1 Introduction

Helicobacter pylori is a Gram-negative, microaerophilic, rod or spiral in shape, typically found to colonize the mammalian stomach. Warren and Marshall described the causal role of this bacterium in 1979 in various human gastrointestinal diseases, including gastritis, peptic ulcers and gastric cancer [1, 2]. The occurrence of gastric cancer is notably high in East Asia, especially in Japan and Korea, as well as in East Europe, and several Latin American countries. Gastric cancer is the 5th most common cause of cancer in the world [3, 4], constituting ~ 80% of mortality among new cases in 2018 [5]. There are about 65–80% of non-cardia gastric cancers attributable to H. pylori infection [6]. World Health Organization listed H. pylori as Group 1 carcinogen in 1994 [7].

Transmission of H. pylori is primarily through close food contact among people; in addition, poor hygiene and consumption of contaminated food or water also contribute to the bacterial spread. As of 2022, one-third of the global population continues to suffer from H. pylori infection [8], with a higher prevalence in develo** countries [9]. A meta-analysis, compiling 14,006 reports that were published from 1970 to 2016 spanning 62 different countries, indicated that Africa exhibited the highest rate of H. pylori infection, with a prevalence of 70%, followed by South America and Western Asia with prevalence of 69% and 67%, respectively [10]. Furthermore, Vilaichone et al. estimated that 2 out of 10 people in Southeast Asian countries, particularly Malaysia, Myanmar, Vietnam and Thailand, are H. pylori-positive [11]. Shockingly, in the regions comprising the Golden Triangle, the prevalence of H. pylori infection exceeds 60%.

For a successful infection, H. pylori needs to adhere to, replicate, and persist in the human stomach. H. pylori recruits its adhesins, that are present on the bacterial surface, to facilitate the attachment of bacteria to the gastric epithelial cells. Upon invading mucosal surfaces, the host immune system is activated, leading to the secretion of virulence factors, such as vacuolating cytotoxin A (VacA) [12, 13] and cytotoxin-associated gene A (CagA) [12, 14], neutrophil-activating protein (NAP) [15]. In addition to established virulence factors, duodenal ulcer promoting gene a (dupA) [16, 17] has emerged as a biomarker for duodenal ulcers, owing to its high association with duodenal ulcer development. To our surprise, the infection with dupA-positive H. pylori resulted in minimal alterations to the gastric microbiome [18], but the infections with dupA-negative H. pylori were shown a higher tendency to trigger precancerous gastric ulcers [19]. Cholesteryl glucoside (CG) [20] and its derivatives, such as cholesteryl 6’-O-acyl -α-D-glucopyranosides (CAGs) [14], were recently discovered to possess immunomodulatory properties [21, 22] and contribute to H. pylori immune escape [20, 23]. Additionally, release of urease by the bacterium neutralizes the stomach’s acidic environment, and facilitates bacterial survival and adaptation to the harsh milieu [24].

H. pylori diagnosis employs five different principles: microscopy (detection of Helicobacter-like organisms), urease activity (urea breath test), DNA detection (polymerase chain reaction), and H. pylori-specific IgG antibody detections (two methods, including enzyme-linked immunosorbent assay and western immunoblotting). The invasive techniques usually require endoscopy [25] and subsequent histological examination [26, 27] (including urease test [28, 29]), while the non-invasive methods comprise serology [30], the urea breath test [26, 31], and detection of H. pylori antigen in stool specimens [32].

H. pylori is recognized for its high production of urease, leading to the formation of ammonia through enzymatic reaction. Marshall and his colleagues have innovatively developed the biopsy urease test [33] and the urea breath test [31], providing advantages in diagnosing and treating H. pylori. In addition, the infection history can be readily traced using the aforementioned methods. However, factors like bacterial density, bacterial shapes (spiral or coccoid), and the presence of antibiotics could influence and reduce the sensitivity of the rapid urease test, potentially leading to false-negative results. The serology and stool antigen tests are always used for initial diagnosis of the H. pylori infection. Both tests are simple and safe methods for detecting H. pylori. Serology test is to detect the presence of anti-H. pylori IgG antibodies in blood samples, by using enzyme-linked immunosorbent assay (ELISA) and western immunoblot (WB). Stool antigen test functions as an enzymatic immunoassay to detect the presence of H. pylori antigen. However, it is important to note that serology test has the limitation in their ability to distinguish between past and current H. pylori infections. Currently, non-biopsy-based testing is set to improve the current clinical approach and management of H. pylori-associated diseases. Although endoscopy continues to be a cost-effective means for directly monitoring gastric cancer, it is not advisable to be used for confirming the H. pylori eradication.

Since H. pylori adhesion represents the initial step in the pathogenesis, this review aims to provide in-depth review to demonstrate how H. pylori leverages various adhesion approaches at molecular basis. In addition, we also review various therapeutic treatments and discuss the promise of anti-adhesion strategies.

2 Current therapeutic treatments for H. pylori eradication

Early treatment for H. pylori eradication can lower the likelihood of H. pylori infection-associated complications, including peptic ulcers, gastritis and gastric cancer. At the current stage, empirical treatment regimens utilize combinations of two antibiotics as the primary therapy. This strategy aims to enhance antibiotic effects. An initial higher dosage of antibiotics is typically prescribed to maximize drug efficacy and achieve complete bacterial eradication. However, antibiotic doses are tapered over the time, ultimately compromising the effectiveness of antibiotic treatments. Research indicated that during the tapering of antibiotic doses, not only can the antibiotics exert selective pressure on bacterial population [34], but the sublethal concentrations of antibiotics can also stimulate the development and selection of genetic mutations that confer resistance to the antibiotic [35]. Already, strong metronidazole resistance has been identified in Malaysian isolates, 75% in average [36], and up to 30% of strains in Vietnam exhibit clarithromycin resistance [11]. Additionally, an extensive meta-analysis across 65 countries indicated an alarming level of resistance to metronidazole, levofloxacin, and clarithromycin [37].

In scenarios where both single and dual antibiotic treatments demonstrated ineffectiveness, Graham and Fischbach recommended further evaluating the therapies that include furazolidone or nitazoxanide [38]. Furthermore, hybrid regimens combining sequential or concomitant therapies with proton pump inhibitor (PPI)-amoxicillin-containing dual therapy were also subjected to assessment. In general, the effectiveness of antibiotics is correlated with the bacterial state, indicating that antibiotic-mediated eradication is more likely to succeed when the bacterial cells are actively dividing. Clarithromycin and amoxicillin impede the biosynthesis of bacterial proteins and cell walls, respectively, so that they primarily target actively dividing bacteria [39, 40]. Including a PPI in the regimen can prevent the gastric environment from acidifying (i.e. decreasing the pH), which is in favor of bacterial growth [41]. PPIs therefore reduce the minimum inhibitory concentration of the administered antibiotic(s). H. pylori can exist in a non-dividing state when staying in acidic gastric mucin, making it resilient to antibiotics designated to target actively growing bacteria [42, 43]. Meanwhile, deployment of bismuth in quadruple therapies is effective in increasing bacterial susceptibility to antibiotics, thereby enhancing bacterial clearance. Bismuth not only exerts a bactericidal effect on H. pylori, but also protects the damaged tissue and stimulates the secretion of gastric mucus.

Although including a PPI in the regimen contributes to the reduction of metronidazole resistance [44, 45], massive bismuth use in medical treatments resulted in an unexpected epidemic of bismuth neurotoxicity in France [46, 47], prompting public anxiety. Consequently, many countries have announced plans to remove bismuth from their formularies, including colloidal bismuth, so bismuth-containing compounds are not universally available for pharmacopeias. From a medical point of view, Reynolds et al. claimed that the bismuth case serves as a stark reminder to physicians about a serious consequence associated with a commonly used and easily accessible medication [48]. Unfortunately, the anticipated high eradication rate was not attained, primarily due to the emergence of clarithromycin-resistant H. pylori strains. Clarithromycin resistance in H. pylori is predominantly attributed to point mutations in the domain of the 23S rRNA gene notably the A2143G and A2142G subtypes, prevalent in 81% of resistant strains. Moreover, poor adherence to quadruple therapy also poses a significant challenge.

The conventional triple therapy and bismuth quadruple therapy are no longer recommended due to their unacceptably low success rates, failing to achieve an 80% eradication rate as reported by Graham and Fischbach [38] and Gao et al. [49]. An alternative empirical therapy option involves the use of vonoprazan, a novel potassium-competitive acid blocker. A 7-day triple regimen, which includes vonoprazan, amoxicillin, and metronidazole, has recently been endorsed as a primary treatment for eradicating clarithromycin-resistant H. pylori infections [50,51,52]. Vonoprazan-based treatments have shown higher efficacy (~ 3%) compared to PPI-based therapies [52]. Unlike PPIs, vonoprazan has ability to accumulate within the acidic environment of gastric parietal cells, which is attributed to its relatively high pKa value and stability in acidic conditions [53]. Furthermore, vonoprazan is not influenced by CYP2C19 (cytochrome P450 2C19, a drug-metabolizing enzyme) variants that were shown to affect the plasma concentration of PPIs and the subsequent efficacy of antibiotics [54, 55]. However, concerning vonoprazan-based treatments, most studies (12 out of 13) lack information on metronidazole resistance, and a majority of studies (11 out of 13) are retrospective in nature [52].

There are primary factors contributing to the ineffectiveness of H. pylori therapy: inadequate patient adherence, fast metabolism of PPIs, and antimicrobial resistance. The swift increase in worldwide resistance to clarithromycin, levofloxacin, and metronidazole has significantly reduced cure rates, making these antibiotics inappropriate for empirical therapy. It is undeniable that treatment regimens should be tailored according to local antibiotic resistance patterns. Pichon and coworkers have thoroughly assessed contemporary molecular-based antibiotic susceptibility techniques, particularly regarding clarithromycin and levofloxacin resistance [56, 57]. Transitioning from empirical to susceptibility-guided treatment for H. pylori infection signifies a significant stride, substantiated by numerous H. pylori consensus reports. Meta-analyses have consistently demonstrated the superior efficacy of susceptibility-guided therapy over empirical therapy. The effectiveness of culture-based susceptibility testing-guided therapies has been well-documented in achieving H. pylori eradication. Lee et al. assessed the effectiveness of culture-based, susceptibility‐guided therapy as the first-line eradication approach in areas with elevated antimicrobial resistance [58]. The 10-day sequential culture-based tailored therapy demonstrated notably higher eradication rates in both the intention-to-treat analysis [ITT; 93% (69/74 patients)] and the per-protocol analysis [PP; 100% (69/69 patients)] [58]. Moreover, Furuta’s research team conducted the study involving 300 H. pylori-positive patients, randomly allocated to standard or tailored sensitivity/genetic-based testing groups. Their results showed a significantly higher cure rate (P ≤ 0.001) with the tailored regimen (96.0%, 95% CI 92–98%, 144/150), as compared to the approved standard regimen in Japan (70%, 95% CI 62–77%, 105/150) [59]. In contrast, Liou et al. found no significant difference in their randomized controlled trials. Genotypic resistance-guided therapy (78%, 160/205) showed only a marginal improvement over empirical therapy (72%, 148/205) for patients with multiple failed eradication attempts (P = 0.170) [60]. Despite this, susceptibility testing or genotypic resistance-guided therapy remains the preferred option for refractory H. pylori infection. However, its effectiveness may diminish in populations with exceedingly high antibiotic resistance rates (> 90%) [60].

Research has yielded inconsistent findings on the comparative efficacy of susceptibility-guided therapy. One previous analysis included 16 studies with 2374 patients under susceptibility-guided therapy and 2451 patients under empirical treatment. Susceptibility-guided therapy showed a slight edge over empirical therapy in treatment-naïve individuals (RR 1.14, 95% CI 1.07–1.21, P < 0.0001, I2 = 75%). However, it did not outperform empirical quadruple therapy (RR 1.02, 95% CI 0.92–1.13, P = 0.759, I2 = 80%). Also, three randomized controlled trials targeting previously treated subjects showed high heterogeneity [61].

The national utilization of this regimen needs reassessment 1–2 years after implementation to mitigate escalating therapy failures in light of increasing antibiotic resistance. As a consequence, it is necessary to explore alternative antibiotic-free therapies, particularly placing an emphasis on the initial step in the pathogenesis: bacterial interactions with the host. Therefore, anti-adhesion therapies represent a promising alternative to antibiotic treatments. Notably, bacterial adhesion hinges on specific or non-specific hydrophobic interactions between host cell receptors and the bacterial membrane surface. Numerous studies have provided proof-of-concept for anti-adhesion, which aims to hinder bacterial attachment by disrupting such host-microbe interactions. To date, a suite of anti-adhesive compounds have been identified, the properties of which were also demonstrated in vitro and in vivo. These studies indicated that competitive inhibition of bacterial attachment holds promise in impeding disease progression by mimicking the binding partners of either the bacterial cells or host receptors (see Table 1). In this comprehensive review, we explore H. pylori pathogenesis-related adhesions. Based on recent in vitro and in vivo findings, we summarize the anti-adhesion attributes of agents targeting H. pylori, with a particular emphasis on the membrane-regulating features of CAGs.

Table 1 List of anti-adhesive agents and their mechanism
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3 Bacterial adhesion is crucial for H. pylori colonization

Attachment of H. pylori to gastric epithelial cells represents a crucial step in successful infection. Typically, adhesion is facilitated by specific receptors on host cells that selectively bind to surface proteins on the bacterial cell (see Fig. 1). The specificity of H. pylori adhesion has been a significant focus of multiple in vitro and in vivo studies. Notably, H. pylori demonstrates a strong binding preference for carbohydrates, specifically sialylated oligosaccharides such as mucins [62], sulfated glycolipids [63], and blood group antigens [64]. These molecules are frequently present on gastric epithelial cell surfaces, where they play a pivotal role in acting as initial anchor points for bacterial colonization and, in turn, enabling chronic infection and disease onset.

Fig. 1
figure 1

H. pylori adheres to the surface of gastric epithelial cells in different ways, representing the initial critical step in the bacterial colonization. H. pylori have diverse adhesins, including BabA (Blood group antigen-binding adhesin), SabA (Sialic acid-binding adhesin), AlpA/B (Adherence-associated lipoproteins), and others. Each adhesin displays specific binding affinity for different host cell receptors, allowing H. pylori to interact with a range of cell surface molecules. Furthermore, the bacteria also uses the type IV secretion system (T4SS), a protein channel complex across the cell membrane, to deliver CagA (a bacterial virulence factor) into the host cells. T4SS also interacts with the host integrins to enhance the bacterial adhesion. With the actions of these adhesins and T4SS, H. pylori establishes close association with the host cells, which is essential for further colonization and persistence within the stomach. Created with BioRender.com

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Progressive bacterial colonization is completely dependent on direct contact between H. pylori and host cells. So far, a total of 63 genes encode outer membrane proteins in the H. pylori genome database [65]. Subsequent studies have identified at least 30% proteins in the family of Helicobacter outer membrane protein (Hop) are important adhesins in bacteria-host interactions [66]. Adhesins specifically recognize and bind to particular receptors expressed on the host cell surface. The well-known H. pylori adhesins include sialic acid-binding adhesin (SabA) [67], blood-group-antigen-binding adhesin (BabA) [68], adherence-associated lipoproteins A and B (AlpA/B) [69], outer inflammatory protein A (OipA) [70], Helicobacter outer membrane protein HopZ [71] and HopQ [72]. Furthermore, recent studies by Hsu et al. and Jan et al. have unraveled the correlation between cholesterol-α-glucosyltransferase (CGT) [73] and cholesteryl α-d-glucopyranoside 6′-acyltransferase (CGAT) [14] with H. pylori adhesion, respectively. These two enzymes are not directly involved in the bacterial adhesion. Instead, they are utilized specially for the derivatization of cholesterol. H. pylori is auxotropic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria use CGT to catalyze the glucosylation of cholesterol to form CG (see Fig. 2). The next enzyme CGAT is responsible for the 6′-acylation of CG, leading to the formation of CAG. CAG was shown to be critical for the bacterial adhesion (see the following text).

Fig. 2
figure 2

The biosynthetic pathway of CAG in all H. pylori strains involves consecutive reactions catalyzed by CGT and CGAT, resulting in the formation of CG and CAG, respectively. The R group of CAG denotes a fatty acyl chain.

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Gastric epithelial cells are covered by a mucous gel predominantly comprising the MUC5AC and MUC6 mucins. H. pylori adhesin BabA has been found to interact with those gastric mucins [74], implying that it acts as the main receptor for bacteria to initiate gut colonization. Furthermore, BabA exhibits a preference for interacting with carbohydrate antigens present on small intestinal epithelium [75]. The bacterium can then transfer its virulent CagA into the host cells via its Type IV secretion system (T4SS) [76], the large protein complexes forming a channel to traverse the bacterial cell envelope [77]. T4SS is responsible for the translocation of bacterial proteins or protein-DNA complexes into host cells, which is driven by a number of ATPases [78, 79]. T4SS is also critical for H. pylori adhesion. The interaction of T4SS with the host cell membrane is required to facilitate the formation and anchorage of the T4SS apparatus. Once translocated by T4SS, CagA is phosphorylated to further disrupt cellular processes and elicit an inflammatory response. Moreover, H. pylori also secretes VacA which induces vacuole formation and disrupts the integrity of the stomach epithelium, ultimately causing inflammation.

Many studies have endeavored to decipher the underlying mechanism of bacterial attachment, mostly focused on SabA and BabA. H. pylori SabA recognizes sialyl-Lewisa and sialyl-Lewisx antigens to mediate binding of H. pylori to the gastric mucosa. H. pylori BabA binds to the Lewisb antigen on the surface of stomach epithelial cells. Like SabA, BabA is important for H. pylori colonization and it promotes the development of gastritis and peptic ulcer disease. Furthermore, H. pylori has evolved an extraordinary biosynthetic way in which the enzymes CGT (encoded by the hp0421 gene) [20] and CGAT (encoded by the hp0499 gene) [14] are involved as previously mentioned (see Fig. 2). Since cholesterol is known to modulate membrane fluidity, CG and CAG, and other cholesteryl glucoside derivatives are considered modified versions of cholesterol with the additional attachment of glucose, acyl and other functional groups. It is likely that the production of these molecules represents the bacterial sophisticated strategy to well manipulate and fine-tune membrane features. In particular, CAG was found to promote lipid rafts clustering [80, 81], gathering adhesion molecules such as integrins and Lewis antigens in the raft [14], thereby enhancing bacterial adhesion. Hence, interfering with the bacterial formation of CAG is thought to be an ideal strategy for inhibiting infection.

4 Strategies of anti-adhesion

The mucosal layer lining throughout the gastrointestinal tracts, serves as frontline interface between bacteria and the host cells. Modifying the surface properties of either bacteria or host cell is thought to be an ideal way to diminish bacterial infection. Anti-adhesion strategies have emerged as a promising approach to prevent bacterial colonization within the gastrointestinal tract, where initial attachment and disease onset occur. Various studies have sought potential candidates that can mimic or compete with specific receptor molecules either on the bacterial membrane or the host cell surface. Anti-adhesion agents targeting H. pylori can operate via several inhibitory mechanisms (summarized in Table 1 and Fig. 3), and many of them have been shown effective and efficient. Because anti-adhesion is not bactericidal, it presumably does not induce any resistant mechanism. Thus, manipulating anti-adhesive agents is considered a feasible and desirable strategy to combat bacterial infections, including H. pylori infection.

Fig. 3
figure 3

Overview of anti-adhesion mechanisms to prevent the bacterial attachment to the host cells. (I) Neutralization by natural antibodies: Natural antibodies present in breast milk are able to bind with H. pylori antigens for neutralization, providing immediate protection to infants’ gastrointestinal tract. The antigens include bacterial surface proteins or lipopolysaccharides. (II) Gut microbiome balance: A sustainable gut microbiome refers to a balanced and diverse community of commensal/beneficial bacteria that produce substance(s) (e.g., lactic acid) or/and create an environment less favorable for H. pylori adhesion and colonization. (III) To disrupt lipid rafts clustering: H. pylori adhesins usually require the lipid rafts clustering to gather involved adhesion molecules, leading to enhanced binding interactions. Therefore, to impair lipid raft clustering can reduce the bacterial ability to firmly attach to gastric epithelial cells. (IV) Carbohydrate-based mimicry: Unique oligosaccharides or glycoconjugates, isolated from breast milk or extracted from natural products/substances, structurally resemble the host cell receptors of H. pylori adhesins. The molecular mimicry can provide strong competition to decrease or prevent the bacterial adhesion. (V) Damage on bacterial cell membranes: this approach is to directly change the integrity of bacterial membranes, which can be achieved by several ways. For instance, chitosan, a β1,4-GlcNAc-repeating polysaccharide, is known to perforate bacterial cell membranes. The damage is even more once the N-acetyl groups of chitosan are partially hydrolyzed to produce ammonium ions. The resulting positive charges display electrostatic interactions with the negative charges of membrane phospholipids. Created with BioRender.com 

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Recent in vitro studies indicated that many suitable candidates are able to inhibit H. pylori adhesion to host epithelial cells, including crude extracts and bioactive compounds from natural sources, or probiotic substances/compounds. For instance, human/bovine breast milk, which is high in carbohydrates and immunoglobulins, significantly impairs bacterial adhesion, indicating its potential in preventing and managing H. pylori infection [82,83,84]. Use of natural extracts as bacterial anti-adhesives not only helps to prevent or reduce bacterial attachment to host cell tissues, but also likely stands for a more efficient means to curtail the severity of bacterial infections. Such ‘natural’ anti-adhesion therapy is widely considered safer and more sustainable than antibiotic treatment. In the following sub-sections, we discuss the various anti-adhesion methods currently being exploited to tackle H. pylori infection.

4.1 Use of anti-adhesion antibodies as anti-H. pylori agents

One advantage of using anti-adhesion antibodies as anti-adhesives against H. pylori is that they are highly specific, solely binding to the adhesion structures of H. pylori without affecting the normal gut flora. Additionally, the antibody use does not promote antibiotic resistance. The first evidence that natural antibodies are ideal inhibitors of H. pylori infection was established in the study of immunoglobulin-enriched breast milk [82]. Both human and bovine breast milk were shown to protect humans from H. pylori infection by blocking H. pylori from binding to gastric mucosal cells [83, 85]. Thomas et al. reported a high prevalence of H. pylori infection in countries of West Africa, with new borns being at particular risk, yet breastfed infants presented no or delayed onset of H. pylori infection [82]. Since antibody transfer is a feature of breastfeeding, it has been assumed that high IgA concentrations in breast milk may supress bacterial attachment. However, a study by Rothenbacher and coworkers found no evidence that breast-feeding limits H. pylori infection among pre-school children [86]. Gold et al. reported that breastfed infants may have a high risk of H. pylori IgG seroconversion [36]. Thus, it seems that the IgA in breast milk is insufficient alone to ensure H. pylori infection-free status (see the next sub-section).

Similarly, Figueroa et al. reported an inhibitory effect of anti-adhesion antibodies on H. pylori adhesion to fixed monolayer of human larynx epithelioma cancer (HEp-2) cell lines [87]. It seems likely that anti-adhesin immunity help to prevent infection by stimulating the production of IgA antibodies on mucosal surfaces. Moreover, serum IgG antibodies may contribute to hindering infection by recognizing and attaching to H. pylori antigens, including adhesins, thereby interfering with the bacteria's ability to attach to host cells.

4.2 Use of receptor analogues as a strategy of receptor-adhesin mimicry

In general, glycoproteins- and glycosphingolipids-hosting carbohydrate moieties (such as sulfatides, sialyloligosaccharides or gangliosides) exposed on host cell surfaces serve as bacterial adhesion receptors. Deploying a receptor analogue to inhibit bacterial infection functions by mimicking and outcompeting the binding between bacterial adhesins and host receptors [85]. The earliest work using a receptor analogue as an anti-adhesion antigen was reported in the nineteenth century. Many specific carbohydrates show promise as agents for combating bacterial infections, such as Lewisb (Leb) blood antigens or similar fucosylated carbohydrates. Other well-known carbohydrate-based inhibitors include natural extracts from blackcurrant seeds (Ribes nigrum L.) [88] and human breast milk [85]. A research team led by A. Hensel (University of Münster) used anion-exchange chromatography (AEX) to fractionate the polysaccharides from blackcurrant seeds. Their analysis revealed that the AEX-purified fractions exhibited slight differences in the sugar content, with a subsequent in situ adhesion test on H. pylori demonstrating a variable degree of inhibition. The authors postulated that fractions possessing high molecular weight and negatively-charged glycans can interact with sialic acid-specific Helicobacter adhesins to block bacterial adhesion [88]. Strömqvist and colleagues have recently discovered a fucosylated colostral protein, κ-casein, that inhibits bacterial adhesion [85]. This inhibitory effect was eliminated by metaperiodate oxidation and bacterial adhesion was restored by α-L-fucosidase, but not affected by α-N-acetylneuraminidase or endo-β-galactosidase [85]. These observations also serve as evidence suggesting that the inhibition is likely attributed to interference with Leb group–related receptors of breast milk. The milk with rich content of glycoproteins and surface glycolipids serves as mimics of binding partners for H. pylori adhesins [85]. Of interest bovine κ-caseins were found to be ineffective in inhibiting H. pylori adhesion, in contrast to human κ-caseins [85]. This discrepancy is likely attributed to the heterogeneity of H. pylori adhesins and their cell-type specificity [85]. Nevertheless, evidence indicates that bovine milk retains the ability to neutralize the H. pylori cytotoxin [89]. Bitzan et al. [83] and Hata et al. [89] respectively demonstrated that bovine milk can block the binding to sulfatide [89] or competitively occupy the bacterial adhesin receptor, thereby preventing the interaction with phosphatidylethanolamine (PE) and lyso-PE [83]. Furthermore, Mysore et al. identified 3′-sialyllactose sodium salt (3’SL), a natural oligosaccharide in human breast milk, to reduce bacterial loads in an in vivo monkey model of H. pylori infection [90]. However, infection relapse occurred in several animals with mid- or post-3’SL treatment. 3’SL-treatment regimens plus a PPI or bismuth were seemingly ineffective.

4.3 Use of adhesin analogues as a competition-based strategy

Adhesin analogues can be isolated naturally or engineered as recombinant fragments to bind with host cell receptors, thereby competitively blocking bacterial adhesion. H. pylori adhesin A (HpaA) has been implicated in enabling some H. pylori strains to interact with erythrocytes via sialic acid-sensitive agglutination [91]. Indeed, there is evidence demonstrating that HpaA specifically binds to 3’SL on the surface of mammalian cells [92]. Chaturvedi et al. synthesized a peptide containing the sequence of a host receptor-binding site. Their findings revealed that the synthesized peptide exhibited a dose-dependent inhibition of bacterial adhesion on HEp-2 cell surfaces [93]. Results from 2D-NMR and molecular modelling suggest that this peptide fragment may have a role in receptor recognition, a critical aspect of H. pylori binding to gastric epithelium. Notably, the peptide maintained a suitable binding fold to accommodate the 3'-SL molecule. The binding mechanism is likely facilitated through hydrogen bonding, in which two lysine and one threonine residues are within the hydrogen bonding distance of NeuAcα2,3Galβ1,4Gluc.

Sialylated lactose in bovine milk can prevent H. pylori from adhering to host cells, which was first reported by Evans and coworkers [94]. The same report suggested that the surface antigens of H. pylori function as hemagglutinins because of the binding to erythrocytes, particularly with the binding specificity of NeuAc-lactose. In addition, Chaturvedi et al. showed that fetuin prevents H. pylori from attaching to HEp-2 cell monolayers [93], the observation is in accordance with study of Dunn et al. [95]. However, in some cases, fetuin exhibited weak or no inhibition of bacterial binding or haemagglutination activity, potentially because of differences in the growth media employed. For instance, both Falk et al. and Miller-Podraza et al. reported no bacterial binding to sialylated glycoproteins, including fetuin [96, 97]. Since H. pylori SabA adhesin is the only factor to date found to react with those sialylated proteins, Unemo et al. proposed that the presence of SabA is subjected to phase variation, a significant attribute of bacterial strains [98], to explain these contradictory observations. Apart from fetuin, glycolipid sulfatides [63, 99] and gangliosides [100] represent other targets for H. pylori SabA-mediated attachment in the human stomach. Rivas-Serna and coworkers claimed that ganglioside treatment can restrict H. pylori from adhering to glycoconjugate receptors throughout the gastrointestinal tract [100], with ganglioside variability eliciting varying extents of inhibition, implying that numerous sialic acids may have contributed to the reduced H. pylori adherence they observed.

4.4 Anti-H. pylori adhesion via sustainable gut microbiome

Among the plant extracts listed in Table 1, polysaccharides from cranberry (Vaccinium macrocarpon) have demonstrated anti-adhesion activities in vitro [101] and in vivo [104]. According to the report of Li et al. regular intake of PAC-standardized cranberry juice twice daily demonstrated a notable increase in H. pylori eradication rates over time. Specifically, individuals consuming high-PAC cranberry juice experienced a significant 20% reduction in infection rates compared to other dosage groups and the placebo (P < 0.05). Cranberry PAC inhibits H. pylori adhesion, as previously reported. These PACs prevented the adhesion of H. pylori sialic acid-specific strains to human gastric mucus and stomach cells [103]. Özcan and coworkers confirmed that the human gut microbiome, particularly probiotic strains like Bifidobacterium longum and Lactobacillus plantarum, use cranberry xyloglucan to promote gut microbe growth [105]. Small-intestinal microbes like Lactobacillus, Bacillus, Streptococcus, and Lactococcus [106] are associated with gastric adenocarcinoma risk [107, 108]. A Taiwanese study reported abundant Lactobacillus presence in the gastric microenvironment of gastric cancer patients, further highlighting this association [109]. Crosstalk between H. pylori and other gut flora considerably modulates the sustained physiological changes in the gastrointestinal tract induced by H. pylori infection. The study indicated the importance to maintain the integrity and functionality of the integrity and functionality of the epithelial mucosal barrier is maintained and thereby creating an adverse infection environment that aids in H. pylori eradication [108].

Several studies have reported that certain probiotic strains exhibit inhibitory activity against H. pylori (see Table 1), representing a potential anti-H. pylori treatment. Probiotics typically work through nonimmunological pathways. For example, Bifidobacterium bifidum competes with H. pylori for adhesin receptors in the gut, limiting H. pylori adhesion to the mucosa and thus preventing its colonization and growth [110]. Lactobacillus sp. [111, 112] and Bacillus subtilis [113] were identified to exert bactericidal effects by producing antimicrobial substances. Lactobacillus acidophilus was found to impair H. pylori growth after 24-h incubation, likely by secreting antibacterial lactic acid [111] or hydrogen peroxide [114]. Lactic acid also strengthens the mucosal barrier and alters acid levels in the stomach, further challenging H. pylori [111]. Lactobacillus strains have been shown in vitro to limit H. pylori urease activity [111, 112, 115], a fundamental attribute of its infectivity, but this inhibitory activity was not matched in human [112] and mouse models [115] for in vivo testing. Aiba et al. reported that lactic acid production is weak in L. acidophilus relative to that in L. casei or L. salivarius, potentially explaining discrepancies in the outcome of in vivo tests [116]. Although evidence from in vivo studies indicated that the role of lactic acid is essential for inhibiting H. pylori adherence, the impacts of probiotics on H. pylori infection appear host- or strain-specific [117]. Further in vivo assessments are thus required to establish the mechanisms of suppressive action.

4.5 Anti-H. pylori adhesion by targeting the bacterial membrane

In recent years, attention in research has shifted towards natural extracts from plants or seafood waste that display bactericidal or bacteriostatic effects, impacting the hydrophobicity of bacteria-host cell surfaces [118] or directly causing harm to the bacterium [119]. A study employing a fluorescein isothiocyanate (FITC)-labelling approach investigated various anti-adhesive agents in terms of their impact on H. pylori adhesion to human stomach epithelial cells [120]. The analysis indicated that polysaccharides from liquorice root (Glycyrrhiza glabra) [121] and immature okra fruit (Abelmoschus esculentus) [120] display significant anti-adhesive effects in vitro against H. pylori, attaining inhibition rates of > 60%, inhibition was not shown as the Panax ginseng root extract which was associated with sialic acid-dependent hemagglutination activity [122]. Notably, Messing and coworkers did not detect any change in expression of bacterial adhesins upon treatment with fresh okra extract, but reported that it is the charge distribution on polysaccharides that affect the binding to bacterial surfaces [123]. Despite non-specific binding of freshly extracted okra polymers, this approach still effectively prevented H. pylori adherence [123].

Chitosan, a linear polysaccharide found in crustacean shells consisting of repeated monomers of N-acetyl-D-glucosamine (GlcNAc) residues connected through β1,4-linkages, perforates the bacterial cell membrane to cause the bactericidal effect [124, 125]. Due to its biologically safe, biocompatible, biodegradable, sustainable, and non-toxic characteristics, chitosan is considered a potentially useful anti-microbial material and it has been already widely applied as a drug delivery agent. H. pylori tolerates the low pH environment of the stomach, likely attributable to its urease activity. Chitosan activity is also pH-dependent [126]. Chitosan becomes more positively charged (i.e., the N-acetyl of GlcNAc is hydrolyzed to produce ammonium ion) in acidic conditions, which enhances its antibacterial efficacy [127], with electrostatic interactions between the ammonia ions (NH3+) of chitosan and the negatively-charged phosphoryl groups of the phospholipids in the bacterial cell membranes, leading to the observed damage [119]. Recently, Chang et al. [127] used FITC-labeling to study the impact of deacetylated chitosan (DD95) on adhesion of H. pylori to human gastric cancer cells (TSGH 9201). DD95 was found undeniably successful to suppress H. pylori bacterial proliferation and urease production. Moreover, chitosan microspheres have been shown to efficiently bind to H. pylori in vivo [128].

4.6 Disrupting lipid raft clustering to impair H. pylori adhesion

The bacterial enzyme CGAT and its reaction product CAG have multifaced features regarding their role in the virulence of H. pylori. First, CGAT was found to exist mainly in the outer membrane of H. pylori and secreted extracellularly in the form of outer membrane vesicles (OMVs). The OMVs collected from bacterial culture were found to contain the activities of CGT and CGAT [14], offering three advantages. (1) The bacteria can dispatch OMVs for the delivery of the whole biosynthetic machinery to the host cells. (2) Without bacterial adhesion, both enzymes can directly access the host cholesterol, UDP-glucose (required for the cholesteryl glucosylation), and phospholipids (for the acyl transfer to produce CAG). (3) The translocated CGT and CGAT (by fusion of the bacterial OMVs with the host cells) produce CAG for the lipid raft-mediated enhancement of H. pylori, representing a unique strategy to manipulate the host cell membranes in favor of the bacterial adhesion.

Second, CAG was identified as a compound to promote H. pylori internalization to human gastric epithelial cells. However, the same compound prevents lysosomal clearance of autophagosomes and reduce lysosomal biogenesis [129], supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Additionally, by performing the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells, the CGAT activity was shown mainly present in autophago-lysosomal compartments [129]. Especially, CGAT activity is maximal in an acidic environment such as that of the autophagosome and lysosome. The elevated CGAT activity clearly explains how intracellular bacteria leverage their lipid and lipid-biosynthesis enzymes to regulate degradation of endosomes in host cells.

Third, previous studies indicated that CGAT is critical for the bacterial adhesion. Jan et al. examined the potential of inhibiting CGAT for therapeutic intervention and discovered amiodarone, a drug commonly used as an antiarrhythmic agent, to be a potent inhibitor of CGAT with a IC50 value of 13.8 μM [14]. To study how amiodarone influenced the bacterial infectivity, the co-culture of H. pylori-AGS cells was treated with amiodarone. The result indicated that amiodarone effectively blockaded the bacterial adhesion in a dose-dependent manner, resulting a 74% reduction in bacterial adhesion to AGS gastric epithelial cells upon the treatment of 50 μM amiodarone. Meanwhile, a simultaneous decrease was observed in the translocation and phosphorylation of the bacterial cytotoxin CagA at the same study, demonstrating that amiodarone-mediated inhibition of CGAT significantly reduced the bacterial adhesion to gastric epithelial cells. The use of hp0499-knockout further evidenced the importance of CGAT to the H. pylori infectivity. Since CAGs were rarely produced, less lipid rafts were clustered, resulting in little or no H. pylori adhesion to AGS cells.

Furthermore, the CAG level was found surprisingly abundant in MDR strains of H. pylori that displayed resistance to the treatment of levofloxacin, metronidazole, and clarithromycin. The level was 10–150 times higher than that of the standard strain H. pylori 26695 [14], suggesting that the CAG abundance is likely associated with the membrane barrier to avert the drug entry. To verify if amiodarone is also useful to inhibit the CGAT activity and the related pathogenesis in MDR strains, one MDR strain producing the highest CAG level had been incubated with amiodarone before it infected AGS cells. Amiodarone significantly reduced not only the CAG level at the concentration of 50 μM, but also the corresponding bacterial adhesion, CagA translocation, and the related tyrosine phosphorylation [14]. These results pave the way for further development of CGAT inhibitors for therapeutic intervention. The amiodarone-based anti-adhesion appears to offer an alternative for antimicrobial therapy.

5 Acyl chain of CAG is critical to regulate the bacterial adhesion

The role of H. pylori in the biotransformation of cholesterol into various derivatives is considered crucial for the bacteria to evade the host immune system. CG and CAG, representing two cholesteryl glucosides in H. pylori, are incorporated into the bacterial cell membrane, and were reported essential for the bacterial pathogenesis, including adhesion, colonization and virulence [73, 130]. Previous report indicated that the acyl chain of CAG is critical to manipulate the membrane properties [14, 131]. A suitable acyl-chain-containing CAG was shown to either enhance or inhibit the adhesion of H. pylori, according to our preliminary data [132]. H. pylori CGAT catalyzes the acyl transfer from phospholipids to the 6′-OH of CG. Therefore, the usage of phospholipids is an intriguing approach to generate desirable CAG(s) in vivo to blockade the bacterial adhesion. Nonetheless, there are several factors for consideration. For instance, the idea of introducing exogenic phospholipids produces changes not only in H. pylori, but also in the host cells or animals at the same time, which likely causes profound alterations in the compositions of cell membranes. It remains to be investigated if membrane lipids display any feedback regulation or compensatory effect, not mentioning whether there is occurrence of undesired side effect. In addition, bacterial cells and human cells are known to show significant differences in the lipid metabolisms. Even if any phospholipids are eventually identified to inhibit H. pylori adhesion, there will be a growing concern about how to obtain them at a large scale or/and at reasonable cost.

Ismaili et al. previously demonstrated that increasing plasma membrane fluidity effectively inhibits the adhesion of enteropathogenic Escherichia coli (EPEC) to HEp-2 cells [133]. Steroids can significantly alter membrane structure, reduce the membrane fluidity, and increase the stability [134]. Differences in membrane lipid fluidity are known to affect regional heterogeneity and receptor plasticity [134, 135]. The molecular dynamics simulations by Pandit and coworkers [136] suggested that the interaction between phospholipids and cholesterol requires a specific packing environment [137]. In accordance with the “push and pull” mechanism, the steroid–phospholipid interaction can cause thermodynamic changes [138]. Therefore, less fluidic membranes may initiate lipid phase separation, leading to membrane protein segregation, which increases the energetic cost of membrane domain interfaces [139]. Hydrophobic matching between steroid or phospholipids in lipid bilayers would offer energetic advantages and thus become important in the membrane lateral organization [137, 140].

6 Conclusion

In summary, the rise of MDR in bacterial infections, including H. pylori, has posed serious concern for current antibiotic therapies. Among patients infected by H. pylori, 10–20% of patients suffer from the problem of MDR, indicating that traditional treatments are not always effective, or do require the dosage increase. This scenario underscores the necessary development for alternative approaches to find better solutions. Anti-adhesion strategies hold considerable promise because they primarily target the adhesion-receptor binding, the very first step of bacterial colonization/pathogenesis. Several strategies are available and have been discussed in detail in this review, as illustrated in Fig. 3. The strategies of disrupting lipid rafts clustering and carbohydrate-based mimicry likely show a higher potential owing to better understanding at molecular basis. Related agents can be thus improved and optimized with rationale. The aforementioned studies of CAG and CGAT appear to be in line with this direction. With further research and corresponding clinical examination, new advances of anti-adhesion strategies will pay the way for effective anti-H. pylori therapies.