Helicobacter pylori has been widely recognized as a crucial risk factor for developing gastric cancer (Touati et al., 2003; Graham, 2015; Hartung et al., 2015; Mégraud et al., 2015), as highlighted by the World Health Organization (WHO), which classifies it as a Group I carcinogen (IARC, 2003; Bouvard et al., 2009). The prevalence of H. pylori infection is over 50% worldwide. In China, over 80% of non-cardia gastric cancer cases and over 60% of cardia gastric cancer cases each year can be attributed to H. pylori infection (Yang et al., 2021). The bacterium achieves successful colonization in the human gastric mucosal layer through various mechanisms, as follows: (1) using urease and α-carbonic anhydrase to produce ammonia and HCO₃²⁻ to neutralize gastric acid and raise the pH of local tissues, creating a favorable environment for overproliferation of itself and other bacteria in the stomach (Scott et al., 1998; Celli et al., 2009); (2) enhancing its penetration into the gastric mucosal layer through flagella (Suerbaum, 1995; Martínez et al., 2016); and (3) through bacterial virulence proteins, such as vacuolar cytotoxin (VacA) protein and cytotoxin-associated gene A (CagA) protein. The structure and function of immune cells are regulated in various ways to suppress the body’s immune response to H. pylori (Bakhti et al., 2020; Xi et al., 2023). Moreover, Helicobacter pylori has the ability to manipulate the physiological functions of gastric epithelial cells by expressing mucins such as Muc1, Muc4, and Muc5b, resulting in loss of cell polarity and release of nutrients and chemokines, including interleukin-8 (IL-8) (Navabi et al., 2013).

The virulence factors CagA and VacA of H. pylori are closely associated with GC occurrence, and the carcinogenesis mechanisms among them have been extensively explored. It has been demonstrated that individuals infected with CagA-positive strains are at a higher risk of developing gastric cancer (Blaser et al., 1995; Parsonnet et al., 1997; Huang et al., 2003; Nell et al., 2018). CagA is an oncoprotein that activates multiple signaling pathways, including RAS/ERK, WNT/β-linked protein, JAK/STAT, and PI3K/AKT, and inhibits tumor suppressors, promoting GC (Palrasu et al., 2021). CagA can act as an anti-apoptotic protein that inhibits pro-apoptotic factors such as SIVA1, BIM, and BAD; it also regulates autophagy and induces inflammation (Vallejo-Flores et al., 2015; Palrasu et al., 2020). Humans infected with CagA-positive H. pylori strains are known to exhibit an intense inflammatory response and severe damage to gastric tissues (Yamaoka et al., 1997; Figura et al., 1998). The interaction of CagA with Lactobacillus enhances maturation of human monocyte-derived dendritic cells (DCs) and induction of inflammatory mediators other than H. pylori (Castaño-Rodríguez et al., 2017). This suggests that bacteria capable of producing lactic acid may increase the inflammatory response induced by H. pylori, thus promoting gastric carcinogenesis. CagA was also found to promote the Ye s-Associated-Protein (YAP) signaling pathway, thereby promoting the epithelial-mesenchymal transition (EMT) and gastric carcinogenesis (Li et al., 2018). The EMT causes epithelial cells to lose their characteristic cell–cell contact and become more migratory and invasive by a process that may contribute to the ability of H. pylori to penetrate deeper into the gastric mucosa. VacA, another virulence factor of H. pylori, is associated with a variety of functions including disruption of the gastric mucosal barrier, interference with antigen presentation pathways, and downregulation of autophagy and phagocytosis, which contribute to bacterial inhibition of immune cells and establishment of persistent infection (Atherton et al., 1995; Boncristiano et al., 2003; Sundrud et al., 2004). The ability of VacA to downregulate autophagy and lysosomal degradation contributes to accumulation of CagA in gastric epithelial cells (Abdullah et al., 2019).

Moreover, H. pylori significantly inhibits colonization of the stomach by other bacteria, resulting in lower gastric microbiota diversity (Gao et al., 2018). In female BALB/c mice without specific pathogens, colonization by H. pylori leads to an increase in the gastric microbiota of Clostridia, Bacteroides/Prevotella spp., Eubacterium spp., Ruminococcus spp., Streptococci, and Escherichia coli, with decreases in lactobacilli (Aebischer et al., 2006). An early study used 16S rRNA microarrays to analyze gastric biopsies from subjects infected or not with H. pylori (Maldonado-Contreras et al., 2011). This study found that H. pylori-positive patients had a higher abundance of Proteobacteria, Firmicutes, Bacteroides and Actinobacteria in the stomach and a lower abundance of Actinobacteria, Bacteroidetes, and Firmicutes. All the above studies reveal a significant effect of H. pylori colonization on the gastric microbial community. However, other studies have clearly shown that the gastric microbiota is not altered by chronic H. pylori infection (Tan et al., 2007; Khosravi et al., 2014; Coker et al., 2018). Thus, the relationship between H. pylori and other flora remains controversial (Table 1).

Recent research has suggested that the presence of microbes other than Hp may also contribute to the occurrence of GC. Insulin-gastrin-secreting (INS-GAS) mice are genetically susceptible to GC and are therefore commonly used in developing mouse gastric cancer models. Tumorigenesis is relatively delayed in mice infected with H. pylori alone compared to INS-GAS mice infected with H. pylori and other gastric microbiota (Lee et al., 2008). Furthermore, H. pylori-infected germ-free (GF) INS-GAS mice exhibit less severe gastric mucosal lesions and slower tumor progression than H. pylori-infected specific pathogen-free (SPF) INS-GAS mice (Lofgren et al., 2011). These findings suggest that GC may be promoted by microorganisms in addition to H. pylori, with other gastric microbial communities also playing a potential role in the carcinogenesis and progression of GC in mice.

In another study, the stomachs of H. pylori-infected INS-GAS mice were colonized with different types of intestinal microorganisms, including restricted altered Schaedler flora (rASF; consisting of Clostridum species ASF356, Bacteroides species ASF519 and Lactobacillus murinus ASF361) and pathogen-free (complex IF) mice (Lertpiriyapong et al., 2014). The results showed that the mice colonized by either rASF Hp or IF Hp exhibited severe pathology. The IF Hp-colonized mice showed the strongest inflammatory response, with 40% developing invasive gastrointestinal intraepithelial neoplasia (GIN). The same phenomenon was observed in 23% of the rASF Hp mice. Moreover, it was found that ASF and H. pylori coinfection leads to gastric mucosal changes in mice; gastritis in mice was accompanied by reduced colonization of Clostridium perfringens ASF356 and Bacillus mimicus ASF519 and overgrowth of Lactobacillus murinus ASF361.

A recent study conducted by Kwon et al. (2022) inoculated gastric tissue and gastric fluid from patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM) or gastric cancer (GC) into GF C57BL/6 mice. The gastric microbiota of the mice was analyzed by amplicon sequencing and immunohistochemical analysis of the histopathological features of the stomach of the mice. The results revealed that when microbiomes from IM or GC patients were transplanted into the stomachs of GF mice, precancerous features were induced, including increased inflammation, decreased mural cells, and increased cell proliferation. In addition, long-term observations of mice inoculated with the microbiota from IM or GC patients led to a relatively high incidence of features of gastric dysplasia in mice. From the above evidence, it appears that commensal microorganisms in the stomach other than H. pylori are associated with development of GC.

Fungal species can be detected in the gastrointestinal tract of approximately 70% of healthy adults. The number of fungi in the human stomach is 0–10² cfu/ml, and Candida is the most common species (Schulze and Sonnenborn, 2009; Zwolinska-Wcisło et al., 2009). A study examined gastric fluid from 25 patients undergoing clinical indications using PCR amplification of the internal transcribed spacer region, with Candida and Phaalemonium found to be the only two genera present in all gastric fluid samples (von Rosenvinge et al., 2013). Candida albicans can survive under acidic conditions of pH 1.4 and above, and specific genotypes such as DST1593 may exacerbate the severity of gastric mucosal lesions (Gong et al., 2012). Candida albicans was detected in 54.2% of gastric ulcer cases and in 10.3% of chronic gastritis cases in a fungal analysis of biopsies from 293 patients with clinical manifestations of dyspepsia or ulcer disease (Karczewska et al., 2009). Gastric fungal infections and colonization are common in patients with GC, and chronic ingestion of exogenous mycotoxins from spoiled foods is a common cause of GC due to fungi. Candida infection is present in 20% of patients with gastric cancer (Scott and Jenkins, 1982). A study performed ITS rDNA genetic analysis of GC-associated fungal composition in cancerous lesions and paracancerous and noncancerous tissues from GC patients, reporting identification of 17 fungal species with significant differences between the two groups at the family level. In the GC group, Pseudeurotiaceae, Trimorphomycetaceae, Chaetomiaceae, and Aspergillaceae were significantly decreased and Saccharomycetales_fam_Incertae_sedis and Pleosporaceae increased compared to the control group. In addition, at the genus level, there were 15 different fungi between the two groups, and two fungal genera were enriched in the GC group, including Candida and Alternaria, whereas Saitozyma and Thermomyces were depleted (Zhong et al., 2021).

In addition to the microorganisms mentioned above, EBV can also cause an imbalance in gastric microecology and promote gastric cancer (Martínez-López et al., 2014). More than 90% of adults are infected with EBV, and EBV-associated gastric cancer (EBVaGC) accounts for 5–20% of all gastric cancer cases worldwide (Takada, 2000; Iizasa et al., 2012). Recent studies have described a synergistic role between Helicobacter pylori and EBV in gastric carcinogenesis. Individuals coinfected with H. pylori and EBV exhibit more severe inflammatory lesions than those infected with H. pylori alone (Cárdenas-Mondragón et al., 2013). In addition, some studies have shown that colonization by H. pylori induces reactivation of latent EBV in gastric epithelial cells (Minoura-Etoh et al., 2006; Shukla et al., 2012). Proliferation of lymphocytes after EBV infection and their ability to interact with immune effects may be directly influenced by the presence of bacterial or other microbial components at the site of infection (Iskra et al., 2010). These reports suggest that infections caused by dysbiosis may activate latent EBV, thereby increasing risk of developing cancers associated with EBV infection (Matsusaka et al., 2014; Nishikawa et al., 2014; Rickinson, 2014). Although EBV has been suspected of causing several upper gastrointestinal diseases, most studies to date have been case reports, and large-scale studies to support a causal relationship between EBV and these diseases are lacking (Hisamatsu et al., 2010; Owens et al., 2011). In addition to EBV, various other viruses have been found to contribute to tumorigenic changes in the stomach. These viruses include John Cunningham virus (JCV) (Murai et al., 2007), human cytomegalovirus (HCMV) (Jin et al., 2014; Fattahi et al., 2018; Kosari-Monfared et al., 2019), hepatitis B (HBV) or C (HCV) viruses (Song et al., 2019; Cui et al., 2020; Huang et al., 2020), human immunodeficiency virus (HIV) or human T-cell lymphophilic virus (HTLV) (Matsumoto et al., 2018; Kang et al., 2019; Schierhout et al., 2020), and papillomavirus (HPV) (Zeng et al., 2016; Bozdayi et al., 2019).

The presence of hydrochloric acid within the gastric fluid serves as a barrier to many microorganisms, protecting the stomach against potential infections (Yoshiyama and Nakazawa, 2000; Beasley et al., 2015). Over the past decade, acid-suppressing drugs such as proton pump inhibitors (PPIs) and H2 receptor antagonists (H2RAs) have been frequently used to treat gastrointestinal disorders, which have led to significant changes in the microbial diversity of the stomach (Fisher and Fisher, 2017). This shift can trigger hypochlorhydria, a condition that reduces microbial diversity, fosters proliferation of genotoxic microorganisms, and heightens activity of bacterial nitrate/nitrite reductases, ultimately leading to conversion of nitrite and other nitrogenous compounds in gastric fluid to cancer-associated N-nitroso compounds (Correa, 1992; Ahn et al., 2013; Ferreira et al., 2018). Some bacteria have been isolated from the stomachs of patients with hypochlorhydria, including Lactobacillus, Streptococcus, Pseudomonas, Xanthomonas, Proteus, Klebsiella, Neisseria, E. coli, and Campylobacter jejuni (Williams and McColl, 2006).

Acid-suppressing drugs have also been shown to impact progression of H. pylori, thereby promoting gastric carcinogenesis. In the case of reduced gastric acid secretion (caused by acid-suppressive drugs or chronic atrophy), H. pylori leads to a shift from sinusoidal to gastric body-dominated gastritis (Malfertheiner et al., 2017), reducing the lining cells and potentially enhancing RONS-mediated gastric mucosal damage (Suzuki et al., 1992), all of which are associated with increased risk of gastric cancer (Moayyedi et al., 2000; Sanduleanu et al., 2001). Acid-suppressing drugs have been shown to have negative effects on gastric function and host defense mechanisms, ultimately leading to delayed gastric emptying, decreased gastric mucus viscosity, increased gastric pH, increased bacterial load, and increased bacterial translocation (Wandall, 1992; Scarpignato et al., 2016). Several studies have documented changes in the gastric microbiota of patients treated with PPIs versus those not treated, with reduced bacterial clearance noted in the former (Tsuda et al., 2015; Paroni Sterbini et al., 2016). As the duration of PPI treatment increases, there is a gradual rise in the number of culturable bacteria in the gastric lumen and mucosa (Sanduleanu et al., 2001; del Piano et al., 2012), with maintenance treatment for more than 1 year resulting in a 10⁶-fold increase in CFU counts (del Piano et al., 2012). Other studies have reported an increased risk of gastric cancer in long-term PPI users, with or without H. pylori eradication, up to 2.4 times higher than in nonusers (Ahn et al., 2013; Brusselaers et al., 2017; Cheung et al., 2018).

Interestingly, two recent meta-analyses suggest that current evidence does not support that maintenance PPI can cause or accelerate development or progression of gastric precancerous lesions such as gastric atrophic changes, intestinal chemosis, intestinal chromophobic (ECL) cell hyperplasia and heterogeneous hyperplasia (Eslami and Nasseri-Moghaddam, 2013; Song et al., 2018). Therefore, further prospective studies are needed to elucidate how maintenance antacids lead to alterations in the microbiota and whether such alterations ultimately increase risk of GC.

In general, application of antibiotics fundamentally alters the normal microbial community of the body, and gastric microecology is no exception. For example, treatment with cefoperazone sodium/sulbactam sodium disrupts gastric microecology, resulting in overproliferation of enterococci and a marked decrease in lactobacilli. A meticulous investigation of bacterial and fungal microbiota conducted in 25 dyspeptic patients demonstrated that antibiotics lead to reduced bacterial colonization while having a negligible effect on fungal diversity (Eslami and Nasseri-Moghaddam, 2013). Nevertheless, exposure to penicillin causes yeast overgrowth in gastric epithelial cells in mice (Nardone and Compare, 2015), whereas germ-free mice exposed to cefoperazone develop gastritis due to an increase in Candida albicans (Mason et al., 2012). A 15-year long-term study comprising 3,365 individuals diagnosed with H. pylori infection revealed that short-term treatment with amoxicillin and omeprazole reduces the incidence of gastric cancer by 39% overall (OR = 0.61, 95% CI = 0.38–0.96, p = 0.032) (Ma et al., 2012). Interestingly, eradication of H. pylori failed in more than half of the individuals treated with antibiotics at the 15-year follow-up, suggesting that antibiotic therapy may curb development of gastric cancer by instigating alterations in the non-H. pylori microbiota. It is worth noting that the eradication rate of H. pylori during the follow-up period was only 47%, highlighting that antibiotic intervention can possibly mitigate progression of gastric cancer by inducing modifications in the non-H. pylori microbiota.

Alterations in the anatomy of the gastric environment can also affect its microbiota composition. Roux-en-Y gastric bypass surgery, a common treatment for morbid obesity, involves division of the stomach into two parts, a small proximal pouch and a larger bypass chamber. Postoperative comparison of pH, microbial counts, and mucosal cytokine levels between these two gastric pouches revealed a neutral pH in the proximal gastric capsule (pH 7.0 ± 0.2), with a significant increase in the number of aerobic and anaerobic bacteria, whereas the pH of the anastomotic capsule decreased to 3.3 ± 2.2, with a clear decrease in the total number of bacteria (Faintuch et al., 2007; Ishida et al., 2007). Although no differences in microbiota composition were detected between the two pouches, this is likely due to the limited number of isolates obtained. Residual gastric cancer frequently occurs after distal gastrectomy of benign lesions, with an incidence rate of 1–7% among all gastric cancers (Lagergren et al., 2012). EBV infection has been associated with residual gastric cancer, and atrophic changes in residual gastritis often accompany EBV-positive residual gastric cancer. Moreover, Billroth-II anastomosis (Nishikawa et al., 2002; Kaneda et al., 2012) can significantly alter the gastric microbiota by reducing levels of nitrate and nitrite reductase and expression of nitrosation-related genes, which greatly increases risk of gastric cancer. The diversity of the microbiota in the stomach also becomes richer after subtotal gastrectomy (Clyne and May, 2019). Preoperatively, Ralstonia and Helicobacter were the two dominant genera identified in gastric cancer, and Streptococcus and Prevotella were the two most abundant genera in the gastric mucosal microbiome after gastric lesion resection. Overall, these findings suggest that gastric surgery is a key factor in determining the composition of the gastric microbiota.

The gastric environment is characterized by an acidic and hypoxic setting, accompanied by robust gastric acid secretion and peristaltic motility. The composition and abundance of the gastric microbiota are influenced by various factors, including dietary patterns, lifestyle habits, and medication usage. These factors may impede the survival and colonization of transplanted microbial communities, thereby increasing the challenges associated with establishing a stable microbial population within the stomach. Moreover, unlike the intestinal microbiota, the gastric microbiota exhibits lower diversity and abundance, making it challenging to precisely regulate and restructure the microbial community. Introducing the donor microbiota into the recipient’s gastric environment necessitates ensuring the survival of microorganisms under adverse conditions such as gastric acid, while adapting to the ecological milieu within the stomach. The viability and colonization capacity of the gastric microbiota pose significant challenges.

The transplantation techniques associated with the gastric microbial community are comparatively intricate, requiring a high level of technical expertise and precise execution. In contrast, fecal transplantation can be prepared through simple procedures such as centrifugation and filtration, with relatively accessible sample sources. However, the acquisition of gastric microbiota necessitates invasive procedures such as endoscopic biopsy or other specialized collection methods, which are intrusive operations involving patient fasting and sedation or anesthesia. During the process of preparing gastric samples, stringent quality control is imperative to ensure the purity, activity, and stability of the microbiota, thereby rendering the attainment of microbiota that meets the required standards a challenging endeavor. The transplantation modality may encompass the introduction of the donor microbiota into the recipient’s stomach through means like oral capsules, nasogastric tubes, or gastroscopy. Different transplantation methods may influence the survival rate and colonization efficacy of microorganisms. Additionally, determining the appropriate dosage of microbiota for transplantation presents a complex issue that necessitates consideration of the gastric capacity for accommodation and the survival rate of the microbiota.

The research on GMT remains in its nascent stages, lacking standardized operational procedures and therapeutic protocols. Optimal practices concerning the sourcing and quality control of transplant material, transplantation dosage, frequency, and routes are yet to be definitively established. Currently, there is a dearth of standardized methods for preserving microbial samples to ensure their viability and efficacy during transplantation. The preservation and freezing processes of microorganisms require precise control of temperature, oxygen levels, and other environmental factors to prevent microbial inactivation or damage. Furthermore, the lack of standardization has led to variations in transplantation methods among different medical institutions and practitioners, compromising the comparability and reproducibility of outcomes.

Due to the transplantation of diverse microorganisms involved in GMT, potential safety concerns such as infection, allergic reactions, and rejection responses may arise. Additionally, GMT may give rise to adverse reactions and side effects. These reactions can include allergic responses, gastrointestinal discomfort, and digestive disturbances. Further research is needed to evaluate and understand the specific occurrence rates and severity of adverse reactions and side effects. Currently, limited knowledge exists regarding the long-term safety of GMT. Long-term follow-up and monitoring studies are crucial for assessing the long-term safety of GMT, enabling the identification and resolution of any potential long-term safety issues.

GMT is still in its preliminary research stage, and ensuring that the experimental process aligns with ethical principles and safeguards the rights and safety of participants is a crucial challenge. Additionally, acquiring transplant material involves obtaining gastric microbiota samples from donors. When selecting donors, careful consideration must be given to their health status, screening for infectious diseases, and ethical considerations. GMT may potentially benefit the health of recipients, but it also entails inherent risks and uncertainties. Ethical considerations require a balance between the benefits and risks of transplantation, ensuring that the process is based on scientific data and clinical practice.

Gastric microbiota transplantation technology is still in the research stage, and the cost of treatment is high. More economic investment and research are needed to reduce the cost of treatment and increase the penetration rate.

Currently, there is a lack of standardized evaluation criteria and methods to assess the therapeutic efficacy of GMT. Microbiome analysis can provide information about the composition of microbial communities, but further research is needed to determine how to interpret this data and relate it to treatment outcomes. Additionally, quantifying improvements in patient symptoms, gastric mucosal histopathological changes, and other relevant indicators poses a challenge. There are significant variations in the composition and functionality of microbiota among individuals. Individual factors, including genotype, lifestyle, and dietary habits, contribute to potential variations in the therapeutic effects of GMT among different patients. Therefore, predicting the long-term effects of GMT and the duration of microbial community stability post-transplantation remain challenging.

Recently, only one study on GMT has been reported by a Korean researcher. This study entailed transplantation of the gastric microbiota from individuals with diverse gastric states into germ-free (GF) mice, whereby it was discovered that the gastric microbiota from individuals with intestinal chemosis or gastric cancer (GC) selectively colonized the mouse stomach and induced precancerous lesions (Kwon et al., 2022). Conversely, if healthy human gastric microbiota is transplanted into the stomachs of mice with distinct gastric diseases, what alterations would be made to the disease status of the mice? This is an issue that requires investigation. To understand this issue, we are conducting a clinical study (ChiCTR2200066339) on the effect of GMT for eradicating H. pylori infection. Probiotics and FMT/GMT represent current areas of focus in the field of microecological therapy, with these microbial transplantation techniques projected to serve as effective therapies for gastric cancer and other diseases in the future. To enhance the success of probiotic or FMT/GMT therapy, it is vital for researchers to gain a comprehensive understanding of the systemic impact of the microbiota on the immune system, as well as the relationship of the microbiota with cancer treatment.