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Received: 2015.06.28
Accepted: 2015.09.08
Published: 2015.XX.XX
6427 2 2 130
The Human Microbiota: Composition, Functions,
and Therapeutic Potential
Rick Conrad
EF Alexander V. Vlassov
Corresponding Author: Alexander V. Vlassov, e-mail: sasha.vlassov@thermofisher.com
Source of support: Self financing
The human body – primarily (but not solely) the gut – is populated by 100 trillion bacteria and other members
of the microbiota community, which play a fundamental role in our well-being. Deviations from healthy micro-
bial compositions have been linked with many human diseases, including inflammatory bowel disease, obesi-
ty, cancer, asthma, diabetes, and allergies. This review provides a high-level summary of human microbiome
composition and known health effects, and highlights the typical workflows and tools used in microbiome re-
search – from sample collection and storage to isolation and analysis of DNA. We particularly focus on multi-
ple novel microbiota-based therapeutic approaches, including fecal microbiota transplantation (FMT) and tar-
geted bacteriophage engineering. Although our understanding of the microbiome and its interaction with the
host is still in the nascent stages, it is becoming increasingly clear that we need to treat it as a sophisticated
system, much like the circulatory and immune systems, that exists in harmony with homeostasis, playing mul-
tiple roles within the human body.
MeSH Keywords: Bacteriophages • DNA Sequencing • Microbiome • Microbiota
Full-text PDF: http://www.medscirev.com/abstract/index/idArt/895154
Authors’ Contribution:
Study Design A
Data Collection B
Statistical Analysis C
Data Interpretation D
Manuscript Preparation E
Literature Search F
Funds Collection G
Department of Research and Development, Thermo Fisher Scientific, Austin TX,
U.S.A.
e-ISSN 2373-2490
© Med Sci Rev, 2015; 2:
DOI: 10.12659/MSRev.895154
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Background
The bacterial load in our intestinal tract has long been recog-
nized as an inevitable by-product of exposure to our environ-
ment. Although the lay public and even some physicians may
have considered any microorganism present in our digestive
system either as an inert burden or a health threat, microbiolo-
gists have known for the last half-century that a certain amount
of bacterial load is actually necessary for its proper function-
ing [1–3]. But knowledge about how they help and what roles
specific members of the population play has been slow in com
-
ing until recently. Much of our understanding was based on
methods of culturing bacteria on various sorts of rich media,
which provided fairly coarse information about the types of
bacteria present and their collective biochemistry, and no in-
formation on the genomes of the bacteria in the population.
As the ability to characterize microbes present without cultur-
ing grew, primarily through PCR-based methods, we came to
understand more about the individuals in the commensal pop-
ulation of microbes that are normally present in the human
body and potentially the roles they play in our health. These
techniques have also allowed us to determine the presence
of a native microbiome in other than gut organs. In the past
few years, as microbiome research has become a hot topic of
research, it turns out that the story is not as simple as “good
bacteria = good health”. As we find more information in this
area, it becomes clear that knowing the genomes of each strain
present does not in and of itself provide insight into the perti-
nent biochemistry of the community as a whole and its actual
physiological effects on health. Indeed, despite over 19,000 pa-
pers published on the human microbiome/microbiota to date
(~5000 of which were published in 2015 alone), and even new
human microbiome-centric journals that have been launched
recently [4–6], most of the current studies are descriptive, pro-
viding identification of the microbiome members to family or
genus level. Much less is understood about what the microbi-
ome is actually doing, in terms of its cross-interaction and in-
teraction with the human host, and its roles in maintaining a
healthy state or disease development. Here, we present a back-
ground of data to provide a reasonably objective view of what
the current state of knowledge and expectations is. First, the
summary of human microbiome composition and known func-
tions is provided, focusing on bacteria residing within the gut.
The typical workflows and tools used in microbiome research
are described next, from sample collection and storage to iso-
lation and analysis of DNA, to point out numerous challenges
with this type of study. We then focus on several unique ap-
proaches that translate the knowledge of the human microbi-
ome into next-generation therapies. Taking into account that
microbiome research is a very broad area, with tremendous
amount of data generated over the last decade, it is impossible
to include every aspect here, and we apologize to the authors
whose work was not cited due to size limitations.
What is the Human Microbiota/Microbiome
and where does it come from?
A microbiota is a community of microbes (or microorganisms), in-
cluding bacteria, archaea, fungi, protists, and viruses that live in
a particular environment – anything from a hot spring to the hu-
man body. The term Microbiome was coined by Joshua Lederberg
to “signify the ecological community of commensal, symbiot-
ic, and pathogenic microorganisms that literally share our body
space and have been all but ignored as determinants of health
and disease” [7]. In common usage this term has evolved, re-
flecting the state of our identification methods. Initially, all char-
acterization was through classical microbiological methods: cul-
turing through a series of selective and differential media, with
reculturing of colony isolates, and performing standardized bio-
chemical tests on these isolates. Sometimes isolates would be
examined under the microscope, providing identifications based
on shape, presence of flagella, and staining characteristics. While
this provided information on the types of microbiota present, it
did not parse down into the genomics (with its inferred molecu-
lar biology) of the various members that is possible with current
PCR-based amplification and sequencing methods. This meth-
od of performing wholesale extraction of DNA from a sample
containing a community of microorganisms and deconvoluting
it after sequencing is termed Metagenomics. It seeks to identi-
fy specific sequences that are diagnostic of families, genera, or
species of the individual bacteria present. Previous methods re-
quired an intermediary growth step in defined media. This could
skew the proportional representation of the member species,
as the media used is more favorable to growth of some organ-
isms than others, and even eliminating those that grow slowly
or not at all. Hence, the metagenomic approach is of great val-
ue in that it allows comprehensive examination of the entire
genomic content of the microbial community, including those
organisms that are uncultivable [8].
Bacteria are the best-studied members of the microbial com-
munity. The human microbiome includes approximately 100
trillion bacterial cells [9,10]. Although the human body is made
up of about 10–40 trillion cells [11], bacterial cells outnumber
human cells by 3–10 fold. In addition to this bacterial popula-
tion, the microbiome includes trillions of viruses (including bac-
teriophages), archaea, fungi and other single-celled eukaryotes.
Viruses might actually outnumber bacterial cells, while fun-
gal cells are present at levels orders of magnitude lower [12].
The amount of diversity at a gene level that this introduces
is thought-provoking, whether one is thinking about horizon-
tal gene transfer potential [13] or just the extra proteins func-
tioning in our gut. Using the current assumption that the hu-
man genome includes about 20 000–25 000 protein-coding
genes [14], all the bacteria, fungi, and viruses in one person’s
microbiome would be estimated to add another ~2–20 mil-
lion [8,15]. Thus, for every human gene there are up to 1000
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non-human genes. The mass this represents is also impressive.
Currently, it is thought the human body contains 2–5 pounds
of microbes, constituting ~1–3% of our body mass [16,17]. As
much of this is in our gastrointestinal tract, which represents
~10% of our mass, it is not surprising that it can exert pro-
found effects on our physiology and well-being.
This article will concentrate on the microbiome in the diges-
tive tract; however, there are also micorbiomic niches on the
skin surface, the linings of the nasal passages, oral cavity, eyes,
lungs, and mammary glands. The reader is referred to Figure 1,
which summarizes the references that can be consulted to find
out more about all these microbiome reservoirs. The digestive
tract, our gut, contains the largest and most diverse ecosys-
tem compared to other sites (Figure 2) and for this reason it
is the most thoroughly studied microbiome system in humans
and other mammals. Although we show some representatives
of the genera found in the small intestine, there are a num-
ber of potential niches in the functional surface of this organ,
and we have yet to learn the roles individuals play in each of
them, knowing only the relative representations in each re-
gion at this point. The densest representation of microbes is
in the human large intestine, which contains 1011–1012 cells
per milliliter of fluid contents [26].
Knowing that the presence of these microbes is necessary for
maintenance and development of a functional gastroenter-
ological tract, an obvious question is where do the seeds of
this population arise from? The genesis of the microbiome is
actively being investigated, and again, the ability to identify
bacteria, at least at the family or genus level, from samples so
dilute that detection was heretofore undetectable, has shak-
en many of our assumptions. Until recently it was thought
that the fetus resided in basically sterile conditions in utero,
but this concept now appears to be not precisely true [32,33].
With some microbiota present prior to birth, it is interesting
to speculate how these microbes act with and influence those
that are known to be picked up as the baby is being passed
through the birth canal – it is coated with microbes from the
mother, presumably picked up from the adjacent urogenital
and digestive tracts. Knowing this now raises questions about
what perturbations occur in the digestive microbiome of neo-
natals who are born via cesarean section. Of course newborns
will acquire antibodies and bacteria from the process of breast
feeding, both from the milk and the skin surface. But, as most
parents know, a young child uses its mouth to freely sample
the environment, from random objects like rocks to toys, pets,
and other people. Although introduction of various microbes
from the food and environment are random, their establish-
ment is keyed to a final configuration optimized for existence
and symbiosis in specific conditions, through a continuous
selection process. The microbiome changes dramatically over
the first few months of life, and by the age of about 2 years, it
seems to reach some sort of equilibrium, resembling what we
currently understand as a typical adult microbiome [34]. From
this point on, what is ingested by the child in the diet has less
Skin: [18]
Ear: [21]
Mammary gland/milk: [23]
Gastrointestinal tract (GIT):
[26,27]
Bladder/urine: [29]
Vagina: [31]
Eye: [19]
Nose: [20]
Mouth: [22]
Lung: [24]
Esophagus and stomach: [25]
Gut-kidney influences: [28]
Penis: [30]
Figure 1. The human body has many
physiological niches that each host
their own individually adapted
microbiome. The Figure highlights
microbiomes for skin, eye, nose, ear,
mouth, mammary gland/milk, lung,
esophagus and stomach, GIT, kidney,
bladder/urine, penis, vagina and
provides representative references –
so that the reader can consult to find
out more about these microbiome
reservoirs (illustration by A. Conrad).
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effect on the ecological balance, unless profoundly aggressive
(and hence, pathogenic) bacteria or poison is ingested [35–37].
Composition of Healthy Human Microbiomes
Our understanding of components of the microbiomes rep-
resented in the ‘healthy’ general population has been en-
abled by the advent of next-generation sequencing (NGS).
The most ambitious program of this type so far is the Human
Microbiome Project (HMP), funded by the National Institutes
of Health. Phase I of this program (2008–2013) examined the
microbiomes of 300 healthy subjects [38,39]. Although the
precise approaches have varied, the basic experimental de-
sign has followed a standard general workflow. First, sam-
ples are collected from several body sites: primarily nasal pas-
sages, oral cavity, skin, gastrointestinal tract, and urogenital
tract. Then the DNA is extracted from the microbes present
(the presence of host DNA will depend on the robustness of
the sample preparation). These nucleic acid samples are then
prepared for NGS using specific workflows that result in a li-
brary of amplified fragments with sequence tags that allow
sequencing on the platform of choice. These small amplicons
can be totally random (a shotgun approach) or semi-selective.
Each approach provides a massive amount of short reads in
parallel. The final stage is the processing and interpretation of
this highly diverse set of short sequences. Much of the anal-
ysis to date has relied on small ribosomal subunit RNA (‘16S
rRNA’) sequences to generate identification at the family and
genus levels. Greater detail at the species level needs sequence
data from other, more diagnostic, genes. At first blush, it ap-
pears the number of different genera of microbes routine
-
ly found in human microbiomes represents only a fraction of
those previously characterized in the master tree of life phy-
logeny [39,40]. The microbial communities within each body
site tested are “optimized” for their specific niche – the gas-
trointestinal tract, urogenital tract, skin, oral cavity, nasal cav-
ity, or other specific sites. There is dramatic variation between
individuals when microbiomes are classified by the species
they contain. However, when classified by function (e.g., abil-
ity to digest different kinds of carbohydrates or synthesize cer-
tain vitamins), they look more similar from person to person
[41]. It appears that each individual’s own microbiome is fair-
ly stable over time unless radical changes are made by inges-
tion or other introduction of agents drastically affecting the
balance, such as chemicals killing or altering the nutritional
balance, or inocula of aggressive, potentially pathogenic, mi-
crobes [35–37,42,43].
To provide a simple description of an extremely complex and
dynamic community, it was proposed to stratify the gut micro-
biomes into 1 of 3 principal clusters or enterotypes, defined by
a dominant presence of 1 of 3 genera: Bacteroides, Prevotella,
or Ruminococcus [37]. Wu et al. [36] found that Bacteroides
group was prevalent with long-term protein and animal fat di-
ets, while Prevotella group was associated with long-term car-
bohydrate diets. Neither enterotype was substantially affected
by 10 days of dietary restrictions, suggesting that they are more
influenced by long-term eating trends [36]. The oral cavity con-
tained the greatest phylogenetic diversity, the stomach had the
lowest diversity, and diversity increased down the gastrointes-
tinal tract from the stomach to the terminus. Certain taxa were
dominant in specific body sites: Bacteroidetes in the GI tract,
Lactobacillus in the vagina, Streptococcus in the oral cavity, and
Actinobacteria/Proteobacteria/Firmicutes on skin [27,44–48].
The studies also identified a number of “outliers” – subjects
Lactobacillus
Clostridum
Streptococcus
Figure 2. Image of the gut microbiome in the
lower region of the small intestine,
in the jejunum/ileum segments.
Three main genera are shown in a
representational manner, as detailed
in the secondary legend. The image is
intended to emphasize how much is
still not known about the microniches
filled by the component bacterial
families. The spaces between the
villi have different roles in digestion
and absorption of our food, and the
separate bacteria may have very
different operating sites at this level.
The size comparisons are accurate,
to indicate the myriad of potential
niches for various bacteria to fit in.
Identification of which bacteria fill
which microniches in humans will
provide a challenge for microbiome
researchers. The microbial community
in the human large intestine is
extremely dense, containing 1011–1012
cells per milliliter of fluid; the small
intestine can vary from 10- to 1000-
fold less than this (illustration by
A. Conrad).
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that had abnormal microbiomes – for example, dominated by
Firmicutes in the gut. Clearly, studies on the microbiome com-
position and normal fluctuation have only scratched the sur-
face at this point. More useful knowledge is certain to be gained
through the recruitment of large cohorts of donors who can
be stratified across a wide spectrum of health classifications.
As we obtain this knowledge, our understanding should sur-
pass the simplistic view of “normality” versus “pathology,” and
lead us to a more subtle understanding of the qualitative ef-
fects these shifts can have.
It should be emphasized that the diversity of bacteriophage
and fungal species in the human microbiome has not yet been
studied as thoroughly as that of bacteria. The field is nascent,
and difficulties at both the sample preparation and bioinfor-
matics stages have to be addressed. However, it is clear that
these populations also include hundreds to thousands of dif-
ferent species, and are necessary components of and contrib
-
utors to the microbiome ecosystems. Their study is every bit
as important as analysis of the bacterial members. Without
the context of the entire microbial ecosystem, information
gained solely from identifying and quantifying known bacte-
ria would be misleading.
The initial thrust of the HMP effort focused solely on healthy
individuals with the objective to determine if a “normal” mi-
crobiome did indeed exist across a wide population. The on-
going work of HMP will complement current and completed
studies by independent laboratories, which are beginning to
look at correlations between particular diseases or responses
to drugs or other treatments with particular microbiome com-
positions [49]. These correlative studies are the beginning for
providing a knowledge base that has the potential for predict-
ing disease risks and developing next-generation diagnostics.
For example, the above-mentioned Bacteroidetes and Firmicutes
phylotypes are also shown to be switched when subjects are
put on a weight-reduction diet [50]. Other examples are dis-
cussed with their potential applications in the next paragraph.
As these studies progress, it must be kept in mind that rigor-
ous (and numerous) controls are required to parse microbiome
changes related to specific diseases from those changes that
occur naturally between healthy members of the same popu-
lation or even in the same individual over time due to aging
or lifestyle changes [42].
HMP is not the only large project dedicated to the character-
ization and diagnostic implications of the human microbiome.
Some others are the American Gut Project [51], MetaHIT [52],
My.Microbes [53], Human Longevity [54], Earth Microbiome
Project [55] and Human Food Project [56], and each is mak-
ing steady progress in its particular endeavor in the microbi-
ome field. Although most current microbiome studies are per-
formed in the framework of identification and categorization
of microbial species, the inevitable evolution of these stud-
ies should lead to understanding the biological system as a
whole, enabling prediction of the net effect of a particular mi-
crobiome composition on an individual with a particular ge-
netic background, and potentially providing the ability to gen-
erate “custom microbiomes” as therapeutics.
Functions and Beneficial Effects of the
Normal Human Microbiota
Of the presumably large number of functions and benefi-
cial effects various microbes exert within the human body,
only a fraction are understood at the moment. For instance,
it is known that human microbes (1) prevent colonization by
pathogens by competing for attachment sites and for essen-
tial nutrients [57,58]; (2) antagonize competitors and foreign
bacteria through the production of substances, ranging from
relatively nonspecific fatty acids and peroxides to highly spe-
cific bacteriocins [59]; (3) perform carbohydrate fermentation
and absorption, enabling the host to utilize some normally in-
digestible carbohydrates [60,61]; (4) synthesize and excrete vi
-
tamins in excess of their own needs, which can be absorbed
as nutrients by their host (e.g. human enteric bacteria secrete
Vitamin K and Vitamin B12, and lactic acid bacteria produce
certain B-vitamins) [62,63]; (5) provide a continuous and dy-
namic effect on the host’s gut and systemic immune systems
[64]; and (6) stimulate the development of certain tissues [1,65].
There is at least preliminary evidence showing that in these,
as well as other yet-to-be-discovered interactions, the micro-
biome can influence multiple diseases, including inflammatory
bowel disease [66–68], malnutrition [69], celiac disease [70],
obesity [50,71,72], vaginosis [46], asthma [73], diabetes [74],
cancer [75], pancreatic disease [76], allergies [77], neurological
disorders [78], and heart disease [79]. Whether dysbiosis or im-
balance in our microbial populations is the cause, or effect, of
each particular disease is a question in which future research
will hope to gain insight. However, current experiments have
shown that in some cases, manipulating the microbial popula-
tion artificially to restore the balance can lead to novel cures.
All microbes constantly excrete metabolites and secrete sub-
stantial amounts of small and large molecules, as well as ves-
icles containing RNA, DNA, and protein [80–82], which allows
them to influence and communicate with their neighbors and
their host. For the gut microbial community, many of these en-
tities can pass through the gut wall and enter the bloodstream
to be distributed throughout the body, causing effects on lo-
cal and distal organs. There are intriguing indications that the
gut microbiome may affect our sleep patterns, mood, eating
behavior, and dietary choices [83,84].
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As stated above, for every human gene within our body, there
are up to 1000 non-human genes and their products, meaning
that the microbiome provides us with an enormous reservoir of
genetic content with the capability to provide ‘niche’ metabo-
lisms beyond those of the human genome itself. Theoretically,
because the microbiome’s metagenome can change much fast-
er than the human genome, the microbiome provides a rapid
and potentially localized means for specific metabolic niches
to adapt when external environmental conditions change. One
example of such an adaptation is a recent discovery of a gene
for digesting seaweed in the microbiome of Japanese people.
This gene is found in environmental bacteria that feed on sea-
weed in the ocean. At some point, these bacteria, while pass-
ing through someone’s gut on a piece of seaweed, transferred
a gene to normal bacteria residing within human gut. The gene
conferred the ability to digest the seaweed that is a common
part of the Japanese diet, and it is now part of the genetic ca-
pacity of the human microbiome in Japan [85].
Overall, the entire microbiome can be viewed as a very so-
phisticated additional organ (or set of organs – taking into
account that there are many ecosystems throughout human
body) having multiple local and systemic effects on the host.
Workflows and Tools for Microbiome Projects
The typical human microbiome workflow includes the follow-
ing steps: (1) sample collection: collecting stool, saliva, urine,
milk, or swabbing various surfaces or orifices; (2) storage: freez-
ing or preservation in stabilizing solutions; (3) extraction and
concentration of aggregate microbial DNA (sometimes RNA);
and (4) sequencing and subsequent data analysis.
A number of sample collection and preservation kits for the
analysis of microbiome in stool, urogenital, oral samples are
offered by various vendors. In addition to basic plastics and
swabs, the more advanced kit options are available with stabi-
lizing solutions that are claimed to preserve DNA and RNA for
up to 2 years at room temperature. Freezing (–80°C) is routinely
used by many scientists for sample preservation; however, the
limitations with this approach include: (i) nucleic acid integrity
can be compromised upon multiple defrosting cycles, and (ii) it
cannot be readily utilized for samples collected in the field, al-
though some companies are working on developing specialized
instruments for making this process easier and more mobile.
Kits for extraction and isolation of microbial nucleic acid from
stool and swabs are available from a number of companies.
The majority of studies utilize bacterial DNA in sequencing.
RNA is used less commonly. It has the potential advantage of
multiple copies per cell (e.g., rRNA should be present at thou-
sands of copies per genome or more), but the drawback of
greater instability. For low throughput applications – up to 24
samples at a time – the most widely used format is solid-phase
extraction on glass fiber columns (e.g. PureLink Microbiome
DNA Purification kit). For high throughput applications solid-
phase extraction with magnetic bead-based kits (e.g. MagMax)
is a more appropriate choice as it can be readily automated.
The major challenge with microbiome samples is not the abil-
ity to extract nucleic acids per se, but the isolation of a DNA
sample that accurately reflects the representation of the di-
verse microbes in the community sampled. Microbes substan-
tially differ in the compositions of their cell walls, which are
the major impediments to lysis. For instance, Gram-negative
bacteria can often be efficiently lysed with heat alone or heat
plus a potent amidase/protease, while Gram-positive bacteria,
with their thicker and more complex cell walls (most conspicu-
ously, the addition of teichoic acid into the peptidoglycan lay-
er), often require an additional enzymatic boost, to hydrolyze
the glycans or peptide portion. Using a procedure that lyses
the former efficiently may inefficiently lyse the latter, intro-
ducing a bias that presents an inaccurate view of the propor-
tional representations for the genera. The research community
agrees that protocols including upfront mechanical disruption
of bacteria by physical means (bead beating, sonication, shear-
ing, and/or pressure-differential lysis) are the best options, as
this ensures the breakage of very thick cell walls of durable mi-
crobes such as mycobacteria. However, these are known only
as pathogens, not commensals, and theoretically a carefully
optimized enzymatic digestion in combination with special-
ized detergents might enable lysis of all the bacteria present
in the human body without this labor-intensive step, result-
ing in sample processing more amenable to high-through-
put. A number of enzymes are effective on certain species of
bacteria: Lysozyme, Labiase, Lysostaphin, Achromopeptidase,
Mutanolysin, and Proteinase K. Certain combinations of these
enzymes will not function together in the same reaction mix-
ture. However, through careful design, sequential additions of
one or several enzymes can be made to lyse microbes of dif-
ferent species substantially more effectively than a single en-
zyme alone, approaching the efficiency of mechanical lysis.
In addition to the efficiency of lysis, the purity of the DNA or
RNA can affect the downstream analysis, as some methods
carry over more inhibitors of the enzymes employed in the
assays being used (e.g., polymerases, ligases, and phospha-
tases). Such inhibitors include bile, bilirubin, food digestion
products, heme, and humic acids. These can often be dimin-
ished by additional washing or precipitation steps. An alter-
native approach is to incorporate sequestering agents to the
sample prior to adding it to the assay, or designing the assay
with enzymes in the downstream assays that have been se-
lected or engineered to be particularly robust, and functional
in the presence of high levels of inhibitors.
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A number of laboratories compared various commercially avail-
able kits as well as “homebrew” protocols for isolation of mi-
crobial DNA, and analyzed the obtained community profiles
for any kind of biases [86–90]. There is a clear need to consol-
idate all these findings into a set of best practices for sample
collection, storage, recovery and analysis of microbial DNA.
The world scientific community is making a steady progress
at standardizing the microbiome workflows that will allow di-
rect comparison of studies performed by different institutions.
For the downstream analysis, qPCR [91] and microarrays (such
as Phylochip [92]) are routinely used for detection and quanti-
tation of predicted microbial species in the samples, while NGS
is utilized for discovery purposes. The microbiome projects (in-
cluding HMP) are using both the metagenomic whole genome
shotgun (WGS) strategy and 16S rRNA sequences to identify and
rank preponderance. With the WGS DNA sequencing method,
the random fragments of genomes from bacteria and other mi-
croorganisms, as well as contaminating DNA from the host, are
sequenced, identified, and classified. Using 16S rRNA sequenc-
es is a very common approach which involves PCR amplifica-
tion of taxonomically informative regions of the 16S ribosomal
RNA (rRNA) gene by using mixtures of primers corresponding
to conserved regions flanking the informative variable regions.
These are then subjected to next-generation DNA sequencing,
enabling the classification of individual reads to specific taxa
at various levels from phylum down to genus. In this case, am-
plicon sequencing can be targeted specifically against bacteria,
does not require the availability of reference genome sequenc-
es, and can be utilized in cases when only small amounts or low
quality DNA is available. Currently, the 2 most popular next-gen-
eration sequencing technologies are benchtop sequencers de-
veloped by Illumina (MiSeq [93]) and Life Technologies (PGM,
Proton [94–97]), both of which have developed sufficiently long
reads now so as to permit data generation from highly infor-
mative sections of the 16S rRNA gene. Although improvements
in sequencing methodologies has led to a decreasing cost per
sample of performing NGS, for instance with the introduction
of sample barcoding, the level of useful information is still lim-
ited by the current bioinformatics tools, especially available
databases. Public databases have an immense problem with
variable and often poor annotation. Since this is the only con-
nection between sequence and actual bacterial information,
improving these annotations and in turn the databases will be
a huge step in allowing researchers to make rapid progress in
the categorization of human microbiomes from various sources.
After DNA sequences of microbiome samples have been ac-
quired, they must be analyzed and interpreted. The tremen-
dously large amount of data produced in now days studies
require sophisticated analysis tools. The key bioinformatics
approaches and tools for analyzing such data are reviewed in
detail in Hamady & Knight [98] and Kuczynski et al. [99]. Again,
parsing the sequences and correlating them to database entries
might not lead to fruitful information due to poor annotation.
As we gain more understanding of the microbial DNA make-
up, the scientific community is initiating more sophisticated
types of analysis, including: microbial Proteome – analysis of
individual peptides in a mixture of proteins by a combination
of liquid chromatography and mass spectroscopy after enzy-
matic digestion of the protein mixture; Interactome: Analysis
of protein-protein interactions between members of the mi-
crobiota or between the microbiota and the host; Lipidome:
measuring lipid profiles using ultra performance liquid chro-
matography combined with electrospray ionization tandem
mass spectroscopy; Metabolome: Measure of metabolomic
profiles using untargeted and targeted LC-MS methods; anal-
ysis of live microbes in order to understand their functions
and networks; analysis of the host molecular profiles. There
is a growing need for development of new technologies and
tools to enable these advanced studies of microbial commu-
nities and interaction with the human host.
Translating the Human Microbiome into Next
Generation Therapeutics
As discussed above, the studies to correlate certain microbial
distributions with certain syndromes are just beginning. But
it is already apparent that alterations in the human microbi-
ome are linked to a wide range of human diseases. Often, it is
seen that specific genera or even species predominate in the
gastrointestinal tract of individuals with specific syndromes.
Despite the lack of clarity in many cases on whether the dom-
inance of certain taxa of bacteria are the primary cause of dis-
ease or the consequence, modulation of microbial communi-
ties is already being proposed and tested from the therapeutic
development standpoint. Modulation can be in the form of
addition of ‘desired’ species and/or elimination of ‘problem’
ones. Unfortunately at the moment the only means to accom-
plish the latter effectively is through the use of broad-spec-
trum antibiotics, which usually eliminate most of the benefi-
cial bacteria in the gut, and the body, along with the problem
pathogen(s). This then creates the problem of repopulating
the gut microbiome. Even with very specific microbiotic pop-
ulations being introduced (as with probiotics), the body is also
getting repopulated from sources in its present environment,
and none of these precisely reproduces the one that generat-
ed the previous biome. The assumption has been that a rea-
sonable approximation will reside after repopulation, and this
is adequate, but current research may lead us to question this.
In addition to the problem of re-establishment of eliminat-
ed commensals, antibiotic-resistant but previously innocu-
ous members of the original microbiome can be cast into the
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role of pathogen. This is exactly the case with opportunistic
Clostridium difficile infections. The antibiotic-induced disruption
of normal microbiota in the intestine allows an overgrowth of
the C. difficile bacteria, which is normally present at very low
levels within a human body; it produces a toxin damaging the
lining of the large intestine, causing severe diarrhea, and some-
times fever, abdominal pain, nausea and vomiting [100–102].
Treatment requires an additional antibiotic to eradicate the C.
difficile bacteria so the healthy bacteria can repopulate the in-
testine. Unfortunately, about 20% of patients with C. difficile
infection have a recurrence of the infection after they finish a
course of appropriate treatment, and another round of anti-
biotic treatments only makes the situation worse. Colectomy
(surgical removal of a part of the colon) is the last resort, but
the procedure is risky, expensive, and does not guarantee that
the problem will be solved. There are >300 000 patients with C.
difficile infections in the US alone, resulting in 14 000 deaths
each year (especially adults over the age of 65) [100–102].
Surprisingly, it has been found that introduction of the exist-
ing intestinal microbiome from a healthy individual can actu-
ally restore the balance and reduce the C. difficile to normal
levels. Since the transferred material is prepared from stool
samples of a healthy donor, the procedure has been coined
Fecal Microbiota Transplant (FMT). The infusion has been per-
formed via different modalities, several of which are referenced
in Allegretti et al. [103]. The success rate of this non-canon-
ical and unique medical treatment is >90% with a single ad-
ministration [104]. In September 2013 the FDA began permit-
ting its use as an experimental drug in cases where C. difficile
infection fails to respond to antibiotic therapy [105]. Today,
there are multiple organizations routinely performing FMT
to treat C. difficile infections [106–108], and there is a Fecal
Transplant Foundation that is raising awareness of and sup-
porting FMT in many ways, including continuous process opti-
mization and standardization [109]. There is a non-profit stool
bank, OpenBiome, that collects fecal samples, screens them
for parasites and pathogens, stores them at –80°C, and ships
to hospitals on demand [110]. Several companies are develop-
ing next-generation solutions – capsules loaded with bacteria
derived from human stool, or mass produced in fermenters –
that are intended to make the FMT technology more appeal-
ing and standardized [107,111]. A number of ongoing clinical
trials are using FMT to treat chronic diseases that may have
their basis in microbiome imbalances and the body’s respons-
es to them, such as ulcerative colitis, Crohn’s disease, obesi-
ty, metabolic syndrome, and type 2 diabetes mellitus [112].
The use of FMT should be seen as a first iteration of therapeu-
tic approaches aimed at adjusting the balance of gut microbi-
omes. Although it has some documented successes, it is still a
very blunt tool for providing a healthy community of microbes
to displace the dysfunctional microbiome of a patient suffering
from dysbiosis. The hope is that finer tools can be designed as
studies provide us with more understanding of the roles indi-
vidual members and/or groups play in a healthy microbial ho-
meostasis. Knowing that this is an interactive balancing act, it
would be foolish to assume the desired microbiome would be
the same for all individuals – there will presumably be different
optimal microbiomic compositions for different hosts. The hope
would be that, much like organ transplants find matches within
key antigenic groups, there will be microbiome types (roughly
like the phylotypes described above) that are compatible with
key important components from the host’s genetic/metabol-
ic makeup. Tools would provide specific ways to both deplete
and augment specific niches of the microbiome being target-
ed. Augmentation will be a matter of identifying key members,
then developing methods for propagation and efficient deliv-
ery into specific patients’ gastrointestinal tracts or other tar-
gets. For depletion, a highly specific agent suppressive towards
pathogenic bacteria and not affecting the ‘good’ bacteria or the
host would be required. A promising approach is using natural
or engineered bacteriophages (phages) – unique viruses that
infect and kill specific bacteria. This specificity can be very nar-
row or broader, and it can also be shifted through currently de-
veloped in vitro techniques. In addition, since phages are tar-
geted for specific components of the bacterial cell membrane,
they are completely safe for humans [113–115]. Multiple ad-
vantages of phage therapy over conventional antibiotics are
listed in Table 1. A number of companies have started devel-
oping bacteriophage-based solutions for pathogenic bacteria,
especially targeting antibiotic-resistant species.
AmpliPhi Biosciences is building a drug development and man-
ufacturing platform designed to allow rapid development and
production of multiple phage-based therapies. The scope in-
cludes chronic lung, sinus and gastrointestinal (GI) infections,
with particular focus on controlling Pseudomonas aeruginosa
infections. Pseudomonas aeruginosa is responsible for long-
term deleterious effects in humans, infecting burns, wounds,
and body cavities. It is the leading complication in patients with
cystic fibrosis, resulting in damage to the lungs that often lead
to respiratory failure. AmpliPhi Biosciences has 3 Ampliphage
drug candidates in the preclinical stage of development [116].
OmniLytics is developing bacteriophage solutions for pathogen
control in the pharmaceutical, agricultural, food and water, in-
dustrial, and defense markets. One of their primary focuses is
development of phage therapies for antibiotic-resistant bacte-
ria, which are causing devastating problems worldwide such as
the shut-down of cardiac units and even entire hospitals due to
outbreaks of methicillin-resistant Staphylococcus aureus (MRSA).
This problem is developing into a crisis because there are several
strains of the disease that have become resistant to all known an
-
tibiotics and have become extremely difficult to eliminate [117].
One organization has years of experience treating challenging
bacterial infections with phages. The Phage Therapy Center
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provides effective lytic phage-based treatment solutions for
patients with bacterial infections that are recalcitrant to treat-
ment: non-healing, long term chronic, drug-resistant infections
that do not respond to conventional therapies. They have de-
veloped novel technologies and protocols for the treatment of
chronic maladies, such as chronic urinary tract infection, chron-
ic prostatitis, chronic sinusitis and non-healing wounds, as well
as acne, bronchitis, cystic fibrosis, lung infections, colitis, skin
infections, intestinal infections, and general dysbiosis [118].
In addition to the above described strategies, there are other
elegant tools that will allow selective modulation of the un-
desired components of the microbial community. Small mol-
ecules, including peptides, are still an active pursuit of many
biotech companies, but being used to alter microbial balanc-
es through more subtle effects than simply poisoning unde-
sired members of the community. Also, the addition of individ-
ual or subsets of microorganisms should be able to be tuned
to both suppress the growth of pathogenic microbes and pro-
mote the growth/establishment of beneficial microbes. A num-
ber of companies are translating the knowledge of microbi-
omes into the next generation of highly specific therapeutics
(see Table 2 and references within). It is worth noting that
some of the drug candidates already completed Phase 2 clin-
ical trials. But the field is still in its inception; to achieve the
optimal and most healthy homeostasis for the individual with
these advanced therapies, the medical research community
must keep pursuing a deeper understanding of the microbio-
ta population and its functions and networks, and interaction
with the host. Although the microbiome is a particularly com-
pelling therapeutic target, because it is something that can be
readily altered (in contrast to human DNA), this ease of ma-
nipulation can lead to disastrous consequences if too much is
attempted without a sufficient knowledge base.
Besides smaller companies listed in Table 2, some major phar-
maceutical companies such as Glaxo Smith Kline, Pfizer, Bayer,
and Johnson & Johnson, as well as cosmetic and personal care
company L’Oreal and food-products company Dannone, have
started investing in programs around GI, skin, oral, and uro-
genital microbiomes, and it is expected that the first series of
microbiome-modulating drugs should become widely available
within the next 5–10 years. According to a report published
by MarketsandMarkets, the emerging microbiome market will
reach $658 million in size by 2023 [124], excluding probiotics,
which is a multi-billion dollar market already.
Thus far we have concentrated on the effects of the micro-
biome on human health in general. In addition to effects on
homeostasis, it can exert another very important, recently
discovered effect: on the metabolism of orally administered
medications. The microbes in the gut have been found to af-
fect the metabolism of some drugs, such as digoxin (a heart
medication often used to treat atrial fibrillation) [125] and ac-
etaminophen (used to treat pain and fever) [126]. A drug used
to treat advanced colorectal cancer, irinotecan (CPT-11), which
is normally detoxified by the liver, is turned back into its ac-
tive (toxic) form by certain gut microbes, causing extensive
cell death in the intestines, leading to severe diarrhea [127].
Prescribing an additional drug that inhibits the microbial en-
zyme responsible for this re-activation may allow cancer pa-
tients to be given more directed doses of the anticancer drug.
Overall, a growing number of studies [128–130] suggest that
to make decisions regarding the best personalized medicine,
in addition to sequencing the patient’s genomic DNA, it is ex-
tremely important to sequence and understand the patient’s
microbiome as well.
Conclusions
In the past several years, the presence of bacteria, archaea, fun-
gi, protists, and viruses in the gastrointestinal tract and other
sites of human body has become to be known as an essential
Bacteriophages Antibiotics
Very specific towards bacterial species Antibiotics target both pathogenic bacteria and normal human microbiota.
This sometimes results in dysbiosis and serious secondary infections (e.g.
yeast)
Replicate exactly at the site of infection Antibiotics do not concentrate at the site of infection and are metabolized
and eliminated from the body
No major side effects Multiple side effects, including intestinal disorders, allergies, and secondary
infections
Selecting new phages takes few weeks Developing a new antibiotic takes several years
Phage-resistant bacteria remain susceptible to
other phages
There is a number of bacterial strains resistant to all known antibiotics
Table 1. Advantages of bacteriophage therapy vs. conventional antibiotics.
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component of human health. It is a finely balanced community
of microorganisms, tuned to the metabolism of its host, now
referred as the human microbiome. What has become clear is
that not one, but multiple microbiomes exist at different sites
in and on the body. Researchers are currently primarily in a dis-
covery phase – what are the compositions of microbial com-
munities in various niches in the human body, and which are
beneficial versus detrimental in their consequences? This has
provided a base of knowledge to build on, so that some re-
searchers are already investigating the roles of various mem-
bers within these communities in regard how it contributes to
that microbiome’s aggregate effect on our general health. It is
fascinating to look at how our perceptions of microorganisms
has evolved, from Pasteur and Koch’s initial demonstrations
showing ‘germs’ as the basis of disease in the late 1800’s, to
the realization in the mid-twentieth century that a microbial
load is tolerated as a natural part of existence, to the current
concept that our bodies not simply tolerate, but rely on nu-
merous collections of microorganisms to provide critical sup-
port for our well-being. As over 20 companies are applying
knowledge of human microbiomes in a clinical setting, next
generation therapies – ranging from introduction of synthet-
ic- and microbe-derived molecules to live bacteria and phag-
es – should soon become a reality.
Acknowledgement
The authors are grateful to Alan Conrad for producing the il-
lustrations in conjunction with the authors.
Company Programs and Status
Rebiotix [111] Developing a new kind of biological drug designed to reverse pathogenic processes
responsible for disease through the transplantation of live human-derived microbes
into a sick person’s intestinal tract. Rebiotix focus is new solutions for challenging
gastrointestinal diseases. The company has completed the Phase 2 open-label clinical trial
to assess the safety of RBX2660 (microbiota suspension) for the treatment of recurrent
Clostridium difficile infection
Enterome [119] Development of novel drugs and diagnostics to support personalized therapies in
microbiome-related diseases such as Inflammatory Bowel Diseases (IBD), metabolic
diseases, and related disorders. Three programs at the validation phase: IBD 120 Ulcerative
colitis-relapse prediction; MET 220 Bariatric surgery-outcome prediction; MET 230
Stratified nutrition test. One program at discovery phase: IBD 110 Crohn’s disease-activity
monitoring
Second Genome [120] Development of microbiome modulators, which are bioactive therapeutics that benefit
human health by altering the composition and activities of the microbial communities in
the body to regulate host pathways. Second Genome’s focus is on microbiome modulators
(small molecules, peptides and bacteria) that impact infection, immunity and metabolic
disease. The company is currently pursuing three preclinical programs and additional
discovery efforts
4D Pharma [121] Developing biotherapeutics using live bacteria, as opposed to traditional drugs based
on chemically synthesized small molecules and antibodies. 4D Pharma has two ongoing
programs. Thetanix: for treatment of paediatric Crohn’s disease. Blautix: for treatment of
Irritable Bowel Syndrome (IBS)
Seres Health [122] Developing Ecobiotic® therapeutics (combinations of a small number of selected discrete
organisms) to treat a range of important medical conditions based on the microbiome
biology at their core. SER-109 therapeutic for recurrent C. difficile is in Phase 3 trials. Pre-
clinical pipeline includes SER-262 for primary C. difficile and SER-155 for drug-resistant
bacteria. Therapeutics for inflammatory and metabolic diseases are in discovery phase
Microbiome therapeutics [123] Development of microbiome modulators- products designed to alter bacterial populations
and their environment in the gastrointestinal tract to prevent and treat serious health
conditions. The company initial research and products are focused on metabolic conditions
including prediabetes, diabetes and obesity. The lead microbiome modulator, NM505 is
in clinical development to assess its efficacy and safety as a reformation of metformin,
the most widely prescribed drug for the treatment of type 2 diabetes. NM504 is in clinical
development as a prescription for treatment of prediabetes and diabetes
Table 2. Companies developing microbiome therapies*.
* Not comprehensive.
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References:
1. Dubos RJ, Schaedler RW: The effect of the intestinal flora on the growth
rate of mice, and on their susceptibility to experimental infections. J Exptl
Med, 1960; 111: 407–17
2. Brownlee A, Moss W: The influence of diet on lactobacilli in the stomach
of the rat. J Pathol Bacterial, 1961; 82: 513–16
3. Dubos R: The Microbiota of the gastrointestinal tract. Gastroenterology,
1996; 51(5): 868–74
4. http://www.microbiomejournal.com/ (Microbiome journal)
5. http://www.tandfonline.com/loi/kgmi20#.VJvpPl4A4 (Gut Microbes journal)
6. http://www.wageningenacademic.com/BM (Beneficial Microbes journal)
7. Lederberg J, McCray AT: Genealogical treasury of words. Scientist, 2001; 15: 8
8. Turnbaugh PJ, Ley RE, Hamady M et al: The human microbiome project.
Nature, 2007; 449: 804–10
9. Savage DC: Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol,
1977; 31: 107–33
10. Whitman WB, Coleman DC, Wiebe WJ: Prokaryotes: the unseen majority.
Proc Natl Acad Sci USA, 1998; 95: 6578–83
11. Bianconi E, Piovesan A, Facchin F et al: An estimation of the number of cells
in the human body. Annals Human Biol, 2013; 40: 463–71
12. Huffingale GB, Noverr MC: The emerging world of the fungal microbiome.
Trends Microbiol, 2013; 21: 334–41
13. Boto L: Horizontal gene transfer in the acquisition of novel traits by meta-
zoans. Proc Biol Sci, 2014; 281: 20132450
14. Pertea M, Salzberg SL: Between a chicken and a grape: estimating the num-
ber of human genes. Genome Biol, 2010; 11: 206
15. The Human Microbiome Project Consortium: Structure, function and diver-
sity of the healthy human microbiome. Nature, 2012; 486: 207–14
16. MacDougall R: NIH Human Microbiome Project defines normal bacterial
makeup of the body. NIH, 2012
17. Reid A, Greene S: FAQ human microbiome – 2013
18. Schloss PD: Microbiology: An integrated view of the skin microbiome. Nature,
2014; 514: 44–45
19. Dong Q, Brulc JM, Iovieno A et al: Diversity of bacteria at healthy human
conjunctiva. Invest Ophthalmol Vis Sci, 2011; 52: 5408–13
20. Wilson MT, Hamilos DL: The nasal and sinus microbiome in health and dis-
ease. Curr Allergy Asthma Rep, 2014; 14: 485
21. Liu CM, Cosetti MK, Aziz M et al: The otologic microbiome: a study of the
bacterial microbiota in a pediatric patient with chronic serous otitis media
using 16SrRNA gene-based pyrosequencing. Arch Otolaryngol Head Neck
Surg, 2011; 137: 664–68
22. Zaura E, Nicu EA, Krom BP, Keijser BJ: Acquiring and maintaining a normal
oral microbiome: current perspective. Front Cell Infect Microbiol, 2014; 4: 85
23. Cabrera-Rubio R, Collado MC, Laitinen K et al: The human milk microbiome
changes over lactation and is shaped by maternal weight and mode of de
-
livery. Am J Clin Nutr, 2012; 96: 544–51
24. Beck JM, Young VB, Huffnagle GB: The microbiome of the lung. Transl Res,
2012; 160: 258–66
25. Lawson RD, Coyle WJ: The noncolonic microbiome: does it really matter?
Curr Gastroenterol Rep, 2010; 12: 259–62
26. Sears CL: A dynamic partnership: celebrating our gut flora. Anaerobe, 2005;
11: 247–51
27. Qin J, Li R, Raes J et al: A human gut microbial gene catalogue established
by metagenomic sequencing. Nature, 2010; 464: 59–65
28. Ramezani A, Raj DS: The gut microbiome, kidney disease, and targeted in-
terventions. J Am Soc Nephrol, 2014; 25: 657–70
29. Hilt EE, McKinley K, Pearce MM et al: Urine is not sterile: use of enhanced
urine culture techniques to detect resident bacterial flora in the adult fe-
male bladder. J Clin Microbiol, 2014; 52: 871–76
30. Price LB, Liu CM, Johnson KE et al: The effects of circumcision on the pe-
nis microbiome. PLoS One, 2010; 5(1) e8422
31. Huang B, Fettweis JM, Brooks JP et al: The changing landscape of the vag-
inal microbiome. Clin Lab Med, 2014; 34: 747–61
32. Rautava S, Luoto R, Salminen S, Isolauri E: Microbial contact during preg-
nancy, intestinal colonization and human disease. Nat Rev Gastroenterol
Hepatol, 2012; 9: 565–76
33. Aagaard K, Ma J, Antony KM et al: The placenta harbors a unique microbi-
ome. Sci Transl Med, 2014; 6: 237
34. Koenig JE, Spor A, Scalfone N et al: Succession of microbial consortia in
the developing infant gut microbiome. Proc Natl Acad Sci USA, 2011; 108:
4578–85
35. Muegge BD, Kuczynski J, Knights D et al: Diet drives convergence in gut
microbiome functions across mammalian phylogeny and within humans.
Science, 2011; 332: 970–74
36. Wu GD, Chen J, Hoffmann C et al: Linking long-term dietary patterns with
gut microbial enterotypes. Science, 2011; 334: 105–8
37. Arumugam M, Raes J, Pelletier E et al: Enterotypes of the human gut mi-
crobiome. Nature, 2011; 473: 174–80
38. http://www.hmpdacc.org/
39. http://commonfund.nih.gov/hmp/index
40. Claesson MJ, O’Sullivan O, Wang Q et al: Comparative analysis of pyrose-
quencing and a phylogenetic microarray for exploring microbial communi-
ty structures in the human distal intestine. PLoS One, 2009; 4: e6669
41. Turnbaugh PJ, Hamady M, Yatsunenko T et al: A core gut microbiome in
obese and lean twins. Nature, 2009; 457: 480–84
42. Ursell LK, Clemente JC, Rideout JR et al: The interpersonal and intraperson-
al diversity of human-associated microbiota in key body sites. J. Allergy Clin
Immunol, 2012; 129: 1204–8
43. Lozupone CA, Stombaugh JI, Gordon JI et al; Diversity, stability and resil-
ience of the human gut microbiota. Nature, 2012; 489: 220–30
44. Gao Z, Tseng CH, Pei Z, Blaser MJ: Molecular analysis of human forearm su-
perficial skin bacterial biota. Proc Natl Acad Sci USA, 2007; 104: 2927–32
45. NIH HMP Working Group, Peterson J, Garges S, Giovanni M et al: The NIH
Human Microbiome Project. Genome Res, 2009; 19: 2317–23
46. Ravel J, Gajer P, Abdo Z et al: Vaginal microbiome of reproductive-age wom-
en. Proc Natl Acad Sci USA, 2011; 108: 4680–87
47. Grice EA, Kong HH, Conlan S et al: Topographical and temporal diversity of
the human skin microbiome. Science, 2009; 324: 1190–92
48. Costello EK, Lauber CL, Hamady M et al: Bacterial community variation in
human body habitats across space and time. Science, 2009; 326: 1694–97
49. http://hmp2.org/
50. Ley RE, Turnbaugh PJ, Klein S, Gordon JI: Microbial ecology: human gut mi-
crobes associated with obesity. Nature, 2006; 444: 1022–23
51. http://humanfoodproject.com/americangut/ (American Gut)
52. http://www.metahit.eu/
53. http://my.microbes.eu/
54. http://www.humanlongevity.com/
55. http://www.earthmicrobiome.org/
56. http://humanfoodproject.com/
57. Candela M, Perna F, Carnevali P et al: Interaction of probiotic Lactobacillus
and Bifidobacterium strains with human intestinal epithelial cells: adhe-
sion properties, competition against enteropathogens and modulation of
IL-8 production. Int J Food Microbiol, 2008; 125: 286–92
58. Fukuda S, Toh H, Hase K et al: Bifidobacteria can protect from enteropatho-
genic infection through production of acetate. Nature, 2011; 469: 543–47
59. Guarner F, Malagelada J: Gut flora in health and disease. Lancet, 2003; 361:
512–19
60. Sonnenburg JL, Xu J, Leip DD et al: Glycan foraging in vivo by an intestine-
adapted bacterial symbiont, Science, 2005; 307: 1955–59
61. Yatsunenko T, Rey FE, Manary MJ et al: Human gut microbiome viewed
across age and geography. Nature, 2012; 486: 222–27
62. Burkholder PR, McVeigh I: Synthesis of vitamins by intestinal bacteria. Proc
Natl Acad Sci USA, 1942; 28: 285–89
63. LeBlanc JG, Milani C, DeGiori GS et al: Bacteria as vitamin suppliers to their
host: a gut microbiota perspective. Curr Opin Biotechnol, 2013; 24: 160–68
64. Olszak T, An D, Zeissig S et al: Microbial exposure during early life has per-
sistent effects on natural killer T cell function. Science, 2012; 336: 489–93
65. Kanther M, Tomkovich S, Xiaolun S et al: Commensal microbiota stimulate
systemic neutrophil migration through induction of serum amyloid A. Cell
Microbiol, 2014; 16(7): 1053–67
11
Conrad R. et al.:
The human microbiota: Composition, functions, and therapeutic potential
© Med Sci Rev, 2015; 2:
PROOF © INTERNATIONAL SCIENTIFIC INFORMATION
1
5
10
15
20
25
30
35
40
45
50
53
1
5
10
15
20
25
30
35
40
45
50
53
66. Frank DN, St Amand AL, Feldman RA et al. Molecular-phylogenetic char-
acterization of microbial community imbalances in human inflammatory
bowel diseases. Proc Natl Acad Sci USA, 2007; 104: 13780–85
67. Dicksved J, Halfvarson J, Rosenquist M et al: Molecular analysis of the gut
microbiota of identical twins with Crohn’s disease. ISME J, 2008; 2: 716–27
68. Shreiner AB, Kao JY, Young VB: The gut microbiome in health and in dis-
ease. Curr Opin Gastroenterol, 2015; 31: 69–75
69. Kau AL, Ahern PP, Griffin NW et al: Human nutrition, the gut microbiome
and the immune system. Nature, 2011; 474: 327–36
70. Lorenzo Pisarello MJ, Vintiñi EO, González SN et al: Decrease in lactobacilli
in the intestinal microbiota of celiac children with a gluten-free diet, and
selection of potentially probiotic strains. Can J Microbiol, 2014; 1: 1–6
71. Turnbaugh PJ, Backhed F, Fulton L, Gordon JI: Diet-induced obesity is linked
to marked but reversible alterations in the mouse distal gut microbiome,
Cell Host Microbe, 2008; 3: 213–23
72. Cox LM, Blaser MJ: Pathways in microbiome-induced obesity. Cell Metab,
2013; 17: 883–94
73. Gibson PG, Foster PS: Asthma 2014: from monoclonals to the microbiome.
Lancet Respir Med, 2014; 2: 956–58
74. Qin J1, Li Y, Cai Z, Li S et al: A metagenome-wide association study of gut
microbiota in type 2 diabetes. Nature, 2012; 490: 55–60
75. Lupton JR: Microbial degradation products influence colon cancer risk: the
butyrate controversy J. Nutr, 2004; 134: 479–82
76. Farrell JJ, Zhang L, Zhou H et al: Variations of oral microbiota are associated
with pancreatic diseases including pancreatic cancer. Gut, 2012; 61: 582–88
77. Stefka AT, Feehley T, Tripathi P et al: Commensal bacteria protect against
food allergen sensitization. Proc Natl Acad Sci USA, 2014; 111: 13145–50
78. Gonzalez A, Stombaugh J, Lozupone C et al: The mind-body-microbial con-
tinuum. Dialogues Clin Neurosci, 2011; 13: 55–62
79. Tang WH, Hazen SL: The contributory role of gut microbiota in cardiovas-
cular disease. J Clin Invest, 2014; 124: 4204–11
80. Sekirov I, Russell SL, Antunes LC, Finlay BB: Gut microbiota in health and
disease. Physiol Rev, 2010; 90: 859–04
81. Vlassov AV, Magdaleno S, Setterquist R, Conrad R: Exosomes: Current knowl-
edge of their composition, biological functions, and diagnostic and thera-
peutic potentials. Biochim Biophys Acta, 2012; 1820: 940–48
82. Smythies LE, Smythies JR: Exosomes in the gut. Front Immunol, 2014; 5: 104
83. Foster JA, McVey Neufeld KA: Gut-brain axis: how the microbiome influ-
ences anxiety and depression. Trends Neurosci, 2013; 36: 305–12
84. Alcock J, Maley CC, Aktipis CA: Is eating behavior manipulated by the gas-
trointestinal microbiota? Evolutionary pressures and potential mechanisms.
Bioessays, 2014; 36: 940–49
85. Hehemann JH, Correc G, Barbeyron T et al: Transfer of carbohydrate-active
enzymes from marine bacteria to Japanese gut microbiota. Nature, 2010;
464: 908–12
86. Maukonen J, Simo C, Saarela M: The currently used commercial DNA-
extraction methods give different results of clostridial and actinobacterial
populations derived from human fecal samples. FEMS Microbiol Ecol, 2012;
79(3): 697–708
87. Wesolowska-Andersen A, Bahl MI, Carvalho V et al: Choice of bacterial DNA
extraction method from fecal matter influences community structure as
evaluated by metagenomic analysis. Microbiome, 2014; 2: 19
88. Yuan S, Cohen DB, Ravel J et al: Evaluation of methods for the extraction
and purification of DNA from the human microbiome. PLoS One, 2012; 7(3):
e33865
89. Santiago A, Panda S, Mengels G et al: Processing faecal samples: a step
forward for standards in microbial community analysis. BMC Microbiology,
2014; 14: 112
90. Cardona S, Eck A, Cassellas M et al: Storage conditions of intestinal micro-
biota matter in metagenomic analysis. BMC Microbiol, 2012; 12: 158
91. https://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-
pcr.html
92. http://www.affymetrix.com/estore/esearch/search.jsp?Ntt=phylochip&basic=1
93. http://www.illumina.com/
94. http://www.lifetechnologies.com/us/en/home/brands/ion-torrent.html
95. Salipante SJ, Kawashima T, Rosenthal C et al: Performance comparison of
illumina and ion torrent next-generation sequencing platforms for 16S
rRNA-based bacterial community profiling. Appl Environ Microbiol, 2014;
80: 7583–91
96. Ardissone AN, DeLaCruz DMDavis-Richardson AG et al: Meconium micro-
biome analysis identifies bacteria correlated with premature birth. PLoS
One, 2014; 9: e90784
97. Petrof EO, Gloor GB, Vanner SJ et al: Stool substitute transplant therapy for
the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut.
Microbiome, 2013; 1: 3
98. Hamady M, Knight R: Microbial community profiling for human microbi-
ome projects: Tools, techniques, and challenges. Genome Res, 2009; 19:
1141–52
99. Kuczynski J, Lauber CL, Walters WA et al: Experimental and analytical tools
for studying the human microbiome. Nat Rev Genet, 2011; 13: 47–58
100. http://www.cdc.gov/hai/organisms/cdiff/Cdiff-patient.html
101. Award MM, Johansen PA, Carter GP et al: Clostridium difficile virulence fac-
tors: insights into an anaerobic spore-forming pathogen. Gut Microbes,
2015; 1: 10
102. Luciano JA, Zuckerbraun BS: Clostridium difficile Infection: Prevention, treat-
ment, and surgical management. Surg Clin North Am, 2014; 94: 1335–49
103. Allegretti JR, Korzenik JR, Hamilton MJ: Fecal microbiota transplantation via
colonoscopy for recurrent C. difficile infection. J Vis Exp, 2014; (94)
104. Matsuoka K, Mizuno S, Hayashi A et al: Fecal microbiota transplantation
for gastrointestinal diseases. Keio J Med, Keio J Med, 2014; 63(4): 69–74
105. http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatory
Information/Guidances/Vaccines/ucm361379.htm
106. http://www.mayoclinic.org
107. http://www.brightmedicineclinic.com/
108. http://taymount.com/
109. http://thefecaltransplantfoundation.org/
110. http://www.openbiome.org/
111. http://www.rebiotix.com/
112. https://clinicaltrials.gov/
113. Viertel TM, Ritter K, Horz HP: Viruses versus bacteria-novel approaches to
phage therapy as a tool against multidrug-resistant pathogens. J Antimicrob
Chemother, 2014; 69: 2326–36
114. Qadir MI: Phage therapy: A modern tool to control bacterial infections. Pak
J Pharm Sci, 2015; 28: 265–70
115. Nilsson AS. Phage therapy – constraints and possibilities. Ups J Med Sci,
2014; 119: 192–98
116. http://ampliphibio.com/
117. http://omnilytics.com/
118. http://www.phagetherapycenter.com/
119. http://www.enterome.com/
120. http://www.secondgenome.com/
121. http://www.4dpharmaplc.com/
122. http://sereshealth.com/
123. http://www.mbiome.com/
124. http://www.marketsandmarkets.com/Market-Reports/human-microbiome-
market-37621904.html
125. Haiser HJ, Gootenberg DB, Chatman K et al: Predicting and manipulating
cardiac drug inactivation by the human gut bacterium Eggerthella lenta.
Science, 2013; 341: 295–98
126. Clayton TA, Baker D, Lindon JC et al: Pharmacometabonomic identification
of a significant host-microbiome metabolic interaction affecting human
drug metabolism. Proc Natl Acad Sci USA, 2009; 106: 14728–33
127. Takasuna K, Hagiwara T, Hirohashi M et al: Involvement of beta-glucuron-
idase in intestinal microflora in the intestinal toxicity of the antitumor
camptothecin derivative irinotecan hydrochloride (CPT-11) in rats. Cancer
Res, 1996; 56: 3752–57
128. Saad R, Rizkallah MR, Aziz RK: Gut Pharmacomicrobiomics: the tip of an ice-
berg of complex interactions between drugs and gut-associated microbes.
Gut Pathog, 2012; 4: 16
129. Li H, Jia W: Cometabolism of microbes and host: implications for drug me-
tabolism and drug-induced toxicity. Clin Pharmacol Ther, 2013; 94: 574–81
130. Haiser HJ, Turnbaugh PJ: Is it time for a metagenomic basis of therapeu-
tics? Science, 2012; 336: 1253–55
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