PMMB 2023, 6, 1; a0000273. doi: 10.36877/pmmb.0000273 http://journals.hh-publisher.com/index.php/pmmb Review Article Exploring the Impact of Helicobacter pylori and Potential Gut Microbiome Modulation Isaac Jingkhai Ong1, Ke-Yan Loo1, Lydia Ngiik-Shiew Law2, Jodi Woan-Fei Law1, Loh Teng-Hern Tan1,3, Vengadesh Letchumanan1* Article History 1Novel Bacteria and Drug Discovery Research Group (NBDD), Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Subang Jaya 47500, Malaysia; iong0002@student.monash.edu (IJO); ke.loo@monash.edu (K-YL); jodi.law1@monash.edu (JW-FL) 2Monash Credentialed Pharmacy Clinical Educator, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, VIC, Australia; lydia19595@gmail.com (LN-SL) 3 Clinical School Johor Bahru, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Johor Bahru 80100, Malaysia; loh.teng.hern@monash.edu (LT-HT) *Corresponding author: Vengadesh Letchumanan, Novel Bacteria and Drug Discovery Research Group (NBDD), Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Subang Jaya 47500, Malaysia; vengadesh.letchumanan1@monash.edu (VL) Received: 08 November 2022; Received in Revised Form: 21 December 2022; Accepted: 30 December 2022; Available Online: 04 January 2023 Abstract: Helicobacter pylori is a highly prevalent bacteria that can harm humans due to its major involvement in developing gastrointestinal diseases, particularly gastric cancer. Therefore, eradicating H. pylori is one of the most important strategies for preventing gastric cancer. Antibiotic treatment has always been the gold standard treatment for H. pylori infection. However, the decreasing efficacy of antibiotic therapy due to the rising antibiotic resistance and high incidence of dysbiosis-related adverse effects resulted in eradication failure. To enhance the effectiveness of antibiotic therapy, strategies that modulate the gut microbiome were proposed to play a positive role. Generally, the integration of probiotics or symbiotic into antibiotic therapy was shown to enhance the eradication rate and reduce the incidence of adverse effects. This review aims to discuss the role and effect of H. pylori in gastric carcinogenesis and gut microbiome modulation in eradicating H. pylori infection. Keywords: Helicobacter pylori; gut microbiome; gastric cancer; carcinogenesis 1. Introduction The gut microbiome is composed of trillions of microorganisms that live within the human gastrointestinal tract (GIT) [1]. The gut microbiome has been extensively researched for its association with human health and diseases. Research has established the gut PMMB 2023, 6, 1; a0000273 2 of 19 microbiome connection in human digestion, nutrition, immune functions, and physiology. Any dysbiosis of the gut microbiome has been linked to various diseases, such as irritable bowel syndrome (IBS) [2], obesity [3], type 2 diabetes [4, 5], autoimmune diseases [6-8], cancer [9], psoriasis [10, 11], obsessive-compulsive disorder [12], and autism spectrum disorder [13]. Hence, modulating the gut microbiome has been proven to manage disease outcomes [14-18]. The GIT is very vulnerable to pathogens, such as Vibrio sp. [19-22], Salmonella sp. [23], Listeria monocytogenes [24, 25], Escherichia coli [26], Staphylococcus aureus [27], Campylobacter sp. [28], Helicobacter pylori [29-31], and many more. Upon invasion, these pathogens release their toxins and colonize the host gut, leading to a dysbiosis of the gut microbiome [32]. Of the mentioned pathogens above, Helicobacter pylori research has been reviewed extensively due to its impact on human health. Helicobacter pylori is a gram-negative, spiral-shaped, microaerophilic bacteria in the gastric mucosa. It is highly prevalent among the world population affecting over half of them [33]. Most people acquire this pathogen in early childhood and can remain asymptomatic throughout their life [34, 35]. H. pylori is among the well-known bacteria due to its role in developing gastrointestinal diseases such as gastritis, gastric ulcer, and gastric cancer [36]. The pathogen was shown to be the primary etiology for gastric cancer and was listed as a Type 1 carcinogen in 1997 by the World Health Organization [37]. Due to its association with gastric cancer, H. pylori remains a high burden to healthcare systems worldwide. According to Global Cancer Statistics 2020, gastric cancer is the sixth most common cancer, with an estimated 1089,103 cases, and the fourth leading cause of death due to cancer, with 768,793 deaths in 2020 [38]. Most gastric cancers are adenocarcinoma, which is then classified into two morphological types: intestinal type and diffuse-type gastric cancer. For intestinal-type cancer: tumour cells form adhesions, arranging in glandular or tubular formation, and a well- defined sequence has been proposed to be the carcinogenesis model, progressing from healthy gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia, and gastric cancer [39, 40]. As for diffuse-type cancer, tumour cells are more scattered and non-cohesive. Carcinogenesis is a direct consequence of chronic active inflammation without any similar defined sequence [39]. The human stomach has always been a sterile organ due to its low pH [41]. However, the significant advancement of the technology in microbiome investigation led to the discovery of a diversified microbial community consisting of multiple commensal microorganisms forming a distinct ecological niche where complex interplay is present among H. pylori and the commensal microorganism [42, 43]. A delicate balance is maintained among the microbial community in the stomach, in which any alterations in the balance may lead to dysbiosis and induction of gastric diseases, particularly gastric cancer [44]. Meanwhile, H. pylori suppress acid production and destroy the parietal cells, resulting in increased stomach pH and enabling the colonization of new microorganisms [40, 45, 46]. Hence, eradicating H. pylori is essential for preventing gastric cancer [47]. PMMB 2023, 6, 1; a0000273 3 of 19 For the past few decades, standard triple antibiotic therapy was the gold standard treatment to eradicate H. pylori. Recently, there has been a decreasing eradication rate by antimicrobial therapy due to the increasing prevalence of antibiotic resistance [48, 49]. Despite developing new antibiotic regimens, they are still associated with adverse effects, leading to poor patient compliance and eradication failure [50]. Antibiotic therapy causes perturbation of the gastric microbiome leading to dysbiosis-induced gastrointestinal side effects [51]. To reduce the incidence of adverse effects from antibiotic-induced dysbiosis, strategies and therapies that can modulate the gut microbiota are being extensively explored [52]. Hence, this review aims to discuss the role and effect of H. pylori in gastric carcinogenesis and potential gut microbiome modulation in eradicating H. pylori infection. 2. Pathogenesis of H. pylori The fact that not every individual infected with H. pylori develops gastric cancer shows that the pathogenesis of the variable clinical outcomes of H. pylori infection is multifactorial, including the virulence factors of the bacteria strain, host gene polymorphism, and environmental influences. Gene polymorphism of pro-inflammatory cytokines such as IL-1B increased gastric cancer risk through different mechanisms, including inflammatory injury, gastric acid secretion inhibition, and angiogenesis promotion [53-55]. Besides IL-1B, genetic variation in another pro-inflammatory cytokine, TNF-α, increased expression is also associated with enhancing gastric cancer risk [56]. With inflammation playing a vital role in cancer progression, polymorphism in inflammatory cytokines, which regulates the intensity of immune response, may augment gastric cancer risk [57]. Environmental factors, including dietary habits, cigarette smoking, and alcohol consumption, also affect gastric cancer risk. The dietary habits consisting of high salt content, red meat, processed meat, and low fiber enhanced gastric cancer risk [58-60]. Interestingly, former and current smokers demonstrated higher rates of gastric cancer than never-smokers, and the risk intensifies with the number of cigarettes per day for current smokers [61]. As for alcohol consumption, heavy drinkers with more than four drinks per day have significantly increased odds of developing gastric cancers compared to abstainers [62]. Besides host genetic vulnerability and environmental influences, virulence factors associated with the bacteria's pathogenicity were involved in gastric carcinogenesis. The human stomach contains gastric fluid with a pH of around 2.0. This highly acidic environment remains a huge challenge for colonizing most invading pathogens, which enables the bacteria to survive in the highly acidic environment, colonize the gastric mucosa, and evade the immune cells. H. pylori possesses urease, one of the main virulence factors that neutralize acidity and enable it to survive and colonize in such a harsh environment [63]. The urease produced by H. pylori plays an alkalizing role as it hydrolyses urea in the stomach and produces ammonia plus carbon dioxide, which neutralizes the stomach's acidity and provides a favorable environment for the survival of H. pylori [63]. Aside from acid neutralization, urease was also involved in the carcinogenesis of gastric cancer. Recently, an in vitro study uncovered that ureases can trigger a specific pathway to initiate angiogenesis in the gastric epithelial cell. This can potentially lead to the carcinogenesis of gastric cancer [64]. Additionally, urease displayed pro-inflammatory PMMB 2023, 6, 1; a0000273 4 of 19 activity in the gastric endothelial cells, inducing alterations in the oxidative profile of the cells and resulting in differentiation which may contribute to gastric carcinogenesis [63]. Two main virulence factors affect the pathogenicity of the host cell, CagA and VacA virulence protein. CagA is the first bacterial oncoprotein shown to be associated with cancer in humans [65]. It is transported into the host gastric epithelial cells by a syringe-like structure called Type 4 secretion system [66-68]. In the host cells, there is phosphorylation of CagA by tyrosine kinase protein. Then, CagA binds to signaling proteins that perturb multiple intracellular signaling pathways leading to the host cells' malignant changes [69]. The expression of CagA leads to morphological changes in the cells, specifically elongation and increased motility, known as the “hummingbird” phenotype [70]. This oncoprotein also causes disruption of the intercellular junction and the polarity of the epithelial cells [71-73]. In addition, this molecule induces anti-apoptotic activity in the gastric epithelial cells leading to decreased turnover of cells. Also, it triggers instability in the genome which both are classic traits of cancer cells [74, 75]. Recently, Choi et al noted that CagA upregulates the CDX1 expression involved in regulation of intestinal function, which may lead to gastric carcinogenesis [76]. Another virulence factor VacA is a pore-forming cytotoxin that can induce vacuolation and promote apoptosis in gastric epithelial cells [77, 78]. VacA exerts its effects on cellular activity through the mitochondrial pathway, triggering the release of cytochrome c leading to autophagy and cell death [79, 80]. A study proposed that VacA induces cell apoptosis by activating the endoplasmic reticulum stress cascade [81]. Moreover, VacA modulates the host immune reaction by preventing the recruitment of immune cells, including T cells and B cells, to ensure the longevity of the infection [82, 83]. VacA also targets macrophages, the first line of defense, by interfering with the maturation of the phagosome and blocking the activation of cytokines induced by macrophages [84, 85]. Recent studies also showed the interplay between VacA and CagA, where the disruption of autophagy by VacA promotes CagA accumulation in gastric cells resulting in the persistence of H. pylori in the gastric mucosa [86]. 3. H. pylori and gastric microbiome The dogma that the human stomach is a sterile organ has been shattered since the revelation of H. pylori. Since then, there has been a hypothesis that the human gut harbors a diversified bacterial community [87]. Bik et al. conducted a critical study that explored the composition of the gastric microbiome through a biopsy of the gastric mucosa and PCR of the samples. This study shows that the gastric microbiota of healthy individuals comprises five major phyla, including Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria, which were then supported by newer studies [88, 89]. Despite the variation of the microbiota composition among individuals, the human stomach contains a diversified microbiome [87, 90]. In the gastric microbiome consisting of a highly diversified bacterial community, H. pylori seemed to be the most dominant bacteria with the highest abundance in the gastric microbiome when present [42, 91]. Schulz et al. noticed that the abundance of H. pylori PMMB 2023, 6, 1; a0000273 5 of 19 comprises more than 50% among H. pylori-positive subjects, and the abundance of the other major species decreased [42]. A recent study in 2019 obtained a more significant result where the bacterial communities of the stomach among H. pylori-positive children were notably dominated by 95.43% of H. pylori in the genus [91]. As for H. pylori-negative individuals, studies have shown that Streptococcus was commonly the prominent genera with the highest abundance in the gastric microbiome [42, 91, 92]. With the dominance of H. pylori in the gastric microbiome, H. pylori were shown to alter the composition and species diversity. Studies showed that the gastric microbiome was significantly less diversified in H. pylori-positive patients than in H. pylori-negative patients. In a study by Anders et al., they identified 262 bacterial phylotypes compared to 33 in H. pylori-positive individuals. The result shows that for individuals with H. pylori infection, their gastric microbiota was significantly less diversified than H. pylori-negative individuals [93]. A recent study in Indonesia supported the findings that bacterial diversity decreased in the H. pylori-positive group [94]. Several other studies further emphasized that the alpha diversity of H. pylori-positive patients is significantly lower than healthy individuals [42, 89]. 3.1. H. pylori and gastric microbiome in gastric carcinogenesis According to Correa’s model of gastric carcinogenesis, persistent colonization of H. pylori triggers an inflammatory process that then initiates the carcinogenesis cascade, starting from atrophic gastritis, intestinal metaplasia, dysplasia, and gastric cancer. Long-term colonization of H. pylori leads to atrophy of the parietal cells, whose main function is the secretion of acid, leading to increased pH of the gastric fluid and atrophic gastritis [40]. As most bacteria cannot live in highly acidic conditions, H. pylori-induced achlorhydria disrupts the acid barrier, colonizing new microbes, and leading to the gastric microbiome alterations. Meanwhile, the proliferation of new microbes may further promote inflammatory reactions, leading to cancer progression [40]. Multiple studies attempted to discover the role of non-H. pylori in the carcinogenesis of gastric cancer. One hypothesis proposed that some of these microbes can reduce nitrate into nitrite, forming N-nitroso compounds that are potentially carcinogenic [95]. Ferreira et al. analyzed the functional composition of the gastric microbiome in individuals with chronic gastritis and gastric cancer. They noticed that the gastric cancer microbiome has a higher nitrate reductase function, promoting the formation of carcinogenic compounds [45]. Jo et al. reported twice as much nitrosating bacteria in individuals with gastric cancer compared to the control groups [96]. Similarly, there was an enrichment of bacteria involved in nitrate metabolisms, such as Escherichia coli, Lactobacillus, and Nitrospirae, in patients with gastric cancer [46, 97]. Another hypothesis stated that oral microbiota plays a role in gastric carcinogenesis. Coker et al. demonstrated that microbiome from the oral origin, including Slackia exigua, Peptostreptococcus stomatis, Streptococcus anginosus, Parvimonas micra and Dialister pneumosintes were significantly enriched and formed strong interactions in the gastric microbiome of gastric cancer [98]. In another study among gastric cancer patients from Malaysia and Singapore, there is an increased abundance of oral bacterial taxa such as PMMB 2023, 6, 1; a0000273 6 of 19 Leptotrichia, Fusobacterium, Haemophilus, Veillonella, and Campylobacter [99]. In a few other studies, there is reportedly a higher abundance of oral bacteria such as Lactobacillus, Streptococcus, and Neisseria in gastric cancer patients [100-102]. Sun et al. developed a scoring system to screen for patients with high suspicion of gastric cancer by detecting oral bacteria. The sensitivity was as high as 97.3%, with a 7.7% false-positive rate. However, this study consists of a small sample size of 50 subjects, and more extensive studies are required to validate the results [103]. Some studies also examined the changes in gastric microbiota diversity along Correa’s carcinogenesis cascade. Most studies reported significant differences in gastric microbiome diversity in gastric cancer compared to precancerous lesions, demonstrating gastric dysbiosis's role in gastric carcinogenesis [45, 98]. Jimenez et al. conducted a study to explore gastric microbiota changes along the carcinogenesis cascade and reported a decreased diversity in gastric cancer [104]. Wang et al. supported these findings, showing that the gastric microbiome diversity decreases along the gastric carcinogenesis cascade [98]. However, studies have proposed contradictory results stating that gastric microbiome diversity increase in gastric cancer. Rodriguez et al. and Eun et al. reported that gastric microbiome diversity increases in subjects with gastric cancer [99, 100]. Wang et al. further supported the findings in a study involving 315 patients showing that gastric microbiota diversity increases in gastric cancer [46]. The discrepancy in the results from different studies could be due to factors influencing the gastric microbiome, such as the variation of gender, ethnic group, and dietary habits among the subjects [87, 98]. 4. Antibiotic therapy in H. pylori infections Various antibiotic therapies have been evaluated to eradicate H. pylori. However, few were highly effective, with consistently high eradication rates and low incidence of adverse effects [105]. The reasons for eradication failure are mostly due to the increasing antibiotic resistance and non-compliance to treatment. According to clinical guidelines, bismuth quadruple therapy replaced standard triple therapy as the first line in areas with known high macrolide resistance. Besides that, multiple alternative drug regimens are recommended as the first-line treatment, including levofloxacin, sequential therapy, concomitant therapy and hybrid therapy, depending on antibiotic resistance and penicillin allergies [49]. Recently, Vonoprazan emerged as one of the most promising drugs for H. pylori infection [106]. Vonoprazan is a highly potent acid-inhibitory drug with rapid onset and longer duration of action than conventional proton pump inhibitors [107, 108]. Studies have reported that antibiotic therapy reduces the diversity and composition of the gut microbiome [109, 110]. In a multicentre randomized trial, Liou et al. explored the effect of standard triple therapy, bismuth quadruple therapy, and concomitant therapy on the gut microbiome. After antibiotic treatment, faecal microbiota analysis was done at 2, 8, and 10 weeks. The results demonstrated significant perturbation of gut microbiota with reduced microbiome diversity at week 2 for all three antibiotic regimens. After one year, the bacterial diversity was fully restored in standard triple therapy but not in bismuth quadruple and PMMB 2023, 6, 1; a0000273 7 of 19 concomitant therapy. There was an increased abundance of Proteobacteria and decreased Firmicutes and Bacteroidetes in concomitant therapy and quadruple therapy groups [109]. In line with the previous study, Chen et al. noticed a decreased microbial diversity, increased Proteobacteria, and decreased Firmicutes and Bacteroidetes, which persisted for more than six weeks after treatment, suggesting the antibiotic-induced dysbiosis may persist without complete restoration after a long period [110]. The abundance of butyrate-producing bacteria, Lachnospiraceae, decreased after antibiotic treatment, increasing the risk of Clostridium difficile infection [110, 111]. Few other studies supported the findings that gut microbiome diversity decreased after eradication therapy with increased Proteobacteria and decreased Firmicutes, Bacteroidetes and Actinobacteria after antibiotic therapy [112, 113]. Interestingly, there was a reduced abundance of Bifidobacterium in a few studies [114-116]. Bifidobacterium prevents colonization of commensal pathogens and assists in the metabolism of carbohydrates, suggesting that decreased Bifidobacterium may be linked to antibiotic- induced adverse effects [115, 117]. 5. Modulation of the gut microbiome Probiotics are live microorganisms that may provide health benefits if given in optimum quantity [118]. Today, probiotics have gained people's interest as an effective therapeutic option for managing digestive and immune health [119]. Probiotics have proven effective in treating various diseases, from gastrointestinal issues to recent COVID-19 infections [120-122]. Lactobacillus, Bifidobacterium, and other lactic acid-producing bacteria (LAB) are common probiotics used in dairy products and yogurt. As human microbiome research expands, a range of potential probiotics with good benefits to the host has been discovered. Among those new potential probiotic candidates is Streptomyces sp. Recent studies have revealed that Streptomyces sp. has strong antibacterial, anti-Vibrio activity and probiotic properties for aquaculture usage [123-127]. Further studies should be conducted to strengthen these findings before they can be administrated to humans. Based on the properties of different probiotics, several proposed mechanisms of action against H. pylori include immunological and non-immunological mechanisms. The first line of defense against foreign pathogens is its high acidity and intact stomach mucosa. Probiotics can synthesize antimicrobial substances, including lactic acids, short-chain fatty acids, and bacteriocins. Certain probiotic strains, such as Bifidobacterium longum and Lactobacillus casei were found to inhibit the activity urease in H. pylori, hindering its ability to colonize the stomach [128, 129]. In a study by Kim et al., mice model gastric pH decreases significantly after administering lactic acid bacteria, eliminating H. pylori [129]. Bacteriocins are proteinaceous toxins that possess anti-H. pylori activity and its antimicrobial activities vary with different probiotic strains. Bacteriocin and lactic acid secreted by Lactobacillus bulgaricus and Lactobacillus pentosus strains were shown to inhibit antibiotic sensitivity and antibiotic resistance H. pylori strains [130, 131]. Other than Lactobacilli, Lim et al. reported that the bacteriocins produced by Enterococcus faecalis BK61 exhibit anti-H. pylori activity [132]. A recent in vitro study by Sacacino et al. reported several probiotic strains, including Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Bifidobacterium PMMB 2023, 6, 1; a0000273 8 of 19 lactis and Streptococcus thermophilus strains possess both bactericidal and bacteriostatic against H. pylori [133]. Additionally, reuterin, a nonpeptide antimicrobial substance secreted by Lactobacillus reuteri was seen to exert effect through downregulation of H. pylori virulence factors, VacA and flaA [134]. Probiotics can adhere to binding sites of gastric epithelial cells and provide competition for the adhesion of H. pylori. Mukai et al. reported that L. reuteri strains have a specific affinity for two glycolipid binding sites on epithelial cells thus competing and occupying the adhesion site H. pylori, preventing the pathogen from colonizing the gastric mucosa [135]. A newer study by Holz et al. added that L.reuteri decreases the mobility of H. pylori by entangling and forming aggregate with H. pylori which will eventually be flushed out from the gut [136]. Additionally, Lactobacillus gasseri and Lactobacillus brevis strain was noted to reduce the adherence of H. pylori to the host cell by inhibiting the expression of sabA gene [137]. A probiotic yeast named Saccharomyces boulardii was noted to inhibit adherence of H. pylori to the host cell by removing α‐2,3‐linked sialic acid, the ligand for H. pylori adhesin with its neuraminidase activity [138]. Probiotics reduce the inflammation in the gastric cells through modulation of the immune reaction and inhibit the production of pro-inflammatory cytokines. Studies have shown that L. bulgaricus, L. acidophilus and L. rhamnosus inhibit the production of inflammatory cytokine, particularly IL-8, in the cells infected with H. pylori [139, 140]. Besides, Yu et al. demonstrated that a probiotic cocktail consisting of E.faecalis, L.acidophilus and B.longum plays a significant role in the downregulation of inflammatory chemokines and cytokines, including TNF-α, IL-10, IL-1β, IL-6, MIP-2 and G-CSF which eventually leading to improvement of H. pylori-induced gastritis [141]. Lactobacillus strain probiotics have been widely studied in terms of their role in enhancing the eradication rate and improving gastrointestinal symptoms. A randomized controlled trial by Poonyam et al. evaluated the effect of probiotic supplementation on antibiotic therapy by prescribing L.reuteri during bismuth-based triple therapy to eradicate H. pylori. Results showed no significant difference in eradication rates but a significantly lower incidence of side effects, including nausea, diarrhea, black stools, and abdominal discomfort [142]. Francavilla et al. assigned 100 H. pylori-positive individuals into two groups: one group received standard triple therapy plus two L.reuteri strain, and another group received the same antibiotic treatment and placebo. In contrast to the previous study, the probiotic group's eradication rate was significantly increased compared to the placebo group (75% vs 65.9%). Similarly, lower side effects were reported in the probiotic group compared to the placebo (40.9% vs 62.8%) [143]. The probiotic yogurt containing L. gasseri was seen to reverse the dysbiosis and restore the balance in the gastric microbiome [144]. Besides Lactobacillus, Bifidobacterium was also commonly used as supplementation for anti-H. pylori antibiotic therapy. In a study by Jiang et al., 232 patients were randomly prescribed bismuth quadruple therapy plus live combined Bifidobacterium probiotic or bismuth quadruple therapy without probiotic. Significantly higher eradication rates and lower PMMB 2023, 6, 1; a0000273 9 of 19 incidences of side effects were noted in the probiotic group [145]. Cekin et al. compared the eradication rate between one group receiving sequential eradication therapy with probiotics containing Bifidobacterium animalis subsp. lactis B94 and one group without probiotics. The eradication rate was significantly higher with probiotics than with the controls (86.8% vs 70.8%). The probiotic group also experienced a lower incidence of diarrhea (1.88% vs 12.26%), leading to better compliance with treatment [146]. Several studies have explored the role of yeast probiotics such as S. boulardii. Seddik et al. reported that adding S.boulardii to anti-H. pylori sequential therapy resulted in an increased eradication rate compared to sole sequential therapy (86.0% vs 76.7%). The probiotic group also reported a significantly lower incidence of antibiotic-induced diarrhea (2.0% vs 46.4%) and overall side effects (17.0% vs 55.7%). This led to a significantly higher compliance rate in the probiotic group (95.0% vs 91.2%) [147]. In 2019, He et al. administered S.boulardii during bismuth-based quadruple therapy. No significant difference was noted in the eradication rate. Still, the incidence of nausea, diarrhea, and side effects was significantly lower compared to the control group. This study also noted that supplementation of probiotics simultaneously with quadruple therapy recorded a lower incidence of adverse effects than administration on the 14th day after commencing quadruple treatment, suggesting the best timing for probiotic supplementation [148]. A recent study by Cardenas et al. revealed that S. boulardii supplementation increased bacterial diversity in the gastric microbiome [149]. Additionally, Clostridium was also considered a potential candidate for probiotic supplementation. Mukai et al. demonstrated that anti-H. pylori therapy regimen consisting of probiotic C. butyricum and standard triple therapy recorded a higher eradication rate of 87.1% compared to 70.1% in standard triple therapy without probiotics [150]. Chen et al. also noted that C.butyricum supplementation improves gastrointestinal symptoms by restoring gut microbiota composition [139]. Furthermore, there are also studies exploring the role of other probiotics. Tang et al. prescribed bismuth quadruple therapy to 162 patients, with one group receiving probiotics consisting of Enterococcus faecium plus Bacillus subtilis and another group receiving a placebo. The result was consistent with the previous study, as the probiotic group's eradication rates were higher at 87.01% compared to 82.43% in the placebo group [151]. Furthermore, beneficial bacteria were enriched with probiotic supplementation, including Oscillospira and Lactobacillales, known for producing short fatty acid chain butyrate, regulating immune reactions, and alleviating gastrointestinal symptoms [152, 153]. Inflammation-linked harmful bacteria, such as Collinsella, Dialistera and Sutterella were also reduced in the probiotic group [154]. Jung et al. compared the eradication rate and side effects of one group receiving concomitant therapy and another group receiving standard triple therapy plus probiotics containing either freeze-dried L. casei rhamnosus or B.subtilis combined with E. faecium. In contrast to previous studies, there were no significant differences in the eradication rates between groups, but the probiotic group reported a lower incidence of adverse effects [155]. Aside from probiotics, prebiotics and synbiotics also play a role in gut modulation. Prebiotics are compounds that are non-digestible but can be metabolized by gut microbiota PMMB 2023, 6, 1; a0000273 10 of 19 to allow benefits for the host [156]. In contrast to probiotics, limited studies were done to investigate the role of prebiotics on anti-H. pylori therapy. Instead, more studies researched synbiotics, a combination of probiotics and prebiotics. A study by Sirvan et al. explored the impact of Bifidobacterium lactis based synbiotics on standard triple therapy. Results showed a higher eradication rate in the triple therapy plus synbiotics group compared to triple therapy alone (88% vs. 72%). In terms of the adverse effects, the synbiotics group experienced a significantly lower incidence of adverse effects, including nausea, vomiting, diarrhea, and abdominal pain [157]. Contrary to this study, two other trials used the same B.lactis strain and inulin during standard triple therapy. There was no significant difference in the eradication rate compared to the control group. Ustundag et al. reported no significant difference in the side effects in both groups. In contrast, a study by Islek et al. reported significantly lesser side effects in the symbiotic group than in the triple therapy group (63% vs. 17%) [158, 159]. Fecal microbiota transplant (FMT), which is the transplantation of fecal material from a healthy donor into the patient’s intestines, has been proven to restore the gut microbiota and treat gastrointestinal disorders, including irritable bowel syndrome, inflammatory bowel disease, and Clostridium difficile infection [160]. Additionally, FMT was shown to be more cost-effective than antimicrobial therapy [33]. Therefore, researchers hypothesized that fecal microbiota transplantation could play a role in eradicating H. pylori through the restoration of gut microbiota. To verify this hypothesis, Ye et al. conducted a study in 2020 where they administered washed microbiota transplant (WMT) to 32 patients. WMT was performed in this study due to its lesser adverse effects and higher efficacy as compared to conventional FMT [161, 162]. 32 patients diagnosed with H. pylori infection for the past year and not on eradication therapy were administered WMT once a day for three consecutive days. The result showed an eradication rate of 40.6% four weeks post-WMT [162]. This positive result warrants future randomized controlled trials with a larger sample size to determine the optimal dosage and frequency of WMT and to confirm the safety and efficacy of WMT. 6. Conclusion Antibiotic therapy has been the gold standard for the eradication of H. pylori. Nevertheless, antibiotic therapies have limitations, including increasing antibiotic resistance and antibiotic-induced dysbiosis, leading to a high incidence of adverse effects and poor patient compliance. The few factors combined lead to decreased efficacy of antibiotic therapy and reduced eradication of H. pylori, contributing to a higher prevalence of gastrointestinal diseases, particularly gastric cancer. This review shows that integrating probiotics into antibiotic therapy enhances antibiotics' efficacy and restores the gastric microbiome's homeostasis. Probiotics extensively studied and shown to be effective include Lactobacillus, Bifidobacterium, Saccharomyces, and Clostridium strains. However, there is insufficient evidence to determine the best probiotic strain for eradicating H. pylori. Therefore, large sample trials should be conducted to determine the selection, optimum dosage, and duration of antibiotics and probiotics to improve the efficacy of antibiotic therapy. Besides probiotics, there is also an increased eradication rate and reduced adverse effects with symbiotic supplementation. However, relatively fewer trials were done on symbiotic, prompting more PMMB 2023, 6, 1; a0000273 11 of 19 studies to investigate the efficacy of symbiotic supplementation and the possible combination of probiotics and prebiotics for the best effect. Lastly, fecal microbiota transplant is potentially a highly cost-effective therapy for H. pylori. Further studies should be conducted to evaluate its safety, effectiveness, and synergistic effect when combined with the current eradication regimen. Author Contributions: The literature search, data extraction, and manuscript writing were performed by IJO. The manuscript was formatted and edited by K-YL. This review was critically reviewed, proofread, and edited by LS-NL, JW-FL, LT-HT and VL. VL conceptualized this review writing project. Funding: No external funding was provided for this research. Conflicts of Interest: The authors declare no conflict of interest. References 1. Lau AWY, Tan LT-H, Ab Mutalib N-S, et al. 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