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The Gastric Microbiome in Benign and Malignant Diseases
Chapter 10
The Gastric Microbiome in Benign and Malignant Diseases Thais Fernanda Bartelli1, Luiz Gonzaga Vaz Coelho2 and Emmanuel Dias-Neto1 1
Lab. Medical Genomics, A.C.Camargo Cancer Center, São Paulo, Brazil; 2Alfa Institute of Gastroenterology, Clinics Hospital, Federal University
of Minas Gerais, Belo Horizonte, Brazil
THE GASTRIC MICROBIAL COMMUNITY For many years, the concept of an endogenous stomach microbiota was dismissed by the notion that the stomach is a harsh and inhospitable environment. The low pH, a consequence of hydrochloric acid (HCl) production, the occasional reflux of bile acids, and the presence of nitric oxide (a potent antimicrobial agent), as well as the constant peristalsis of its muscularis layers, led to the belief that the stomach was a sterile organ [1]. In the early 1980s, this belief was challenged by the discovery of several bacteria, including Campylobacter pyloridis (later renamed Helicobacter pylori), Streptococcus, Lactobacillus, and Neisseria, which were identified by techniques based on microorganism growth [1]. Current estimates point out that whereas the large intestine harbors up to 1012 colony-forming units (CFU)/mL, the stomach lodges around 103e104 CFU/mL [2,3]. Although significantly smaller, this microbiota operates as an important barrier against the colonization by exogenous and pathogenic bacteria that could find its way through the gastrointestinal tract and infect the host. Additionally, recent studies found that the stomach actively releases oxidizing chemicals and may be able to recognize hazardous foreign bodies, thus triggering this response [4,5]. It was only in 2006 that the first molecular study using 16S rDNA by Bik and collaborators [6] would identify at least 128 different bacterial phylotypes on the gastric mucosa of healthy individuals. The five most abundant phyla in both children and adults are Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria [6e10]. This gastric microbiota is relatively stable and maintained over different populations, possibly because of the high selective pressure exerted by the stomach environment, rather than cultural individual habits [8]. Specifically, the five most common genera, found in healthy and antral gastritis patients without H. pylori infection were Streptococcus (phylum Firmicutes), Prevotella and Porphyromonas (phylum Bacteroidetes), and Neisseria and Haemophilus (phylum Proteobacteria) [8]. H. pylori, a Proteobacteria present in almost 50% of the world population, is the most studied gastric bacteria, with a well-recognized role in gastritis, preneoplastic, and neoplastic lesions. Lactobacilli, another common stomach microorganism, is commonly used as probiotic due to its health-promoting properties. Nonetheless, growth-independent methods have identified a vast number of commensals, including Rothia, Actinobacillus, Caulobacter, Corynebacterium, Gemella, Leptotrichia, Porphyromonas, Capnocytophaga, TM7, Flexistipes, and Deinococcus [6].
TECHNICAL LIMITATIONS IN THE GASTRIC MICROBIOME ANALYSIS A relevant limitation of the traditional molecular microbiome studies may arise by the essentially one-and-only analysis of the bacterial DNA content present in the stomach environment, by 16S rDNA sequencing, which is unable to distinguish between transcriptionally active or inactive, dead or alive bacteria [10]. As a consequence, the sequencing of this bacterial DNA present may not represent the true active community, as it may also contemplate bacterial DNA derived from dead bacteria or migrating organisms derived from the upper aerodigestive tract and/or from the diet.
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Additionally, due to the complexity of the still unexplored interactions between bacteria and the gastric environment, the study of gastric juice may lead to inconclusive data, since it can be more representative of the stomach transient bacteria population, rather than the bacterial cells that closely interact with the gastric tissue and mucus layer [1]. This occurs because, while the gastric juice tends to restrict the growth of the majority of the microorganisms, the mucus layer has a protective role allowing a more hospitable environment and the growth of a richer and more diverse tissue-associate microbiome [11]. In the gastrointestinal tract, the mucus barrier is variable among the population and formed by different patterns of host-specific glycosylation products and glycoproteins, which are capable of shaping the identity and the composition of the indigenous microbiota [12].
EUKARYOTES AND VIRUSES IN THE UPPER GASTROINTESTINAL TRACT Whereas the gut bacteriome is usually the center of the attention, it is important to keep in mind that viruses and eukaryotes such as fungi, helminths, and protozoa are also important members of the human microbial community [13]. A comprehensive analysis using traditional culture methods and molecular study techniques detected a rich eukaryotic community in the human gastrointestinal tract, composed by more than 15 different protozoan and 50 helminthic genera linked to commensalism or parasitism [13,14]. Among them are known human parasites such as Blastocystis spp., with high prevalence in healthy individuals, Cryptosporidium spp., and Entamoeba histolytica [13]. Bacteriaeprotozoa interactions and host immune system modulation are possible in this context [13]. Fungal communities have also been detected in the upper portion of the gastrointestinal tract as a possible resident, or otherwise transient, members of the human gastric environment in healthy individuals [10,15]. Fungi represent only 0.1% of the microorganisms found in the human microbiota [16e18]; nonetheless, the mycobiome is believed to play a greater role than previously expected in mucosal health, especially when the bacteriome is disturbed [15]. Fungi of the genus Candida, known members of the human microbiome and resident members of the intestinal tract, are also frequently identified in the stomach environment [15,19]. Analysis of the gastric fluid of 25 patients with clinically recommended upper endoscopy found Candida sp. in all samples, along with the opportunistic fungus Phialemonium [10], also involved in infections in immunocompromised hosts. Viruses also inhabit the stomach. Examples of their importance to the gastric health can be given by phage and the EpsteineBarr virus (EBV). Phages, a group of bacteria-infecting viruses, have an important role in infecting and controlling bacterial populations; however, they also have a role in horizontal gene transfer and antibiotic resistance [20]. EBV is known to play a major role in approximately 10% of stomach cancer cases [21]. The identification of EBV as an important player in gastric carcinogenesis opens the possibility that other viruses may inhabit the stomach and play a role in gastric diseases.
HELICOBACTER PYLORI AND GASTRIC CANCER H. pylori is thought to be colonizing humans for at least 50,000 years [22]. Chronic infection by H. pylori has been established as a major risk factor for gastric cancer (twofold risk), particularly non-cardia and nondiffuse tumor subtypes, with most gastric cancer cases (80%) associated with the chronic presence of this bacterium, making H. pylori a definite carcinogen by the World Health Organization, since 1994 [23,24]. The causal or mechanistic links associated to H. pylori stomach carcinogenesis are due to putative virulence genes, such as (1) cagPAI (cytotoxin-associated gene pathogenicity island), which encodes proteins that are members of the type IV secretion system, which eventually leads to dephosphorylation of host cell proteins and destabilization of cellular morphology; (2) genes encoding outer-membrane proteins (oipA, sabA, sabB, babA, babC, and hopZ), responsible mainly for bacterium binding to the gastric mucosal cells or promotion of inflammatory processes; and (3) vacA (vacuolating cytotoxin gene), which encodes a cytotoxic protein capable of inducing vacuolation on the host cells [25]. Other gut bacteria, such as Escherichia coli, are also capable of producing virulence factors that directly target the host DNA, such as colibactins, genotoxins capable of inducing DNA double-strand breaks, associated with genomic instability and colon cancer tumorigenesis [26]. Despite the clear role of H. pylori in gastric cancer, the occurrence of gastritis, and disease progression to metaplasia and dysplasia, is irrespective of the presence, eradication, or absence of the pathogen [8,27e29]. Only 1%e3% of H. pylori-infected patients effectively develop gastric cancer [30].
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GENERAL DYSBIOSIS IN GASTRIC CANCER Other taxa enriched in gastric cancer patients are Lactococcus, Veillonella, Fusobacterium, and Leptotrichia. Analysis of microbial metabolic output in those patients identified an increase in lactic acideproducing bacteria, enrichment of shortchain fatty acid production pathways, and enrichment of proinflammatory oral bacterial species [31]. Dysbiosis in intestinal metaplasia and gastric cancer subjects has been characterized, as compared to superficial or atrophic gastritis. Additionally, oral bacteria, such as Peptostreptococcus stomatis and Dialister pneumosintes, had significantly higher abundance in gastric cancer as compared to the other stages analyzed, and together with Streptococcus anginosus, Parvimonas micra and Slackia exigua, could differentiate gastric cancer patients from superficial gastritis cases [32]. Whether the gastric mucosal dysbiosis is the causative agent or a consequence of altered mucosal physiology/tumor microenvironment is yet to be fully uncovered. Controlled in vivo experiments, through the introduction of specific known bacteria, will be required to elucidate their role [33]. Nonetheless, gastric cancer has been subdivided into four molecular subtypes by the TCGA (The Cancer Genome Atlas) [34], one of them defined according to the presence of EBV. Future studies might well consider the results of gastric microbiome analysis [33].
GASTRIC MICROORGANISMS AND EXTRAGASTRIC DISEASE Campylobacter concisus, transcriptionally active in the stomach [10], has a significantly higher prevalence in children with Crohn’s disease in comparison to healthy controls [35,36]. H. pylori, aside from being a known risk factor for stomach carcinogenesis, has a massive influence overall the gastrointestinal tract, including modulation of the resident microbiota and the host immunological response. In the stomach, colonization by H. pylori leads to increased pH through mucosal atrophy and changes in nutrient availability, favoring the growth of bacterial species that would otherwise not survive gastric physiology, leading to conditions that alter the stomach homeostasis, consequently favoring carcinogenesis [7,37], and influencing the whole gut. Analysis of the active microbiome, determined by sequencing 16S rRNA transcripts of stomach biopsies, indicated that gastric cancer patients had increased bacterial richness and phylogenetic diversity as compared to functional dyspepsia patients, independent of their H. pylori serum-positivity. However, the composition of the gastric microbiota and global microbial metabolic output, in patients testing positive for this bacterium, was different from those tested as negative [31].
EPSTEINeBARR VIRUS AND GASTRIC CANCER EBV is present in about 95% of the population in a latent stage [38], yet its oncogenic activity has been associated with the occurrence of several human tumors, comprising about 10% of gastric cancer cases worldwide [39], predominantly nonantrum carcinomas [39e41]. The virus enters the stomach via the oral route, where it is capable of infecting the epithelial mucosal cells, through unknown mechanisms that are different from those used for EBV B-lymphocyte infection. A series of biological mechanisms operate in EBV-induced tumors, including the interference of EBV with cell-cycle checkpoints as well as cell death pathways and the immortalization and proliferation of EBV-infected cells [14]. EBV-immortalized B-cells are in the basis of B-cell lymphomas, where EBV-infection appears to act as a somatic mutator, due to the activation of DNA and RNA editing enzymes. Some authors have also suggested that gastritis (related or not to H. pylori infection) will recruit EBV-infected B-lymphocytes in a process that may increase the epithelial frequency of EBV-infected cells [41]. As EBV-positive tumors have been classified by TCGA as a distinct molecular subtype of gastric cancer (alongside the microsatellite unstable, genomically stable and those with chromosomal instability subtypes), EBV-positive gastric tumors have specific molecular alterations such as mutations in the PIK3CA gene, DNA hypermethylation and amplification of JAK2, CD274 and PDL1 and PDL2 [34]. These EBV-positive tumors have been associated with the increased expression of programmed cell death 1 ligand (PDL1/2). For this reason, the presence of this virus may indicate good candidates for therapy with immune checkpoint inhibitors [34], with evidence of better outcomes for those patients [42,43].
LIFESTYLE HABITS AND STOMACH DYSBIOSIS Even though well-defined microbial communities colonize the gastrointestinal tract, its composition is dynamic and can be shaped by several factors, such as lifestyle and dietary habits, use of medications, and eventually predisposition by genetic
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Exogenous elements Diet, alcohol, tobacco, protonpump inhibitors, drugs, others
Modulation of immune cells
absorption modify
shape
Modified dietary compounds
Modulate local mucosa immunity
Microbiome Microbiome monitoring modify
shape
Host genome
Cancer
Endogenous FIGURE 10.1 Microbiota and cancer: an integrated view. The central role of the microbiota in the general homeostasis is presented, together with some of its interactions with exogenous and endogenous elements that impact mutation burden and cancer development. Relevant to gastric cancer, the microbiota is shaped by dietary elements such as a salted diet, consumption of red meat, vegetables, as well as alcohol, tobacco, and pH-elevating drugs. The microbiota, including viral infections (such as the EBV), presumably triggers DNA-modifying enzymes, leading to the accumulation of mutations in the cancer genome. On the other hand, these exogenous elements are also able to directly lead to mutations in the host genome and are modified by the microbiota, such as the breakdown of ethanol to acetaldehyde or the synthesis of vitamins and cofactors produced by the microbiota. Modified dietary compounds are important modulators of the immune system that affects the local immune status and impact the development of cancer.
factors. In turn, the microbiota will metabolize diet components and contribute to the shaping of the immune system, with remarkable influence over the microbiota itself and also over the development of diseases such as cancer (Fig 10.1). Proton pump inhibitors (PPI) are among the top five drugs used by the Western population. Together with antibiotics, PPIs are especially used for the eradication of H. pylori, through direct targeting of the bacterial proton pumps or by promoting changes in the pH of the gastric microenvironment, affecting its growth [44]. However, long-term PPI use has been associated with an at least 2.4-fold increase in the risk of gastric cancer development, in a dose- and time-dependent manner, especially risky for daily users for a period 3 years [45]. Aside from drastic changes in the stomach pH by PPIs, which influence the gastric microbiome toward inflammation and cancer predisposing bacteria [46,47], colonization by H. pylori also compromises the stomach acidity by the active release of urease, hydrolyzing urea, and increasing the pH in the surroundings [48]. How does the pH change caused by these drugs affect the gastric microbiota in medium/long term? H. pylori control is likely to make available an ecological niche in the stomach that, together with the modification of gastric pH due to PPIs use, is still an open question. The gastric mucosa of patients under the use of PPI usually presents a lower microbial diversity, enriched with a few specific bacteria, particularly Streptococcaceae, whose abundance is known to exacerbate or maintain the symptoms associated to dyspepsia [47,49]. In addition, all samples analyzed by Paroni et al. [47] had increased Capnocytophaga (Bacteroidetes) and Actinobacillus (a Proteobacteria like H. pylori). Increased Streptococcaceae has also been described in a cohort of 1827 healthy individuals using PPIs, where additionally, the change in the gastric pH has led to an abundance of oral and upper gastrointestinal tract commensals in their feces [50]. In this sense, the use of PPIs is associated with an increased risk of intestinal infections, including Clostridium difficile colitis [51,52]. Aside from the increase in Streptococcaceae, authors have recently observed a decrease in the abundance of Faecalibacterium [49,53] in patients using PPIs. This genus is expected to be highly abundant in the healthy gut and has important roles aiding the digestion of dietary fibers, or as a potential probiotic, with anti-inflammatory properties capable of boosting the immune system. Diminished Faecalibacterium has been linked to intestinal inflammation-associated diseases (i.e., colitis and Crohn’s disease). The use of diet components or additives may also be associated with dysbiosis and the emergence of epidemics or to the virulence of some bacteria. Some components of the Western diet have been modified by the industry for a series of reasons. An example is the disaccharide trehalose, a sweetening/texturizing ingredient employed to decrease the freezing
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point of certain processed food, with added properties of reducing sudden glycemic increment after ingestion. In 2018, a group of Baylor College of Medicine showed that the recent epidemics of C. difficile could be associated with the rise in the use of the artificial sweetener trehalose, by the food industry. Epidemic C. difficile ribotypes are able to metabolize trehalose, which increases C. difficile virulent strains [54]. As expected, antibiotics are also modifiers of the stomach microbiome. Although their use has unquestionable benefits to the control of bacterial pathogens, it does not come without some prejudice to the indigenous microbiota. The effect of oral cefoperazone, a third-generation cephalosporin antibiotic, on the gastric bacterial microbiota was observed at least 3 weeks after cessation of its use, causing an especially significant disturbance associated, among other effects, to an increase in the presence of the yeast Candida albicans in the stomach, responsible for gastritis during the postantibiotic recovery phase [55]. Immunosuppression linked to elderly patients and the use of PPI also seems to be important risk factors favoring yeast colonization in the stomach. This microbial unbalance and association of Candida yeasts and H. pylori are believed to be linked to peptic diseases [19] and the development of gastric malignancies [56]. In a study with over 150 patients, at least 17% of them had the fungus Candida detected in their gastric mucosa and 11% of those patients were co-colonized with both H. pylori and Candida [57]. The importance of this coinfection in the rise of gastric diseases is still poorly explored, yet this bacteriumeyeast association or yeast infection alone could be causing dyspeptic diseases [19]. H. pylori has been inversely associated with a series of immune-associated diseases, including asthma [58,59], due to direct or indirect influences on gastric and gut microbial community, or the patient immune system [37]. Studies show that the gastric microbiome is influenced by H. pylori infection in children but not in adults, as children infected with H. pylori tend to have a more diverse bacterial community in the stomach than noninfected ones, with a smaller abundance of Firmicutes and increased richness of non-Helicobacter Proteobacteria. On the other hand, H. pylori-infected adults have similar bacterial profiles as those noninfected. H. pylori in the early life of these children possibly suppress the mucosal inflammation by a T-regulatory response, reducing the symptoms of mucosal damage. Meanwhile, it is still not clear whether this early-life presence of H. pylori has an impact on the gastric disease symptoms in adults [58], although in a murine model, the age at which H. pylori was acquired influenced, for example, whether they would develop or not precancerous lesions [60]. Aside from having an active role in carcinogenesis, the gut bacteriome may indirectly, through its metabolites, influence the stomach. Conditions that alter the stomach pH knowingly favor the growth of non-H. pylori, especially nitrosating bacteria, which convert nitrate, a common food additive, to carcinogenic N-compounds [7,61]. In the same manner, a recent meta-analysis reinforced the notion that ethanol consumption leads to an elevated risk of gastric cancer development (odds ratio: 1.39; 95% confidence interval 1.20e1.61) [62], as ethanol metabolization generates the carcinogen acetaldehyde, a process that can be exacerbated by bacteria, making ethanol-induced carcinogenesis also dependent of the host microbiota. Some natural interventional strategies, such as the use of probiotics, can help in restoring the healthy microbiome. Although this approach is more constantly used aiming the low gastrointestinal tract, the ingestion of probiotic-rich food such as yogurt may improve symptoms linked to gastric dyspepsia [63,64] bringing benefits to the host. Lactic acid bacteria, such as Lactobacillus, have shown to be beneficial in preventing or helping eradicate H. pylori infection [65,66] or even effectively suppressing dyspeptic symptoms in H. pylori-infected patients [64]. Nonetheless, the literature concerning gastric probiotics is still limited and need further investigation. The importance of endogenous and exogenous factors to shape and to determine the microbiome composition is broadly recognized. However, until recently, the relative contribution of the host genetic versus the environmental factors have not been evaluated. A recent study genotyped 696 healthy individuals who live in similar environments but derived from distinct ancestral origins. The authors found no statistically significant association between microbiome composition and ethnicity. In contrast, genetically unrelated individuals sharing the same household had very similar microbiome compositions. This publication strongly suggests that the human microbiome composition is most importantly determined by environmental factors rather than host genetics [67].
CONCLUSIONS AND FUTURE PERSPECTIVES A significant challenge that remains is the establishment of a causal link explaining the roles of bacterial populations and their derived metabolites in the development of diseases in humans, including gastric cancer, gastritis, and others. Once this causal link is well established, it raises the possibility of modulating the stomach microbiome, aiming to prevent or intervene in the course of a disease [1]. As the microbiota knowledge advances, including the molecular basis of
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microbialehost interaction, microbial populations will be translated in a sort of library of chemical/biochemical compounds that may have the potential of revolutionizing a series of human diseases and conditions. The impact of the gastric microbiota in the future of gastric cancer therapy is expected, as compelling evidence demonstrates its determinant role in tumor response to chemotherapy and immunotherapy. Specifically, there is increasing interest in the synergistic combination of microbiome in the modulation of the host immune system [68]. The literature started to document that the blockage of immune checkpoint inhibitors that target molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death 1 (PD1) and programmed cell death 1 ligand 1 (PDL1) can benefit from a healthy microbiome status during cancer therapy [33]. In general, higher microbiota diversity is associated with better response to cancer treatment. Recent investigations targeting hundreds of patients, with diverse tumor types (bladder, kidney, lung), clearly show that an antibiotic-disrupted microbiota is correlated with faster tumor relapse and reduced overall survival in patients treated with anti-PD1 immunotherapy. These observations were reinforced by the finding that germ-free mice receiving fecal transplantation from chemotherapy responders versus nonresponders showed the same response as the donors [67] and the same was observed with regard to melanoma patients, treated by the same drug [68]. Microorganisms that are capable of priming T-cells populations toward better recognition of tumor neoantigens and inducing the release of IL-12 showed to be beneficial for immunotherapy response. However, bacteria pointed out by these two articles were different: Akkermansia muciniphila in the first study [67] and Faecalibacterium and Clostridiales in the second one [68], a difference that can be explained by diet and lifestyle habits of the different populations enrolled. While the right composition for fecal transplantation is still being vetted, the study of Zitvogel’s group is clear in suggesting that by simply avoiding antibiotics during immunotherapy, response rates can jump from 25% to 40% [67]. In this sense the importance of a better understanding of the human microbiome, including the gastric microbiome, to better treat human diseases is becoming a reality.
ACKNOWLEDGMENTS The authors acknowledge the financial support received from PRONON and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - 2014/26897-0).
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[47] Paroni Sterbini F, Palladini A, Masucci L, Cannistraci CV, Pastorino R, Ianiro G, et al. Effects of proton pump inhibitors on the gastric mucosa-associated microbiota in dyspeptic patients. Appl Environ Microbiol November 15, 2016;82(22):6633e44. [48] Walker MM, Talley NJ. Review article: bacteria and pathogenesis of disease in the upper gastrointestinal tractebeyond the era of Helicobacter pylori. Aliment Pharmacol Ther April 2014;39(8):767e79. [49] Takagi T, Naito Y, Inoue R, Kashiwagi S, Uchiyama K, Mizushima K, et al. The influence of long-term use of proton pump inhibitors on the gut microbiota: an age-sex-matched case-control study. J Clin Biochem Nutr January 2018;62(1):100e5. [50] Jackson MA, Goodrich JK, Maxan M-E, Freedberg DE, Abrams JA, Poole AC, et al. Proton pump inhibitors alter the composition of the gut microbiota. Gut May 2016;65(5):749e56. [51] Janarthanan S, Ditah I, Adler DG, Ehrinpreis MN. Clostridium difficile-associated diarrhea and proton pump inhibitor therapy: a meta-analysis. Am J Gastroenterol July 2012;107(7):1001e10. [52] Kwok CS, Arthur AK, Anibueze CI, Singh S, Cavallazzi R, Loke YK. Risk of Clostridium difficile infection with acid suppressing drugs and antibiotics: meta-analysis. Am J Gastroenterol July 2012;107(7):1011e9. [53] Tsuda A, Suda W, Morita H, Takanashi K, Takagi A, Koga Y, et al. Influence of proton-pump inhibitors on the luminal microbiota in the gastrointestinal tract. Clin Transl Gastroenterol June 11, 2015;6:e89. [54] Collins J, Robinson C, Danhof H, Knetsch CW, van Leeuwen HC, Lawley TD, et al. Dietary trehalose enhances virulence of epidemic Clostridium difficile. Nature January 18, 2018;553(7688):291e4. [55] Mason KL, Erb Downward JR, Falkowski NR, Young VB, Kao JY, Huffnagle GB. Interplay between the gastric bacterial microbiota and Candida albicans during postantibiotic recolonization and gastritis. Infect Immun January 2012;80(1):150e8. [56] Wang ZK, Yang YS, Stefka AT, Sun G, Peng LH. Review article: fungal microbiota and digestive diseases. Aliment Pharmacol Ther April 2014;39(8):751e66. [57] Karczewska E, Wojtas I, Sito E, Trojanowska D, Budak A, Zwolinska-Wcislo M, et al. Assessment of co-existence of Helicobacter pylori and Candida fungi in diseases of the upper gastrointestinal tract. J Physiol Pharmacol Off J Pol Physiol Soc December 2009;60(Suppl. 6):33e9. [58] Brawner KM, Kumar R, Serrano CA, Ptacek T, Lefkowitz E, Morrow CD, et al. Helicobacter pylori infection is associated with an altered gastric microbiota in children. Mucosal Immunol September 2017;10(5):1169e77. [59] Arnold IC, Dehzad N, Reuter S, Martin H, Becher B, Taube C, et al. Helicobacter pylori infection prevents allergic asthma in mouse models through the induction of regulatory T cells. J Clin Investig August 2011;121(8):3088e93. [60] Arnold IC, Lee JY, Amieva MR, Roers A, Flavell RA, Sparwasser T, et al. Tolerance rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia. Gastroenterology January 2011;140(1):199e209. [61] Mowat C, Williams C, Gillen D, Hossack M, Gilmour D, Carswell A, et al. Omeprazole, Helicobacter pylori status, and alterations in the intragastric milieu facilitating bacterial N-nitrosation. Gastroenterology August 2000;119(2):339e47. [62] Ma K, Baloch Z, He T-T, Xia X. Alcohol consumption and gastric cancer risk: a meta-analysis. Med Sci Monit Int Med J Exp Clin Res January 14, 2017;23:238e46. [63] Ianiro G, Pizzoferrato M, Franceschi F, Tarullo A, Luisi T, Gasbarrini G. Effect of an extra-virgin olive oil enriched with probiotics or antioxidants on functional dyspepsia: a pilot study. Eur Rev Med Pharmacol Sci 2013;17(15):2085e90. [64] Takagi A, Yanagi H, Ozawa H, Uemura N, Nakajima S, Inoue K, et al. Effects of Lactobacillus gasseri OLL2716 on Helicobacter pylori-associated dyspepsia: a multicenter randomized double-blind controlled trial. Gastroenterol Res Pract 2016;2016:7490452. [65] Lu C, Sang J, He H, Wan X, Lin Y, Li L, et al. Probiotic supplementation does not improve eradication rate of Helicobacter pylori infection compared to placebo based on standard therapy: a meta-analysis. Sci Rep March 21, 2016;6:23522. [66] Sakamoto I, Igarashi M, Kimura K, Takagi A, Miwa T, Koga Y. Suppressive effect of Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in humans. J Antimicrob Chemother May 2001;47(5):709e10. [67] Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science January 5, 2018;359(6371):91e7. [68] Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science January 5, 2018;359(6371):97e103.
The Gastric Microbiome in Benign and Malignant Diseases Thais Fernanda Bartelli1, Luiz Gonzaga Vaz Coelho2 and Emmanuel Dias-Neto1 1
Lab. Medical Genomics, A.C.Camargo Cancer Center, São Paulo, Brazil; 2Alfa Institute of Gastroenterology, Clinics Hospital, Federal University
of Minas Gerais, Belo Horizonte, Brazil
THE GASTRIC MICROBIAL COMMUNITY For many years, the concept of an endogenous stomach microbiota was dismissed by the notion that the stomach is a harsh and inhospitable environment. The low pH, a consequence of hydrochloric acid (HCl) production, the occasional reflux of bile acids, and the presence of nitric oxide (a potent antimicrobial agent), as well as the constant peristalsis of its muscularis layers, led to the belief that the stomach was a sterile organ [1]. In the early 1980s, this belief was challenged by the discovery of several bacteria, including Campylobacter pyloridis (later renamed Helicobacter pylori), Streptococcus, Lactobacillus, and Neisseria, which were identified by techniques based on microorganism growth [1]. Current estimates point out that whereas the large intestine harbors up to 1012 colony-forming units (CFU)/mL, the stomach lodges around 103e104 CFU/mL [2,3]. Although significantly smaller, this microbiota operates as an important barrier against the colonization by exogenous and pathogenic bacteria that could find its way through the gastrointestinal tract and infect the host. Additionally, recent studies found that the stomach actively releases oxidizing chemicals and may be able to recognize hazardous foreign bodies, thus triggering this response [4,5]. It was only in 2006 that the first molecular study using 16S rDNA by Bik and collaborators [6] would identify at least 128 different bacterial phylotypes on the gastric mucosa of healthy individuals. The five most abundant phyla in both children and adults are Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria [6e10]. This gastric microbiota is relatively stable and maintained over different populations, possibly because of the high selective pressure exerted by the stomach environment, rather than cultural individual habits [8]. Specifically, the five most common genera, found in healthy and antral gastritis patients without H. pylori infection were Streptococcus (phylum Firmicutes), Prevotella and Porphyromonas (phylum Bacteroidetes), and Neisseria and Haemophilus (phylum Proteobacteria) [8]. H. pylori, a Proteobacteria present in almost 50% of the world population, is the most studied gastric bacteria, with a well-recognized role in gastritis, preneoplastic, and neoplastic lesions. Lactobacilli, another common stomach microorganism, is commonly used as probiotic due to its health-promoting properties. Nonetheless, growth-independent methods have identified a vast number of commensals, including Rothia, Actinobacillus, Caulobacter, Corynebacterium, Gemella, Leptotrichia, Porphyromonas, Capnocytophaga, TM7, Flexistipes, and Deinococcus [6].
TECHNICAL LIMITATIONS IN THE GASTRIC MICROBIOME ANALYSIS A relevant limitation of the traditional molecular microbiome studies may arise by the essentially one-and-only analysis of the bacterial DNA content present in the stomach environment, by 16S rDNA sequencing, which is unable to distinguish between transcriptionally active or inactive, dead or alive bacteria [10]. As a consequence, the sequencing of this bacterial DNA present may not represent the true active community, as it may also contemplate bacterial DNA derived from dead bacteria or migrating organisms derived from the upper aerodigestive tract and/or from the diet.
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Additionally, due to the complexity of the still unexplored interactions between bacteria and the gastric environment, the study of gastric juice may lead to inconclusive data, since it can be more representative of the stomach transient bacteria population, rather than the bacterial cells that closely interact with the gastric tissue and mucus layer [1]. This occurs because, while the gastric juice tends to restrict the growth of the majority of the microorganisms, the mucus layer has a protective role allowing a more hospitable environment and the growth of a richer and more diverse tissue-associate microbiome [11]. In the gastrointestinal tract, the mucus barrier is variable among the population and formed by different patterns of host-specific glycosylation products and glycoproteins, which are capable of shaping the identity and the composition of the indigenous microbiota [12].
EUKARYOTES AND VIRUSES IN THE UPPER GASTROINTESTINAL TRACT Whereas the gut bacteriome is usually the center of the attention, it is important to keep in mind that viruses and eukaryotes such as fungi, helminths, and protozoa are also important members of the human microbial community [13]. A comprehensive analysis using traditional culture methods and molecular study techniques detected a rich eukaryotic community in the human gastrointestinal tract, composed by more than 15 different protozoan and 50 helminthic genera linked to commensalism or parasitism [13,14]. Among them are known human parasites such as Blastocystis spp., with high prevalence in healthy individuals, Cryptosporidium spp., and Entamoeba histolytica [13]. Bacteriaeprotozoa interactions and host immune system modulation are possible in this context [13]. Fungal communities have also been detected in the upper portion of the gastrointestinal tract as a possible resident, or otherwise transient, members of the human gastric environment in healthy individuals [10,15]. Fungi represent only 0.1% of the microorganisms found in the human microbiota [16e18]; nonetheless, the mycobiome is believed to play a greater role than previously expected in mucosal health, especially when the bacteriome is disturbed [15]. Fungi of the genus Candida, known members of the human microbiome and resident members of the intestinal tract, are also frequently identified in the stomach environment [15,19]. Analysis of the gastric fluid of 25 patients with clinically recommended upper endoscopy found Candida sp. in all samples, along with the opportunistic fungus Phialemonium [10], also involved in infections in immunocompromised hosts. Viruses also inhabit the stomach. Examples of their importance to the gastric health can be given by phage and the EpsteineBarr virus (EBV). Phages, a group of bacteria-infecting viruses, have an important role in infecting and controlling bacterial populations; however, they also have a role in horizontal gene transfer and antibiotic resistance [20]. EBV is known to play a major role in approximately 10% of stomach cancer cases [21]. The identification of EBV as an important player in gastric carcinogenesis opens the possibility that other viruses may inhabit the stomach and play a role in gastric diseases.
HELICOBACTER PYLORI AND GASTRIC CANCER H. pylori is thought to be colonizing humans for at least 50,000 years [22]. Chronic infection by H. pylori has been established as a major risk factor for gastric cancer (twofold risk), particularly non-cardia and nondiffuse tumor subtypes, with most gastric cancer cases (80%) associated with the chronic presence of this bacterium, making H. pylori a definite carcinogen by the World Health Organization, since 1994 [23,24]. The causal or mechanistic links associated to H. pylori stomach carcinogenesis are due to putative virulence genes, such as (1) cagPAI (cytotoxin-associated gene pathogenicity island), which encodes proteins that are members of the type IV secretion system, which eventually leads to dephosphorylation of host cell proteins and destabilization of cellular morphology; (2) genes encoding outer-membrane proteins (oipA, sabA, sabB, babA, babC, and hopZ), responsible mainly for bacterium binding to the gastric mucosal cells or promotion of inflammatory processes; and (3) vacA (vacuolating cytotoxin gene), which encodes a cytotoxic protein capable of inducing vacuolation on the host cells [25]. Other gut bacteria, such as Escherichia coli, are also capable of producing virulence factors that directly target the host DNA, such as colibactins, genotoxins capable of inducing DNA double-strand breaks, associated with genomic instability and colon cancer tumorigenesis [26]. Despite the clear role of H. pylori in gastric cancer, the occurrence of gastritis, and disease progression to metaplasia and dysplasia, is irrespective of the presence, eradication, or absence of the pathogen [8,27e29]. Only 1%e3% of H. pylori-infected patients effectively develop gastric cancer [30].
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GENERAL DYSBIOSIS IN GASTRIC CANCER Other taxa enriched in gastric cancer patients are Lactococcus, Veillonella, Fusobacterium, and Leptotrichia. Analysis of microbial metabolic output in those patients identified an increase in lactic acideproducing bacteria, enrichment of shortchain fatty acid production pathways, and enrichment of proinflammatory oral bacterial species [31]. Dysbiosis in intestinal metaplasia and gastric cancer subjects has been characterized, as compared to superficial or atrophic gastritis. Additionally, oral bacteria, such as Peptostreptococcus stomatis and Dialister pneumosintes, had significantly higher abundance in gastric cancer as compared to the other stages analyzed, and together with Streptococcus anginosus, Parvimonas micra and Slackia exigua, could differentiate gastric cancer patients from superficial gastritis cases [32]. Whether the gastric mucosal dysbiosis is the causative agent or a consequence of altered mucosal physiology/tumor microenvironment is yet to be fully uncovered. Controlled in vivo experiments, through the introduction of specific known bacteria, will be required to elucidate their role [33]. Nonetheless, gastric cancer has been subdivided into four molecular subtypes by the TCGA (The Cancer Genome Atlas) [34], one of them defined according to the presence of EBV. Future studies might well consider the results of gastric microbiome analysis [33].
GASTRIC MICROORGANISMS AND EXTRAGASTRIC DISEASE Campylobacter concisus, transcriptionally active in the stomach [10], has a significantly higher prevalence in children with Crohn’s disease in comparison to healthy controls [35,36]. H. pylori, aside from being a known risk factor for stomach carcinogenesis, has a massive influence overall the gastrointestinal tract, including modulation of the resident microbiota and the host immunological response. In the stomach, colonization by H. pylori leads to increased pH through mucosal atrophy and changes in nutrient availability, favoring the growth of bacterial species that would otherwise not survive gastric physiology, leading to conditions that alter the stomach homeostasis, consequently favoring carcinogenesis [7,37], and influencing the whole gut. Analysis of the active microbiome, determined by sequencing 16S rRNA transcripts of stomach biopsies, indicated that gastric cancer patients had increased bacterial richness and phylogenetic diversity as compared to functional dyspepsia patients, independent of their H. pylori serum-positivity. However, the composition of the gastric microbiota and global microbial metabolic output, in patients testing positive for this bacterium, was different from those tested as negative [31].
EPSTEINeBARR VIRUS AND GASTRIC CANCER EBV is present in about 95% of the population in a latent stage [38], yet its oncogenic activity has been associated with the occurrence of several human tumors, comprising about 10% of gastric cancer cases worldwide [39], predominantly nonantrum carcinomas [39e41]. The virus enters the stomach via the oral route, where it is capable of infecting the epithelial mucosal cells, through unknown mechanisms that are different from those used for EBV B-lymphocyte infection. A series of biological mechanisms operate in EBV-induced tumors, including the interference of EBV with cell-cycle checkpoints as well as cell death pathways and the immortalization and proliferation of EBV-infected cells [14]. EBV-immortalized B-cells are in the basis of B-cell lymphomas, where EBV-infection appears to act as a somatic mutator, due to the activation of DNA and RNA editing enzymes. Some authors have also suggested that gastritis (related or not to H. pylori infection) will recruit EBV-infected B-lymphocytes in a process that may increase the epithelial frequency of EBV-infected cells [41]. As EBV-positive tumors have been classified by TCGA as a distinct molecular subtype of gastric cancer (alongside the microsatellite unstable, genomically stable and those with chromosomal instability subtypes), EBV-positive gastric tumors have specific molecular alterations such as mutations in the PIK3CA gene, DNA hypermethylation and amplification of JAK2, CD274 and PDL1 and PDL2 [34]. These EBV-positive tumors have been associated with the increased expression of programmed cell death 1 ligand (PDL1/2). For this reason, the presence of this virus may indicate good candidates for therapy with immune checkpoint inhibitors [34], with evidence of better outcomes for those patients [42,43].
LIFESTYLE HABITS AND STOMACH DYSBIOSIS Even though well-defined microbial communities colonize the gastrointestinal tract, its composition is dynamic and can be shaped by several factors, such as lifestyle and dietary habits, use of medications, and eventually predisposition by genetic
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Exogenous elements Diet, alcohol, tobacco, protonpump inhibitors, drugs, others
Modulation of immune cells
absorption modify
shape
Modified dietary compounds
Modulate local mucosa immunity
Microbiome Microbiome monitoring modify
shape
Host genome
Cancer
Endogenous FIGURE 10.1 Microbiota and cancer: an integrated view. The central role of the microbiota in the general homeostasis is presented, together with some of its interactions with exogenous and endogenous elements that impact mutation burden and cancer development. Relevant to gastric cancer, the microbiota is shaped by dietary elements such as a salted diet, consumption of red meat, vegetables, as well as alcohol, tobacco, and pH-elevating drugs. The microbiota, including viral infections (such as the EBV), presumably triggers DNA-modifying enzymes, leading to the accumulation of mutations in the cancer genome. On the other hand, these exogenous elements are also able to directly lead to mutations in the host genome and are modified by the microbiota, such as the breakdown of ethanol to acetaldehyde or the synthesis of vitamins and cofactors produced by the microbiota. Modified dietary compounds are important modulators of the immune system that affects the local immune status and impact the development of cancer.
factors. In turn, the microbiota will metabolize diet components and contribute to the shaping of the immune system, with remarkable influence over the microbiota itself and also over the development of diseases such as cancer (Fig 10.1). Proton pump inhibitors (PPI) are among the top five drugs used by the Western population. Together with antibiotics, PPIs are especially used for the eradication of H. pylori, through direct targeting of the bacterial proton pumps or by promoting changes in the pH of the gastric microenvironment, affecting its growth [44]. However, long-term PPI use has been associated with an at least 2.4-fold increase in the risk of gastric cancer development, in a dose- and time-dependent manner, especially risky for daily users for a period 3 years [45]. Aside from drastic changes in the stomach pH by PPIs, which influence the gastric microbiome toward inflammation and cancer predisposing bacteria [46,47], colonization by H. pylori also compromises the stomach acidity by the active release of urease, hydrolyzing urea, and increasing the pH in the surroundings [48]. How does the pH change caused by these drugs affect the gastric microbiota in medium/long term? H. pylori control is likely to make available an ecological niche in the stomach that, together with the modification of gastric pH due to PPIs use, is still an open question. The gastric mucosa of patients under the use of PPI usually presents a lower microbial diversity, enriched with a few specific bacteria, particularly Streptococcaceae, whose abundance is known to exacerbate or maintain the symptoms associated to dyspepsia [47,49]. In addition, all samples analyzed by Paroni et al. [47] had increased Capnocytophaga (Bacteroidetes) and Actinobacillus (a Proteobacteria like H. pylori). Increased Streptococcaceae has also been described in a cohort of 1827 healthy individuals using PPIs, where additionally, the change in the gastric pH has led to an abundance of oral and upper gastrointestinal tract commensals in their feces [50]. In this sense, the use of PPIs is associated with an increased risk of intestinal infections, including Clostridium difficile colitis [51,52]. Aside from the increase in Streptococcaceae, authors have recently observed a decrease in the abundance of Faecalibacterium [49,53] in patients using PPIs. This genus is expected to be highly abundant in the healthy gut and has important roles aiding the digestion of dietary fibers, or as a potential probiotic, with anti-inflammatory properties capable of boosting the immune system. Diminished Faecalibacterium has been linked to intestinal inflammation-associated diseases (i.e., colitis and Crohn’s disease). The use of diet components or additives may also be associated with dysbiosis and the emergence of epidemics or to the virulence of some bacteria. Some components of the Western diet have been modified by the industry for a series of reasons. An example is the disaccharide trehalose, a sweetening/texturizing ingredient employed to decrease the freezing
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point of certain processed food, with added properties of reducing sudden glycemic increment after ingestion. In 2018, a group of Baylor College of Medicine showed that the recent epidemics of C. difficile could be associated with the rise in the use of the artificial sweetener trehalose, by the food industry. Epidemic C. difficile ribotypes are able to metabolize trehalose, which increases C. difficile virulent strains [54]. As expected, antibiotics are also modifiers of the stomach microbiome. Although their use has unquestionable benefits to the control of bacterial pathogens, it does not come without some prejudice to the indigenous microbiota. The effect of oral cefoperazone, a third-generation cephalosporin antibiotic, on the gastric bacterial microbiota was observed at least 3 weeks after cessation of its use, causing an especially significant disturbance associated, among other effects, to an increase in the presence of the yeast Candida albicans in the stomach, responsible for gastritis during the postantibiotic recovery phase [55]. Immunosuppression linked to elderly patients and the use of PPI also seems to be important risk factors favoring yeast colonization in the stomach. This microbial unbalance and association of Candida yeasts and H. pylori are believed to be linked to peptic diseases [19] and the development of gastric malignancies [56]. In a study with over 150 patients, at least 17% of them had the fungus Candida detected in their gastric mucosa and 11% of those patients were co-colonized with both H. pylori and Candida [57]. The importance of this coinfection in the rise of gastric diseases is still poorly explored, yet this bacteriumeyeast association or yeast infection alone could be causing dyspeptic diseases [19]. H. pylori has been inversely associated with a series of immune-associated diseases, including asthma [58,59], due to direct or indirect influences on gastric and gut microbial community, or the patient immune system [37]. Studies show that the gastric microbiome is influenced by H. pylori infection in children but not in adults, as children infected with H. pylori tend to have a more diverse bacterial community in the stomach than noninfected ones, with a smaller abundance of Firmicutes and increased richness of non-Helicobacter Proteobacteria. On the other hand, H. pylori-infected adults have similar bacterial profiles as those noninfected. H. pylori in the early life of these children possibly suppress the mucosal inflammation by a T-regulatory response, reducing the symptoms of mucosal damage. Meanwhile, it is still not clear whether this early-life presence of H. pylori has an impact on the gastric disease symptoms in adults [58], although in a murine model, the age at which H. pylori was acquired influenced, for example, whether they would develop or not precancerous lesions [60]. Aside from having an active role in carcinogenesis, the gut bacteriome may indirectly, through its metabolites, influence the stomach. Conditions that alter the stomach pH knowingly favor the growth of non-H. pylori, especially nitrosating bacteria, which convert nitrate, a common food additive, to carcinogenic N-compounds [7,61]. In the same manner, a recent meta-analysis reinforced the notion that ethanol consumption leads to an elevated risk of gastric cancer development (odds ratio: 1.39; 95% confidence interval 1.20e1.61) [62], as ethanol metabolization generates the carcinogen acetaldehyde, a process that can be exacerbated by bacteria, making ethanol-induced carcinogenesis also dependent of the host microbiota. Some natural interventional strategies, such as the use of probiotics, can help in restoring the healthy microbiome. Although this approach is more constantly used aiming the low gastrointestinal tract, the ingestion of probiotic-rich food such as yogurt may improve symptoms linked to gastric dyspepsia [63,64] bringing benefits to the host. Lactic acid bacteria, such as Lactobacillus, have shown to be beneficial in preventing or helping eradicate H. pylori infection [65,66] or even effectively suppressing dyspeptic symptoms in H. pylori-infected patients [64]. Nonetheless, the literature concerning gastric probiotics is still limited and need further investigation. The importance of endogenous and exogenous factors to shape and to determine the microbiome composition is broadly recognized. However, until recently, the relative contribution of the host genetic versus the environmental factors have not been evaluated. A recent study genotyped 696 healthy individuals who live in similar environments but derived from distinct ancestral origins. The authors found no statistically significant association between microbiome composition and ethnicity. In contrast, genetically unrelated individuals sharing the same household had very similar microbiome compositions. This publication strongly suggests that the human microbiome composition is most importantly determined by environmental factors rather than host genetics [67].
CONCLUSIONS AND FUTURE PERSPECTIVES A significant challenge that remains is the establishment of a causal link explaining the roles of bacterial populations and their derived metabolites in the development of diseases in humans, including gastric cancer, gastritis, and others. Once this causal link is well established, it raises the possibility of modulating the stomach microbiome, aiming to prevent or intervene in the course of a disease [1]. As the microbiota knowledge advances, including the molecular basis of
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microbialehost interaction, microbial populations will be translated in a sort of library of chemical/biochemical compounds that may have the potential of revolutionizing a series of human diseases and conditions. The impact of the gastric microbiota in the future of gastric cancer therapy is expected, as compelling evidence demonstrates its determinant role in tumor response to chemotherapy and immunotherapy. Specifically, there is increasing interest in the synergistic combination of microbiome in the modulation of the host immune system [68]. The literature started to document that the blockage of immune checkpoint inhibitors that target molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death 1 (PD1) and programmed cell death 1 ligand 1 (PDL1) can benefit from a healthy microbiome status during cancer therapy [33]. In general, higher microbiota diversity is associated with better response to cancer treatment. Recent investigations targeting hundreds of patients, with diverse tumor types (bladder, kidney, lung), clearly show that an antibiotic-disrupted microbiota is correlated with faster tumor relapse and reduced overall survival in patients treated with anti-PD1 immunotherapy. These observations were reinforced by the finding that germ-free mice receiving fecal transplantation from chemotherapy responders versus nonresponders showed the same response as the donors [67] and the same was observed with regard to melanoma patients, treated by the same drug [68]. Microorganisms that are capable of priming T-cells populations toward better recognition of tumor neoantigens and inducing the release of IL-12 showed to be beneficial for immunotherapy response. However, bacteria pointed out by these two articles were different: Akkermansia muciniphila in the first study [67] and Faecalibacterium and Clostridiales in the second one [68], a difference that can be explained by diet and lifestyle habits of the different populations enrolled. While the right composition for fecal transplantation is still being vetted, the study of Zitvogel’s group is clear in suggesting that by simply avoiding antibiotics during immunotherapy, response rates can jump from 25% to 40% [67]. In this sense the importance of a better understanding of the human microbiome, including the gastric microbiome, to better treat human diseases is becoming a reality.
ACKNOWLEDGMENTS The authors acknowledge the financial support received from PRONON and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - 2014/26897-0).
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