Dear Editor

Tamboli et al[1] have initiated the discussion about dysbiosis that is rather a forgotten term.

Most of the Medical Dictionaries have yet to define this term. Dysbiosis as described by Metchnikoff (1910),[2] a colleague of Louis Pasteur, can be explained as the process rendering abnormal condition of the native gut micro-biota. Circumstances suggest that dysbiosis preceding the rotavirus infection in infants and children determine the severity of disease.

Professor Stig Bengmark, Lund University, Sweden, regards the native gut microbiota as the non-somatic organ of the body (personal communication).[3] Could we use a term gut eco-organ for this? The non-pathogenic native (and autochthonous as explained by Dubos et al.[4] and not the transient/ allochthonous) gut micro-biota can safely be regarded as an eco-organ (or micro-biota organ). This organ is made up of diverse micro-biota that follows the rules of open ecosystem. It is not made up of body cells hence the prefix eco- is used. It produces metabolites useful for the body, participates in the metabolism of the body, and is a part of body’s immune system hence the suffix –organ is used. Similarly, introducing the terms vaginal eco-organ or skin eco-organ would simplify the discussion of pathogenesis of diseases associated with these.

In human species, the microbial population exceeds by at least 10- fold the population of human cells in the body. Hence the human animal can be regarded as the vessel that has evolved to permit the survival and propagation of microorganisms. Alternatively, the human can be regarded as a biological organism composed of both eukaryotic animal cells and eukaryotic and prokaryotic microbial cells.[5] The concept is supported by the lifetime work of the R. Dubos and R. W. Schaedler from Rockfeller University, New York, USA; B. Gustafsson and T. Midtvedt from Karolynska Institute, Stockholm, Sweden; T.Mitsuoka, from University of Tokyo, Japan and P. Raibaud and R. Ducluzeau from Institut National de Recherches Agronomiques, Jouy-en-Josas, France.[6]

The latter concept anticipated by Savage has been examined. In simpler words, human animal has genetic plus somatic plus micro-biotic cells or human is a macroorganism having micro-biotic cells.

Here is a model given by Bry et al.[7] to show how a bacterium behaves like an organ of the mammalian physiological system. Bacteroides thetaiotaomicron was the component of the normal gut microbiota of conventionally housed experimental NMRI mice. These mice produced fucosylated glycoconjugates and an alpha1, 2-fucosyltransferase messenger RNA in the epithelial cells of small intestine. The epithelial cells of small intestine of germ free NMRI mice lack this fucosylation program. The fucosylation program was restored in the gnotobiotes produced from the germ free animals using Bacteroides thetaiotaomicron. When the mice were modified by transgenesis (insertion of transposon) they lost their ability to use L-fucose as a carbon source. Gnotobiotes produced using such a modified mice did not restore the small intestine epithelium fucosylation program. This indicates that particular bacteria may be suitable for a particular host.

The intestinal niches are unique.[8] Hence the autochthonous microorganisms evolved in those niches are also unique. A microorganism native to one niche may be transient to another.[9] It is felt that isolation of autochthonous microorganisms from these niches and their cultivation in vitro is difficult by conventional methods and appropriate environment resembling the niche in the gut is yet to be created through the application of tissue engineering. The microorganisms isolated from the gut and propagated in vitro and used as probiotics, are most likely to be transient microorganisms present in the gut and does not seem to be true to most of the niches in the intestine.

The terms probiotic, symbiotic or antibiotic are used to denote the relationship between the life forms. The eco-organ has to be considered as the part of the body or macroorganism. The body is a single (although complex) life presentation. If the eco-organ concept is recognized then the relationship of eco-organ with the body as a whole is a self-dialogue. The emotional stress cause disturbance in the stable composition of gut microbiota i.e. gut eco-organ [10-12] and the physical exercise done by the individual may have impact on the health of the organs including eco-organ. Recognizing the concept of eco-organ makes the subject understandable as discussed below.

The body (inclusive of eco-organ) and probiotics can have a dialogue. These elements either have symbiotic relationship i.e. the probiotics exert symbiosis on the body by producing metabolites that can be called as symbiotic (e.g. short chain fatty acids, other fatty acids, amino acids, peptides, polyamines, carbohydrates, vitamins, numerous antioxidants and phytosterols, growth factors, coagulation factors, various signal molecules such as cytokine-like bacteriokines) and the results are beneficial for the body alone or the probiotic relationship where both the body and probiotics are benefited mutually by each other by probiosis. The term symbiotic was coined for the combined action (synergy) of prebiotics and probiotics and preferably spelled as synbiotics, however it is now used in the sense as described above.[13] The amino acid residues like glutamine that are heat labile may be produced in vivo by symbiotic supplement.

It appears most of the time that the benefits provided by the probiotics to the body outweigh the benefits provided by the body to the probiotics. Therefore, the probiotics are not likely to be permanent residents of the gut. After seeding of probiotics in the gut, they may adhere to mucus and may or may not proliferate to some extent and reach to plateau and then decline for the convenience of the autochthonous microbiota. It is quite possible that some bacteria are evolved for performing the role of probiotics however; work in this direction is still not carried out. The body (inclusive of eco-organ), the probiotics and the pathogenic microorganisms can have a trilogue. Both the body and the probiotics may exert antibiosis against the pathogenic microorganisms independently and/or through their interaction.

It would be interesting to observe whether microbiota of probiotics preparation mutates or adapts to become a member of the body. Bacteria influence other bacteria either by the release of signal molecules [14] or by the transfer of genetic material between bacteria.[15] It is known that antibiotic resistant bacterial species evolve in the gut.[16-18]

Food must nourish the native microbiota for prevention of disease:

Food contains several nutrients that nourish body including eco- organs. Malnutrition weakens immune system. However, food may also contain certain functional ingredients. Some of them are discussed.

Fresh fruits and vegetables are source of dietary nitrate. Some of the dietary nitrates are absorbed and re-secreted in the saliva. The bacterial enzymes produced in the mouth reduce some of the nitrate to nitrite. When saliva is swallowed the acidic gastric juice reduces nitrite to nitric oxide.[19] Human milk is a source of nitrate [20,21] and nitrate-reducing microorganisms.[22] A high Concentration of nitrite in the stomachs of breastfed neonates is suggested to enable a high production of Nitric Oxide (NO). It is proposed that breast milk is important in regulating the mucosal blood flow and gastric motility and in achieving bacteriostasis via induction of NO generation in the neonatal stomach.[21] The nitric oxide is antibacterial and antiviral.[23,24] Whether it has any role in controlling gastroenteritis due to rotavirus is not known. It appears that some of the weaning diets might be reducing supply of nitrate. The severity of acute infectious gastroenteritis could be related to this supply. Nitric Oxide is also an acute phase reactant. It is also thought to be mutagenic.[25]

The human milk contains array of protective substances. The anti- rotavirus effect of lactoferrin [26-28] and lactadherin [29-34] are known. Human milk may also contain antirotaviral antibodies.[35-44] In addition food is source of prebiotics and probiotics.

Understanding the dysbiosis with reference to the weaning:

We now understand that dysbiosis in our species could be due to the abnormal food (drifting away from diets having Palaeolithic pattern), or anti-metabolites produced by pathogenic microorganisms or by antibiotics taken as drugs. The dysbiosis is not a unique condition. The dysbiosis caused by eating high meat low fibre diet may be different than the one caused due to intake of antibiotics. Similarly, dysbiosis expressed by flatulence and bloating may be different than dysbiosis expressed by chronic diarrhoea or dysentery. The dysbiotic body has dysfunctional immune system.[45,46] Some of the dysbiotic condition in the gut promotes translocation of gut microorganisms that may have serious consequences like organ failure.[47,48]

We are living the modern life with a body that is genetically the same like body of a human in Palaeolithic era. As an adult we may be adapted to modern foods based mostly on cereal and milk products but the babies undergoing weaning may be facing such food as a strange one. How the Palaeolithic children were weaned is difficult to say but certainly cereal and milk based diets were not available to them. Rotavirus diarrhoea occurs in neonate animals but in humans it occurs mostly during weaning indicating the possibility that an animal virus had an opportunity created by typical dysbiotic condition due to modern weaning foods to evolve as a human pathogen.

Piglets are used as an animal model for human rotavirus gastroenteritis. The piglets don’t show susceptibility to the human rotavirus unless they are derived in germ free state by caesarean operation and raised with restricted and known microbiota as gnotobiotes [49] a condition that can be called as induced dysbiosis.

Diarrhoea associated with rotavirus:

Breast-feeding helps in preventing the nosocomial rotavirus infection and prevention of diarrhoea in infected infants. Asymtomatic virus shedding was also seen in non-breast-fed infants.[50] It is recommended that breast-feeding should continue for the first two years [51] and that should be exclusive for the first 6 months of life.[52] The natural age of complete weaning for human children is thought to be between 2.3 and 7 years of age.[53] Ideally, child should guide the mother when to stop the breast-feeding completely and not the vice versa. Cooperstock and Zedd [54] divided the development of the intestinal microbiota in infants and children into 4 phases as follows:

Phase-1:
Acquisition of microbiota from transient one over the first 1–2 wk occurs during phase 1. This phase extends 2 weeks after the birth.

Phase-2:
Period of exclusive breast-feeding after the phase 1 till the start of food supplementation is phase 2.

Phase-3:
Period of supplementation after the phase 2 till weaning is phase 3

Phase-4:
From weaning till the growth of biota resembling adult is phase 4.

Nutrition of a child up to phase-2 i.e. exclusive breast-feeding is ultimate. The infection of rotavirus causing acute disease is uncommon during this period. Although the composition of breast milk of a mother of Palaeolithic era could be somewhat different than that of the modern mother by and large pattern of nutrients would be similar. The child receives a supplemental diet during phase 3 and a weaning diet during phase 4. These diets may range from Palaeolithic pattern to a complete non -Palaeolithic pattern/ modern pattern made up of highly processed foods. The extent of dysbiosis could depend upon the type of food pattern of these diets.

Bottle-feeding is common and may happen quite early in some infants. Lactic acid bacteria dominate the microbiota of breast-fed infant as evidenced by presence of lactic acid in the faeces. The lactic acid was absent in the faeces of formula-fed infants. The formula-fed infants also had higher faecal ammonia and other potentially harmful bacterial products.[55] The authors have asked 3 questions that could be answered as follows:

Should we retain a breast-fed style flora with limited ability to ferment complex carbohydrates?
The answer is probably "No" as it is difficult to maintain the breast-fed style flora. It is also impractical.

Can pro- and prebiotics achieve a flora with adult characteristics but with more lactic acid bacteria in weaned infants?
Supplementation of food and weaning food should have natural pre- and probiotics. It must be understood that the infant has a body similar to a Palaeolithic infant. Modern and processed food for supplementation and weaning is an artefact. The character of weaning diet having Palaeolithic- like human diet pattern should be worked out.

Are there any health risks associated with such manipulations of the flora?
Health risks are likely to be low if natural and hygienic food having high prebiotic value [56] is planned carefully for stabilization of flora. Fresh fruits and nuts may carry lesser health risks than animal milks.

In the infant or child the dysbiotic condition created due to faulty weaning as described above may prevail causing loss of resistance to rotavirus leading to severe acute diarrhoea on exposure to the virus. However, the infection of rotavirus itself may induce dysbiosis.[57] The dysbiotic conditions prevailing before and after infection are naturally different.

The products generated by dysbiosis could be toxic, carcinogenic, mutagenic or teratogenic and may affect the whole body. The abnormal digestive products present in the gut are known as Ama in Indian medicine.[58] These products may be called as dysbiotics (The term is not listed in the dictionary or medical texts) and could be the markers of dysbiosis. The diet having pattern of Palaeolithic human diet and/or predominantly raw vegan diet and life-style found on natural living could be the preventive measure against dysbiosis.

Standardization of the assay of dysbiosis is required:

Nugent score is a simple and convenient method for quantitative estimation of vaginal dysbiosis.[59] It requires only smear of vagina and Gram staining. Unfortunately, a method for simple and quantitative estimation of gut dysbiosis giving appropriate index is yet to be developed and approved by the experts. It is the need of the day.

Methods to be devised for statistical bioassay of gut dysbiosis index could be molecular, biochemical, clinical or their combinations. The methods for field studies, particularly in developing countries, need to be simple and rapid.

Culture independent methods for stool analysis for the study of intestinal microbiota are preferred [60] for the simple reason that the outcome of culture depends on representative stool sample that is difficult to obtain. Cultural conditions promote growth of certain species of bacteria and may not support the growth of some fastidious autochthonous bacteria. Culture of mucosal microbiota is different than the stool microbiota as mentioned by Tamboli et al.[1]

However stool cultures have been tried and may be essential for identification of toxigenic Clostridium difficile.[61] Stool may not be uniform and a randomly picked up sample of a part of stool is not a desirable material particularly for a quantitative assay of metabolic markers of mucosal microbiota. A better sample of total bowel content for dysbiotic studies of a person appearing normal may be obtained by collection of complete morning stool followed by an enema by inert material, mixing thoroughly and then picking up the proportional sample.

Rectal swab samples have been used successfully for studying molecular epidemiology of antibiotic resistance Gram-negative bacilli in a neonatal intensive care unit during a non-outbreak period [62] and for isolation of Staphylococcus aureus strains from healthy infants.[63] Although these are convenient samples these have yet to be demonstrated as an acceptable for quantitative dysbiosis studies.

Blood and urine provide better samples for the quantitative assay of dysbiosis than stool or rectal swab. The metabolic activity of gut microbiota is determined by the amount of enzymes produced by them and their activity results in certain products that can be detected in faeces,[64], blood [65] or urine.[66]

Urease producing microorganisms hydrolyse urea and produce ammonia that raises the pH of the stool. This action is linked with the colon cancer.[67] The urease in the stool, stool ammonia and stool pH when raised are the markers of intestinal dysbiosis. Urease may be produced by several bacterial species and thus the amount of urease rather than the individual population of bacteria producing it carries significance. Moreover, it is easier to study stool urease than studying individual bacterial populations. The fresh stool samples give correct values for the stool urease assay. The values may decline during storage of stool sample.

Such procedures are usually used in controlled experiments and quantitative values for routine clinical application are yet to be defined. Another important thing in this regard is that quantitative rather than qualitative test carry significance.[68]

The bacterial tryptophanase degrades tryptophan to phenol, which is a nephrotoxin[69] and can be detected in the urine. Thus urinary phenol is a marker of gut dysbiosis.[70] It is interesting to see e.g. what would be the course of rotavirus in terms of pathogenesis that has crossed the intestinal barrier in the presence or absence of agents like phenol in the blood.

Similarly, degradation of amino acid residues by bacterial decarboxylase produces amines that are toxic. Metchnikoff recognized this toxicity almost a century ago.[2] The free primary amines particularly p-tyramine present in the faeces was more in the infants (1-18 months) with gastroenteritis than healthy ones. The amine concentration was significantly related to the diet and was low in breast fed infants than infants fed cow milk.[71] Since the amines are deaminated in the liver, stool sample rather than blood sample is preferred for the study of dysbiosis in this case.

Activities of gut bacterial azoreductase [72] and nitroreductase [73] are harmful to the body and the quantitative assay of these enzymes also provides information on gut dysbiosis.

Oestrogen is conjugated in the liver and excreted in the bile. However its deconjugation occurs in the gut due to bacterial beta- glucuronidase releasing oestrogen that is reabsorbed raising the level of this hormone in the blood. This is implicated in the breast cancer. The same enzyme also deconjugates the bile releasing carcinogenic secondary bile products.[74,75]

Isolation of nucleic acid, Community finger printing by Denaturing Gradient Gel Electrophoresis (DGGE)/ Rapid Analysis of random amplified polymorphic DNA, Sequence analysis using rDNA database and preparation of phylogenic tree, Hybridisation in situ / whole cells/ Dot Blot- Southern blot /Quantitative Slot Blot is a protocol suggested by [76] and McCartney [77] for the study of gut microbial populations. Favier et al.[78] reported a method of molecular monitoring of succession of bacterial communities from neonate to infant using faecal samples. Recently, Vaughan et al. [79] and Tannock [80] have reviewed the Molecular methods for exploring the intestinal ecosystem.

These methods may be modified for detection and quantitation of dysbiosis. Having standardized these techniques to calculate a sort of Dysbiotic Index, a great asset for research would be developed. Predisposing conditions of not only rotavirus but also several other diseases may be seen clearer. At this juncture no technique may be called as the best but most of them are complementary to each other.

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74. Hill MJ, Melville DM, Lennard-Jones JE, Neale K, Ritchie JK. Faecal bile acids, dysplasia, and carcinoma in ulcerative colitis. Lancet. 1987 Jul 25;2(8552):185-6.

75. Bennet JD. Ulcerative colitis: the result of an altered bacterial metabolism of bile acids or cholesterol. Med Hypotheses. 1986 Jun;20 (2):125-32.

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80. Tannock GW. Molecular methods for exploring the intestinal ecosystem. Br J Nutr. 2002 May;87 Suppl 2:S199-201.

Dear Editor

We thank Dr Szilagyi for his very interesting comments regarding dysbiosis in IBD.[1]

The main question remains as to why beneficial bacteria such as Bifidobacteria might be lacking in IBD.[2] Dr Szilagyi describes an interesting hypothesis of colonic prebiotic deficiency as a possible mechanism for dysbiosis. A suggestion is made that this deficiency could be linked to increased proximal small-intestinal permeability with enhanced absorption of prebiotic substrate, causing a relative deficit of prebiotics distally. Certainly, the phenomenon of increased small bowel permeability has been documented in Crohn's disease; it's importance in ulcerative colitis is less clear, however. Since lactulose is not a major component of the normal human diet, long-term epidemiologic dietary trends should also have to be consistent with a significantly decreased intake of common prebiotic substrates, if this hypothesis is correct. Unfortunately, there is currently little clinical evidence to support or refute this theory, as dietary studies in IBD have been subject to many biases inherent in their study design.

Bifidobacteria are strongly glucidolytic and show nearly no growth in the absence of fermentable sugars or polysaccharides. One group of good substates are mucins, which are often increased in Crohn's disease. Perhaps differences in ease of glycosylation between some substrates affect the flora's ability to metabolize them. This has not been well studied in IBD. There are likely to also be other influences on the host's dominant flora, including genetic factors. Observations among some of our healthy cohorts with low levels of Bifidobacteria have revealed highly variable responses of Bifidobacterial counts to prebiotic treatments. Whether a poor response might represent a risk factor for IBD is also an interesting question.

References

(1) Szilagyi A. Dysbiosis as a prerequisite for IBD [electronic response to Tamboli et al. Dysbiosis in inflammatory bowel disease] gutjnl.com 2004http://gut.bmjjournals.com/cgi/eletters/53/1/1#244

(2) CP Tamboli, C Neut, P Desreumaux, and JF Colombel. Dysbiosis in inflammatory bowel disease. Gut 2004; 53: 1-4

Dear Editor

The excellent synopsis by Tamboli et al raises the issue of the possible role of dypbiosis on the pathogenesis of IBD.[1] Although not clinically proven yet multiple studies do show benefit of the use of probiotics in both Crohn’s disease (CD) and ulcerative colitis (UC).[2] These findings coupled with studies showing a reduction of lactic acid producing bacteria (LAB) in both major forms of IBD suggest that such bacteria may have a protective role against disease.

The authors do pose the question when dysbiosis sets in and whether it is a secondary phenomenon or is it truly involved in pathogenesis. We have previously shown that a group of patients with IBD colitis in remission cannot be induced to colonically adapt to lactulose intolerance by consuming the candidate prebiotic lactulose over 3 weeks.[3] This phenomenon was particularly evident in patients with CD. Colonic bacterial adaptation can be clinically tested by measuring breath Hydrogen before and after exposure for a limited time in subjects intolerant to larger doses of a sugar (e.g. lactulose).[4] Since lactulose specifically expands populations of Bifidobacteria [5,6] and CD in particular, maybe a Bifidobacteria deficient condition our results supported the notion of dysbiosis in CD (and possibly UC as well). However the mechanism of loss of Bifidobacteria and other LABs as pointed out by Tamboli et al. remain unclear. One possible explanation we postulated in our study was that the small amounts of lactulose given to IBD patients in our study might not have reached the colon at all. It has been previously shown in both UC and CD that when lactulose, a universally malabsorbed disaccharide, is given to such patients blood levels of lactulose are nevertheless measurable.[7] In other words, this previously malabsorbed disaccharide is absorbed intact in IBD patients. Intestinal permeability defects in IBD have been clearly described.[8] It is thus possible that the failure to adapt to lactulose was due to short circuiting of the prebiotic away from intestinal flora. How long such a possible process continues pre illness is unclear. To date the longest demonstrated change in intestinal permeability to time of development of clinical CD was 8 years.[8] It is thus possible that reduction of protective LABs precedes the onset of IBD permitting the emergence subsequently of a more aggressive flora. This sequence of events could trigger consequent disease development. In addition, all the factors mentioned by Tamboli et al. could contribute to the development of dysbiosis as well. Therefore, particularly in westernized societies dysbiosis may be an early event preceding subsequent development of IBD.

References

(1) Tamboli CP, Neut C, Desreumaux P, Colombel JF. Dysbiosis in inflammatory bowel disease. Gut 2004;53:1-4.

2. Hamilton-Miller JMT. A review of clinical trials of probiotics in the management of inflammatory bowel disease. Infect Dis Rev 2001;3:83-87.

3. Szilagyi A, Rivard J, Shrier I. Diminished efficacy of colonic adaptation to lactulose occurs in patients with inflammatory bowel disease in remission. Dig Dis Sci 2002;47:2811-2822.

4. Flourie B, Briet F, Florent C, Pellier P, Maurel M, Rambaud JC. Can diarrhea induced by lactulose be reduced by prolonged ingestion of lactulose? Am J Clin Nutr 1993;58:369-375.

5. Terada A, Hara H, Kataoka M, Mitsuoka T. Effect of lactose on composition and metabolic activity of the human fecal flora. Microbial Ecol Health Dis 1992;5:43-50.

6. Clausen MR, Mortensen PB. Lactulose disaccharides and colonic flora. Drugs 1997;53:930-942.

7. Oriishi T, Sata M, Toyonaga A, Sasaki E, Tanikawa K. Evaluation of intestinal permeability in patients with inflammatory bowel disease using lactulose and measuring antibodies to lipid A. Gut 199;36:891-896.

8. Hollander D, Vadheim CM, Brettholz E, Petersen GM, Delahunty T, Rotter JI. Increased intestinal permeability in patients with Crohn’s disease and their relatives. Ann Int Med 1986;105:883-885.

9. Irvine EJ, Marshall JK. Increased intestinal permeability precedes the onset of Crohn’s disease in a subject with familial risk. Gastroenterology 2000;119:1740-1744.