The Human Microbiota in Health and Disease

Abstract

Trillions of microbes have evolved with and continue to live on and within human beings. A variety of environmental factors can affect intestinal microbial imbalance, which has a close relationship with human health and disease. Here, we focus on the interactions between the human microbiota and the host in order to provide an overview of the microbial role in basic biological processes and in the development and progression of major human diseases such as infectious diseases, liver diseases, gastrointestinal cancers, metabolic diseases, respiratory diseases, mental or psychological diseases, and autoimmune diseases. We also review important advances in techniques associated with microbial research, such as DNA sequencing, metabonomics, and proteomics combined with computation-based bioinformatics. Current research on the human microbiota has become much more sophisticated and more comprehensive. Therefore, we propose that research should focus on the host-microbe interaction and on cause-effect mechanisms, which could pave the way to an understanding of the role of gut microbiota in health and disease, and provide new therapeutic targets and treatment approaches in clinical practice.

Full text

Engineering 3 (2017) 71–82
Research
Microecology—Review
The Human Microbiota in Health and Disease
Baohong Wang, Mingfei Yao, Longxian Lv, Zongxin Ling, Lanjuan Li*
National Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of
Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China
a r t i c l e i n f o a b s t r a c t
Article history:
Received 20 December 2016
Revised 9 January 2017
Accepted 12 January 2017
Available online 20 February 2017
Trillions of microbes have evolved with and continue to live on and within human beings. A variety of
environmental factors can affect intestinal microbial imbalance, which has a close relationship with hu-
man health and disease. Here, we focus on the interactions between the human microbiota and the host
in order to provide an overview of the microbial role in basic biological processes and in the develop-
ment and progression of major human diseases such as infectious diseases, liver diseases, gastrointesti-
nal cancers, metabolic diseases, respiratory diseases, mental or psychological diseases, and autoimmune
diseases. We also review important advances in techniques associated with microbial research, such as
DNA sequencing, metabonomics, and proteomics combined with computation-based bioinformatics.
Current research on the human microbiota has become much more sophisticated and more comprehen-
sive. Therefore, we propose that research should focus on the host-microbe interaction and on cause-
effect mechanisms, which could pave the way to an understanding of the role of gut microbiota in
health and disease. and provide new therapeutic targets and treatment approaches in clinical practice.
© 2017 THE AUTHORS. Published by Elsevier LTD on behalf of the Chinese Academy of Engineering and
Higher Education Press Limited Company. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords:
Microbiome
Health
Infectious disease
Liver diseases
Gastrointestinal malignancy
Metabolic disorder
Microbiota technology
Probiotics
1. Introduction
More than 100 trillion symbiotic microorganisms live on and
within human beings and play an important role in human health
and disease. The human microbiota, especially the gut microbiota,
has even been considered to be an “essential organ” [1], carrying
approximately 150 times more genes than are found in the entire
human genome [2]. Important advances have shown that the gut
microbiota is involved in basic human biological processes, in-
cluding modulating the metabolic phenotype, regulating epitheli-
al development, and influencing innate immunity [3–6]. Chronic
diseases such as obesity, inflammatory bowel disease (IBD), di-
abetes mellitus, metabolic syndrome, atherosclerosis, alcoholic
liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), cir-
rhosis, and hepatocellular carcinoma have been associated with
the human microbiota [7,8] (Fig.1).
In recent decades, a tremendous amount of evidence has strong-
ly suggested a crucial role of the human microbiota in human
health and disease [7,9–23] via several mechanisms. First, the
microbiota has the potential to increase energy extraction from
food [24], increase nutrient harvest [9,10], and alter appetite
signaling [25,26]. The microbiota contains far more versatile
metabolic genes than are found in the human genome, and pro-
vides humans with unique and specific enzymes and biochemical
pathways [9]. In addition, a large proportion of the metabolic
microbiotic processes that are beneficial to the host are involved
in either nutrient acquisition or xenobiotic processing, including
the metabolism of undigested carbohydrates and the biosynthe-
sis of vitamins [10]. Second, the human microbiota also provides
a physical barrier, protecting its host against foreign pathogens
through competitive exclusion and the production of antimicro-
bial substances [11–13]. Finally, the microbiota is essential in the
development of the intestinal mucosa and immune system of the
host [14,16]. For example, germ-free (GF) animals have abnor-
mal numbers of several immune cell types, deficits in local and
systemic lymphoid structures, poorly formed spleens and lymph
* Corresponding author.
E-mail address: ljli@zju.edu.cn
http://dx.doi.org/10.1016/J.ENG.2017.01.008
2095-8099/© 2017 THE AUTHORS. Published by Elsevier LTD on behalf of the Chinese Academy of Engineering and Higher Education Press Limited Company.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/eng
Engineering

72 B. Wang et al. / Engineering 3 (2017) 71–82
nodes, and perturbed cytokine levels [16]. Studies on GF animals
have suggested that the immune modulation functions of the
microbiota are primarily involved in promoting the maturation of
immune cells and the normal development of immune functions
[14]. In addition, studies have revealed the central role of micro-
bial symbiosis in the development of many diseases [17], such as
infection [18], liver diseases [19], gastrointestinal (GI) malignancy
[20], metabolic disorders [7], respiratory diseases [21], mental or
psychological diseases [22], and autoimmune diseases [23].
In this article, we provide an overview of the role of the human
microbiota in health and disease, the advent of microbiome-wide
association studies, and potential and important advances in the
development of clinical applications to prevent and treat human
disease.
2. The human microbiota in health
The human microbiota affects host physiology to a great ex-
tent. Trillions of microbes colonize the human body, including
bacteria, archaea, viruses, and eukaryotic microbes. The body
contains at least 1000 different species of known bacteria and
carries 150 times more microbial genes than are found in the
entire human genome [2]. Microbiotic composition and function
differ according to different locations, ages, sexes, races, and diets
of the host [27].
Commensal bacteria colonize the host shortly after birth. This
simple community gradually develops into a highly diverse eco-
system during host growth [28]. Over time, host-bacterial asso-
ciations have developed into beneficial relationships. Symbiotic
bacteria metabolize indigestible compounds, supply essential nu-
trients, defend against colonization by opportunistic pathogens,
and contribute to the formation of intestinal architecture [29]. For
example, the intestinal microbiota is involved in the digestion of
certain foods that cannot be digested by the stomach and small
intestine, and plays a key role in maintaining energy homeosta-
sis. These foods are primarily dietary fibers such as xyloglucans,
which are commonly found in vegetables and can be digested by
a specific species of Bacteroides [30]. Other non-digestible fibers,
such as fructooligosaccharides and oligosaccharides, can be uti-
lized by beneficial microbes, such as Lactobacillus and Bifidobac-
terium [31]. Studies have clarified the role of the gut microbiota
in lipid and protein homeostasis as well as in the microbial syn-
thesis of essential nutrient vitamins [32]. The normal gut micro-
biome produces 50–100 mmol·L
-1
per day of short-chain fatty acids
(SCFAs), such as acetic, propionic, and butyric acids, and serves as
an energy source to the host intestinal epithelium [33]. These SC-
FAs can be quickly absorbed in the colon and serve many diverse
roles in regulating gut motility, inflammation, glucose homeostasis,
and energy harvesting [34,35]. Furthermore, the gut microbiota has
been shown to deliver vitamins to the host, such as folates, vita-
min K, biotin, riboflavin (B
2
), cobalamin (B
12
), and possibly other B
vitamins. A previous study demonstrated that B
12
can be produced
from delta-aminolevulinate (ALA) as a precursor [36].
In addition, gut-colonizing bacteria stimulate the normal de-
velopment of the humoral and cellular mucosal immune systems
[37]. The signals and metabolites of microorganisms can be sensed
by the hematopoietic and non-hematopoietic cells of the innate
immune system and translated into physiological responses [38].
Studies comparing normal mice with GF mice have found that GF
mice show extensive defects in the development of gut-associated
lymphoid tissue and antibody production [29,39]. A report has
also demonstrated that the gut microbiota generates a tolerogenic
response that acts on gut dendritic cells and inhibits the type 17
T-helper cell (Th17) anti-inflammatory pathway [40]. However, not
all microbiota lead to health benefits. Some induce inflammation
under certain conditions.
3. The human microbiota in disease
3.1. The human microbiota and infectious diseases
Infection is one of the most common diseases caused by dys-
biosis of the microbiota. Importantly, infectious disease and its
treatment have a profound impact on the human microbiota,
which in turn determines the outcome of the infectious disease in
the human host (Fig. 2). Offending pathogens colonize the intesti-
nal mucosa, thus resulting in the induction of a strong inflamma-
tory response, followed by the translocation of the intestinal bac-
teria [41,42]. Numerous studies have demonstrated the intimate
relationship between infection and dysbiosis of the microbiota,
and have shown that infection is associated not only with the
microbiome, but also with viruses [43,44]. For example, the intes-
tinal microbiota of patients with Clostridium difficile (C. difficile)
infection (CDI) is significantly altered [45,46]. Disturbance of the
microbiota is also associated with the progression of human im-
munodeficiency virus (HIV) [44,47], hepatitis B virus (HBV) [48],
and other diseases [49,50].
3.1.1. Infection with Clostridium difficile
The pathological overgrowth of C. difficile is usually related to
antibiotic-associated diarrhea, which is one of the most frequent
complications following antibiotic administration and which is
now a growing public health threat [45]. C. difficile is an anaero-
bic, gram-positive, spore-forming bacillus that is a component of
the human gut microbiota. Antibiotics disturb intestinal mucosa
homeostasis, thus decreasing resistance against toxin-producing
C. difficile and promoting the progression of CDI [45]. Gu et al. [45]
found that fecal bacterial diversity is reduced and the microbial
composition dramatically shifts in patients following antibiotic ad-
ministration, whether or not CDI is present. A decrease in putative
butyrate-producing anaerobic bacteria and an increase in endo-
toxin-producing opportunistic pathogens and lactate-producing
phylotypes have been detected in patients following antibiotic
administration, whether or not CDI is present [45]. Putative
butyrate-producing anaerobic bacteria are significantly depleted
Fig. 1. Human microbial symbiosis has a close relationship with diseases of differ-
ent systems.

73B. Wang et al. / Engineering 3 (2017) 71–82
effective highly active anti-retroviral therapy (HAART), the diver-
sity and composition of the fecal microbiota are not completely
restored, and the dysbiosis remains [47]. South African teenage
girls and young women have extremely high rates of HIV infec-
tion, a phenomenon that has been considered to be associated
with biological factors. Recently, one study found that the vaginal
microbiome might affect the risk of HIV infection. A bacterium in
the vagina named Prevotella bivia has been identified as causing
inflammation. A microbicidal gel is available that decomposes the
anti-HIV drug tenofovir, thus leading to tenofovir treatment fail-
ure [44]. Through close examination of the vaginal microbiome,
Cohen [44] recently found an unusual bacterium named Gard-
nerella in the vagina; this finding potentially explains the high
infection rates in South African women and strongly suggests that
the vaginal microbiome affects HIV risk. Cohen [44] found that
Gardnerella “gobbles up” tenofovir, thus rapidly decreasing the
levels of the drug and leading to tenofovir treatment failure.
3.2. The human microbiota and liver diseases
Growing evidence demonstrates the close interaction of the
GI tract (GIT) and the liver, as well as the chronic exposure of the
liver to gut-derived factors including bacteria and bacterial com-
ponents, thus fostering the use of the term “gut-liver axis” [51].
The intestinal microbiota produces ethanol, ammonia, and acet-
aldehyde; these products may influence liver function through
endotoxin release or liver metabolism [52].
Alterations in the intestinal microbiota play an important role
in inducing and promoting liver damage progression as well as
in direct injury resulting from different causal agents (e.g., viral,
toxic, and metabolic agents) [53] through mechanisms such as
the activation of Kupffer cells by bacterial endotoxins. The gut
microbiota participates in the pathogenesis of liver cirrhosis
complications, such as infections, spontaneous bacterial peri-
tonitis, hepatic encephalopathy, and renal failure. Patients with
liver cirrhosis have an altered bacterial composition in their gut;
patients in Child-Pugh classes B/C have a higher prevalence of
bacterial overgrowth than those in class A [54,55]. Fecal microbi-
al communities are distinct in patients with cirrhosis compared
with healthy individuals. By bacterial 16S rRNA gene sequencing,
microbial diversity, and especially Bacteroidetes species, is shown
to be reduced in cirrhotic patients, while the number of species
of Proteobacteria and Fusobacteria are increased [56]. In line with
the previous study, Bacteroides has also been found to decrease at
in patients with antibiotic treatment when compared with healthy
controls. The above changes in microbial communities may in-
crease susceptibility to C. difficile colonization. Ling et al. [46] found
that different toxigenic C. difficile strains have different effects on
fecal microbiota in children. C. difficile strains that are both toxin
A-positive and toxin B-positive reduce fecal bacteria diversity to a
greater degree than strains that are only toxin B-positive.
3.1.2. Infection with Helicobacter pylori
Helicobacter pylori (H. pylori) is a pathogen that induces pep-
tic disease. It was recently found to be related to the progress of
periodontitis [49]. Hu et al. [49] investigated the correlation of
H. pylori infection with periodontal parameters, periodontal path-
ogens, and inflammation. Their study showed that the frequen-
cies of Porphyromonas gingivalis, Prevotella intermedia, Fusobacte-
rium nucleatum, and Treponema denticola are significantly higher
in patients infected with H. pylori than in those without infection,
whereas the frequency of Aggregatibacter actinomycetemcomitans
is lower. The results indicate that patients with H. pylori show sig-
nificantly higher probing depth and attachment loss, and that H.
pylori might promote the growth of some periodontal pathogens
and aggravate the progress of chronic periodontitis [49].
3.1.3. Bacterial vaginosis
Another important infection called bacterial vaginosis (BV) is
associated with numerous adverse health outcomes including
pre-term birth and the acquisition of sexually transmitted infec-
tions. BV is regarded as an ecological disorder of the vaginal mi-
crobiota. Using culture-independent polymerase chain reaction
(PCR) denaturing gradient gel electrophoresis (DGGE) and bar-
coded 454 pyrosequencing methods, Ling et al. [50] observed a
profound shift in the absolute and relative abundances of bacte-
rial species present in the vagina. In a comparison of populations
associated with healthy and diseased conditions, three phyla and
eight genera were clearly and strongly associated with BV. These
genera may be used as targets for clinical BV diagnosis by means
of molecular approaches [50].
3.1.4. Infection with HIV
At present, HIV continues to be a major global public health
issue. The gut microbiomes in patients with HIV are significantly
disturbed, and there are significant increases in the Firmicutes/
Bacteroidetes ratio of patients infected with HIV-1 [47]. Although
the viral loads of HIV-1 are reduced after a short-term course of
Fig. 2. Infectious diseases have a profound impact on the human microbiota. The wide use of antibiotics, immunosuppressive drugs, and other new treatment technologies
for infectious diseases such as frequently emerging infectious diseases, HIV infection, and CDI has a profound impact on the human microbiota, which in turn determines
the outcome of the infectious disease in the human host.

74 B. Wang et al. / Engineering 3 (2017) 71–82
the genus level, according to a metagenomics technique. In this
study, a gene catalog of the gut microbiomes of Chinese patients
with liver cirrhosis was constructed for the first time. Further-
more, Veillonella, Streptococcus, and Clostridium were found to be
enriched in patients with liver cirrhosis [57]. On the basis of our
previous findings, we further detected dysbiosis of the duodenal
mucosal microbiota in liver cirrhosis patients. In this research,
we found that cirrhotic patients were colonized by a remarkably
different duodenal mucosal microbiota in comparison with the
controls. Twelve operational taxonomic units (OTUs) were iden-
tified as the key microbes contributing to the differentiation be-
tween the cirrhosis and control duodenal microbiota. Regarding
the etiology of cirrhosis, two OTUs were found to discriminate
between types of liver cirrhosis, with different etiology results for
HBV-related cirrhosis and primary biliary cirrhosis (PBC). These
findings indicated that duodenum dysbiosis might be related to
alterations in the oral microbiota and to changes in the duodenal
microenvironment [58]. The oral microbiota is one of the most
important microbial communities in the human body. This study
was also the first to show that the diversity and composition of
the oral microbiota in patients with liver cirrhosis are signifi-
cantly different from those of healthy controls and from those of
patients with HBV-related chronic diseases. Harmful bacteria may
be derived from the oral cavity. In addition, patients with chronic
liver disease show oral diseases [43].
Acute-on-chronic liver failure (ACLF) syndrome is character-
ized by the acute decompensation of cirrhosis, with high 28-d
mortality. Based on the final clinical outcome at 90 d, we were
the first to identify gut dysbiosis in ACLF patients, and to demon-
strate its predictive value for mortality. We found a marked dif-
ference between the gut microbiota of the ACLF group and that of
the control group. Our study indicated that there are correlations
between specific bacterial families and inflammatory cytokines
in ACLF patients. We have demonstrated that the relative abun-
dance of Pasteurellaceae and the model of end-stage liver disease
(MELD) score are independent factors that predict the mortality
rate, thus indicating that gut dysbiosis is associated with the
mortality of patients with ACLF [59].
Although the exact reason for these changes in liver cirrho-
sis remains unclear, these changes are certainly associated with
reduced intestinal motility and pancreatobiliary secretions, an
impaired intestinal barrier, and decreased gastric acidity. In ad-
dition, 80% of hepatocellular carcinoma (HCC) develops in a mi-
croenvironment of chronic injury, inflammation, or fibrosis [60].
Changes in the composition of the gut microbiota promote HCC
by contributing to hepatic inflammation through increased intes-
tinal permeability and the activation of Toll-like receptors [60].
The incidence and prevalence of primary sclerosing cholangitis
(PSC), PBC, and autoimmune hepatitis increases every year [61].
Autoimmune liver diseases are presumed to involve environmen-
tal factors in individuals with genetic susceptibility; however, the
gut flora is relevant to pathogenesis. Recently, a study showed
that patients with PSC-IBD have distinct gut microbiota and a
significant increase in the abundance of Escherichia, Lachnos-
piraceae, and Megasphaera, along with a near-absence of Bacte-
roides, as compared with IBD patients and control patients [62].
Another study found patients with PBC-altered gut bacterial taxa
that exhibited potential interactions through their associations
with altered metabolism, immunity, and liver-function indicators
[63]. There is evidence that bacterial antigens translocate across
a leaky and inflamed gut wall into the portal and biliary system;
thus, they may induce an abnormal immune response and initiate
autoimmune liver disease [64].
NAFLD is a multifactorial disorder comprising a group of dis-
eases. Genetic, epigenetic, and environmental factors interact with
one another during the development of these diseases. Nonalco-
holic steatohepatitis (NASH) is a hepatic feature of metabolic syn-
drome. Obesity and insulin resistance are often factors promoting
NASH. The accumulation of triglycerides in hepatocytes is the
most commonly observed phenotype in NAFLD [65]. Alterations
in the gut microbiota are considered to be a key factor contribut-
ing to NAFLD, and the interplay of metabolic syndrome, diabetes,
and liver disease in NAFLD patients influences the microbiota in
complementary ways [66]. Because the body mass index (BMI)
may be a major determinant of compositional changes in micro-
bial communities [7], Wang et al. [8] directly assessed the fecal
microbial composition and its correlation with liver biochemistry
in non-obese adult patients with NAFLD. In a human study of
gut dysbiosis across the spectrum of NAFLD lesions, comprising
57 patients with biopsy-proven NAFLD, significant fibrosis was
found to be associated with large amounts of Bacteroides and Ru-
minococcus and decreased levels of Prevotella. Along with metab-
olite information from patients, a microbiota analysis is useful for
predicting NAFLD classes and severity. For example, Bacteroides
abundance is independently associated with NASH severity, and
Ruminococcus abundance is associated with significant fibrosis
[67].
Thus, liver disease is usually accompanied by an increase in
Enterobacteriaceae and a decrease in Bifidobacterium. Gut dysbio-
sis can lead to endotoxemia in patients through bacterial translo-
cation (BT). Endotoxemia may induce immune dysfunction, thus
leading to further liver cell necrosis and liver failure. Therefore,
we propose the development of new probiotics specifically for
the prevention and treatment of the progression of liver diseases
(Fig. 3).
3.3. The human microbiota associated with gastrointestinal
malignancy
GI malignancy is a leading cause of human morbidity and
mortality worldwide. Aside from widely accepted genetic fac-
tors, non-genetic factors for cancer risk, especially the residential
microbes in the GIT, exert a broad impact on the development of
cancers that arise within the GIT. Recent advances in microbial
research on GI malignancies, such as gastric cancer, colorectal
cancer, and esophageal cancer, provide new insight into the role
of the human microbiota in tumorigenesis.
3.3.1. Gastric cancer
H. pylori-associated chronic inflammation is considered to be the
strongest risk factor for gastric cancer. Each year, approximately
660 000 new cases of gastric cancer are caused by H. pylori in-
fection, which results in the loss of acid-producing parietal cells,
thus leading to the development of gastric atrophy, metaplasia,
dysplasia, and, finally, carcinoma formation [68]. It is interest-
ing that the elimination of H. pylori before the onset of chronic
atrophic gastritis may protect against gastric cancer [69]. As a
distinct causative factor for gastric cancer, H. pylori has been clas-
sified as a class I carcinogen by the World Health Organization
(WHO). However, only 1%–2% of people with an H. pylori infection
develop stomach cancer [70]. The carcinogenic risk may be relat-
ed to the genetic diversity of the H. pylori strain, variations in host
responses, and specific host-microbe interactions [71]. Impor-
tantly, the phylogenetic origin of H. pylori is a good predictor of
the risk for gastric cancer [72].
The two best-studied H. pylori determinants, cytotoxin-associated
antigen A (CagA) and vacuolating cytotoxin (VacA), have been
shown to be associated with a higher risk of cancer [73]. It
has been reported that VacA promotes the apoptosis of gastric
epithelial cells, in a specific host response to H. pylori that may be

75B. Wang et al. / Engineering 3 (2017) 71–82
responsible for gastric carcinogenesis by interfering with mito-
chondrial function [74]. In addition, VacA suppresses the host im-
mune response by inducing dendritic cells to express and release
the anti-inflammatory cytokines interleukin (IL)-10 and IL-18.
This compromised immune response promotes H. pylori evasion
and enhances tumor survival [75]. Unlike the VacA gene, the cag
pathogenicity island (PAI), which is present in some strains of H.
pylori, is associated with a significantly increased risk of devel-
oping adenocarcinoma of the stomach [76]. Genes within the cag
PAI encode proteins that form a bacterial type IV secretion system
(T4SS). T4SS exports CagA and peptidoglycan from adherent H.
pylori into host cells, thus activating the PI3K pathway, stimu-
lating cell migration, and contributing to carcinogenesis [77].
After tyrosine phosphorylation, CagA interacts with and activates
several host cell proteins, thereby leading to morphological alter-
ations, including cell scattering and elongation [78]. Beyond H.
pylori, Lertpiriyapong et al. [79] noted that the synergetic coloni-
zation of altered Schaedler’s flora (ASF) causes more pronounced
gastric pathology in insulin-gastrin (INS-GAS) mice, including gas-
tric corpus inflammation, epithelial hyperplasia, and dysplasia.
3.3.2. Colorectal cancer
The interaction of the gut microbiome with the development
of colon cancer has recently become a major focus of research.
Microbial dysbiosis has been implicated in the etiology of
colorectal adenomas and colorectal cancer (CRC). A pathological
imbalance in the microbial community has been observed in
subjects with adenomas compared with normal controls [80,81].
Despite the varied results among different studies, the microbiota
in cases of adenomas or CRC is characterized by a high proportion
of potential pathogens, such as Pseudomonas, Helicobacter, and
Acinetobacter, and by a lower richness of beneficial bacteria, such
as butyrate-producing bacteria [80]. Zackular et al. [82] observed
that the gut flora from tumor-bearing mice promotes inflamma-
tion and tumorigenesis in recipient animals, thus directly contrib-
uting to CRC. This study has provided mechanistic insight into the
relationship between the gut microbiome and CRC development.
However, it is still unclear from human studies whether the al-
teration in the microbial community is a cause or consequence of
adenomas and CRC.
In addition, the contribution of specific bacterial species to
cancer risk remains to be fully established. Fusobacterium nuclea-
tum, a periodontal pathogen, has been suggested to be overabun-
dant during disease progression from adenomas to cancer [83]. A
significant increase in Bacteroides massiliensis, Bacteroides ovatus,
Bacteroides vulgatus, and Escherichia coli (E. coli) has also been ob-
served from advanced adenoma to carcinoma [84]. The potential
mechanism underlying this development includes the promotion
of inflammation and the induction of tumorigenesis [85,86].
Kostic et al. [86] observed that Fusobacterium nucleatum modu-
lates the tumor-immune microenvironment by promoting the
myeloid infiltration of intestinal tumors in an adenomatous poly-
posis coli (APC) multiple intestinal neoplasia (Min) mouse model
of CRC and increasing the expression of pro-inflammatory genes
such as PTGS2 (COX2), SCYB1(IL8), IL6, TNF (TNFα), and MMP3. In
addition, many studies have elucidated a link between bacterial
antigens, virulence factors, and colon malignancy. Enterotoxigenic
Bacteroides fragilis (ETBF) produces a toxin known as fragilysin (B.
fragilis toxin, BFT), which activates the Wnt/β-catenin signaling
pathway and NF-κB; consequently, it increases cell proliferation
and induces the production of inflammatory mediators [87–89].
The role of ETBF in colorectal carcinogenesis was further illustrat-
ed by Wu et al. [90], who showed that mice colonized with ETBF
exhibit a marked increase in colon adenomas and tumors as com-
pared with normal controls. Enterococcus faecalis and E. coli may
induce DNA damage by promoting the release of extracellular
superoxide in host cells and encoding the enzymatic machinery
that generates colibactin via the polyketide synthase (PKS) geno-
toxic island [91,92]. Although these observations show an etiolog-
ical contribution by intestinal microbiota in colorectal neoplasia,
additional investigations are needed to determine their potential
as CRC biomarkers, or their utility as diagnostic and therapeutic
targets.
Moreover, many bacteria-derived metabolites have been im-
plicated in the suppression of colon cancer development; these
include SCFAs, which are produced through the microbial fermen-
tation of complex polysaccharides, including acetate, propionate,
and butyrate, which serve as energy sources for colonic epithelial
cells. Butyrate, which is primarily produced by species within the
Lachnospiraceae and Ruminococcaceae, has been shown to be
protective against colonic neoplasia. A high fiber intake reported-
ly leads to a reduction in the risk of developing colon malignancy
because of the production of butyrate [93,94]. In an in vitro study
of cancer lines, butyrate was found to exert a tumor-suppressing
Fig. 3. Our hypothetical pathway for the role of gut microbiota dysbiosis in liver diseases. Evidence shows that chronic liver disease is usually accompanied by intestinal
dysbiosis, which is characterized by the increase of Enterobacteriaceae and the decrease of Bifidobacterium; this can lead to BT, then to endotoxemia and even spontaneous
bacterial peritonitis (SBP), and finally to progression of the liver disease. Importantly, the maintenance of the normal microbial community by means of probiotics/prebiotics
could greatly improve the prevention and treatment effect of liver disease.

76 B. Wang et al. / Engineering 3 (2017) 71–82
effect by inducing apoptosis, inhibiting proliferation, causing
epigenetic changes in gene expression, and modulating inflam-
matory responses and cytokine levels [95]. Therefore, modulation
of the gut microbiome through dietary control or antibiotic treat-
ment may offer great therapeutic potential. The manipulation
of gut microbiota to favor and enhance the production of SCFAs
through the use of prebiotic or non-digestible food ingredients
may be a promising approach to program host metabolism, and
may consequently influence cancer risk.
3.3.3. Esophageal cancer
Recent studies confirm that chronic inflammation at the end
of the esophagus that is caused by gastroesophageal reflux is
closely related to esophageal adenocarcinoma (EA). The overall
pathophysiology of this development process can be described as
“gastroesophageal reflux disease–Barrett’s esophagus–esophageal
adenocarcinoma” (GERD–BE–EA) [96–98]. Regional differences
in its incidence appear to be correlated with economic develop-
ment. Therefore, researchers have suggested that the morbidity
from EA may be related to the use of antibiotics worldwide.
Long-term changes in esophageal microecology after frequent
antibiotic exposure may lead to a higher incidence of GERD, thus
resulting in an increasing morbidity from EA [99]. A large num-
ber of descriptive studies have reported observing esophageal
microecological changes in patients with GERD [100]. However,
the local microbiome does not distinguish between squamous
cell carcinoma and adenocarcinoma [101]. In addition, the role
of H. pylori in the pathogenesis of GERD and EA remains unclear
and controversial. H. pylori was first identified by the WHO as a
carcinogen associated with gastric cancer in the 1990s, and erad-
ication treatments against this bacterium are widely performed.
In addition, researchers have found that, with the decline in H.
pylori infection, GERD incidence has increased [102]. A series of
case-control studies also suggested that H. pylori may play a pro-
tective role in the development of GERD and associated EA. How-
ever, the eradication of H. pylori treatment does not worsen GERD
or increase new GERD [103].
3.4. The human microbiota and metabolic disorders
The composition of the gut microbiota is influenced by the use
of antibiotics and by the lifestyle of the human host, including
exercise, diet, and hygiene preferences. In turn, the dysbiosis of
intestinal flora affects the production of immune mediators and
induces both chronic inflammation and metabolic dysfunction
[104]. Obesity and its associated metabolic complications, such as
type 2 diabetes (T2D) and cardiovascular disease, have become a
global epidemic health problem and are considered to be the con-
sequences of a complex multidirectional interaction among host
genetics, diet, environment, and the gut microbiota [105].
3.4.1. Obesity
An increasing number of in vivo and human studies have in-
dicated that interactions between the gut microbiota and host
genotype or dietary changes may be crucial factors that contrib-
ute to obesity and related metabolic disorders [106,107]. Ridaura
et al. [108] demonstrated that the microbiota from lean or obese
co-twins induces similar adiposity and metabolic phenotypes in
mice. Moreover, the lean co-twin’s microbiota can prevent adi-
posity gain in obese-recipient mice, if the mice are fed with an
appropriate diet [108]. Several studies on the gut microbiota in-
dicated that diet modulates the composition and function of mi-
crobes in humans [109] and rodents [110]. For example, a mouse
study revealed that mice that were fed with lard for 11 weeks
exhibited increased Toll-like receptor activation and white adi-
pose tissue inflammation, along with reduced insulin sensitivity,
compared with mice that were fed with fish oil [110]. However,
phenotypic differences between the dietary groups can be partly
attributed to differences in microbiota composition. Increasing
evidence shows that the gut microbiota is an important mod-
ulator of the interaction between diet and the development of
metabolic diseases [111]. Furthermore, recent studies have shown
that the gut microbiota influences the circadian clock and under-
goes circadian oscillations [112]. Disruption of the host circadian
clock induces dysbiosis, which is associated with host metabolic
disorders [113]. Obesity, which is associated with gut microbiota
dysbiosis and altered metabolic pathways, induces impaired gut
epithelial barrier function and has significant influences on phys-
iological processes [114], such as gut and immune homeostasis
[115], energy metabolism [116], acetate [25] and bile acid metab-
olism [117], and intestinal hormone release [118].
3.4.2. Type 2 diabetes
T2D is a prevalent metabolic disease worldwide; the link be-
tween the gut microbiome composition and the development of
T2D is gradually being uncovered [119–121]. Growing numbers
of studies indicate that an altered gut microbiome characterized
by lower diversity and resilience is associated with diabetes. The
mechanisms that cause the disease may be related to the trans-
location of microbiota from the gut to the tissues, thus inducing
inflammation [122]. Pedersen et al. [123] recently demonstrated
that the human gut microbiome may affect the serum metab-
olome and induce insulin resistance through species such as
Prevotella copri and Bacteroides vulgates. Metformin is one of the
most widely used antidiabetic drugs and is thought to confound
the results of metagenomics data analysis [121]. The gut microbi-
ota may directly affect T2D through its effect on the metabolism
of amino acids; thus, future antidiabetic treatment strategies may
target bacterial strains that cause imbalances in amino acid me-
tabolism [121,124] . Therefore, obesity and its associated metabolic
complications may be a result of complex gene-environment
interactions. Microbiome interventions aimed at restoring the
homeostasis of the gut microbiome have recently emerged, such
as the ingestion of specific fibers or therapeutic microbes. These
are promising strategies to reduce insulin resistance and related
metabolic diseases.
3.5. The human microbiota and other diseases
Growing evidence indicates that alterations in the microbiota
are implicated in the pathogenesis of a number of other dis-
eases, such as severe asthma, food allergies, autism, and major
depressive disorder (MDD) [125–130], all of which have recently
received considerable scientific interest. Interestingly, these dis-
eases may not involve direct interactions with the microbiota.
However, the regulating function of the microbiota, such as the
microbiota-gut-brain axis, may participate in the specific path-
ways of the diseases. The complex microbiota-host interactions
are dynamic, involving a variety of mechanisms that include im-
mune, hormonal, and neural pathways. Therefore, changes in the
microbiota may result in the dysregulation of host homeostasis
and in an increased susceptibility to these diseases. On the basis
of these well-established connections between disease and the
disruption of homeostatic interactions in the host, microbiota-
targeted therapies may alter the community composition, and
microbiota restoration might be used for treating these diseases.
3.5.1. The microbiota and allergic diseases
An early-life, antibiotic-driven low diversity in gut microbiota
enhances susceptibility to allergic asthma [131], and thus may

77B. Wang et al. / Engineering 3 (2017) 71–82
also affect asthma development in childhood after long-term
follow-up. Of course, the mode, place of delivery, and infant feed-
ing also affect the GI microbiota composition and subsequently
influence the risk of atopic manifestations [132]. Bunyavanich
et al. [128] found that infants with a gut microbiota enriched in
Clostridia and Firmicutes at a host age of 3–6 months are associ-
ated with the resolution of cow’s milk allergy (CMA) by the age
of 8 years. Because the intestinal microbiota of an infant evolves
rapidly in the first year, the early-life gut microbiota composition
may be one of the determinants for CMA outcomes in childhood.
The gut microbiota interacts with the immune system intimately,
providing signals to promote the maturation of regulatory anti-
gen-presenting cells and regulatory T cells (Tregs), which play a
crucial role in the development of immunological tolerance. The
specific members of the microbiota, such as Clostridium species,
interact with Treg and regulate immunoglobulin E (IgE) levels
[133]. Saarinen et al. [127] showed that the clinical course and
prognosis of CMA are highly dependent on the milk-specific IgE
status. A previous study also found that specific microbiotic sig-
natures, such as that of Clostridium sensu stricto, can distinguish
infants with IgE-mediated food allergies from those with non-IgE-
mediated ones, and that Clostridium sensu stricto is positively cor-
related with specific IgE level in serum [126].
3.5.2. The microbiota and psychiatric diseases
Psychiatric diseases have posed a severe threat to human health
throughout history [134]. They are caused by a combination of bi-
ological, psychological, and environmental factors [135–137]. The
existence of a gut-brain axis has been acknowledged for decades.
The gut-brain axis plays a key role in maintaining normal brain
and GI function. More recently, the gut microbiota has emerged
as a critical regulator of this axis. The concept of this axis has
been extended to the “microbiota-gut-brain axis,” and is now
seen to involve a number of systems, including the endocrine sys-
tem, neural system, metabolic system, and immune system, all of
which are engaged in constant interaction [138]. Gut microbiota
dysbiosis may increase the translocation of gut bacteria across the
intestinal wall and into the mesenteric lymphoid tissue, thereby
provoking an immune response that can lead to the release of
inflammatory cytokines and the activation of the vagus nerve and
spinal afferent neurons [139,140]. Autism spectrum disorder (ASD)
has been reported as correlated with an altered gut microbiota,
and low relative abundances of the mucolytic bacteria Akkerman-
sia muciniphila and Bifidobacterium spp. have been found in the
feces of children with autism [125]. Our previous study found an
altered fecal microbiotic composition in patients with MDD. Most
notably, the MDD groups had increased levels of Enterobacte-
riaceae and Alistipes, but reduced levels of Faecalibacterium [130].
These studies suggest the role of the gut microbiota in autism and
MDD as a part of the gut-brain axis; this suggested role should
form a basis for further investigation of the combined effects of
microbial, genetic, and hormonal changes in the development
and clinical manifestation of autism and MDD.
4. Advancements in microbiota technology
Over the past few decades, human microbiome research has
been revolutionized by high-throughput sequencing technology.
High-throughput sequencing provides an opportunity for studies
to focus on complex microbial systems without the need to clone
individual genes. Initially, microbiota studies focused on compo-
sitional studies (i.e., answering the question: what is there?) and
functional studies (i.e., what are they doing?). With the develop-
ment of sequencing technology and bioinformatics analysis, it has
become increasingly interesting to study the activity of microbes
within microbial communities. It is widely accepted that the mi-
crobes with the highest abundance are not always the most active
ones. RNA sequencing (RNAseq) permits the analysis of gene ex-
pression, adding valuable expression data to compositional data
sets. Gosalbes et al. [141] performed the first metatranscriptomic
analysis of the healthy human gut microbiota in 2011. The anal-
ysis of 16S transcripts showed the phylogenetic structure of the
active microbial community. Lachnospiraceae, Ruminococcaceae,
Bacteroidaceae, Prevotellaceae, and Rickenellaceae were the pre-
dominant families detected in the active microbiota. The primary
functional roles of the gut microbiota were found to be carbohy-
drate metabolism, energy production, and the synthesis of cellu-
lar components. A systematic comparison of the gut metagenome
and metatranscriptome revealed that a substantial fraction (41%)
of microbial transcripts was not differentially regulated relative to
their genomic abundances. The metatranscriptional profiles were
significantly more individualized than the DNA-level functional
profiles but were less variable in their microbial composition
[142]. A transcriptome analysis of bacteriophage communities
in the periodontal microbiota was recently performed using
RNAseq. Oral phages were found to be more highly expressed in
individuals with relative periodontal health [143].
To achieve precise microbiome-based medicine in the future,
it is necessary to understand which individual microorganisms
mediate vital microbiome-host interaction(s) under health or
disease conditions. Most gut microbes are currently uncultivable.
Even with the use of recent technologies, such as gnotobiotic
mice and anaerobic culturing techniques, it is possible to culture
only approximately half of the bacterial species identified by 16S
rDNA high-throughput sequencing [144]. In addition, species-
level identification may not reflect the real situation because
most of the functional diversity can be reflected only at the strain
level. Therefore, it will be crucial to develop technologies to iden-
tify and isolate these microorganisms and/or microbial consortia.
Compared with traditional microbiology approaches, the use
of anaerobic conditions and gnotobiotic animals largely facilitates
the cultivation of difficult-to-grow microbes. Numerous previously
uncultivable microbes can now be cultured in a laboratory set-
ting [145]. A chip-based isolation device (the iChip) was recently
developed and was specifically designed to identify uncultivable
microbes within complex microbial ecosystems [146]. The iChip
is composed of hundreds of miniature diffusion chambers, each
of which is inoculated with a single environmental cell. The ca-
pacity for microbial recovery using the iChip is many times high-
er than that of standard cultivation, and the resulting species are
of significant phylogenetic novelty [146]. A new device for in situ
cultivation (the I-tip) was subsequently developed. The principal
of the I-tip is similar to that of the iChip; however, the I-tip traps
individual microbes within a gel, thus allowing for the passage of
metabolites and nutrients. The in situ isolation of microbes from
invertebrate organisms using the I-tip has recovered isolates from
34 novel microbial species [147].
Simulating GIT conditions can greatly facilitate in vitro cultiva-
tion. The Simulator of the Human Intestinal Microbial Ecosystem
(SHIME) has succeeded in establishing stable, reactor-grown GIT
microbial communities. More importantly, this system is able
to precisely simulate different regions of the human GIT, thus
allowing the diversity of the community to be studied in vitro
in a different niche. The power of SHIME has been estimated
by numerous studies [148–150]. For example, it has been found
that different regions of SHIME are colonized by different unique
microbial communities when cultured with microbes. The distri-
bution is highly similar to that of the living host, such as the prev-
alence of Bacterioides/Prevotella spp. and Lactobacillus spp. in the
colon [148].

78 B. Wang et al. / Engineering 3 (2017) 71–82
Identifying single cells that produce metabolites of interest
within complex microbial ecosystems is very important for un-
derstanding microbiome-host interactions. With this purpose, a
flexible high-throughput approach using a combination of micro-
fluidics and fluorescence-activated cell sorting has recently been
developed [151]. This system has successfully identified xylose-
overconsuming Saccharomyces cerevisiae and L-lactate-producing
E. coli cells from a population. This system also allows for the
screening of mutants in known pathways.
The use of traditional animal models in microbiome studies
continues to provide insight into host-microbiota interactions.
However, animal models often do not predict the results obtained
in humans, thus posing a particular problem when considering
challenges relating to the oral absorption of drugs and nutrients.
Here, we introduce two methods of in vitro simulation based on
stem cells: the gut-on-a-chip system and colonic stem cell con-
struction. The gut-on-a-chip system takes advantage of biomaterial
engineering and provides an optional approach to study the com-
plex interactions occurring within the gut microbiome. This system
is an in vitro living cell-based model of the intestine that mimics
the properties of the human gut along with crucial microbial sym-
bionts. Biomimetic human gut-on-a-chip micro-devices are usually
composed of microfluidic channels and a porous flexible mem-
brane that are coated with an extracellular matrix and lined with
human intestinal epithelial cells [152]; such devices mimic the
complex structure and physiology of a living intestine. Microfluidic
devices can also be used to study microbe-microbe interactions,
such as chemotaxis/chemical attraction and quorum sensing [153];
such interactions have been shown to be more effectively studied
using microfluidic devices than using traditional capillary-based
assays [154]. In addition, given the recapitulation of many complex
functions of the normal human intestine, it may also become an
essential platform for drug screening and toxicology testing.
Colonic stem cell construction is a recently developed in vitro
system that is used to grow 3D organoid colonic epithelium
structures that are guided by microstructures without the utiliza-
tion of microfluidics technology [155]. Within a Matrigel overlay,
spherical 3D structures grown from colonic stem cells or intesti-
nal stem cells are collected from an array containing individually
grown structures. These membrane-free 3D stem-cell-derived
organoids, which contain various differentiated cell types, form a
barrier similar to that of intestinal or colonic epithelia [156,157].
These organoids have recently been used to demonstrate that the
Salmonella enterica serovar Typhimurium can successfully invade
the epithelial cell layer [157], and that C. difficile can disrupt the
epithelial barrier function [158]. This technology also provides
more novel and valuable methods for higher throughput micro-
biome studies than existing models, although this technique is
still in its infancy.
5. Application of the human microbiota
The human microbiome can be considered as an important
origin of resources for genetic diversity, a modifier of disease, an
essential component of immunity, and a functional entity that
influences metabolism and modulates drug interactions. On one
hand, there are many potential probiotics or beneficial bacteria
that may prevent or treat certain diseases, although most of them
cannot be cultivated at present [159]. For example, some of these
gut microbes belong to genera that contain many probiotics such
as Lactobacillus and Bifidobacterium. Some are novel potentially
beneficial bacteria, such as Faecalibacterium prausnitzii for treat-
ing IBD and irritable bowel syndrome (IBS), and Akkermansia
muciniphila for improving metabolic health [160]. On the other
hand, as our second genome, the human microbiome must pro-
duce a large number of metabolites. Some isolated metabolites
have important potential applications, although it still remains a
great challenge to isolate and identify all the metabolites of the
human microbiome. For example, Chu et al. [161] discovered me-
thicillin-resistant Staphylococcus aureus-active antibiotics by using
primary sequencing from the human microbiome.
With the increased understanding of the relationship between
the human microbiome and a variety of diseases, the use of these
findings to predict or diagnose diseases has attracted a great deal
of attention [162]. Enrichments of some microbes are noted as
potential biomarkers in some research; however, these altera-
tions are often observed in other research as well, and cannot be
distinguished among different diseases. In contrast, clinical mod-
els based on tens of genes within a metagenome analysis perform
better in diagnostics and predicting diseases. In addition, we
found that the Bifidobacterium/Enterobacteriaceae (B/E) ratio indi-
cates the microbial colonization resistance of the bowel, and that
this ratio is considered to be an indicator of human microbiome
heath. The B/E ratio is higher than 1 in people with healthy mi-
crobiomes, whereas it is far below 1 in patients with cirrhosis and
patients with the avian influenza H7N9 infections [163,164].
The prevention and treatment of diseases by targeting the
microbiome have been widely investigated, and some therapies
have been successfully applied in the clinic. The administration of
probiotics is reported to help restore the health of H7N9 patients
more quickly [164]. Fecal microbiome transplantation has exhib-
ited better clinical efficacy than antibiotics in the treatment of C.
difficile infections [165]. Substantial progress has also been made
in the treatment of liver diseases by modulating the gut micro-
biome. A clinical trial showed that probiotic VSL#3 reduces liver
disease severity and hospitalization in patients with cirrhosis
[166]; the administration of Lactobacillus salivarius LI01 or Pedi-
ococcus pentosaceus LI05 improves the acute liver injury induced
by D-galactosamine in rats [167]. Furthermore, the regulation of
the human microbiome plays important roles in the treatment
of GI diseases, such as infectious diarrhea, antibiotic-associated
diarrhea, inflammatory bowel disease, and necrotizing enter-
ocolitis. For example, the oral administration of a mixture of
17 Clostridia strains from the human microbiota to adult mice
was found to attenuate disease in models of colitis and allergic
diarrhea [168]. Modulation of the gut microbiome may also con-
tribute to the treatment of cancer. Iida et al. [169] reported that
optimal responses to cancer therapy require an intact commensal
microbiota that mediates the therapy effects by modulating mye-
loid-derived cell functions in the tumor microenvironment. Viaud
et al. [170] reported that the gut microbiota helps to shape the
anticancer immune response of cyclophosphamide. In addition,
many clinical studies have shown that probiotics and their prod-
ucts have outstanding effects on the treatment of allergic diseas-
es, especially those in infants [171].
6. Future perspectives
The human microbiota plays an important role in the well-
being of the human host, and participates actively in the develop-
ment of a wide variety of diseases. Given the extensive influence
of microorganisms throughout the human body, we propose that
research on host-microbiota interactions should go beyond a char-
acterization of the community composition and an investigation
of the community members’ associations. From the structure to
the function of the microbiota, future research should move mi-
crobiome investigations toward providing explanations of causal-
ity. With new techniques for microbiota function prediction, new
microbiota interaction models, and novel analytical and simulation
approaches, future advances will help to clarify the interactions

79B. Wang et al. / Engineering 3 (2017) 71–82
between the microbiota and human development, and the po-
tential roles of those microbiota involved in the mechanisms of
various diseases, such as liver diseases, bacterial infection, cancer,
psychiatric diseases, and metabolic diseases. The crucial roles of
the human microbiota should be investigated at a much deeper
level, and microbiome-based diagnosis and treatment strategies
will be used for future personalized medicine work.
Acknowledgements
This study was supported by grants from the National Basic
Research Program of China (973 Program, 2013CB531401), the
Major Science and Technology Program of Zhejiang Province
(2014C03039), and the Natural Science Foundation of Zhejiang
Province (R16H260001). We acknowledge Drs. Chunlei Chen, Bo
Li, Jing Guo, Ding Shi, Qiongling Bao, Silan Gu, Yanfei Chen, Kai
Zhou, Qixiang Luo, Ruiqi Tang, and Xiangyang Jiang for the lit-
erature search and the preparation for the manuscript. We also
thank the reviewers for their thoughtful and helpful comments.
Compliance with ethics guidelines
Baohong Wang, Mingfei Yao, Longxian Lv, Zongxin Ling, and
Lanjuan Li declare that they have no conflict of interest or finan-
cial conflicts to disclose.
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