2015/2016
Emanuel Alexandre Otero Dias Jácome
Gastric Microbiota and Carcinogenesis:
The Role of Non-Helicobacter Pylori Bacteria - a Systematic Review
Mestrado Integrado em Medicina
Área: Gastrenterologia Tipologia: Monografia
Trabalho efetuado sob a Orientação de: Professor Doutor Pedro Pimentel Nunes
Trabalho organizado de acordo com as normas da revista: Revista Española de Enfermedades Digestivas Emanuel Alexandre Otero Dias Jácome
Gastric Microbiota and Carcinogenesis:
The Role of Non-Helicobacter Pylori Bacteria -a Systematic Review
Dedicatória
GASTRIC MICROBIOTA AND CARCINOGENESIS: THE ROLE
OF NON-HELICOBACTER PYLORI BACTERIA - A
SYSTEMATIC REVIEW
Running title: Gastric microbiota and gastric cancer
Key words: Stomach, Microbiota, Bacteria, Carcinogenesis, Helicobacter pylori.
Emanuel Dias-Jácome 1, Diogo Libânio 2, Marta Borges-Canha 3, Ana Galaghar 4,
Pedro Pimentel-Nunes 1,2,3.
1. CINTESIS - Center for Health Technology and Services Research, Faculty of Medicine, University of Porto, Porto, Portugal
2. Gastroenterology Department, Oncology Portuguese Institute of Porto, Porto, Portugal.
3. Department of Physiology and Cardiothoracic Surgery, Cardiovascular Research & Development Unit, Faculty of Medicine,
University of Porto, Porto, Portugal.
4.Pathology Department, Oncology Portuguese Institute of Porto, Porto, Portugal.
Correspondence address:
Emanuel Dias-Jácome
CINTESIS - Center for Health Technology and Services Research, Faculty of Medicine
Rua Dr. Plácido da Costa, 4200-450, Porto, Portugal.
Telefone: +351 225 513 622
Email: diasj0310@gmail.com
Disclosures:
The authors have no potential financial, professional or personal conflicts relevant to the manuscript.
Abstract
Background and Aim: Helicobacter pylori is the strongest risk factor for gastric cancer. However, recent advances in DNA sequencing technology have unraveled a complex microbial community in the stomach that can also participate in the development of gastric cancer. The aim of this study is to present recent scientific evidence regarding the role of non-Helicobacter pylori bacteria in gastric carcinogenesis.
Methods: A systematic review of original articles published in PubMed in the last ten years related to gastric microbiota and gastric cancer was performed.
Results: Thirteen original articles were included. The constitution of gastric microbiota appears to be significantly affected by gastric cancer and premalignant lesions as well as by Helicobacter pylori infection. In addition, a gradual shift in the gastric microbiota profile from non-atrophic gastritis to intestinal metaplasia, and then to intestinal type cancer appears to exist. Gastric carcinogenesis can be associated with an increase in many bacteria (such as Lactobacillus coleohominis, Klebsiella pneumoniae or
Acinetobacter baumannii) as well a decrease in others (such as Porphyromonas spp, Neisseria spp, Prevotella pallens or Streptococcus sinensis). However, no conclusive
data suggest if these changes in microbiota are cause or consequence of the process of carcinogenesis.
Conclusions: Gastric cancer may be the result of a complex cross-talk between gastric microbiota and Helicobacter pylori. Gastric microbiota appears to play an important role in gastric carcinogenesis, beyond the well-known influence of Helicobacter pylori. Further studies are needed to elucidate the specific role of different microorganisms in the development of gastric cancer.
Introduction
Human beings are colonized by a myriad of microorganisms that interact with each other and with the host in a symbiotic manner, constituting an integrated and functional ecosystem (microbiota) that can influence health as well as disease (1, 2). In particular, gastrointestinal microbiota can modulate cancer risk along the gastrointestinal tract (3). Most researchers today have focused on colon microbiota and its relationship with colorectal cancer (4) and rarely examined gastric microbiota. Historically, the stomach was considered a sterile organ due to its acid production.
The discovery of Helicobacter pylori (H. pylori) demolished this belief and, for many years, this bacterium was thought to be the only able to survive in the stomach. However, recent evidence suggests that other bacteria besides H. pylori flourish despite the hostile environment and gastric microbiota is, actually, more diverse and complex than once thought; its composition can be shaped by the host physiology as well as by external factors, such as diet, H. pylori infection, enteral feeding, proton pump inhibitors, antibiotics and disease (5).
Correa’s model of gastric carcinogenesis postulated that chronic H. pylori infection of the gastric mucosa progresses through the stages of chronic active gastritis, atrophic gastritis, intestinal metaplasia, dysplasia and, ultimately, gastric cancer (6). Today, H. pylori is clearly identified as the strongest risk factor for gastric cancer (7, 8), the fourth most common malignancy and the second leading cause of cancer-related mortality worldwide (9, 10).
However, only 1-2% of infected persons develop gastric cancer (11), and its pathogenic mechanisms are unclear. It is possible that gastric microbiota constitutes the link between H. pylori and gastric carcinogenesis, when other bacteria colonize the stomach under conditions of decreased acidity (such as chronic atrophic gastritis), creating reactive oxygen and nitrogen species and modulating inflammatory responses (12, 13). Therefore, it is important to unravel the significance of specific microbiota to determine which are causative of gastric cancer and which are present merely as a consequence in order to develop novel prevention and treatment strategies.
This systematic review aims to provide an update of recent findings related to the role of gastric microbiota in gastric carcinogenesis, focusing on microorganisms besides H. pylori to understand how the constitution of microbiota can influence cancer risk and how it is affected by H. pylori status, possibly explaining its pathogenesis.
Methods
A comprehensive search was performed using PubMed and the following query (date of last search 5th August 2015): ("microbiome" OR "microbiota" OR "microflora" OR "bacterial flora" OR "bacterial community") AND ("gastric" OR "stomach" OR "upper digestive tract" OR "upper gastrointestinal tract") AND ("cancer" OR "neoplasia" OR "malignancy" OR "tumour" OR "carcinoma" OR "adenocarcinoma" OR "lymphoma" OR "premalignancy" OR "premalignant" OR "tumorigenesis" OR "carcinogenesis" OR "intestinal metaplasia" OR "atrophic gastritis" OR "Helicobacter pylori" OR "H. pylori").
In the first phase, titles and abstracts were screened by two reviewers, accordingly to specific criteria defined in order to guide this systematic review. Any disagreement was solved by a third independent reviewer. We included studies presenting information about the role of gastric microbiota beyond H. pylori in gastric carcinogenesis or presenting information about the influence of H. pylori in the constitution of gastric microbiota. The exclusion criteria were: articles published before 2005 (because the constitution of gastric microbiota has been defined more accurately approximately since this year, as previous studies were based in methods with limitations in sensitivity and coverage when compared with more recent approaches); articles not written in English, Portuguese or Spanish; studies performed in animals; studies performed in cell culture models; non-original studies (reviews and case reports).
In the next phase, full-texts of the selected references were assessed and references of included studies were checked to identify additional articles.
Data about bacterial species studied and results obtained were extracted of each article, mentioning methods and main limitations when relevant. The information was then synthesized and organized in the present systematic review.
Results
1. Characteristics of included studies
A total of 300 articles were obtained from database searching. After screened by title and abstract, 16 studies were selected for full-text reading, where 5 studies were further excluded and 2 studies included by cross-reference. A total of 13 articles were finally included (Figure 1). Their general characteristics are summarized in Table 1.
2. Influence of normal gastric microbiota by H. Pylori
The interaction between gastric microbiota and H. pylori is likely to influence risk of gastric cancer. Thus, characterization of gastric microbiota in subjects with and without H. pylori could provide new insight into the mechanisms behind this complex cross-talk and its possible link with gastric carcinogenesis. The effect of H. pylori on the microbiota of the human stomach has been examined in a limited number of studies.
2.1. H. Pylori and gastric microbiota in adults
In 2006, Bik et al characterized bacterial diversity within the human gastric mucosa using a small subunit 16S rDNA clone library approach. This group analyzed 1833 sequences generated by broad-range bacterial PCR from gastric biopsy samples of 23 individuals. A diverse gastric bacterial community of 128 phylotypes was dominated by 5 major phyla: Proteobacteria (952 clones, 32 phylotypes), Firmicutes (464 clones, 36 phylotypes), Bacteroidetes (193 clones, 35 phylotypes), Actinobacteria (164 clones, 12 phylotypes), and Fusobacteria (56 clones, 10 phylotypes). H. pylori was the only member of the genus Helicobacter found, his sequences were found in 19 of the 23 samples and constituted 42% of all sequences analyzed (777 clones), making this the most abundant genus followed by Streptococcus (299 clones) and Prevotella (139 clones). Interestingly, its presence did not affect significantly the composition of gastric microbiota, although H. pylori-positive subjects demonstrated a relative lack of
non-Proteobacteria phylotypes, especially Bacteroidetes (p>0.05) (14).
A similar constitution of gastric microbiota was reported by Li et al, who analyzed gastric microbiota of 10 subjects without H. pylori infection by cloning and sequencing 16S rRNA. A total of 1223 non-H. pylori sequences were obtained and, similarly, a diverse bacterial community of 133 phylotypes was dominated by the same 5 phyla: the most abundant was Firmicutes (383 clones), closely followed by
Proteobacteria (345 clones), while Bacteroidetes, Actinobacteria and Fusobacteria
were less abundant. The most common genera were: Streptococcus (254 clones),
Prevotella (243 clones), Neisseria (175 clones), Haemophilus (122 clones) and Porphyromonas (68 clones). All of them constituted 70.5% of all microbial clones (15).
Nevertheless, once only H. pylori-negative subjects were included, the impact of H.
pylori in the constitution of microbiota was not evaluated.
Although these two studies analyzed two geographically (Hong Kong vs California) and ethnically (Chinese vs Caucasian, Hispanic and African American) distinct populations, the overall microbiota constitution was surprisingly similar. Both identified approximately 130 phylotypes from the same dominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria) and the two most abundant genera (Streptococcus and Prevotella) were also identical (14, 15). This highlights the selective pressure for the microbiota under the harsh stomach environment.
However, contradicting findings have been reported and other studies suggest that, actually, H. pylori can change the composition of gastric microbiota (16, 17).
In 2008, Andersson et al analyzed gastric biopsy samples from six Swedish patients (three H. pylori-infected and three uninfected) by tagged 454-pyrosequencing, finding higher gastric diversity in patients of negative H. pylori status (262 phylotypes) than in patients of positive H. pylori status (33 phylotypes) (16).
In 2011, Maldonado-Contreras et al characterized the structure of the human gastric bacterial community in relation to H. pylori status from 12 adult patients (10 Amerindians and 2 non-Amerindians) using a high-density 16S rRNA gene microarray. A total of 44 bacterial phyla were detected in an uneven ecosystem, with a strong dominance of only four phyla: Proteobacteria, Firmicutes, Actinobacteria and
Bacteroidetes (in descending order). Positive H. pylori status was significantly
associated with increased relative abundance of non-Helicobacter bacteria from the
Proteobacteria, Spirochetes and Acidobacteria, and with decreased abundance of Actinobacteria, Bacteroidetes and Firmicutes. In fact, about 28% of the total variance in
the gastric microbiota of the 12 subjects was explained by H. pylori status, although a larger fraction (44%) was related to the ethnicity of the subjects, stressing the need for conducting larger studies of different human populations (17).
Very recently, other studies were based in bacterial culture analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), allowing high accuracy and rapid analysis of a large number of samples (18, 19).
Hu et al adopted this approach to analyze 103 gastric biopsy specimens from H.
pylori infected patients. A total of 201 non-H. pylori bacterial isolates were cultured
from samples of 67 (65%) patients and all the isolates were identified using MALDI-TOF MS. Overall, a total of 18 non-H. pylori bacterial genera (43 species) were isolated, mostly gram-positive bacteria (73.8%). The dominant species were: Neisseria
flavescens (13.7%), Streptococcus salivarius (9.5%), Rothia mucilaginosa (8.9%) and Streptococcus pneumoniae (6.6%). This study demonstrates a high prevalence of the
non-H. pylori bacteria concurrent with H. pylori infection, indicating that upper gastrointestinal disorders may enable non-H. pylori bacteria to survive and colonize the stomach (18). However, this approach excludes non-cultivable bacteria and does not include H. pylori-uninfected subjects, so it does not evaluate its impact in the constitution of gastric microbiota.
In an attempt to overcome this limitation, Khosravi et al adopted a similar approach to compare culturable gastric microbiota between H. pylori positive and negative gastric disease patients in a large sample of 215 Malaysian patients (131 H.
pylori-positive and 84 H. pylori-negative). Notably, H. pylori colonization did not
significantly affect the constitution of human gastric microbiota (19), consistent with the results reported by Bik et al (14). Similarly, Maldonado-Contreras et al did not report a variation in the absolute number of non-H. pylori bacteria present but found that H. pylori resulted in differences in the relative abundance of several phyla (17).
Predominant phyla were Firmicutes (270 colonies from 208 positive biopsies) and Proteobacteria (220 colonies from 160 biopsies) while Actinobacteria were rare (53 colonies from 43 biopsies). At the genus level, Streptococcus were predominant (121 positive biopsies) followed by Neisseria (44 positive biopsies), Klebsiella (42 positive biopsies) and Lactobacillus (42 positive biopsies) (19). These results are consistent with previous studies reporting that Proteobacteria, Firmicutes,
Actinobacteria and Bacteroidetes are the predominant phyla in human gastric
microbiota (14-17), overcoming the important limitation of the small number of human samples (6-23 individuals) in these studies. Nevertheless, the approach adopted by Khosravi et al. still excludes non-cultivable bacteria.
Eun et al also analyzed gastric microbiota profile according to H. pylori status using high-throughput 16S rRNA sequencing. The relative abundances of family
Bradyhizobiaceae, Caulobacteraceae, Lactobacillaceae, and Burkholderiaceae were
significantly higher in H. pylori-negative patients, whereas the relative abundance of
Helicobacteriaceae family was significantly higher in H. pylori-positive patients (20).
2.2 H. pylori and gastric microbiota in children
Information about gastric microbiota in children is even more limited; to date, only Kato et al addressed this question by comparing gastric microbiota in 10 children and 10 adults in relation to H. pylori status (5 H. pylori-infected and 5 uninfected in each group) using both culture and RT-PCR. An overall colonization rate of
non-Helicobacter bacteria in gastric mucosa was higher in adults (100%) than in children
(10%) (p<0.001). Also, there was no significant difference between adults with H. pylori infection and those without it (p=0.056). This demonstrates that, regardless of H.
pylori status, bacteria reside on the gastric mucosa of adults, although such
microorganisms rarely colonize the stomach of children (21).
3. Roles of microbiota in the development of gastric cancer
The inhospitable acid environment in the stomach provides an effective barrier against colonization by microbes that enter the gastrointestinal tract. However, a change of the physiological conditions of the stomach, as occurs during corpus atrophy or gastric cancer, provides an opportunity for foreign microbes to enter and colonize the stomach. This raises the question whether gastric microbiota plays a role in the progression from chronic atrophic gastritis to gastric cancer or simply develops as a consequence of decreased acidity caused by these mucosal changes. The very few studies addressing this question are analyzed in this section.
3.1 Global gastric microbiota diversity and gastric cancer
Aviles-Jimenez et al analyzed the constitution of gastric microbiota in gastric cancer and premalignant lesions (chronic atrophic gastritis and intestinal metaplasia) and refers low gastric microbiota diversity in all three disease groups: two phyla,
Firmicutes and Proteobacteria, represented almost 70% of microbial community while
9 families represented on average 55.6% of each sample’s operational taxonomic units (OTU), with Lachnospiraceae and Streptococacceae representing over 20% of the
microbiota. In addition, although significant differences in bacterial diversity were noted between non-atrophic gastritis and gastric cancer (p=0.004) but not between intestinal metaplasia and any other group, a trend to decrease from non-atrophic gastritis to intestinal metaplasia to gastric cancer was noticed (22).
A cross-sectional study performed by Yu et al supports the idea that decreased gastric microbial diversity could favor the development of gastric cancer. This study found a linear relation between microbial richness and pepsinogen I/pepsinogen II ratio. Therefore, lower gastric microbial richness is associated with lower pepsinogen I/pepsinogen II ratio, which is a marker for chronic atrophic gastritis, a cancer-predisposing condition in the stomach (23).
However, Eun et al compared gastric microbiota between patients with gastric cancer, intestinal metaplasia and chronic gastritis and reported that the evenness and diversity of gastric microbiota was significantly increased in gastric cancer (20).
3.2 Specific changes in microbiota possibly related to gastric carcinogenesis Dicksved et al contributed the first DNA-based description of a complex gastric bacterial community in individuals with gastric cancer with potential as a maintainer or bystander in the progression of this neoplasia. This group compared gastric microbiota of 10 patients with gastric cancer with that of 5 subjects with normal mucosa using terminal restriction fragment length polymorphism (T-RFLP) in combination with 16S rRNA gene cloning and sequencing. Sequencing of 140 clones revealed 102 phylotypes, with representatives from five bacterial phyla: Firmicutes (60%), Bacteroidetes (11%),
Actinobacteria (7%), Proteobacteria (6%) and Fusobacteria (3%). The abundance of H. pylori was very low and gastric cancer microbiota was instead dominated by different
species of the genera Streptococcus, Lactobacillus, Veillonella and Prevotella. Remarkably, the constitution of gastric microbiota was not significantly different between gastric cancer patients and controls (p=0.12) (24).
In contrast to the aforementioned results, Eun et al reports significant differences in the composition and diversity of gastric microbiota among patients with gastric cancer, intestinal metaplasia and chronic gastritis, especially in Helicobacter-dominant group. The gastric cancer group showed a significant decrease in Epsilonproteobacteria class and Helicobacteriaceae family, while the Bacilli class and Streptococacceae family increased significantly. These alterations may play an important, as-yet-undetermined role in gastric carcinogenesis of Helicobacter predominant patients (20).
Supporting this possible influence of gastric microbiota in the development of gastric cancer, a gradual shift in the gastric microbiota profile from non-atrophic gastritis to intestinal metaplasia, and then to intestinal type gastric cancer was recently reported by Aviles-Jimenez et al. Further analyses revealed 27 operational taxonomic units (OTU) with significant abundance difference across groups. From the 12 OTU with the most significant p-values (as selected by the authors), 10 displayed a clear decreasing trend from non-atrophic gastritis to intestinal metaplasia to gastric cancer (2 TM7, 2 Porphyromonas spp, 1 Neisseria spp, 1 Haemophilus spp, 1 Bulleidia spp,
Prevotella pallens, Streptococcus sinensis and Bergeriella denitrificans), while 2
revealed an increasing trend (Lactobacillus coleohominis and Lachnospiraceae) (22). The approach taken by Khosravi et al also aimed to provide an insight into the contribution of disease to the constitution of gastric microbiota. Gastric microbiota was compared between patients with different gastric diseases, including non-ulcer dyspepsia (87.6%), peptic ulcer disease (10.2%) and gastric cancer (3.7%). Interestingly, the prevalence of Klebsiella pneumoniae and Acinetobacter baumannii in gastric cancer patients was significantly higher than that in the non-ulcer dyspepsia and peptic ulcer disease groups (50% vs.18.9% and 9.10%; 25% vs. 3.2% and 4.5%, respectively). Nevertheless, these results should be considered with caution because of the low number of gastric cancer patients compared with the two other groups (19).
Occurrence of Bifidobacteriaceae in hypochlorhydria stomach of 13 patients with atrophic gastritis and 10 patients receiving omeprazole was recently studied by Matarelli et al. Bifidobacteriaceae presence correlated significantly only with the omeprazole patients. However, bacteria belonging to the Actinomycetales order were also present and correlated significantly with both conditions (p<0.01) (25). It is surprising that previous studies on gastric cancer and chronic atrophic gastritis failed to detect these bacteria (20, 22, 24). The significance of this atypical microbiota colonization remains to be studied.
The presence of Helicobacter DNA was investigated by Han et al using a
Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing
analysis in 51 resected gastric adenocarcinomas. H. pylori was present in 47 (92.2%) of the 51 samples. In the four of H. pylori-negative cases, were detected two cases of
Helicobacter cinaedi, one case of Helicobacter mustelae and one case of Campylobacter hyointestinalis. However, their role in the pathogenesis of gastric
Taken together, these results suggest that a gradual shift in the constitution of gastric microbiota might play a role in the progression of pre-malignant lesions to gastric cancer. In fact, gastric carcinogenesis could be related to an increase in many bacteria as well as a decrease in others, as summarized in Table 2.
It is interesting to note that, globally, there is a decrease in bacteria belonging to the phyla Proteobacteria (Neisseria spp (22), Haemophilus spp (22), Bergeriella denitrificans (22), Epsilonproteobacteria (20) and Helicobacteriaceae (20)) and
Bacteroidetes (Porphyromonas spp (22) and Prevotella pallens (22)), whereas bacteria
belonging to the phyla Firmicutes are either increased (Streptococacceae (20),
Lachnospiraceae (22) and Lactobacillus coleohominis (22)) or decreased
(Streptococcus sinensis (22)). Klebsiella pneumoniae and Acinetobacter baumannii are two notable exceptions, as they belong to the phylum Proteobacteria, although they tend to increase along gastric carcinogenesis (19). It is intriguing that one study found that Streptococcus sinensis was associated with a trend to decrease from premalignant lesions to gastric cancer (22), whereas another study reported that its family,
Streptococacceae, was increased in gastric cancer compared with intestinal metaplasia
and chronic gastritis (20).
Bacteria belonging to the other two dominant phyla, Actinobacteria and
Fusobacteria, appear to play a less significant role in the process of carcinogenesis, as
only one study reported a significant increase in bacteria belonging to the phyla
Actinobacteria (Actinomycetales (25)) in atrophic gastritis, whereas there are no studies
reporting significant variations involving Fusobacteria.
4. Considerations about methods used in included studies
Studies included in this systematic review used different methods to evaluate the relation between H. pylori, gastric microbiota and gastric carcinogenesis. The purpose of this section is to compare the strengths and limitations of these approaches in order to try to explain some of the conflicting results obtained.
Studies regarding the role of H. pylori in the constitution of gastric microbiota have provided conflicting results. In fact, some of the included studies did not find significant differences using cloning (14) and culture-based approaches (19, 21), whereas other studies report significant changes in gastric microbiota accordingly to H.
pylori status based in DNA microarray (17) or 454-pyrosequencing (16), which allow to
Dicksved et al contributed the first DNA-based description of a complex gastric microbiota composition in individuals with gastric cancer although it did not differ significantly between gastric cancer patients and controls. However, this study has considerable limitations, including small sample size and lack of the most current high-throughput sequencing technology. Also, the control group was hospital-based, and the individuals were relatively elderly, with a possible deficiency in acid production that may have influenced the results (24). In fact, more recent studies reported significant differences in the constitution of gastric microbiota between patients with gastric cancer and premalignant lesions (20, 22), which could be related to the method used: the study conducted by Dicksved et al was based on cloning, sequencing and T-RFLP of 16S rRNA (24), which has important limitations in sensitivity and coverage compared with more recently developed 16S rRNA sequencing techniques, including G3 PhyloChip (22) or 454 GS FLX Titanium (20).
It is also interesting that some studies suggest that gastric carcinogenesis is related to low gastric microbial diversity (22, 23), which contrasts with higher evenness and diversity in gastric cancer patients reported by Eun et al (20). This could be partially explained by the fact that Yu et al used an array primarily designed to assess oral microbiota (HOMIM), possibly excluding some bacteria from the stomach and the lower microbial richness could actually reflect an increased abundance of these (23). Nevertheless, this is not the case of the results reported by Aviles-Jiménez et al, who used a G3 PhyloChip array (22), while Eun et al used a 454 high-throughput sequencer (20). Both approaches are reliable to characterize gastric microbiota and this discrepancy is intriguing.
Discussion
The present systematic review demonstrates that gastric microbiota may play an important role in gastric carcinogenesis, beyond the well-known influence of H. pylori. Recent advances in DNA sequencing technology have unraveled a complex microbial community in the stomach that is likely to interact with H. pylori and participate in the development of gastric cancer.
Other reviews (27-31) and recent studies on animal models (32-34) strongly support an important role for gastric microbiota in gastric carcinogenesis. Using germ-free transgenic insulin-gastrin (INS-GAS) mice, a mouse model of gastric cancer, Lofgren et al demonstrated that mice infected by H. pylori alone developed gastric intraepithelial neoplasia with a considerable time delay when compared with those infected by H. pylori plus a complex gastric microbiota (32). Similarly, Lertpiryapong et al reported that the colonization of the stomach by an artificial intestinal microbiota (‘Altered Schaedler’s Flora’, including Clostridium, Lactobacillus murinus and
Bacteroides species) increased the incidence of gastric intraepithelial neoplasia up to
69% in male INS-GAS mice, 7 months after H. pylori infection (33). In addition, antibiotic treatments significantly reduced the severity of dysplasia and delayed the development of gastric intraepithelial neoplasia in INS-GAS mice (34).
The impact of H. pylori in the constitution of human gastric microbiota is more controversial, as some studies reported significant differences between H. pylori-infected and unpylori-infected subjects (16, 17), whereas others did not (14, 19, 21), and animal studies have also yielded contradicting findings. In a BALB/c mice model, H.
pylori infection increased the biodiversity of gastric microbiota, whereas vaccination
against H. pylori prevented changes in normal gastric microbiota (35). Also, long-term infection with H. pylori may disturb the composition of gastric microbiota in Mongolian gerbils (36). Nevertheless, chronic H. pylori infection does not significantly alter the murine gastric microbiota (37). These contradictions could be explained by other factors possibly affecting this interaction, such as the degree of H. pylori-related inflammation, time of infection or presence, type and extension of premalignant lesions (30).
Taking all these results together, one hypothesis is that gastric cancer results from the interaction between H. pylori and the gastric microbiota. H. pylori may be more important in the early stages of gastric carcinogenesis, as a trigger for the development of chronic atrophic gastritis, than as a direct inducer of gastric cancer.
Instead, the decreased acidity secondary to mucosal atrophy would lead to changes in the constitution of gastric microbiota that could play important roles in later stages of gastric carcinogenesis, inducing progression from chronic atrophic gastritis to intestinal metaplasia and, finally, to gastric cancer (Figure 2).
The link between H. pylori and gastric microbiota in the pathogenesis of gastric cancer can be related to Toll-like receptors. In fact, there is evidence that increasing activation of these receptors, initially by H. pylori and later potentially by other microorganisms, may play an important role in gastric carcinogenesis (38).
Although our knowledge about the role of gastric microbiota in gastric carcinogenesis is only in the beginning, it looks clear that this is an area of great future potential. Perhaps further knowledge about the role of specific microorganisms in the progression of gastric premalignant lesions would allow the development of specific microbes and microbiota-based biomarkers for diagnostic and therapeutic purposes. Certain microorganisms with anti-H. pylori activity, like Lactobacillus (39, 40) or
Saccharomyces boulardii (41), could be used as a novel probiotic approach for H. pylori
eradication, overcoming antibiotic-associated problems like adverse effects or resistance. Also, if a gradual shift in the gastric microbiota profile from non-atrophic gastritis to intestinal metaplasia, and then to intestinal type gastric cancer is confirmed, perhaps a novel screening method based in searching specific gastric microbiota profiles could be developed that allowed us to detect premalignant lesions.
This systematic review faces some limitations. There are few studies about the role of gastric microbiota in human patients and most involve a limited number of subjects and follow a cross-sectional design. Also, most studies included do not make a clear distinction between diffuse and intestinal types of gastric cancer. More studies in large prospective cohorts are needed to confirm how human gastric microbiota may affect the development of gastric cancer.
Nevertheless, this study to our knowledge is the first systematic review synthesizing the evidence regarding the influence of non-Helicobacter bacteria in the process of gastric carcinogenesis. We conclusively showed that H. pylori is not the only important bacteria colonizing gastric mucosa and that microbiota changes during the cascade of carcinogenesis. However, current evidence does not allow us to conclude about the specific role of others bacteria in the development of gastric cancer, with many potentially being only innocent bystanders of the process.
In conclusion, the interaction between H. pylori and gastric microbiota may be a crucial step in gastric carcinogenesis. However, the identities of specific microorganisms involved in these processes remain largely unknown and further studies clarifying their carcinogenic mechanisms are needed.
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22. Aviles-Jimenez F, Vazquez-Jimenez F, Medrano-Guzman R, Mantilla A, Torres J. Stomach microbiota composition varies between patients with non-atrophic gastritis and patients with intestinal type of gastric cancer. Sci Rep 2014; 4: 4202.
23. Yu G, Gail MH, Shi J, Klepac-Ceraj V, Paster BJ, Dye BA, et al. Association between upper digestive tract microbiota and cancer-predisposing states in the esophagus and stomach. Cancer Epidemiol Biomarkers Prev 2014; 23(5): 735-41. 24. Dicksved J, Lindberg M, Rosenquist M, Enroth H, Jansson JK, Engstrand L. Molecular characterization of the stomach microbiota in patients with gastric cancer and in controls. J Med Microbiol 2009; 58(Pt 4): 509-16.
25. Mattarelli P, Brandi G, Calabrese C, Fornari F, Prati GM, Biavati B, et al. Occurrence of Bifidobacteriaceae in human hypochlorhydria stomach. Microb Ecol Health Dis 2014; 25.
26. Han HS, Lee KY, Lim SD, Kim WS, Hwang TS. Molecular identification of Helicobacter DNA in human gastric adenocarcinoma tissues using Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis. Oncol Lett 2010; 1(3): 555-8.
27. Brawner KM, Morrow CD, Smith PD. Gastric microbiome and gastric cancer. Cancer J 2014; 20(3): 211-6.
28. Wang LL, Yu XJ, Zhan SH, Jia SJ, Tian ZB, Dong QJ. Participation of microbiota in the development of gastric cancer. World J Gastroenterol 2014; 20(17): 4948-52.
29. Wang ZK, Yang YS. Upper gastrointestinal microbiota and digestive diseases. World J Gastroenterol 2013; 19(10): 1541-50.
30. Nardone G, Compare D. The human gastric microbiota: Is it time to rethink the pathogenesis of stomach diseases? United European Gastroenterol J 2015; 3(3): 255-60. 31. Walker MM, Talley NJ. Review article: bacteria and pathogenesis of disease in the upper gastrointestinal tract--beyond the era of Helicobacter pylori. Aliment Pharmacol Ther 2014; 39(8): 767-79.
32. Lofgren JL, Whary MT, Ge Z, Muthupalani S, Taylor NS, Mobley M, et al. Lack of commensal flora in Helicobacter pylori-infected INS-GAS mice reduces gastritis and delays intraepithelial neoplasia. Gastroenterology 2011; 140(1): 210-20. 33. Lertpiriyapong K, Whary MT, Muthupalani S, Lofgren JL, Gamazon ER, Feng Y, et al. Gastric colonisation with a restricted commensal microbiota replicates the promotion of neoplastic lesions by diverse intestinal microbiota in the Helicobacter pylori INS-GAS mouse model of gastric carcinogenesis. Gut 2014; 63(1): 54-63.
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Tables
Table 1 - Main characteristics of studies included in the present systematic review.
Authors Year Type of study Methods Limitations Main conclusions
Bik EM, et al 2006 Cross-sectional - Gastric biopsy samples; - H. pylori testing (culture, rapid urease test, ELISA, histopathology);
- 16S rDNA gene sequencing.
- Subjects with differing medical conditions;
- Different DNA extraction and amplification methods; - Possibility of biopsy
contamination with H. pylori DNA in tissue processing.
- A diverse community of 128 phylotypes was identified: the majority of sequences were assigned to the Proteobacteria,
Firmicutes, Actinobacteria, Bacteroidetes,
and Fusobacteria phyla.
- Presence of H. pylori did not affect the composition of the gastric community. Kato S, et al 2006 Cross-sectional - Gastric biopsy samples;
- 13-C urea breath test; - Bacterial culture; - Real-time PCR.
- Reduced number of subjects; - Culture-based approach could have excluded uncultivable bacteria from detection.
- Regardless of H. pylori status, bacteria reside on the gastric mucosa of adults, whereas such bacteria rarely colonize the stomachs of children.
Andersson AF, et al
2008 Cross-sectional - Sample collection: gastric biopsies, fecal samples and throat swabs;
- DNA extraction; - 454-pyrosequencing.
- Reduced number of subjects. - Higher gastric diversity in patients of negative H. pylori status in comparison with patients of positive H. pylori status.
Dicksved J, et al
2009 Cross-sectional - Gastric biopsy samples; - DNA isolation, PCR; - T-RFLP;
- 16S rRNA gene sequencing.
- Reduced number of subjects; - Control group was hospital-based and the individuals were relatively old, with a possible deficiency in acid production. - Lack of the most current high-throughput sequencing technology.
- There are no significant differences in the composition of gastric microbiota between gastric cancer patients and controls.
Li XX, et al 2009 Cross-sectional - Gastric biopsy samples; - 16S rRNA gene sequencing;
- Reduced number of subjects; - Only H. pylori negative subjects
- The five most common genera were
- qPCR. were included. Prevotella and Porphyromonas
(Bacteroidetes), as well as Neisseria and
Haemophilus (Proteobacteria).
Han HS, et al 2010 Cross-sectional - Sample collection (resected gastric adenocarcinomas); - DNA extraction;
- PCR;
- Pyrosequencing.
- Small sample size - Gastric adenocarcinoma contains
bacteria and the majority are H. pylori. H.
cinaedi, H. mustelae and C. hyointestinalis
rarely occur and their role in the pathogenesis of gastric adenocarcinoma remains unclear.
Maldonado-Contreras A, et al
2011 Cross-sectional - Gastric biopsy samples; - DNA extraction;
- PCR;
- 16S rRNA microarray (Phylochip G2).
- Reduced number of subjects; - Subjects from different ethnicities;
- No information about previous antibiotic treatments.
- There are marked differences in the constitution of gastric bacterial community according to H. pylori status.
Hu Y, et al 2012 Cross-sectional - Gastric biopsy samples; - Rapid urease test; - H. pylori culture and PCR; - MALDI-TOF MS.
- Only culturable bacteria were examined;
- Only H. pylori positive subjects were included.
- There is a high prevalence of non-H.
pylori bacteria concurrent with H. pylori
infection in the stomach.
Aviles-Jimenez F, et al
2014 Cross-sectional - Gastric biopsy samples; - DNA extraction;
- PCR amplification; - 16S rRNA microarray; - Microarray hybridization (Phylochip G3).
- Reduced number of subjects. - There is a gradual shift in gastric microbiota profile from non-atrophic gastritis to intestinal metaplasia to gastric cancer.
Eun CS, et al 2014 Cross-sectional - Gastric biopsy samples; - DNA extraction;
- PCR amplification;
- 16S rRNA gene sequencing.
- Possibility of selection bias because patients were not enrolled consecutively. - The impact of diet on the bacterial composition of mucosa was not evaluated.
- There are significant differences in the composition of gastric microbiota among patients with gastric cancer, intestinal metaplasia and chronic gastritis, especially in Helicobacter-dominant patients.
Khosravi Y, et al
2014 Cross-sectional - Gastric biopsy samples; - Bacterial culture; - MALDI-TOF MS;
- 16S rRNA gene sequencing.
- This approach only takes into account bacteria cultured under defined laboratory conditions. - Low number of patients with gastric cancer compared with non-ulcer dyspepsia and peptic ulcer disease groups.
- Presence of H. pylori did not significantly affect the diversity of gastric microbiota.
Mattarelli P, et al
2014 Cross-sectional - Gastric juice and mucosa biopsy samples;
- Bacterial culture (BHI and Bif-TPY media);
- F6PPK test;
- PAGE of soluble proteins; - Fermentation test; - G-C DNA content; - DNA-DNA hybridization; - PCR.
- Reduced number of subjects. - This study suggests that the
Bifidobacteriaceae species, typically found
in oral cavity, readily colonize the hypochlorhydria stomach of Omeprazole patients.
Yu G, et al 2014 Cross-sectional - Upper gastrointestinal tract cell sample collection; - DNA extraction; - Microarray analysis (HOMIM);
- ELISA.
- Study design cannot distinguish whether decreased microbial richness is cause or consequence of cancer-predisposing states; - Array designed primarily to assess oral microbiota; - Sampling device may have collected material from esophagus and oral cavity; - Results may not be generalizable to Western populations.
- Lower microbial richness is associated with lower PGI/II ratio and, therefore, with an increased risk of chronic atrophic gastritis.
Table 2- Microbiota changes from normal individuals to atrophic gastritis, intestinal metaplasia and, finally, gastric cancer stages.
AG IM GC References Proteobacteria Neisseria spp ↓ ↓ ↓ (22) Haemophilus spp ↓ ↓ ↓ (22) Bergeriella denitrificans ↓ ↓ ↓ (22) Klebsiella pneumoniae ↑ (19) Acinetobacter baumanii ↑ (19) Epsilonproteobacteria ↓ (20) Helicobacteriaceae ↓ (20) Firmicutes Lachnospiraceae ↑ ↑ ↑ (22) Bacilli ↑ (20) Lactobacillus coleohominis ↑ ↑ ↑ (22) Streptococacceae ↑ (20) Streptococcus sinensis ↓ ↓ ↓ (22) Bacteroidetes Porphyromonas spp ↓ ↓ ↓ (22) Prevotella pallens ↓ ↓ ↓ (22) Actinobacteria Actinomycetales ↑ (25) Other bacteria TM7 group ↓ ↓ ↓ (22) Bulleidia spp ↓ ↓ ↓ (22)
Here, ↑ represents an increase and ↓ a decrease. AG: atrophic gastritis; IM: intestinal metaplasia; GC: gastric cancer.
Figure legends
Figure 1 – Fluxogram describing the process of article selection.
Figure 2 – Possible mechanisms of interaction between Helicobacter pylori and gastric microbiota to induce gastric carcinogenesis. Helicobacter pylori may be more important in the early stages, as a trigger for the development of chronic atrophic gastritis. The subsequent decreased acidity would lead to an increase in certain bacterial species and a decrease in others. These changes in the constitution of gastric microbiota could play important roles in later stages of gastric carcinogenesis, inducing progression from chronic atrophic gastritis to intestinal metaplasia and, finally, to gastric cancer.
Figures
Agradecimentos
A elaboração de uma monografia é uma tarefa árdua e exigente que requer muita disponibilidade. Apesar de individual, a elaboração deste trabalho contou com o contributo fundamental de várias pessoas, directa ou indirectamente. Sem eles, a sua realização não teria sido possível.
Em primeiro lugar, gostaria de deixar os meus mais sinceros agradecimentos ao meu Orientador, o Professor Doutor Pedro Nunes, por todo o apoio demonstrado, com paciência e dedicação, ao longo das sucessivas etapas de realização desta monografia. As suas sugestões foram valiosos contributos para aprimorar a sua qualidade final.
À Dra. Ana Galaghar, pela disponibilidade para ajudar em certas etapas do trabalho, o que foi uma ajuda preciosa.
Ao Bernardo Sousa Pinto, por todas as pequenas dúvidas que me foi esclarecendo com amizade e prontidão e que foram uma enorme ajuda.
Por último, mas não menos importante, deixo ainda um agradecimento aos meus pais por todo o apoio incondicional prestado, não só durante a realização desta monografia, como também ao longo de toda a minha vida. Se é verdade que o seu apoio foi verdadeiramente crucial nos momentos mais difíceis da realização deste trabalho, não é menos verdade que, se tal apoio não tivesse sido iniciado há muito mais tempo, decerto eu não teria chegado até aqui.
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You CH, Lee KY, Menguy R. Electrocardiographic study of patients with unexplained nausea, bloating and vomiting. Gastroenterology 1980; 79: 311-4.
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