2.1. H. pylori and GC

H. pylori is a recognized carcinogen closely related to the development of GC, and it is a microaerobic Gram-negative bacterium that parasitizes the epithelium of the human gastric mucosa [10]. H. pylori is not a native inhabitant of the stomach but is an exogenous microflora infected via the oral–oral or fecal–oral pathway [11]. Continuous exposure of the gastric mucosa to H. pylori infection can trigger an inflammatory cascade, leading to AG and significantly increasing the risk of GC. H. pylori infects approximately half of the world’s population, with a prevalence rate of 80% in developing countries [12]. The carcinogenicity of H. pylori is influenced by the virulence of the strain, the bacterial load, genetic predisposition, lifestyle habits of the host, and other microbiota factors. Early anti-H. pylori treatment and eradication of H. pylori can reduce the incidence of GC, which is more obvious in individuals without precancerous lesions of GC [13,14]. It is not clear that the eradication of H. pylori prevents GC by blocking a stage in the course of the infection. A consensus of experts in some countries recommends that H. pylori should be eradicated preferably before the chronic AG stage to prevent GC [15,16,17]. H. pylori prefers colonizing healthy gastric mucosa, and its abundance is notably reduced in tumor tissues compared to adjacent non-tumor tissues [18,19]. Yang et al. discovered a significant increase in H. pylori within the peritumoral microhabitat of patients with favorable prognoses [20]. As GC advances from conditions like gastritis or gastric ulcers, the presence of H. pylori colonization tends to decrease [21], eventually disappearing as GC progresses [22].

In the process of GC formation, gastric acid secretion gradually decreases [23], and H. pylori is often lost, leading to a relatively mild acid environment in the intestines of GC patients and the formation of a unique intestinal microbiota, which is favorable for the colonization of pathogenic bacteria [24]. On the other hand, treatment for H. pylori eradication could potentially result in heightened bacterial diversity [25]. Nakano et al.’s research found that compared with non-gastric cancer patients, the average abundance of unclassified Oxalobacteraceae, Capnocytophaga, and Haemophilus increased at the genus level in patients with GC treated with H. pylori eradication [26].

Significant differences in gastric microbiota exist between patients infected with H. pylori and those not infected with H. pylori, suggesting that H. pylori may play a role in other microbial dysbiosis [27,28,29,30]. Metagenomic data reveal that as the abundance of H. pylori decreases, the occurrence of other microbes increases [21]. In late-stage gastric cancer patients, a reverse expression relationship is observed between H. pylori and various bacteria, including spirochetes, Neisseria, Prevotella, Veillonella, and Rothia [30]. However, several other studies comparing the taxonomic composition of gut microbiota between H. pylori-positive and H. pylori-negative individuals did not find significant differences [9,31]. Ai et al. analyzed RNA-Seq data from 727 gastric cancer samples and found that H. pylori and Lysobacter were markedly more prevalent in normal tissues, whereas Pseudomonas was notably more prevalent in tumor tissues [32]. To date, conclusive evidence is lacking on whether H. pylori acts as a bacterial driver and interacts with other gastric bacteria. Future research should investigate the precise role of H. pylori within the gastric microbiota and its complex relationships with other microbes.

2.2. Bacteria Other than H. pylori and GC

With the application of second-generation sequencing technology in microbiology, besides H. pylori, many studies have also discovered other acid-resistant bacteria. Kim et al. utilized 16S ribosomal RNA gene analysis to study H. pylori-negative GC tissue and found an increase in the relative abundance of the phyla Actinobacteria, Bacteroidetes, and Firmicutes [33]. Maldonado-Contreras et al. and Kaisa et al. both reported higher levels of Actinobacteria in H. pylori-negative GC [31,34]. Park et al. discovered that gastric fluid samples from GC patients exhibited an increase in Lactobacillus and Veillonella while showing a decrease in Verrucomicrobia and Deferribacteres [35]. Akkermansia, a member of the phylum Verrucomicrobia, has been reported to be linked with GC progression [36]. Png et al. observed that the development of GC was characterized by an elevation in Proteobacteria and a reduction in Bacteroidetes [37]. Dai et al. demonstrated through ultra-high-performance liquid chromatography–mass spectrometry that the levels of Lactobacillus, Streptococcus, Bacteroides, and Prevotella were higher in tumor tissues compared to non-tumor tissues [38]. Pimentel-Nunes et al. demonstrated that GC tissues exhibited enrichment in Firmicutes, Gemella, and Streptococcus, alongside a reduction in Proteobacteria [39]. Zhou et al. found significant enrichments of Streptococcus anginosus (Sa) and Streptococcus constellatus (Sc) in the tumor tissues and feces of patients with intraepithelial neoplasia, as well as early and advanced GC [40]. Additionally, several studies consistently found an enrichment of Lactobacillu [41,42], Streptococcus [28,43], and Bacteroidetes [43,44,45] in GC. Furthermore, Fusobacterium, closely associated with CRC development, has recently been reported to be abnormally enriched in gastric adenocarcinoma [19,28,44,46].

Regarding changes in bacterial alpha diversity in GC, the observed results are not entirely consistent. Francisco et al. found a decreasing trend in bacterial diversity from non-atrophic gastritis (NAG) to intestinal metaplasia (IM) and then to intestinal-type gastric cancer [41,47,48,49,50,51]. However, there are some contradictory reports on this subject [46,50,52]. Olabisi et al. analyzed nearly 200 gastric mucosal samples and found no significant difference in microbial diversity but observed differences in microbial composition [46]. These conflicting results may stem from factors including ethnicity, diet, sequencing techniques, the diverse composition of study populations encompassing both H. pylori-positive and H. pylori-negative individuals, and the potential impact of previous GC surgery. However, gastric surgery can reduce the diversity of the gut microbiota in GC [53,54]. The composition of intestinal flora is also different in different stages of GC. Chen et al. found that Collinsella, Blautia, Anaerostipes, Dorea, and Lachnospiraceae expressed differently between early and advanced GC patients [55]. Additionally, the analysis of microbial communities in different parts of the stomach may also be a potential reason for heterogeneous results [56]. Yang et al.’s research indicates that there are differences in microbial composition and metabolic products between proximal and distal GC, even though there is no significant difference in species diversity and abundance [56]. Therefore, specific changes between different gastric microenvironments may help reveal the true relationship between gastric bacteria and the development of GC.

2.3. Epstein–Barr Virus and GC

Epstein–Barr virus (EBV) is a gamma herpesvirus, and some GCs are closely associated with EBV infection [1]. Studies have reported that patients with EBV-associated GC (EBVaGC) account for approximately 8.7–10% of GC patients [57]. EBV latent membrane protein 2A (LMP2A) is a transmembrane protein expressed by EBV and involved in EBVaGC. LMP2A can contribute to the phosphorylation of signal transducer and activator of transcription 3 (STAT3), activate the transcription of DNA methyltransferase 1 (DNMT1), induce extensive methylation of host genes, and affect the cell cycle and the microenvironment of GC [58]. EBVaGC has unique clinicopathological and molecular features, such as DNA hypermethylation, mutations in the PIK3CA gene, and high expression of programmed death receptor ligand 1 (PD-L1) [1]. Clinically, EBVaGC tends to be located in the proximal region, with a higher incidence in males than females [59], exhibits a relatively low rate of lymph node metastasis [60], and has a better prognosis [61]. Currently, the immunotherapy regimen for EBVaGC is similar to the other three GC subtypes. EBV infection is considered a potential biomarker for the immune therapeutic response, and the efficacy of ICB in EBVaGC patients is superior to that in unselected GC patients [62]. Nevertheless, EBVaGC patients still require precision treatment strategies in the future.

In addition, human immunodeficiency virus type 1 (HIV) is a retrovirus that predominantly infects human CD4+ T cells via the digestive tract. Recent studies have shown that HIV exposure is associated with a high risk of gastric cardia cancer [63]. However, the mechanism is unclear. HIV-infected individuals were more susceptible to active EBV infection [64]. Recently, Wahl et al. found that gut flora residents could increase the odds of HIV and EBV infection, as well as increase the density of HIV-infected CCR5+ CD4+ T cells [65]. These studies suggest that interactions between intestinal viruses, bacteria, and host immune cells may collectively influence the onset and progression of GC.

4.1. Chronic Inflammation

Chronic inflammation has been associated with the development of several malignant diseases, with a particularly strong correlation between H. pylori and GC. Several pathogenic mechanisms of H. pylori include altered host gene expression, infection-induced cell proliferation, elongation and loss of polarity of epithelial cells, degradation of cell–cell junctions, and decreased gastric acid secretion [76]. Although multiple “hits” are required for cancer induction, one of the most critical mechanisms may be the induction of chronic inflammation by H. pylori [75]. H. pylori infection is the classic initiator of chronic non-AG, AG, intestinal epithelial hyperplasia, dysplasia, and ultimately GC [75]. H. pylori infection induces inflammation through a variety of pathways, starting with the initially infected gastric epithelial cells and recruiting circulating immune cells to the site of infection. In patients with H. pylori infection, inflammatory factors, including IL-1, IL-6, IL-8, TNF-α, and NFκB, are significantly elevated [77,78]. Engevik et al. discovered that F. nucleatum secretes outer membrane vesicles (OMVs) to trigger TLR4 and NF-κB activation in colonic epithelial cells, leading to the initiation of downstream proinflammatory factors [79]. Notably, proinflammatory effects were absent in the context of an intact gut microbiota, suggesting the importance of the normal microecology [80].

4.2. Bacterial Genotoxins

Bacterial genotoxins are effectors that cause DNA damage by introducing single- and double-strand DNA breaks in the host cells [81]. Such DNA damage in somatic cells may cause genome damage, cell cycle arrest, and apoptosis; modulate the immune response; and launch carcinogenesis in the host [81]. For example, three genotoxins identified in Escherichia coli in CRC are involved in carcinogenesis, including cytolethal distending toxin (CDT), colibactin, and UshA [82]. It has been shown that H. pylori infection can also mediate genomic instability and altered cell polarity with carcinogenesis through the action of a range of bacterial virulence factors including, but not limited to, urease, vacuolating cytotoxin A (VacA), cag pathogenicity islands, cytotoxin-associated gene A (CagA), peptidoglycan outer membrane proteins (e.g., BabA, SabA, and OipA), and γ-glutamyl transpeptidase (GGT) [83]. H. Pylori strains with different virulence factor differences may end up with different clinical outcomes for H. pylori. The EBV-associated genes LMP1, LMP2A, LMP2B, EBNA3C, and EBNA1 also contribute to altered cellular asymmetry [83]. Testing for microbial genotoxicity facilitates the identification of stronger risk factors for gastric disease.

4.3. Short-Chain Fatty Acid (SCFA) Alteration

SCFA are monocarboxylic acids with a chain length of C2 to C6, which are mainly produced by the fermentation of fiber components in food by intestinal flora, of which more than 95% are composed of acetic, propionic, and butyric acids [84]. SCFA are commonly used as energy substrates for colonocytes, stimulate mucus production, promote mucosal immunity and barrier function, and work together to maintain intestinal homeostasis [85]. Small amounts of SCFAs from the gut enter the systemic circulation and act as important signaling molecules [86]. Bacteroides provide most of the acetate and propionate, and Firmicutes is considered the main producer of butyrate [87]. Studies have shown that butyric acid and propionic acid induce apoptosis and necrosis to varying degrees in GC cells and cause cells to stagnate in the G2-M phase, which may have an inhibitory effect on GC [88]. Plasma levels of propionate and butyrate were significantly lower in GC patients compared to chronic superficial gastritis [89]. The presence of SCFA in the host’s digestive system may positively influence cancer treatment and slow down the cancerous process [86].

4.4. Increased Bile Acids

Bile acids and bile reflux are strongly associated with the development of gastric precancerous lesions and GC [90]. There are two major sites of bile acid biosynthesis: hepatocytes within the host and microorganisms in the gastrointestinal tract [91]. Host hepatocytes synthesize primary bile acids from cholesterol, and once these host-derived primary bile acids enter the gastrointestinal tract, the intestinal microbiota chemically modify them into secondary bile acids. These bile salt hydrolase-rich flora are included in the Gram-positive Lactobacillus, Enterococcus, Clostridium spp., and Gram-negative Bacteroides spp. bacteria and are also present in several bacterial strains (e.g., L. plantarum, L. acidophilus, L. salivarius, C. perfringens, etc.) [92]. Bile acids in the gut undergo a multistep metabolism by the intestinal flora, constituting a rich diversity of bile acid profiles. Bile acid also serves as a host factor that governs the composition of the cecal microbiota [93].

Bile acids promote tumor progression and telomerase activity in mice with GC, and this effect is dependent on the expression of c-Myc [94]. In addition, bile acids induce DNA damage, mutation, and reduce apoptotic capacity. H. pylori infection and exposure to bile acids may act synergistically to enhance the occurrence of GC. A Japanese study indicated that in patients with H. pylori infection, the severity of gastric mucosal atrophy and intestinal metaplasia intensifies with increasing concentrations of bile acids, also leading to an elevated risk of GC [95]. Another animal experiment indicates that in the presence of both iron deficiency and H. pylori infection, there is an increased production of deoxycholic acid [96]. Bile acid exposure contributes to the intracellular movement of cancer-associated protein (CagA), promoting gastric carcinogenesis [96]. In gastric juice, the interaction of bile acid and microbiome promotes gastric precancerous lesions via the STAT3 pathway [90,97].

4.5. Intestinal Epithelial Barrier Dysfunction

The mucus layer serves as a protective barrier, segregating the microbiota from direct contact with the intestinal epithelial lining and preventing an inflammatory response. SCFA produced by Akkermansia muciniphila enters the intestinal epithelial cells via the G protein-coupled receptor (GPCR), increasing tight junction proteins claudin-3 and occludin expression, aiding the intestinal barrier function [98,99,100]. Bifidobacteria reduce intestinal endotoxin formation and increase the production of tight junction proteins, thereby decreasing intestinal permeability and bacterial translocation [101]. Thus, during the development of GC, decreased probiotic colonization and imbalance of intestinal flora homeostasis weaken the integrity of the intestinal epithelial barrier, leading to increased intestinal permeability. Prior to direct contact with endothelial cells, H. pylori secretes proteases and phospholipases to degrade the mucus layer on the surface of the gastric mucosa, which enhances H. pylori adhesion [102]. OMVs released by F. nucleatum can also disrupt epithelial homeostasis by compromising the intestinal mucosal barrier [103].

4.6. N-Nitroso Compounds

The GC microbiota has increased nitrate reductase and nitrite reductase function compared to chronic gastritis [51]. Ferreira et al. noted that bacteria possessing nitrate reductase activity were strongly linked to higher gastric juice pH and positively correlated with increased N-nitrosamines concentrations [104]. A possible hypothesis is that during carcinogenesis, changes in the gastric mucosa lead to a decrease in gastric acid secretion, which allows the growth of bacteria capable of reducing nitrate to nitrite [51,105]. Nitrate reduction leads to the formation of N-nitroso compounds, which in turn promote epithelial cell mutagenesis, angiogenesis, and proto-oncogene expression, ultimately contributing to the development of GC [106,107]. Escherichia coli, Enterococci, Gram-positive cocci, and Lactobacillus are among the bacteria capable of converting nitrate to nitrite [108,109,110]. Certain species present in the oral cavity could also facilitate these reactions [110].

5.1. Chemotherapy

The gastrointestinal microbiota can intricately shape the response to chemotherapeutic agents, with instances such as the antitumor effects of 5-fluorocytosine and regafur (a precursor of 5-fluorouracil) being linked to derivatives of Escherichia coli [120]. Various microbiota exhibit diverse enzyme functions that have a substantial impact on both the efficacy and potential toxicity of chemotherapy [118]. Bacterial-derived nitroreductase plays a role in augmenting the activation of CB1954, a prodrug of gemcitabine, whereas the cytidine deaminase from Aspergillus origin may facilitate the inactivation of gemcitabine [120,121]. Butyrate was associated with enhanced activation of irinotecan [122]. Certain bacterial colonies displaying enzymatic beta-glucuronidase activity can transform the inactive form of irinotecan (SN38G) into its active form (SN38) within the gut, resulting in intestinal toxicity [123].

Oxaliplatin commonly induces peripheral neurotoxicity, affecting over 30% of patients. Studies show that the efficacy of Oxaliplatin is diminished in mice subjected to antibiotic interventions or raised in a sterile environment due to the absence of gut microbiota, leading to reduced inflammatory mediators and impaired ROS production by myeloid cells [124]. Furthermore, Clostridium nucleatum contributes to resistance against oxaliplatin and 5-fluorouracil by targeting TLR4 and MYD88, thereby activating autophagy and stimulating microRNA expression [125]. However, there is a mutual interaction between microbiota and chemotherapy, as chemotherapy can also result in drug toxicity and epithelial damage by disrupting the microbial balance and inducing metabolic changes [126].

5.2. Immunotherapy

Immunotherapies, including immune checkpoint blockade (ICB) treatments, are rapidly reshaping the standard of care for advanced gastric cancer patients by offering the potential for long-term disease control in certain individuals. Multiple studies have validated the pivotal role of the gastrointestinal microbiota in influencing the effectiveness and resistance of immune checkpoint blockade (ICB) therapies [127]. Bifidobacterium has the capacity to stimulate antigen-presenting cells, thus augmenting the effectiveness of PD-1/PD-L1 blockade therapies [128]. The effectiveness of CTLA-4 blockade therapy (Lpilimumab) was associated with specific T-cell responses facilitated by Bifidobacterium thetaiotaomicron or Bifidobacterium fragilis [129]. In addition, Akkermansia muciniphila affects the efficacy of the PD-1 antibody (Nivolumab) against epithelial tumors, is dependent on IL-12, and involves the recruitment of CCR9+ CXCR3+ CD4+ T lymphocytes [130]. However, Mycobacterium methylis has been shown to decrease TGFβ expression and suppress CD8+ tissue-resident memory T cells [47]. In addition, PD-L1 expression in tumors of GC patients was significantly correlated with H. pylori infection status [131]. H. pylori exposure inhibited CD4+ T cell proliferation and induced T cell apoptosis in gastric epithelial cells, and this effect was reversed by a PD-L1 antibody (Pembrolizumab), suggesting a potential candidate for ICB therapy [131,132,133]. The microbiota’s influence on the effectiveness of ICB may also be associated with its metabolic functions [134]. For example, inosine from Bifidobacterium bifidum enhances antitumor immunity [135]. In addition, SCFA originating from intestinal bacteria affects immune cell differentiation and function [136].

6.1. Diet

Tumor growth and survival rely on host-provided nutrition. Modifying dietary intake can potentially alter nutrient availability in the tumor microenvironment, offering a promising strategy for tumor control [143]. A high-fat diet (HFD) could stimulate intestinal stem cells and promote intestinal regeneration and oncogenesis [144]. Animal studies have shown that a diet rich in proteins (casein and whey), L-leucine, fish oil, and oligosaccharides can prevent Pseudomonas aeruginosa translocation, thus mitigating CTX-induced neutropenia [145]. Dietary fiber intake, including oligoisomaltose, oligofructose (FOS), and inulin, has been found to reduce irinotecan-associated toxicity by promoting intestinal butyrate production [146]. The ketogenic diet improved tumor control and survival during radiation therapy [147]. While there is a strong link between diet and gastrointestinal microbial diversity [136], it remains to be determined whether a universally beneficial dietary regimen exists that can simultaneously improve treatment efficacy and minimize toxicity.

6.2. Antibiotics Act as Modulators of Microbiota

Antibiotics are commonly used in solid cancer animal models to simulate the loss of microbial diversity, and evidence for the importance of the microbiome comes from probiotic therapy used to reverse the effects of antibiotics. Compared to mice receiving oxaliplatin treatment without antibiotics, those administered antibiotics alongside oxaliplatin displayed compromised cancer regression, decreased overall survival, and alleviated associated side effects [148].

While antibiotic treatment theoretically has the potential to eradicate bacterial populations linked to immune-related adverse events (irAEs), several clinical studies have noted diminished responses to immunotherapy in patients undergoing antibiotic treatment [149]. This can be attributed to the broad-spectrum antibiotics’ impact on the beneficial gut bacteria required for optimal outcomes in ICB treatment. Patients who refrained from using antibiotics within 42 days prior to starting ICB therapy experienced a 3.43-fold increase in overall survival compared to those who had used antibiotics within 60 days before ICB therapy [150]. This underscores the importance of the timing of antibiotic use.

6.3. Probiotics, Prebiotics, and Synbiotics

Probiotics are defined as living microorganisms that, when ingested in sufficient quantities, can provide health benefits to the body. Xiong et al. demonstrated that administering probiotics preoperatively to gastric cancer patients can lower postoperative inflammation levels and preserve the diversity and abundance of microorganisms after surgery [151]. Prebiotics are defined as indigestible dietary fibers fermented by intestinal bacteria [151] 148 and primarily have a beneficial impact on the gastrointestinal microbiota. Symbiotics consist of a combination of probiotics and prebiotics [152]. Research indicated that the supplementation of a probiotic mixture such as BIO-three, composed of Bacillus, Butyricicoccus, and Faecalibacterium, can mitigate oxaliplatin-induced intestinal damage in GC patients [148]. Postoperative supplementation of probiotics in GC can reduce complications, promote nutritional recovery, and improve prognosis [53,153]. Clinical studies have demonstrated that supplementing certain probiotics in conjunction with antibiotic treatment for H. pylori can improve treatment outcomes [154]. Furthermore, many studies have found that common prebiotics like inulin or oligofructose can increase the abundance of Bifidobacteria, Lactobacilli, and Fecalibacterium, assisting in cancer treatment [155,156]. Dietary inulin has also been shown to exert preventive effects on cancer metastasis by promoting the production of SCFA [157]. Additionally, the use of synbiotics as supplements has been found to ameliorate post-treatment symptoms in cancer patients, including nausea, vomiting, anorexia, diarrhea, and febrile neutropenia [158,159,160].