2. 2 Brandt
cellular processes), metaproteomics (study of all proteins in an eign antigens. (Figures 1 and 2). We know that reduced microbial
environment), and metatranscriptomics (study of all RNA mole- stimulation during infancy results in slowed postnatal maturation
cules produced in a population of cells). Microbial communi- of the immune system and delayed development of an optimal
REVIEW
ties differed remarkably at each of the 15 (male) to 18 (female) balance between TH1 and TH2-like immunity (9). During the first
Human Microbiome Project-sampled body sites (nasal pas- year of life, the total number of IgA-, IgG- and IgM-secreting cells
sages, oropharynx, skin, stool, and vagina) and the diversity of is lower in infants born by vaginal delivery than in those born by
each habitat’s signature microbes varied widely among healthy cesarean section, possibly reflecting excessive antigen exposure
individuals, with further variation not just dependent upon across the vulnerable intestine (7).
ethnicity and host genetics, but also on one’s diet and environ- The numbers and types of our intestinal microbiota increase
ment (2). Stool, representing the distal bowel, showed relatively over the first year of life to assume a relatively stable adult pattern
high intra- and inter-subject diversity. The majority of our at the phylum level, but continue to evolve at the species level with
microbiota is anaerobic, and although more than 50 bacterial subsequent dietary and environmental exposures, including anti-
phyla have been described, only four constitute the majority of microbial therapies (3). In one study, diet inventories of 98 indi-
mammalian intestinal microbiota (Bacteroidetes, Firmicutes, viduals were correlated with participants’ fecal enterotypes to show
Actinobacteria, and Proteobacteria) and only two predominate that the Bacteroides enterotype was highly associated with animal
in our intestinal tract: the Bacteroidetes and the Firmicutes; protein, a variety of amino acids, and saturated fats (western diet),
most of the Firmicutes phyla are members of the Clostridia class whereas the Prevotella enterotype was associated with low values
(4). It is estimated that about 4,000 bacterial species reside in for these groups but high values for carbohydrates and simple sug-
our gastrointestinal tract, and that the human microbiota con- ars (agrarian diet). Moreover, microbiome composition changed
tains as many as 1014 bacterial cells, a number that is 10 times within 24 h of dietary alteration (10).
greater than the number of human cells in our body (5). Per Intestinal microbiota has important roles in the post-natal struc-
gram of contents, there is a marked and progressive distal tural and functional maturation of the gut. Germ-free animals
increase in the number of bacteria: 101 in the stomach, 103 in have, for example, enlarged ceca; increased enterochromaffin cell
the duodenum, 104 in the jejunum, 107 in the ileum, and 1012 area; a reduced intestinal surface area with narrower villi resulting
in the colon. This longitudinal heterogeneity of the microbiota from reduced cell regeneration and prolonged cell cycle time, and
population has a predominance of Firmicutes and Proteobac- a smaller villous capillary network; hypotonic and hyporesponsive
teria (notably Helicobacter pylori in the stomach), Firmicutes mesenteric vasculature; impaired lymphoid organs; impaired peri-
and Actinobacteria in the small intestine and a prevalence of stalsis; and abnormal cholesterol and bile acid metabolism (3). It
Bacteroidetes and the Lachnospirae family of Firmicutes in the has been shown, for example, that Bacteroides thetaiotaomicron
colon; of note, bacteria account for 60% of the dry weight of can induce angiogenesis (11); influence enteric nerve function,
feces. The microbiota within the intestinal lumen differs sig- and, therefore, possibly peristalsis (12); and also modulate intes-
nificantly from that dwelling in close proximity to or within tinal glycocalyx structure (13). Various microbiota, including
the intestinal epithelium (6). Therefore, fecal micro-organisms B.thetaiotaomicron and Lactobacilli, are also involved in maintain-
cannot be used as a surrogate for all communities of the bowel ing intestinal barrier integrity through maintenance of cell-to-cell
microflora. Moreover, luminal microbial communities and junctions and promotion of epithelial repair after injury (1).
surface adherent/associated populations are distinct and fulfill The areas in which microbiota have a major influence are legion,
different roles, only some of which I will mention briefly. growing, and far beyond the scope of this general overview. One
Our intestine becomes colonized with micro-organisms during such area is mucosal immunity with influence on immunocytes,
or shortly after birth and the intestinal microbiota of infants deliv- gut-associated lymphoid tissue, Peyer’s patches, IgA-producing
ered by cesarean section differs from that of vaginally-delivered plasma cells, immunoglobulin secretion, and pattern recognition
infants (7,8). The gastrointestinal tract of the newborn is still sterile receptors including toll-like and NOD-like receptors. As a specific
after caesarian section and its microbiota is initiated with feeding; example, the deficiency of CD4 + T-cells in germ-free mice can
in breast-fed infants Bifidobacteria predominate with minor rep- be completely reversed by mono-contamination with Bacteroides
resentation from lactobacilli and streptococci, whereas in formula- fragilis or administration of its polysaccharide capsular antigen (14).
fed infants, similar amounts of Bacteroides and Bifidobacteria are The gastrointestinal tract needs to coexist with the dense car-
found with minor representation from Staphylococci, Escherichia pet of bacteria that overlies its mucosa without inducing exces-
coli, and Clostridia (8). The first colonization of the intestine is a sive immune reaction, and the intestinal microbiota mediates
profound immunological exposure and early maternal inocula- such antigenic tolerance. As examples, intestinal dendritic cells
tion, as occurs with vaginal delivery, likely has an important role in are conditioned to a tolerogenic phenotype by intestinal epithe-
subsequent immune reactions and our susceptibility or resistance lial cells that are stimulated by Lactobacillus spp and certain E.
to certain diseases. Indeed, this initial exposure heralds the con- coli strains (15); B. thetaiotaomicron prevents activation of the
tinuing intimate roles our microbiota will have with our diet and proinflammatory transcription factor NFkβ (16); and Aeromonas
environment, and initiates the vital interactions of the microbiota or Pseudomonas promote intestinal alkaline phosphatase, which
with our metabolic activities, as well as with the immunological dephosphorylates and inactivates the lipopolysaccharide found
apparatus that constitutes our major defense system against for- in the outer membrane of Gram-negative bacteria thus protecting
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4. 4 Brandt
Bifidobacterium spp.
Clostridium spp.
REVIEW
Bacteroidetes Gm-PG SCFA Conjugation of
SCFAs
B. fragilis (PSA) Gm-LPS metabolism linoleic acid
Cellular immunity Xenobiotics metabolism Oxalate
AMPs IgA Nutrition
Lymphoid organogenesis Drug disposition excretion
Mucosal immunity
Immunocompetence Lap Lipid
Behavior O. fomigenes
Tolerance activation metabolism
NFKB Normalization of
DC tolerization
inactivation HPA stress response
Lactobacillus spp. E. coli B. thetaiotaomicron B. infantis
Peristalsis
Angiogenesis
Glycosylation
Barrier GIT surface maturation
maintenance GIT functional maturation
Figure 2. Some examples of the effects of intestinal microbiota and host physiology. The intestinal microbiota can affect many aspects of normal host
development and function. Members of the microbiota, with their various components or products of metabolism are shown in red. Microbial effects
on the host are shown in green. Affected host phenotypes are shown in blue. AMP, antimicrobial peptides; DC, dendritic cells; Gm, Gram negative;
HPA, hypothalamus-pituitary adrenal; Iap, intestinal alkaline phosphatase; PG, peptidoglycan; PSA, polysaccharide. From Sekirov et al. (3).
appetite regulation, energy utilization, digestion and absorption microbiota transplantation (FMT). Fecal microbiota transplant is
of ingested nutrients, and drug metabolism. Bacterial metabo- the term used when stool is taken from a healthy individual and
lism of dietary fiber to short chain fatty acids, and conversion of instilled into a sick person to cure a certain disease. As the exact
indigestible polysaccharides to absorbable monosaccharides are agent or agents that effect cure is currently unknown, the term
well known examples of such interaction. B. thetaiotaomicron, as fecal microbiota transplant (FMT) presently is preferred to fecal
another example, has been shown to upregulate expression of pan- bacterial transplantation, or fecal bacteriotherapy; stool trans-
creatic co-lipase and an intestinal Na + /glucose co-transporter (12). plant is an accurate but unaesthetic term (21).
Awareness of the interplay between these complex metabolic func- I’ve already reviewed the very early history of FMT, but FMT
tions and the intestinal microbiome sets the stage to study whether also has been used for centuries in veterinary medicine per rectum
manipulation of the microbiome can be used to understand and to treat horses with diarrhea or per os as rumen transfaunation to
treat conditions of obesity and underweight (19). Inter-individual treat a variety of illness in cattle. Its first clinical use in the English
and inter-population differences in intestinal microbiomes with language dates back to a 1958 case series of four patients with
their attendant varied metabolic profiles may explain the differ- pseudomembranous enterocolitis, three of whom were critically
ent toxicities of commonly used therapeutics in varied geographic/ ill. C. difficile had not yet been recognized as a cause of pseu-
cultural populations and set the stage for the development of domembranous colitis and Micrococcus pyogenes (hemolytic,
personalized medicine-based on one’s intestinal microbiome coagulase-positive Staphylococcus aureus) was cultured from each
profile (20). patient’s stool. Fecal enemas were administered as an adjunct to
antibiotic treatment and all four patients had “dramatic” reso-
Clostridium difficile infection and fecal microbiota lution of symptoms within 24–48 h of FMT (22); the first use
transplantation (FMT): introduction of FMT for confirmed recurrent CDI was reported in 1983 by
A perturbed intestinal microbiome has been associated with an Schwan et al. (23), in a 65-year-old woman who thereafter had
increasing number of gastrointestinal and non-gastrointestinal “prompt and complete normalization of bowel function”. Up until
diseases which brings us to C. difficile infection (CDI) and fecal 1989, retention enemas had been the most common technique
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6. 6 Brandt
(a) Homeostasis
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(d) Expansion of commensals (b) Antibiotic perturbation
(c) Transient dysbiosis
(h) C.difficile clearance (e) C.difficile exposure
(g) Disrupting dysbiosis (f) Persistent dysbiosis
Bacteriotherapy
or FMT
Figure 3. Role of antibiotics and C. difficile in perturbing Intestinal homeostasis and the corrective effect of fecal microbiota transplantation (FMT).
Intestinal homeostasis (a) is characterized by a diverse, stable microbiota. Antibiotic perturbation (b–c) kills susceptible bacteria resulting in a less diverse
community structure with loss of colonization resistance. In the absence of opportunistic infection, the microbiota usually recovers its diversity (d) to
re-establish homeostasis and colonization resistance (a). Exposure to C. difficile (e) after antibiotic perturbation (b), however, can lead to persistent
dysbiosis (f). Bacteriotherapy or FMT can disrupt the dysbiosis (g) allowing clearance of C. difficile (h) and re-establishment of intestinal homeostasis (a).
Modified from Lawley et al. (33).
body piercing or tattoo in the previous 3 months or recent incar- blender is to be used for several patients, its parts would have to
ceration are also exclusions. A history of diarrhea, constipation, be sterilized before the next procedure. Some authors use milk
inflammatory bowel disease, colorectal polyps or cancer, irri- as the suspending fluid, others water; saline and milk may give
table bowel syndrome, immunocompromise, morbid obesity, slightly lower resolution (86.2 and 88.6%, respectively) and recur-
metabolic syndrome, atopy, and chronic fatigue syndrome are rence (3.0 and 3.2%, respectively) rates, while water may give
additional donor exclusions because they conceivably may be higher resolution (98.5%) and recurrence (7.8%) rates (28). The
transmittable by inoculation with intestinal microbiota. amount of stool to use has not been standardized, although those
One systematic review provided data to suggest that FMT given to weighing and measuring rather than just “eyeballing” the
using stool from a related donor (spouse, or intimate partner), product’s appearance favor 50 g in 250 cc of diluent. It seems as
yields a somewhat higher rate (93.3%) of CDI resolution than if more is better and most “FMTers” are now using about 300 cc
when stool from an unrelated donor (84%) was used (28). More for colonic FMT and 60 cc for upper tract FMT. An administered
recent experience with frozen/thawed or fresh fecal preparations volume of < 200 ml gave a resolution rate of 80% and a relapse
obtained from “standard” or “universal” donors, however, gave rate of 6.2%, whereas a volume of > 500 ml gave a resolution rate
excellent results (90–92% resolution, 9% recurrence) exceeding of 97.3% and a relapse rate of 4.7%. Use of < 50 g of stool was
those obtained with patient-selected donors (70% resolution, associated with resolution and relapse rates of 8.2% and 3.8%,
30% recurrence), and casting doubt on preference for related or respectively, whereas > 50 g of stool had resolution and relapse
intimate contacts (36). rates of 86.2% and 1.0%, respectively (28). I like to use donor stool
So what are the “nuts and bolts” of FMT? The donor has a rela- within 8 h of passage although this time limit has never been stud-
tively simple job: to provide the stool in a timely fashion. This, I ied rigorously. Stool should not be frozen and need not, but may,
have seen, may cause a level of “performance anxiety” in some be refrigerated for travel. After adding my beverage of choice to
donors. To facilitate passage and to enable me to work with a the stool and getting it to the proper consistency, I filter the mix-
soft stool, I have the donor take a double dose of milk of mag- ture through gauze pads to remove large particulate matter that
nesia before bedtime the night before the procedure. A soft stool may obstruct the colonoscope’s channel and then draw the elixir
is passed into a clean plastic container. I add non-bacteriostatic into 60 cc catheter-tipped syringes. It is recommended that stool
saline to the stool, stir it, shake it, and mix it thoroughly. Oth- preparation be performed under a hood, because stool is rated as
ers have opted for the blender method and some practitioners a Level 2 biohazard, although this recommendation is not practi-
have even had patients bring their own blender. Obviously, if a cal and this is the safest stool we, as gastroenterologists, will ever
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8. 8 Brandt
or genetically refined products derived from stool rather children; and autistic symptoms have sometimes been reduced by
than stool itself to treat CDI. The next step in this journey oral vancomycin treatment (3). Although at first glance it appears
has already been taken with the development of a standard- as if there is no connection with neuropsychiatric disease and
REVIEW
ized filtered, frozen preparation of stool for FMT and use of a intestinal flora, studies now have expanded the original concept
“universal donor” (36). of the brain-gut axis and recognize the brain-gut-microbiota axis
(51). Moreover, the increasing recognition of the role that micro-
When should FMT be done? biota have in affecting mood and thought is actively being worked
When should FMT be done for CDI? The clearest indication, on (52,53).
as discussed above, is recurrent or refractory disease, but even
this is not universally accepted and awaits the results of rand-
omized controlled trials; to quote a reviewer of our NIH grant, CONCLUSION
however, “…we all know it works”. I believe it also has a role as I believe that the intestinal microbiota will be shown to have
first-line treatment for patients with CDI rather than antibiot- important roles in maintaining our health and modulating energy
ics because of its rapid effect, minimal risk, relatively low cost expenditure, inflammation, and resistance or susceptibility to var-
and reestablishment of a “balanced” colonic microbiota (39). I, ious disease, some gastrointestinal and some not. Bacteria should
and others, also have used FMT to treat patients with severe CDI no longer be regarded as just “bad” guys and we have come a long
manifest by toxic megacolon or ileus and have seen the patient’s, way since Hippocrates said,” All disease begins in the gut”; today
family’s, gastroenterologist’s, and even surgeon’s relief as the he might instead say “Our health is determined by the micro-
patient’s abdominal distention, fever, and white blood cell count biota in our gut”. We are witnessing a paradigm shift in the way
decreased, occasionally within hours of the procedure; in none we understand health and treat disease and in its center is our
of these cases was the patient’s condition or course of disease microbiota.
worsened by FMT.
FMT also has been used to treat a variety of other gastro- CONFLICT OF INTEREST
intestinal disorders including ulcerative colitis, Crohn’s disease, Guarantor of the article: Lawrence J. Brandt, MD, MACG.
irritable bowel syndrome, and constipation and there is a grow- Financial support: None.
ing literature on an altered intestinal microbiome in these disor- Potential competing interests: Dr Brandt is on the ‘Speaker’s
ders (40–42) (See Table 1). I now have personal experience with Bureau of Optimer Pharmaceuticals, Inc. and has received a research
FMT in 20 patients with UC, 4 with Crohn’s disease and 20 with grant from Optimer Pharmaceuticals, Inc.
IBS; and in all groups, I have noted remarkable symptomatic
improvement in some individuals. Rigorous studies of FMT in REFERENCES
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