Fecal bacteriotherapy

Proper gut microbiota establishment begins in the moment we are born and is shaped by lifestyle and environmental factors in subsequent years. In some cases, the degree of dysbiosis is so severe that there is not turning back and practical dietary/lifestyle recommendations are useless.

Fecal bacteriotherapy

The logic behind this intervention is simple: it tries to "reset" the gut microbiota. It has shown promising results in intestinal bowel disease (IBD) and resistant Clostridium difficile infections. The following protocol is taken from Silverman et al (1). My intent is to facilitate information, not to encourage the realization of this protocol without medical supervision. Interested persons should consult with their doctors before doing any procedure of this nature. Donors and recipients should be examined carefully before the intervention. The complete set of tests can be consulted in the mentioned study.

Pretreatment

Recipients are initiated on maintenance therapy with oral Saccharomyces boulardii (probiotic), 500mg orally, twice per day. Metronidazole (500mg/3 times per day, PO) or vancomycin (125mg/4 times per day, PO) are also used. Both are antibiotics normally used against C.difficile infections.

Equipment

- 1 bottle of normal saline (200mL)

- 2 standard 2 quart enema bag kits (available at drug stores)

- 3 standard kitchend blenders (1L capacity) with markings for volume

Procedure

- Vancomycin/metronidazole should be stopped 24-48 hours before procedure.

- S.boulardii should be continued during the transplant and 60 days afterwards.

1.     Add 50mL of stool (volume occupied by solid stool) from the healthy donor immediately prior to administration (< 30 minutes) to 200mL of normal saline in the blender.

2.     Mix until getting a "milkshake" consistency.

3.     Pour mixture (approximately 250mL) into the enema bag.

4.     Administer enema to the recipient following the kit instructions. The patient should hold the infusate as long as possible and lie still as long as possible on his/her left side to prevent the urge of defecation. The procedure should be ideally performed after the first bowel movement.

5.     If diarrhea recurrs within 1 hour, the procedure may be immediately repeated.

Modifications and perspectives

This procedure was made to treat C.difficile infections. Accordingly, the antibiotics and the probiotic used aimed to eliminate C.difficile from the gut. However, there are certain modifications which can be useful for treating severe dysbiosis. First, broad-spectrum antibiotics can be used to wash out most bacterial species and reduce colonization resistance. In addition, utilization of probiotics such as Bifidobacteria or Lactobacilli during and after the treatment should help preventing colonization by enteropathogenic species. Why Bifidobacteria? The use of broad-spectrum antibiotics increases the risk for colonization of enteropathogens. Bifidobacteria competes and prevents colonization by these pathogens directly and indirectly, via production of antibacterial molecules (2). In addition, dysbiosis is characterized by low levels and expression of Foxp3+ Tregs, which compromises immune tolerance and promotes inflammation. Oral administration of B.infantis has been shown to increase expression of Foxp3+ and IL-10 in peripheral blood and to drive maturation of dendritic cells towards a regulatory phenotype (3), and certain strains of Bifidobacteria are capable of modulating the plasticity of Th17/Treg populations in human PBMCs (4). On the other hand, Lactobacilli has also shown protective properties (specially against vaginal infections) (5) and competes with enteropathogens for adhesion on intestinal epithelial cells (6). Importantly, the effects over Treg induction and T cell differentiation differ between strains from the same species. I should address this issue in future posts. One thing that is not emphasized in the above protocol is the importance of diet for maintaining a correct microbiota. This, in my opinion, is key to success.

It is worth noting that because of the nature of the procedure, the microbiota of recipient subjects is altered and reduced, but not completely eliminated such as seen with studies in fecal transplantation. The utilization of fecal transplantation in humans is promising and should result in better outcomes. Indeed, positive preliminary results from the FATLOSE trial (7, 8) have been recently published in which patients with metabolic syndrome improved insulin resistance and lipid profiles after feces infusion from healthy donors. The positive results seem to be correlated with increases in colonic butyrate concentrations. These results fit nicely with the ones found previously with fecal transplantation in obese mice.

Turnbaugh et al (9) found astonishing differences in the microbiome of obese mice, compared to lean mice (greater abundance of Firmicutes). Metagenomic analysis revealed that the obese microbiome is enriched for EGT (environmental gene tags) encoding many enzymes invoved in the break down of otherwise indigestable dietary polysaccharides. These included KEGG pathways for starch/sucrose metabolism, galactose metabolism and butanoate metabolism. Increased concentrations of butyrate and acetate were also observed, as the fact that obese mice were able to harvest more energy compared to lean mice (assessed by less energy remaining in feces by bomb calorimetry,). Despite equal amount of food consumed in both groups, colonization of lean mice with obese microbiota led to an increase in bodyfat percentage of approximately 47% after two weeks. The potential for fecal bacteriotherapy in the treatment of several diseases has been observed in different animal models of inflammatory and autoimmune diseases.

Thus, it seems possible that future therapies for obesity, metabolic syndrome and other inflammatory/autoimmune conditions will aim to modulation of the gut microbiota.

Silverman MS, Davis I, & Pillai DR (2010). Success of self-administered home fecal transplantation for chronic Clostridium difficile infection. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association, 8 (5), 471-3 PMID: 20117243

Is phytate really a problem?

As mentioned in a previous post, there is increasing evidence of adaptation to gluten consumption by humans. This adaptation is not genetic, but symbiotic. It appears that we have developed new symbiotic relationships with specific microorganisms to help us degrade gluten, and by doing so, being able to exploit an unnatural food source. 

Aside from resistance to degradation by mammalian enzymes and creation of neo-epitopes from partial gliadin digested peptides, one common reason given for avoiding gluten by paleo advocates is its phytate content (1, 2). Phytate is an anti-nutrient which binds to and form complexes with proteins, lipids, carbohydrates, and metal ions (zinc, iron, calcium and magnesium) thereby reducing their bioavailability. Phytate is the common name for myo-inositol-(1,2,3,4,5,6)-hexakiphosphate (InsP6). 

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From its chemical structure, we can see that it is basically a myo-inositol with six phosphate groups. The ability to degrade InsP6 is conferred by phytases. There are three types of phytases, namely, 3-phytase, 5-phytase and 6-phytase. The differences between these phosphatases is the position on the inositol ring at which the initial attack of a phosphoester bond takes place. Thus, attack by different phytases produce different isomers. Phytase production and activity in humans is relatively low (mainly in the small intestine) (3), so the greatest source of phytases is the gut microbial community . 

Gut flora phytase activity

Of the Bifidobacteria species which predominate in the human gut, the B. catenulatum group (B. catenulatum and B.pseudocatenulatum) is the most common. Haros et al (4) examined the InsP6 degrading capacity of B.pseudocatenulatum ATCC2919, isolated from the human gut. It was found that B.pseudocatenulatum is able to degrade InsP6 in sequential dephosphorylations (starting in the 6-position of the myo-inositol ring, followed by the 5-position). The solubility of mineral chelates of myo-inositol phosphates is related to the number of phosphates per molecule. InsP6 and InsP5 have adverse effects on mineral absorption. On the contrary, breakdown products with 1,2,3-grouping interact specifically with iron, increasing its solubility and preventing its ability to catalyse hydroxyl radical formation. Overall, the mineral-binding strength to inositol phosphates  becomes progressively lower when phosphate are removed from the molecule (with the exception of the 1,2,3-grouping mentioned above). B.pseudocatenulatum also showed selective adhesion to Caco-2 epithelial cells and tolerance to increased concentrations of bile, which reflects its adaptation to the human gut. A previous study (5) found that B.infantis is able to degrade 100% of InsP6, producing InsP3 as the main product. The optimal pH for the phytase activity of B.infantis was 6.0-6.5, with an activity of 51.2% at 37C; similar to that observed for B.pseudocatenulatum. Other Bifidobacteria species present in the human gut have also phytase activity, although to a lesser extent.

InsP6 antinutrient effect

Typically, InsP6 and fiber occur together in whole foods. This is problematic for analyzing the antinutrient effect of InsP6 as there is evidence that fiber also reduces mineral bioavailability (6). When given alone in animal models, InsP6 does not show toxic effects on bone minerals (7):

This suggests that its the combination of fiber and InsP6 which causes the antinutrient effect observed. 

The type of fiber seems to be important on mineral bioavailability. The addition of FOS to a diet high in InsP6 improves cecal absorption of minerals and stimulates bacterial hydrolysis of InsP6 (8, 9), counteracting the negative effects of high doses of InsP6. Inulin has also shown to improve calcium balance and absorption (10). The importance of the fiber type on the effects of phytic acid is highlighted by a study in which healthy women following the recommended daily intake of fiber-rich wheat bread (300g/day) showed impaired iron status independent of the phytic acid content (11).

Anti-cancer properties

InsP6 is a broad-spectrum antioneoplastic agent in vitro and in vivo (12). Structurally, InsP6 is similar to D-3-deoxy-3-fluoro-ptdIns, a potent PI3K inhibitor. Accordingly, InsP6 is able to inhibit PI3K and ERK phosphorylation (13), thereby inhibiting AP-1 activation. InsP6 has also been shown to activate PKC delta and decrease phosphorylation of Erk1/Erk2 and Akt, causing upregulation of p27-Kip1 and reduction of pRb phosphorylation (14). Other protective effects include the induction of apoptosis by inhibiting the Akt-NFkB pathway and increasing cytochrome C release (15), downregulation of constitutive and ligand-induced mitogenic and cell survival signaling (showing different effects on ERK1/2, JNK1/2 and p38 in response to different mitogens) (16), its antioxidant effect (17), enhancement of NK cell activity (18), modulation of expression of TNF-alpha and its receptors genes (19), inhibition of angiogenesis (20) and metastasis, by modulation of integrin dimerization, cell surface expression and integrin-associated signaling pathway (lack of clustering of paxilin and reduced FAK autophosphorylation) (21, 22). Utilization of InsP6 has been shown to offer some benefits during chemotherapy (23) and future trials are on their way.

Are whole grains inherently unhealthy?

Because whole-grains and legumes are high in phytic acid, it is plausible to hypothesize that intake of these foods will reduce to some extent the risk of developing cancer. Whole-grain intake has been associated with reduced risk of cancers (24, 25) as well as intake of legumes (26). However, some studies have found no association (27, 28). Because of the nature of these studies, it is not possible to draw causative conclusions. Most people eating the supposedly healthy foods have low intakes of harmful foods, so the decreased risk in some studies might be due to the exclusion and not the inclusion of some foods. In either case, most studies have not observed an increased cancer risk associated with these foods*. Other food sources rich in phytic acid include nuts and cocoa. 

Conclusions

The dangers of phytic acid have been overestimated. Contrary to popular the paleo belief, phytic acid might be beneficial in small doses and might have anticancer effects. As seen with gluten degradation by Rothia species, the phytase activity present in some exclusive human Bifidobacteria shows that adaptation to wheat/grains is indeed happening. Once again, the microbiota plays a dominant role.

From epidemiological data, foods with high phytate content are not associated with increased risk for several chronic diseases. As association doesnt means causation, we cannot conclude that whole-grains are healthy but we cant also conclude that whole-grains are unhealthy. With the increasing attention to paleolithic and similar diets, it is of utmost importance that all evidence is critically analyzed and reviewed. Making unsupported statements and cherry-picking data would only cause rejection by scientists. Dogma is not good in science (or in anything else, for that matter).

I dont recommend whole-grains and legumes because there are foods more nutritious, as well as because whole-grains and legumes are very high in carbohydrates. The potential benefits of phytate can be obtained by eating other phytate rich foods, such as nuts and cocoa; as well as soluble fiber and oligosaccharides as the main dietary fiber type. The problem with high levels of phytate is only relevant when the diet is deficient in micronutrients and essential food sources. Finally, maintaining a proper gut flora is essential for phytic acid metabolism and adequate mineral absorption. 

*Any evidence of a significant increased risk from these foods would be greatly appreciated.

Haros M, Carlsson NG, Almgren A, Larsson-Alminger M, Sandberg AS, & Andlid T (2009). Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential. International journal of food microbiology, 135 (1), 7-14 PMID: 19674804

Haros M, Bielecka M, Honke J, & Sanz Y (2007). Myo-inositol hexakisphosphate degradation by Bifidobacterium infantis ATCC 15697. International journal of food microbiology, 117 (1), 76-84 PMID: 17462768

Bifidobacteria, butyrate and carbohydrates

In a previous post, john asked:

Regarding your old post on ketogenic diet and microbiota, why do you think bifidobacterium decreased on low carb? I would generally guess this is a negative...?

I cited two studies on low carbohydrate dieting and gut microbiota composition, one by Duncan et al (1) and the other by Brinkworth et al (2). They showed a negative effect of reducing carbs on gut flora, measured by species composition (16S RNA) and SCFA. They both analyzed fecal samples. In general, fecal samples are reliable and make easier to study colonic SCFA metabolism. However, they are an indirect method of quantification. Of the three main SCFA produced in the colon, only acetate has shown a correlation between fecal concentration and absorption (3):

Copyright © 2011 by the American Society for Nutrition

The data shows a negative correlation (r=-0.834) between acetate absorption from an infusion and fecal acetate concentration. This means that the fecal concentration of acetate might reflect absorption rather than production, in an inverse manner (less acetate in fecal samples equals more absorption). In this study, neither propionate or butyrate showed a correlation between absorption and fecal concentration. 

SCFA in the Duncan et al. study

Acetate, butyrate and propionate concentrations in fecal samples from the Duncan et al. study are shown below:

SCFA concentrations (mM) for fecal samples. M=Maintenace; HPMC= High-protein, moderate-carbohydrate; HPLC = High-protein, low-carbohydrate. Mean values.

As the intake of carbohydrate decreased, there was a parallel reduction in all three SCFA. 

SCFA in the Brinkworth et al. study

Acetate, butyrate and propionate concentrations in fecal samples from the Brinkworth et al. study are shown below:

Fecal SCFA concentrations (mM) after 8 weeks of either a low carbohydrate (LC) or high carbohydrate (HC) diet. Mean values. 

As seen in the Duncan et al. study, after 8 weeks with a low-carbohydrate diet, SCFA concentrations were reduced, although not as drastically. 

Analysis and interpretation of the data

Both studies show a clear correlation between carbohydrate intake and SCFA concentration in fecal samples. The magnitude of the changes between individual SCFA might be due to differences in the intervention time (4 weeks vs. 8 weeks). 

As shown by Vogt and Wolever (see above), acetate concentration in fecal samples reflect more precisely acetate absorption rather than production. Thus, lower fecal acetate levels with reduced carbohydrate reflect more acetate absorption (or utilization, see below). 

Most focus has been given to the apparent reduction in butyrate levels, which may compromise colonic health. In this regard, low carbohydrate diets might be detrimental for colonic health because of reduced butyrate production. For assessing the validity of this statement, we must look at colonic butyrate metabolism. 

Colonic butyrate metabolism 

Approximately, 95% of the butyrate produced in the colon is absorbed. This is why fecal concentrations are not a good guide to production rates: a very high proportion of the SCFA is taken up by the colonic mucosa (4). Butyrate is produced from two molecules of acetyl CoA, yielding acetoacetyl CoA, which is further converted to finally butyryl CoA. This metabolite can be converted to butyrate via butyrate kinase or butyryl CoA:acetate CoA transferase.

Butyrogenic substrates include starch, inulin and xylan. But certain species are capable of producing butyrate from acetate. Synthesis of butyrate from acetate is performed via the butyryl CoA:acetate CoA transferase pathway, which seems to be the most prevalent route of butyrate synthesis by human gut bacteria (5). So, while glucose is needed for butyrate synthesis, acetate seems to be the main substrate for butyrate formation. The predominance of butyrate synthesis from the acetate dependent pathway might reflect a selective advantage for bacteria which transform acetate to butyrate in the colon, where acetate concentrations are high. 

Overall, the reduction in acetate and butyrate fecal concentrations may be translated to increased absorption and reduced excretion. Butyrate can be synthesized from acetate, which reduces the concentration of both SCFA in feces. The determined Km for butyrate transport in the colon has been found to be 14.8 +/-3.6 mM (6) and 17.5 +/- 4.5 mM in the proximal colon (7). The apparent saturation kinetics showed by butyrate transport across the colonic luminal membrane could further explain the results seen in the studies mentioned above: increasing the carbohydrate content in the diet would augment the number of glucose-dependent butyrogenic bacteria, increasing the colonic production and concentration of butyrate. Because transport of butyrate is saturable, excess butyrate is excreted, producing increased levels in feces. 

The case for Bifidobacteria

The change in Bifidobacteria concentrations after the low carbohydrate diet is due to the presence of an important number of bacteria capable of degrading glucose/starch. In this scenario, reduced carbohydrate availability would reduce the number of total Bifidobacteria (at least certain species). This does not mean that this is bad per se. It is important to determine the specific Bifidobacteria which are responsive to diet. For instance, B.longum seem to be capable of catabolizing not only dietary oligosaccharides, but also glycoproteins and glycoconjugates from the host; as well as nucleotides (8). Moreover, gut Bifidobacteria (as shown by the genomic analysis of B.longum), are capable of adapting to different carbohydrate substrates depending on their availability (9). In addition, metabolic-crossfeeding occurs between Bifidobacteria and other species. For example, E.hallii, a butyrogenic bacterium, is unable to grow on pure starch by itself. Co-culture of this bacterium with B.adolescentis stimulates its growth and butyrate synthesis, paralleled by a reduction in lactate levels (10). The scheme is pretty simple: B.adolescentis is capable of fermenting starch, producing lactate which serves as substrate to E.hallii. Other lactate-independent mechanisms of cross-feeding have also been observed in FOS and oligofructose-only co-cultures of B.longum with Roseburia intestinalis or Anaerostipes caccae, which are cabaple of producing butyrate and consuming acetate (11).

Because of its complexity, the specific mechanisms by which certain Bifidobacteria could be beneficial are unknown, although there is evidence of health benefits from increasing gut Bifidobacteria (12, 13, 14). There are some issues with interpreting the evidence in this topic:

•  Many authors don't determine the exact species being studied (take all Bifidobacteria as a group).
• Supplementation is done with different strains and the long-term effects are not known, because bacteria supplemented via diet are treated as allochthonous. 
• Genomic inspection has shown that Bifidobacteria are metabolically very flexible. Adaptation to substrate variations might take longer than 8 weeks. 
• There is metabolic-crossfeeding occuring between bacteria. This is a highly complex network of connections for which we are only starting to get an initial picture. 

Having this in mind, I cant assure either that a low carbohydrate diet is not harmful to the gut microbiota. As far as the evidence goes, we can only speculate and formulate hypotheses. And useful hypotheses should be based on logic and evolutionary inference. We should ask not only "how" but also "why". In this case, we are not going to focus on the "how" but on the "why"; to put it formally, why an increased intake of starch is associated with an increase in Bifidobacteria? What is the evolutionary basis?

One important protective role of Bifidobacteria is preventing colonization of enteropathogens by reducing their adhesion to intestinal epithelial cells. This has been shown directly for E. coli and S. typhimurium (15). Other commonly problematic Enterobacteriaceae include Klebsiella and Shigella. Growth of these pathogens is stimulated by high glucose-low oxygen conditions. The selective advantage of having responsive Bifidobacteria in the gut might be protection. As increased glucose concentrations favor the development of an adequate environment for growth of these pathogens, there has to be a mechanism by which the composition of the normal microbiota is maintained. So there is a parallel increase in Bifidobacteria with increasing concentrations of dietary carbohydrates to restrain colonization of pathogenic anaerobes. The fact that certain species of Bifidobacteria can metabolize different oligosaccharides and adapt to the substrate availability supports this hypothesis. I might elaborate more on this in subsequent posts. 

Conclusions

Low carbohydrate diets seem to reduce the fecal concentration of SCFA in the short term. Some adaptation seems to occur, judging by the differences between the study periods (4 weeks vs. 8 weeks). Fecal concentrations of SCFA are not good indicators of SCFA colonic production. Conversely, they rather reflect excretion (butyrate) and absorption (acetate). Butyrate can be produced from different substrates, of which acetate is the main precursor in the human gut. There is a reduction in the levels of Bifidobacteria detected in stool samples, proportional to the decrease in carbohydrate in the diet. Although no individual species where identified, studies have shown that Bifidobacteria are capable of adapting to substrate availability and cross-feed with other bacteria. The evolutionary basis for increased Bifidobacteria in response to sugar might involve a protective mechanism against colonization of enteropathogenic bacteria, such as E. coli, Klebsiella, Shigella and Salmonella.

Duncan SH, Belenguer A, Holtrop G, Johnstone AM, Flint HJ, & Lobley GE (2007). Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate and butyrate-producing bacteria in feces. Applied and environmental microbiology, 73 (4), 1073-8 PMID: 17189447

Brinkworth GD, Noakes M, Clifton PM, & Bird AR (2009). Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty acids and bacterial populations. The British journal of nutrition, 101 (10), 1493-502 PMID: 19224658