactobacilli ingested with yogurt, which can produce the lactase activity missing in lactose-intolerant subjects.[220]
Detoxification. Another important action of the intestinal bacteria is their involvement in the enterohepatic cycle and the detoxification of numerous substances and drugs.[221]
Thus, cholesterol is converted to form coprostanol and the bile salts to form bile acids and then lipocholic acid and other derivatives conjugated with amino acids such as glycine and taurine, facilitating their detoxification and elimination.[218] Rowland and Grasso[222]have investigated the degradation of the N-nitrosamines by the intestinal flora.
Production of Harmful Substances. In contrast, some microorganisms produce substances toxic to the host, notably histamine, tyramine, agmatine, cadaverine, ammonium, phenols, N-nitrosamines, and bacterial toxins.
4. Tumoral Action
Cancer of the colon is the second greatest cause of death in Great Britain and in the United States. It would appear that 90% of human cancers are due to the environment and could therefore be avoided. Major differences have been observed between cancer risks, but it would seem that neither the place where the population live nor their race is responsible, rather that the etiology of the disease is related to diet.[223]Considerable research has been carried out in an attempt to identify carcinogens.
A diet containing low fiber and high quantities of animal fats appears to promote the onset of cancer of the colon, but no directly active carcinogen has been isolated. Aries
Hoang-Dung TRAN and friends et al.[224]therefore believe that carcinogens may be produced in situ, probably as a result
of the enzymatic activity of the bacteria of the digestive flora on a harmless procarcinogen substrate derived from the diet. It is reasonable to think that the intestinal flora could produce or potentiate carcinogens or procarcinogens.
5. Effect of the Anti-infectious Barrier Toward Pathogens
The microbial population of the gastrointestinal tract forms a barrier against proliferation of exogenous pathogens.[224] One explanation may be that the colonization of the endogenous flora maintains the pathogens at a subclinical level by preventing the coloni-zation of the undesirable flora by competition for the substrate or epithelial receptors.[225]
This recently discovered role challenges the theory of the anti-infectious barrier effect suggested by Ducluzeau et al.[220] which holds that the barrier effect can be observed only in bacteria belonging to the dominant flora, and that pathogenicity can be effective only above a certain colonization threshold of the invasive bacteria. In fact, the subdomi-nant flora uses the endogenous substrates for its own metabolism, but also for the domisubdomi-nant population, preventing the proliferation of pathogenic bacteria and the adhesion of other organisms. The intestinal microorganisms inhibit the growth of the invasive pathogens by producing organic acids, particularly volatile fatty acids, deconjugating bile acids, which inhibit pathogenic bacteria in their conjugated forms, and producing bacteriocin and volatile acids, which stimulate peristaltism.[227,228]
XIV. BIFIDUM-INTESTINAL RELATIONSHIPS: PROBIOTIC