Introduction
Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide,
accounting for more than 720,000 deaths annually [1]. The strongest known risk factor
for this devastating disease is infection with Helicobacter pylori, which drives the
development of premalignant lesions (such as gastric atrophy, intestinal metaplasia,
and dysplasia) that can lead to gastric cancer (Fig 1). However, although H. pylori
is the most common bacterial infection worldwide and colonizes greater than 50% of
the global population, only 1%–3% of infected individuals ever develop gastric cancer.
10.1371/journal.ppat.1006573.g001
Fig 1
Alterations in the gastric microbiota following Helicobacter pylori infection and
gastric disease progression.
(A) Schematic representation of the predominant phyla of the gastric microbiota based
on H. pylori infection status. H. pylori-negative individuals harbor a microbiota
that is more complex and highly diverse compared to H. pylori-positive individuals.
(B) Schematic representation of the predominant genera at different stages within
the gastric carcinogenesis cascade. Following infection with H. pylori, Proteobacteria
and specifically H. pylori dominate the gastric microbiota. This leads to the development
of chronic gastritis. In the later stages of the disease, ranging from intestinal
metaplasia to gastric adenocarcinoma, a number of genera are enriched. These include
Escherichia-Shigella and Burkholderia within the Proteobacteria phylum; Lactobacillus,
Lachnospiraceae, Streptococcus, and Veillonella within the Firmicutes phylum; and
Prevotella within the Bacteroidetes phylum.
Drivers of susceptibility to gastric carcinogenesis include H. pylori strain-specific
virulence determinants, host constituents, and environmental factors. Along with these
elements, the microbiota of the stomach may also influence the development of gastric
malignancies. The acidic environment of the stomach in conjunction with low levels
of cultured bacteria from this site previously led to assumptions that the gastric
niche was not able to support a diverse microbial community. However, recent advances
in DNA sequencing of conserved ribosomal RNA genes, phylogenetic analyses, and computational
methods have uncovered a complex microbiota within the human stomach with the potential
for disease induction [2].
The gut microbiota and dysbiosis
The human gut microbiota is critical for maintenance of human health and plays an
integral role in energy metabolism, absorption of nutrients, and defense against invading
pathogens [3–5]. However, this microbiota exists within a delicate balance that, if
altered, becomes dysbiotic and contributes to aberrant proinflammatory immune responses,
susceptibility to invading pathogens, and initiation of disease processes, including
cancer [6]. Dysbiosis contributes to the pathogenesis of gastrointestinal carcinomas
in the esophagus [7] and colon [8, 9], and specific bacterial species are associated
with the development of colorectal cancer (Fusobacteria nucleatum and Escherichia
coli) [10, 11] and gastric cancer (H. pylori) [12].
The relationship between specific microbial pathogens and carcinogenesis has been
the subject of extensive investigation, and historically, the majority of research
has focused on individual pathogens, such as H. pylori, and their ability to initiate
and perpetuate disease. Advances in sequencing technology have greatly enhanced the
ability of scientists to identify additional microbial species that may be potentially
associated with various disease states, such as cancer, although establishing cause
versus effect presents multiple challenges. Therefore, understanding how dysbiosis
impacts aberrant host inflammatory responses and downstream carcinogenic cascades
will be critical to accurately define the role of niche-specific microbiota in oncogenesis.
H. pylori, the human gastric microbiota, and gastric cancer
A first step in establishing causation is to take inventory of a particular resident
microbial population, and several investigations have focused on defining microbial
communities within the human stomach and the interactions of these populations with
H. pylori (Fig 1A). Numerous groups have used PCR- and sequencing-based approaches
to demonstrate that H. pylori-negative individuals harbor a highly diverse gastric
microbiota dominated by 5 predominant phyla: Proteobacteria, Firmicutes, Actinobacteria,
Bacteroidetes, and Fusobacteria [13–15]. In contrast, among H. pylori-positive subjects,
H. pylori is the single most abundant bacterium present in the stomach and accounts
for between 72% and 97% of all sequence reads [13, 14].
These initial studies primarily focused on defining the composition of the gastric
microbiome stratified by H. pylori infection status but not disease diagnosis. Subsequent
human cross-sectional studies have compared the gastric microbiota in patients with
pathologic outcomes that span the gastric carcinogenesis cascade (Fig 1B). One study
demonstrated that H. pylori was present at relatively low abundance in patients with
advanced premalignant lesions and that the microbiota of patients with gastric cancer
were dominated by species of Lactobacillus, Streptococcus, Veillonella, and Prevotella
[16]. Another study demonstrated a steady decrease in bacterial diversity of the gastric
microbiota, with an increasing abundance of Lactobacillus and Lachnospiraceae in patients
progressing along the carcinogenic cascade [17]. The increase in these genera validates
other studies that demonstrated similar increases in the abundances of Lactobacilli
[16] and Lachnospiraceae [18, 19] in tissue samples from gastric cancer patients.
The abundance of Lactobacillus, Lachnospiraceae, Escherichia-Shigella, Nitrospirae,
and Burkholderia is also enriched when gastric cancer patients are compared to controls
[19], supporting previous findings that Lactobacillus and Lachnospiraceae are present
at higher abundance in gastric cancer [16–18, 20] and that Escherichia-Shigella is
enriched in patients with colorectal cancer [21]. It is important to note that these
studies only identified genetic evidence of bacteria and in-depth studies to assess
viability of these organisms have not been performed. However, these results raise
an intriguing hypothesis, namely that gastric colonization by non-H. pylori bacteria,
many of which also colonize the intestine, could impact the risk for gastric cancer.
Most bacteria cannot survive in the acidic environment of the stomach. However, it
has been well established that, in a subset of persons, infection with H. pylori leads
to achlorhydria and decreased acid secretion. Thus, long-term H. pylori colonization
and neutralization of the gastric environment may directly contribute to alterations
in the gastric microbiota. There are also clinical studies that support this concept,
namely that patients treated with acid-suppressive drugs, such as proton pump inhibitors,
exhibit a significant increase in the burden of non-H. pylori bacteria within the
stomach. Of interest, this increase correlates with increased inflammatory responses,
suggesting that non-H. pylori bacteria that colonize an achlorhydric stomach may have
the capacity to promote inflammation that could potentially facilitate the progression
to cancer [22, 23]. However, definitive evidence for this requires careful interventional
studies that have yet to be performed.
Although these studies demonstrate associations between the human gastric microbiota
and H. pylori infection (as well as various H. pylori-induced pathologies), they do
not directly differentiate cause from effect, primarily due to their cross-sectional
study designs. One longitudinal study that supported the role of non-H. pylori species
in the development of cancer assessed the effects of H. pylori eradication therapy
on gastric cancer incidence over a 15-year time period [24]. Despite only a 47% eradication
rate for H. pylori, there were similar reductions in the incidence of gastric cancer
among subjects who received antibiotics and were unsuccessfully eradicated compared
to those who remained H. pylori-free [24]. These results suggest that bacteria in
addition to H. pylori may have been affected by antibiotics, which may have contributed
to attenuated rates of gastric cancer. There are also computational biology studies
that support these concepts. Using a computerized search algorithm designed to identify
the presence of bacterial DNA within interrogated known cancer genomes, these investigators
determined that the type of cancer that harbored the second highest number of bacterial
DNA sequences was gastric adenocarcinoma. However, the most common type of integrated
bacterial DNA was not H. pylori but was instead Pseudomonas [25].
The effect of the gastric microbiota on H. pylori-induced gastric inflammation and
cancer in rodents
The ability to establish causality is greatly enhanced by carefully controlled and
manipulatable animal model systems. Inbred mice with defined genotypes are commonly
used to study the effects of H. pylori infection on gastric diseases such as cancer.
Of interest, the gastric microbiota of C57BL/6 mice is dominated by the same predominant
phyla that have been reported in humans: Firmicutes, Bacteroidetes, Proteobacteria,
and Actinobacteria [26]. Longitudinal studies in mice have provided more direct evidence
of the contribution of non-H. pylori species to H. pylori-induced gastric carcinogenesis.
For example, 1 study demonstrated that INS-GAS mice harboring a complex microbiota
developed gastric cancer within 7 months following H. pylori infection, whereas the
development of gastric cancer was markedly prolonged in germ-free mice that were monocolonized
by H. pylori [27]. Following H. pylori infection, there was an overall decrease in
gastric microbial diversity [27], similar to that observed in human populations, but
there were no significant differences in the intestinal microbiota among any of the
groups. These observations were studied in greater depth in an INS-GAS H. pylori mono-associated
germ-free mouse model, where the addition of a restricted microflora accelerated the
development of gastric cancer in conjunction with H. pylori [28]. Specifically, germ-free
INS-GAS mice supplemented with a gastric and intestinal microbiota containing only
3 species of commensal intestinal bacteria (ASF356 Clostridium species, ASF361 Lactobacillus
murinus, and ASF519 Bacteroides species) were sufficient to promote gastric neoplasia
in H. pylori-infected INS-GAS mice to the same extent as observed in H. pylori-infected
INS-GAS mice harboring a complex microbiota [28]. Importantly, these genera are also
enriched in the stomachs of patients that develop premalignant and malignant lesions.
Further supporting the concept of a contributory role of the gastric microbiota in
promoting disease have been interventions with antibiotic therapy, which were shown
to delay the onset of gastric cancer in INS-GAS mice in a manner that was not dependent
on the presence of H. pylori [29]. Collectively, these results suggest that non-H.
pylori bacteria can colonize the stomach and may represent an additional modifier
of gastric cancer risk, particularly among H. pylori-infected individuals.
In addition to the stomach, bacteria within other microbial niches may exert a role
in modulating H. pylori-induced gastric inflammatory responses. Two studies have shown
that precolonization with intestinal Helicobacters (H. bilis, H. hepaticus, and H.
muridarum) can either increase or decrease the severity of gastric inflammation induced
by H. pylori by altering T-regulatory cell responses [30, 31]. Another study demonstrated
that H. pylori per se is present within the intestine in a coccoid form and that the
interaction between phagocytes and H. pylori within intestinal Peyer’s patches plays
a critical role in modifying the intensity of H. pylori-induced gastritis [32]. However,
other studies have shown that the microflora within the stomach can accelerate the
progression of gastric cancer in the presence of H. pylori and do so with no differences
detected in the composition of the intestinal microbiota [27, 28]. Addressing whether
the resident intestinal microbiome directly contributes to the pathophysiology of
H. pylori-induced gastric diseases is an avenue that requires further investigation,
and it is important to consider that the effects of bacteria and microbial communities
in the intestine and the stomach on gastric pathophysiology may not be mutually exclusive.
Conclusions
Evidence that the host microbiota specifically functions to promote health and prevent
disease and that dysbiosis contributes to inflammation, susceptibility to pathogens,
and diseases (including cancer) is undisputed [3]. As a result, the concept of specific
microorganisms solely driving cancer initiation and progression may need to be modified
in certain circumstances. Although great advances have been made in understanding
the complex interplay between the gastric microbiota and H. pylori in the development
of gastric inflammation and cancer, detailed studies are still needed in well-defined
human populations to compare differences in the microbiota of H. pylori-infected persons
with and without neoplastic lesions. Cross-sectional studies can provide initial insights
into microbial associations with cancer; however, reverse effects are a concern, as
it is difficult to discern whether carcinogenesis leads to changes in the local microenvironment
that creates a new niche for microbes or whether alterations in the microbial population
or its functions contribute to carcinogenesis [33]. Due to the acidic nature of the
stomach, most bacteria cannot survive in this environment. However, infection with
H. pylori leads to achlorhydria of the stomach in a subset of colonized persons; thus,
long-term H. pylori colonization and neutralization of the gastric environment may
directly contribute to alterations in the gastric microbiota.
Since the gastric microbiota is more austere in terms of microbial breadth and depth
compared to the intestinal microbiota, future studies should focus on assessing whether
the composition of the gastric microbiome in different anatomical regions of the stomach
exerts differential effects on cancer risk. This could be done through site-specific
topographical mapping of the microbiota in the presence or absence of H. pylori as
well as assessing differences in relation to different disease states along the gastric
carcinogenesis cascade. Clearly, longitudinal studies that utilize sequential sampling
to elucidate the temporal nature of microbial associations with premalignant lesions
are needed. Details regarding patient populations, including age, gender, diet, and
other comorbidities need to be assessed and compared in a rigorous fashion to discern
whether any of these variables affect the potential for the gastric microbiome to
influence disease. Since studies of the gastric microbiota have largely focused on
bacterial communities, more in depth studies elucidating effects of other microorganisms
that potentially populate the stomach in addition to bacteria, including fungi, protists,
archaea, and viruses, are needed to fully characterize the gastric microbiome and
its relationship with cancer risk. Furthermore, to more definitively determine cause
versus effect, studies may also need to incorporate humanized mouse models to discern
effects of the human gastric microbiome on disease. As we begin to understand and
elucidate the specific role of the gastric microbiota and its effects on human health
and disease, studies of these microbial populations in innovative systems will likely
yield translational opportunities to reduce gastric cancer morbidity and mortality
by improving screening, prevention, and treatment. It is tempting to speculate that
future studies will identify specific combinatorial populations of bacteria that are
predictive of pathologic outcomes, yielding strategies to manipulate the microbiota
to ultimately prevent disease.