Molecular Detection Technologies for Helicobacter pylori Antibiotic Resistance: Current Status and Clinical Implications

Abstract

Helicobacter pylori (H. pylori) infects approximately half of the global population and is closely linked to chronic gastritis, peptic ulcers, and gastric cancer. Antibiotic resistance has become a major global health concern with marked regional variations, severely undermining empirical treatment efficacy. Resistance mechanisms primarily involve target gene mutations, efflux pump overexpression, and altered membrane permeability. This review summarizes resistance mechanisms of commonly used antibiotics against H. pylori, along with diagnostic approaches ranging from phenotypic to molecular methods, the latter offering faster and more precise mutation detection, yet limited by high costs and invasive sampling. Treatment has shifted from clarithromycin-based triple therapy to bismuth-containing quadruple and potassium-competitive acid blocker-based regimens. However, susceptibility-guided therapy faces implementation barriers, and empirical regimens remain prevalent. Future directions emphasize affordable multiplex molecular platforms, resistance surveillance networks, artificial intelligence-driven predictive models, and non-antibiotic adjunctive strategies. Integrating molecular diagnostics with antimicrobial stewardship to transition from empirical to precision-based management is critical for improving eradication rates and curbing further resistance spread.

Introduction

Helicobacter pylori (H. pylori) is a bacterium capable of growing in the human stomach, H. pylori infection is associated with various gastrointestinal diseases, including chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer [1–4]. Numerous therapeutic regimens for H. pylori infection have been established, with their application varying across geographical regions, primarily influenced by local antibiotic resistance patterns. Current clinical practice accepts only therapeutic regimens that attain an H. pylori eradication rate surpassing 90% [5]. International guidelines primarily recommend traditional empiric therapies as first-line treatment regiments; however, their efficacy has significantly declined to below 85% [6]. Traditional therapies comprise triple therapies that include a proton pump inhibitor (PPI), clarithromycin (CLR), and either amoxicillin (AMX) or metronidazole (MTZ). CLR is generally not advised as the first-line empiric therapy for H. pylori infection and should be used solely in regions where the CLR resistance rate is under 15% [7, 8]. Conversely, bismuth-based quadruple therapies, typically comprising bismuth, PPI, MTZ, and tetracycline (TE), are progressively endorsed as alternatives for first-line treatment, exhibiting success rates surpassing 90% [9–11]. In these regimens, antibiotics remain the primary drugs for H. pylori treatment. However, the increasing antibiotic resistance of H. pylori, particularly high resistance to CLR and MTZ, severely limits the therapeutic options in clinical practice, posing significant challenges to existing treatment regimens.

The mechanisms of antibiotic resistance in H. pylori are exhibited through mutations in antibiotic-binding targets or alterations in cell permeability due to external pressures, among other factors [12, 13]. With the advancement of in vitro diagnostic technologies, phenotypic or molecular biological methods can be utilized to specifically detect the antibiotic resistance profiles of H. pylori. Each method possesses distinct advantages and limitations, and the diversity of their results necessitates further investigation into current detection methodologies. Additionally, more considerations must be directed towards factors including the accessibility of sample acquisition, patient safety, and detection costs, in order to provide effective H. pylori antibiotic resistance in clinical practice, improve cure rates, and partially mitigate the prevailing issue of antibiotic misuse during the treatment of H. pylori infections [14]. This article presents a thorough examination of recent studies on H. pylori antibiotic resistance, detection techniques, and clinical treatment strategies.

Regional Variations in H. pylori Antibiotic Resistance

Significant regional variations exist in the characteristics of H. pylori antibiotic resistance, which may be associated with local socio-economic conditions, hygiene practices, healthcare infrastructure, and antibiotic usage patterns. The resistance rates of the primary antibiotics used for H. pylori eradication in selected countries and regions are summarized in Table 1. In Europe, the resistance rates of H. pylori to CLR range from 18% to 21.4%, to levofloxacin (LEV) from 11.0% to 16.3%, and to MTZ reach 39.1% to 56%. Overall, southern European regions generally exhibit higher resistance rates than northern regions, and the resistance rates of H. pylori to these three antibiotics have gradually increased over time [15].

Table1.

Summary of antibiotic resistance profiles in H. pylori across different regions

Antibiotics	Europe	USA	China	South korea	Africa	References
MTZ	39.1%-56.0%	42.1%	77.3%	29.5%	91.0%	[15–19]
CLR	18.0%-21.4%	31.5%	36.7%	17.8%	15.0%	[16, 18]
AMX	0.2%	2.6%	0%	9.5%	38.0%	[18, 20]
LEV	11.0%-16.3%	37.6%	16.6%	37.0%	14.0%	[16, 18]
TE	0%	0.87%	0%	0%	13.0%	[18, 20]

A systematic analysis of antibiotic resistance surveys for H. pylori in the United States from 2011 to 2021 provides an overview of the resistance situation in the country. This survey integrates publicly accessible reports, conference proceedings, and academic papers published over this decade, encompassing a total of 2,660 gastric biopsy samples with a 95% confidence interval. The results indicated that the resistance rates of H. pylori infections in the United States are 42.1% for MTZ, 31.5% for CLR, 37.6% for LEV, 2.6% for AMX, and 0.87% for TE. The dual resistance rate to CLR and MTZ is 11.7%. Notably, the resistance rates for MTZ, LEV, and CLR all exceed 30% in the previously described studies, suggesting that traditional empiric antibiotic treatment regimens are unsuitable without knowledge of the patient’s individual H. pylori resistance profile [21].

In South Korea, the resistance rates of H. pylori to commonly used antibiotics are relatively moderate. The highest resistance rate was observed for LEV at 37.0%, followed by MTZ at 29.5% and CLR at 17.8%, while AMX resistance was relatively low at 9.5%. Notably, no TE resistance has been reported, indicating that TE remains fully active against H. pylori in this region [16, 18].

In contrast, Africa faces a much more severe burden of antibiotic resistance in H. pylori. The resistance rate to MTZ is alarmingly high at 91.0%, and AMX resistance also reaches 38.0%, which is considerably higher than in most other regions. Resistance to CLR, LEV, and TE are moderate at 15.0%, 14.0%, and 13.0%, respectively [18, 20]. These data underscore the critical challenge posed by antibiotic resistance in Africa, particularly for MTZ, which has become nearly ineffective in this region.

The antibiotic resistance rates of H. pylori in China, in descending order, are as follows: MTZ at 77.3%, CLR at 36.7%, and LEV at 16.6% [16]. A drug sensitivity assessment conducted on 157 gastric biopsy samples from pediatric children in southwestern China revealed overall resistance rates of 55.2% for CLR, 71.3% for MTZ, and 18.4% for LEV. No resistance to AMX, TE, or furazolidone (FZD) was found in the isolated strains. In a cohort of 53 primary cases, the resistance rates to CLR, MTZ, and LEV were 45.3%, 73.6%, and 15.1%, respectively. In 34 secondary cases, the resistance rates to these antibiotics were 70.6%, 67.6%,and 23.5%, respectively. Children in southwestern China exhibit elevated resistance rates to CLR, MTZ, and LEV, while exhibiting no resistance to AMX, TE, or FZD. In light of these findings, it is recommended to abandon the standard triple therapies using CLR for empiric treatment, and to consider bismuth-based quadruple therapies (bismuth/PPI/AMX/TE) as a more suitable first-line treatment option for young pediatric patients [20]. A study investigating antibiotic resistance of H. pylori in children from southeastern China collected gastric biopsy samples from 3,111 children between 2015 and 2020, successfully isolating 1,638 H. pylori strains. The resistance rates to CLR, MTZ, and LEV were 32.8%, 81.7%, and 22.8%, respectively. In this investigation, 52.8% of the isolates shown resistant to a single antibiotic, 28.7% displayed dual antibiotic resistance, and 9.0% exhibited triple antibiotic resistance. Compared to the years 2012 to 2014, there was a significant increase in all types of antibiotic resistance, with the exception of single-antibiotic resistance, over the periods from 2015 to 2017 and from 2018 to 2020 [22].

The marked geographical heterogeneity in H. pylori antibiotic resistance arises from a complex interplay of antibiotic consumption patterns, treatment practices, socioeconomic conditions, and regulatory contexts. Regions with higher outpatient macrolide and fluoroquinolone use, such as several southern European countries, exhibit correspondingly elevated resistance levels, whereas northern European countries with more restrictive antibiotic stewardship have maintained lower rates [15]. This north–south gradient is also influenced by differences in empirical treatment practices. Areas that continue to prescribe CLR-based triple therapy despite rising resistance perpetuate a vicious cycle of treatment failure and further resistance selection, while regions adopting bismuth-based quadruple or susceptibility-guided regimens have partially mitigated this pressure [5, 23]. Socioeconomic and sanitary conditions play an additional role, as higher population density, lower socioeconomic status, and inadequate sanitation facilitate both infection acquisition and re-infection, increasing the bacterial population at risk of antibiotic exposure [24, 25]. Pediatric antibiotic exposure constitutes a particularly important determinant, as children in southern and eastern Europe have historically received higher cumulative macrolide doses for respiratory infections compared to their northern counterparts, which may explain the elevated CLR resistance observed in pediatric H. pylori isolates from these areas [26–28]. Furthermore, historical over-the-counter antibiotic availability and weak prescription regulation enforcement in certain regions have created persistently high baseline resistance levels that endure even after regulatory tightening [29]. In summary, the regional divergence in H. pylori resistance reflects the cumulative outcome of differential antibiotic consumption, divergent treatment guidelines, socioeconomic disparities, variable pediatric antibiotic use, and distinct regulatory histories, underscoring the need for regionally tailored interventions that account for local resistance epidemiology and healthcare capacity.

Molecular Mechanisms of Antibiotic Resistance

Previous studies have established that genetic alterations leading to the failure of antibiotic targets contribute to H. pylori to development of antibiotic resistance. In H. pylori susceptible to CLR, CLR inhibits bacterial protein synthesis by interacting with the peptidyl transferase ring within domain V of the ribosomal 23S rRNA subunit (Fig.1A). Point mutations in the V domain of the H. pylori ribosomal 23S rRNA can alter the affinity of CLR to peptidyl transferase, leading to resistance to CLR [30, 31]. Sequencing results of the 23S rRNA gene in H. pylori from the Iranian population revealed that the predominant mutations responsible for CLR resistance are A2143G and A2142 [32].

Fig. 1.

Mechanisms of CLR susceptibility and resistance in H. pylori. (A) In susceptible strains, CLR inhibits protein synthesis by binding to the peptidyl transferase loop within domain V of the 23S rRNA subunit. (B) Resistance mechanisms in CLR-resistant strains include: (a) outer membrane proteins mutations that decrease membrane permeability;(b) point mutations in the 23S rRNA gene that reduce drug–ribosome affinity; and (c) enhanced drug efflux via active efflux pump systems

Outer membrane proteins (OMPs) are implicated in antibiotic resistance in Gram-negative bacteria, and H. pylori is no exception. The H. pylori genome encodes 32 OMPs, classified into five groups: adhesins, porins, iron transporters, efflux proteins, and proteins of unknown function. In CLR-resistant strains, expression of HopT, OMP31, and HofC was upregulated, while iron transporters, ureaseB, elongation factor, and putative OMPs were downregulated; efflux proteins remained unchanged[33, 34]. These findings suggest that OMP alterations may represent a novel mechanism contributing to CLR resistance in H. pylori, although further studies are required to elucidate their specific roles and underlying mechanisms. Some researchers found that the resistance nodulation cell division (RND) family within the efflux pump system is involved in the development of CLR-related resistance. Despite the presence of 23S rRNA point mutations, the minimum inhibitory concentrations (MIC) of CLR-resistant H. pylori strains were significantly reduced upon treatment with the efflux pump inhibitor PaβN in a concentration-dependent manner [34]. A model representing the mechanism of CLR resistance is shown in Fig.1B.

The chemical structure of MTZ contains a nitro group. Under anaerobic or microaerophilic conditions, nitroreductases, primarily encoded by the rdxA gene, within H. pylori reduce this nitro group to reactive free radicals or nitroso derivatives. These active intermediates directly attack the double-stranded DNA of the pathogen, inducing strand breaks and thereby impairing DNA replication and repair, which ultimately suppresses the growth and proliferation of H. pylori [35] (Fig.2A).

Fig. 2.

Mechanisms of MTZ susceptibility and resistance in H. pylori. (A) In susceptible strains, MTZ binds to the nitroreductase RdxA/FrxA and undergoes reductive activation to generate nitro anion radicals, which subsequently cause oxidative damage and disrupt the helical structure of bacterial DNA. (B) In resistant strains, loss of MTZ activation results from point mutations in the RdxA, FrxA genes, which encode key nitroreductases. These mutations impair the production of nitro anion radicals, thereby abolishing the bactericidal effect of MTZ

Mutations in the rdxA gene, and the frxA gene, which encodes a flavin oxidoreductase, are the primary causes of MTZ resistance [36, 37]. These mutations reduce the ability of MTZ to generate intermediate compounds or radicals that are destructive to bacterial DNA structure [38]. The resistance rate to MTZ is the highest among antibiotics [18]. A model representing the mechanism of MTZ resistance is shown in Fig.2B.

LEV interacts with DNA gyrase, encoded by the gyrA and gyrB genes, to inhibit the separation of DNA strands facilitated by DNA gyrase during DNA replication. This inhibition disrupts the DNA synthesis process and bacterial cell replication (Fig.3A). Mutations have been identified at codons 87, 88, 91, and 97 of the gyrA gene, as well as at codon 463 of the gyrB gene in LEV-resistant strains of H. pylori [30, 31, 39]. Whole-genome sequencing of 12 H. pylori isolates obtained from biopsy samples of gastric cancer patients in Colombia revealed that mutations such as N87I/K and D91G/Y in gyrA were associated with resistance to LEV [40], The mutations of gyrA and gyrB result in the inability of LEV to bind, hence allowing H. pylori DNA replication to proceed. A model representing the mechanism of LEV resistance is shown in Fig.3B.

Fig. 3.

Mechanisms of LEV susceptibility and resistance in H. pylori. (A) In susceptible strains, LEV exerts its bactericidal effect by binding to DNA gyrase (composed of GyrA and GyrB subunits), thereby interfering with chromosomal DNA replication. (B) In resistant strains, point mutations within the quinolone resistance-determining region of the gyrA and gyrB genes alter the drug-binding pocket of DNA gyrase, reducing its affinity for LEV and rendering the antibiotic ineffective

AMX exerts its antibacterial effect by interacting with penicillin-binding proteins (PBPs), consequently influencing the mechanical stability of the bacterial cell wall. This leads to the loss of bacterial replicative viability and lysis (Fig.4A).

Fig. 4.

Mechanisms of AMX susceptibility and resistance in H. pylori. (A) In susceptible strains, AMX exerts its bactericidal effect by binding to penicillin-binding proteins (PBPs), thereby inhibiting peptidoglycan synthesis and leading to cell wall weakening, cellular expansion, and subsequent lysis. (B) Resistance mechanisms in AMX-resistant strains involve four distinct strategies: (a) outer membrane protein mutations that decrease membrane permeability and reduce drug influx; (b) gene mutations that induce structural alterations in PBPs, impairing AMX–PBP binding; and (c) enhanced antibiotic efflux mediated by efflux pump systems; (d) β‑lactamase production, which enzymatically degrades and inactivates the chemical structure of AMX

Mutation of PBP genes is a common mechanism of AMX resistance in H. pylori strains. To date, nine distinct PBPs have been identified in H. pylori, including three high-molecular-weight PBPs, designated PBP1, PBP2, and PBP3, as well as six low-molecular-weight PBPs, referred to as PBP4–9[41]. Among these, PBP1A is a member of the PBP1 family possessing both glycosyltransferase and acyltransferase activities—is most closely associated with AMX resistance.

The resistance of H. pylori to AMX involves three main biomolecular mechanisms. The primary mechanism is mutations in PBPs genes, particularly in PBP1A, where amino acid substitutions such as S402G, E406A, S417T, S414R, T555S, and N561Y reduce the affinity of PBPs for AMX, while alterations in PBP2, PBP3, and low-molecular-weight PBPs also contribute to resistance[42–44]. Secondly, production of β-lactamase, specifically the TEM-1 enzyme, can hydrolyze the β-lactam ring of AMX, and the corresponding blaTEM-1 gene may be spread among strains via natural transformation[45]. Thirdly, mutations in outer membrane proteins such as HopB and HopC, along with overexpression of efflux pump components including HefA, HefC, and HofH, reduce membrane permeability and increase drug efflux, thereby decreasing intracellular AMX concentrations [46, 47]. Collectively, these mechanisms, acting independently or in synergy, underlie the development of AMX resistance in H. pylori. A model representing the mechanism of AMX resistance is shown in Fig.4B.

TE exerts its antibacterial effect by binding to the 30S ribosomal subunit through 16S rRNA, interfering with the binding of aminoacyl-tRNA to the ribosome and thereby blocking protein translation, which ultimately inhibits protein synthesis[48] (Fig.5A). Resistance to TE in H. pylori is mainly mediated by two mechanisms. The first is point mutations in the 16S rRNA gene, which alter the TE binding site and reduce drug–ribosome affinity. High-level resistance (MIC > 8 μg/mL) is typically associated with a triple-base-pair mutation (AGA-965-967 to TTC) at the putative binding site, while low-level resistance (MIC ≤ 4 μg/mL) results from double or single mutations; additional mutations such as AGA-926-928 and A939C have also been linked to resistance[49, 50]. The second mechanism involves the proton motive force-dependent efflux pump system. The Hp1165 gene, which encodes an efflux pump protein, is widely expressed in TE-resistant strains and actively transports the antibiotic out of the bacterial cell. The efflux pump inhibitor CCCP has been shown to significantly increase TE accumulation and reduce the minimum inhibitory concentration in resistant isolates, confirming the functional role of this system[51]. Notably, some TE-resistant strains lacking the Hp1165 gene still exhibit efflux pump activity, suggesting that other determinants beyond Hp1165 may also regulate efflux-mediated resistance[52]. OMPs have been confirmed to be involved in TE resistance in Acinetobacter baumannii and Escherichia coli, but whether this mechanism exists in H. pylori requires further investigation[53, 54]. A model representing the mechanism of TE resistance is shown in Fig.5B.

Fig. 5.

Mechanisms of TE susceptibility and resistance in H. pylori. (A) In susceptible strains, TE binds to the bacterial 30S ribosomal subunit, interfering with aminoacyl-tRNA docking at the A-site and thereby inhibiting protein synthesis. (B) Resistance mechanisms in TE-resistant strains include: (a)outer membrane protein mutations that decrease membrane permeability and reduce drug influx; (b) point mutations within the 16S rRNA gene that alter the primary binding site, reducing TE–ribosome affinity; and (c) enhanced drug efflux mediated by efflux pump systems that actively extrude TE from the cell

Beyond antibiotic resistance, the capacity of H. pylori to evade host immune surveillance represents another critical determinant of its chronic persistence. A recent study by Pathirana et al.[55] revealed that H. pylori induces the production of interleukin-37 (IL-37) in human gastric epithelial cells through activation of TLR2, TLR4, and NOD1, as well as the cag pathogenicity island. Once secreted, IL-37 exerts broad immunosuppressive effects by impairing T and B cell activation, migration, immune synapse formation, and proliferation of H. pylori-specific T cells, thereby enhancing bacterial colonization while limiting gastric inflammation and antibody responses. These findings identify gastric IL-37 as a potential therapeutic target for combating chronic H. pylori infection.

Diagnostic Methods for H. pylori Infection and Antibiotic Susceptibility Testing

The increasing antibiotic resistance of H. pylori has led to an overall decline in treatment efficacy, necessitating tailored treatment strategies in clinical diagnosis rather than traditional empirical methods. In clinical practice, in vitro diagnostic procedures commonly employed include serum testing, urea breath test, and fecal antigen test. Among these, serum testing is the most cost-effective, utilizing anti-H. pylori IgG to detect immune responses in serum induced by infection. It is rapid but cannot distinguish between active and past infections [56]. The remaining two procedures are highly specific and require cessation of antibiotics, bismuth, and PPI before testing to avoid false-negative results [57]. Additionally, endoscopy-based testing involves collecting gastric mucosa samples for isolate culture or molecular diagnosis, which can directly detect H. pylori infection and provide information on antibiotic susceptibility. Histological examination of samples can also assess the eradication status of H. pylori, the extent of inflammation, and related pathological features. The choice of diagnostic technique depends on the patient’s clinical characteristics. For instance, endoscopy is recommended for patients presenting signs and symptoms of severe upper gastrointestinal diseases, such as peptic ulcers or gastric cancer. In contrast, asymptomatic relatives of patients with peptic ulcer disease are more suitable for in vitro testing, such as urea breath tests or fecal antigen tests [58]. Beyond the conventional diagnostic approaches, accumulating evidence suggests that serum cytokine profiling may offer additional value in monitoring disease progression in H. pylori-infected patients. Della Bella et al.[59] demonstrated that serum IL-17A levels were significantly elevated in H. pylori-infected patients with gastric intestinal metaplasia and dysplasia compared to those with non-atrophic gastritis and healthy controls. Furthermore, gastric T cells from these patients produced high levels of IL-17A in response to H. pylori stimulation, indicating that H. pylori drives a gastric Th17 inflammatory response that may contribute to the multistep progression from chronic gastritis to gastric cancer. These findings suggest that serum IL-17A measurement could serve as a potential biomarker for identifying H. pylori-infected individuals at higher risk of developing gastric premalignant lesions.

Antibiotic resistance detection methods can be categorized into phenotypic and molecular genetic approaches. The principles, performance characteristics, and clinical applicability of major phenotypic and molecular methods for H. pylori antimicrobial susceptibility testing are compared in Table 2. Phenotypic detection methods include the serial dilution method, disk diffusion method, and E-test.

Table2.

Overview of phenotypic and molecular methods for H. pylori susceptibility testing

Method	Principle	Detection type	Identify specific mutations	Advantages	Limitations	Clinical applicability	References
serial dilution	Serial two-fold dilutions of antibiotics are incorporated into agar medium; following bacterial inoculation, the MIC is determined.	Phenotypic	No	Gold standard; provides precise quantitative MIC.	Time-consuming; labor-intensive	Reference assay for validating other methods	[60–63]
disk diffusion	Antibiotic-impregnated disks are placed onto agar plates inoculated with bacterial suspension; the diameter of the growth inhibition zone is measured to infer susceptibility	Phenotypic	No	Simple operation; low cost.	Lack of standardized quality control; high inter-laboratory variability; no standardized protocol endorsed by CLSI or EUCAST.	Preliminary screening in resource-limited settings	[64, 65]
E-test	A plastic strip impregnated with a predefined gradient of antibiotic concentrations is applied to the agar surface; the MIC is read at the intersection of the inhibition ellipse with the strip.	Phenotypic	No	User-friendly; provides quantitative MIC; superior precision to disk diffusion.	Discrepancies in MIC results for metronidazole compared to agar dilution; relatively high strip cost.	Routine clinical susceptibility testing	[66–68]
RT-PCR	Specific primers amplify target genes; known resistance-associated mutations are detected via fluorescent signals or melting curve analysis.	Molecular	Yes (known sites)	Rapid and sensitive; enables simultaneous detection of infection and resistance mutations.	Limited to known mutations; unable to identify novel resistance determinants	Rapid screening for CLR and LEV resistance mutations	[69–71]
ddPCR	Sample is partitioned into thousands of nanoliter-scale droplets for single-molecule amplification, enabling absolute quantification of target DNA.	Molecular	Yes (known sites)	Ultra-high sensitivity; capable of detecting low-density infections.	Expensive instrumentation; relatively low throughput.	Detection of low-abundance or mixed infections	[72, 73]
PCR-RFLP	PCR-amplified products are digested with restriction endonucleases; mutations are identified based on polymorphic restriction fragment patterns.	Molecular	Yes (known sites)	Low cost; minimal equipment requirements.	Limited to mutations that create or abolish restriction sites; risk of incomplete digestion.	Routine laboratory-based mutation screening	[74, 75]
FISH	Fluorophore-labeled nucleic acid probes hybridize to target sequences in tissue sections or cell smears; signals are visualized under fluorescence microscopy.	Molecular	Yes (known sites)	Enables direct detection in biopsy specimens without prior culture; preserves tissue morphology; allows simultaneous detection of susceptible and resistant populations.	Low throughput; requires fluorescence microscopy.	Direct testing on gastric mucosal biopsy specimens	[76, 77]
Sanger sequencing	Dideoxy chain-termination method enables direct reading of DNA sequences, detecting both known and unknown mutations.	Molecular	Yes (all sites)	Gold standard for nucleic acid sequencing; capable of identifying novel mutations.	Low throughput; low-abundance mutations in mixed infections may be undetected.	Confirmatory single-locus sequencing; validation assay	[78–82]

The serial dilution method is used to determine the MIC, defined as the lowest concentration of an antibiotic that inhibits microbial activity. To determine the MIC, a predetermined concentration of the antibiotic is introduced into the culture medium, followed by the inoculation of bacteria onto this antibiotic-containing medium for cultivation. Following a designed interval, bacterial growth is assessed to determine the requisite antibiotic dosage for inhibitory activity. This method, despite being labor-intensive and time-consuming, is regarded as the gold standard for antibiotic susceptibility testing due to its precise calculation of MIC [61] and is recommended as a reference assay for assessing the accuracy of other methods [62]. H. pylori strains can be classified as sensitive, intermediate or resistant based on their antibiotic susceptibility [63].

The disk diffusion method relies on the diffusion of drugs from a carrier into a dense nutrient medium, inhibiting the growth of the studied culture in areas where the antibiotic concentration exceeds the MIC. This approach utilizes a disk serves as an antibiotic carrier, with the antibiotic efficacy estimated by the width of the growth inhibition zone; a larger diameter indicates a lower MIC and enhanced inhibitory activity against the microorganism under investigation [64]. The disk diffusion method, invented in the 1940s, has been used for antibiotic susceptibility testing of various bacteria and remains a primary method for this purpose today. Despite its long-standing global application, the lack of strict quality control in terms of carrier materials, antibiotic content, and operational procedures may lead to variable or unreliable test results, prompting the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) to refrain from endorsing any standardized method for this purpose [65].

The E-test combines the principles of serial dilution and disk diffusion, using a polymer strip as the antibiotic carrier with a gradient of antibiotic concentrations. When co-cultured with bacteria, the inhibitory zone around the strip gradually narrows as the antibiotic concentration decreases, resulting in a teardrop-shaped inhibitory zone on the culture plate. The concentration at the point where the inhibitory zone touches the strip is considered as the MIC. The E-test has been widely adopted in clinical and microbiological laboratories [64]. However, some reports have raised concerns about inconsistencies in MIC results between the E-test and the serial dilution method for MTZ against H. pylori, may be attributed to changes in redox potential. Consequently, the E-test has been questioned for its application in MTZ susceptibility testing for H. pylori [83].

Molecular genetic methods for detecting antibiotic resistance include real-time polymerase chain reaction (RT-PCR), droplet digital PCR (ddPCR), fluorescence in situ hybridization (FISH), restriction fragment length polymorphism analysis (PCR-RFLP), Sanger sequencing, and next-generation sequencing (NGS). These methods facilitate the identification of specific site mutations that confer antibiotic resistance.

RT-PCR enables the amplification of DNA fragments selected by a pair of specific primers, allowing both qualitative and quantitative analysis of H. pylori DNA. By designing primers targeting certain resistance-associated mutations, one can determine the presence of mutations in the template DNA based on the amplification results. Currently, RT-PCR-based methods for resistance identification exhibit a low error rate and are most commonly employed for detection of resistance mutations in LEV and CLR. However, methods for identifying resistance mutations in AMX and MTZ remain in the developmental stage [71]. A study demonstrated that RT-PCR can be used to detect H. pylori in gastric mucosal biopsy samples before quinolone treatment, with melting curve analysis to determine the presence of DNA mutations in gyrA. The results were consistent with those obtained by Sanger sequencing, indicating its potential use in guiding quinolone therapy [84].

ddPCR is a novel PCR technology that enables absolute quantification of target DNA in samples with higher sensitivity relative to existing PCR methods. In patients with low-density H. pylori infections that are undetectable by conventional procedures, such as the 13C- urea breath test, ddPCR has a detection rate of 36% [72]. ddPCR detects CLR-resistant genotypes of H. pylori by quantifying the 23S rRNA gene. In a study of 102 patients, 45 (44%) had CLR-resistant mutations. The study also found that the prevalence of CLR-resistant mutations varies among ethnic groups, with the highest rate in Asians, where 14 out of 21 patient samples (67%) tested positive for CLR-resistant mutations [85].

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) entails the construction of appropriate amplification primers to ensure that the amplified fragment includes one or more polymorphic recognition sequences for restriction endonucleases. Subsequent to PCR amplification, the PCR products are cleaved using the corresponding restriction enzyme. In the presence of a mutant gene, the sequence of the restriction site may not change, although the relative position of the restriction site within the DNA will alter. Therefore, site mutations can be identified based on electrophoresis patterns. Rawaa A. Hussein et al. conducted PCR-RFLP on 38 isolated CLR-resistant strains, revealing that the A2144G mutation accounted for 41.2%, the A2143G mutation accounted for 17.6%, and strains with both mutations simultaneously accounted for 26.5%. Mutations were absent in 14.7% of the strains, but four A2143G mutant strains had an MIC >32 μg/mL, suggesting that the resistance associated with the A2143G mutation may be more severe [75].

Fluorescence in situ hybridization (FISH) is a detection method based on the principle of base complementarity, where fluorescent labels are used to directly or indirectly mark nucleic acid probes, allowing for qualitative or relative quantitative analysis of mutation sites by observing fluorescent signals in pathological tissue sections and cell smears. A study on drug resistance among 83 isolated and cultivated H. pylori samples from Iran revealed that the E-test identified 21 CLR-resistant strains, while FISH detected genetic mutations in 19 of these, comprising 13 strains with A2143G mutations and 6 strains with A2144G mutations. Comparing the E-test and FISH methods, 69.2% of bacteria with A2143G mutation had an MIC > 8 μg/mL, whereas 83% of strains with the A2144G mutation had an MIC < 8 μg/mL. Therefore, FISH and the E-test showed a certain degree of concordance in drug susceptibility results, indicating that FISH can serve as a method for detecting H. pylori resistance to CLR [77].

Sanger sequencing can simultaneously sequence up to 1000 base pairs (bp) with an accuracy of 98%, making it as the gold standard in nucleic acid sequencing technology [79]. Next-generation sequencing (NGS) provides a vast amount of sequencing information, with modern sequencing instruments capable of sequencing up to 15 billion base pairs, albeit with a read length limited to 700 bp, which is shorter than that of Sanger sequencing. The error rate in the assembly process of multiple sequences is between 0.1% and 15%, indicating that the necessity for the implementation of more stringent and intricate assembly criteria to guarantee precision. Although long-read sequencing can overcome this major limitation of NGS, it is relatively costly and has a lower read length, which constraining its broader application [80]. In H. pylori drug resistance testing, NGS can be used to rapidly identify changes in microbial DNA sequences in biopsy samples, isolates, formalin-fixed samples, and feces. Tran Thanh Binh et al. employed NGS to predict novel resistance loci for MTZ in H. pylori and identified two different missense mutations in the rpsU gene within MTZ-resistant strains. The rpsU gene, related to protein synthesis, has been detected in antibiotic-resistant Escherichia coli, suggesting its potential involvement in H. pylori MTZ resistance through unknown mechanisms [81]. In terms of identifying drug-resistant genotypes, Kristina G. Hulten et al. used NGS to determine the sensitivity of H. pylori to AMX, CLR, MTZ, LEV, TE, and rifabutin, achieving accuracy rates of 95.9%, 97.1%, 77.0%, 89.5%, 98.2%, and 99.4%, respectively, compared to the agar dilution method [82]. The strong consistency with the gold standard serial dilution method renders NGS a promising technology for routine clinical diagnosis. However, NGS detects a large number of unknown genetic variations, and the analysis and interpretation of this unknown data have emerged as a significant difficulty in diagnosis.

Treatment Progress in H. pylori eradication

The rising prevalence of antibiotic resistance has substantially compromised the efficacy of traditional empirical triple therapies for H. pylori eradication. In response, clinical guidelines worldwide have evolved to recommend alternative regimens grounded in up-to-date clinical trial evidence.

The 2024 American College of Gastroenterology (ACG) clinical guideline recommends 14-day bismuth-based quadruple therapy (BQT) as the preferred first-line regimen for treatment-naïve patients when antibiotic susceptibility is unknown; BQT typically comprises a PPI, bismuth, TE, and MTZ [86]. A prospective randomized trial comparing 14-day BQT with 14-day CLR triple therapy in treatment-naïve Chilean patients confirmed that BQT achieved significantly higher eradication rates [87]. Rifabutin triple therapy (rifabutin, AMX, and a PPI) and potassium-competitive acid blocker (P-CAB)-based dual therapy for 14 days are recommended as suitable empiric alternatives for patients without penicillin allergy [86]. The 2025 revised Korean guidelines, reflecting a CLR resistance rate that has risen from 17.8% to 33.3% since 2020, similarly endorse BQT and P-CAB-based regimens as preferred first-line options[88]

Acid inhibitors used in H. pylori eradication have become more diverse in recent years. Vonoprazan, a novel P‑CAB with rapid onset and potent acid suppression, has emerged as a promising alternative to traditional PPIs. A 2025 randomized controlled trial comparing 14‑day vonoprazan‑amoxicilli high‑dose dual therapy with esomeprazole‑AMX high‑dose dual therapy reported per‑protocol eradication rates of 93.0% for vonoprazan-based versus 84.5% for the esomeprazole‑based regimen[89]. Another multicenter randomized controlled trial published in 2026 comparing vonoprazan-based with rabeprazole‑AMX dual therapy found that vonoprazan‑based therapy achieved significantly higher eradication rates in both modified intention‑to‑treat (83.0% vs 78.4%, P=0.018) and per‑protocol (90.1% vs 79.7%, P=0.015) analyses [90].

For patients who fail initial eradication, bismuth quadruple therapy remains the most effective second‑line regimen. A population‑based real‑world study of 7,591 patients in Hong Kong reported that BQT achieved the highest second‑line success rate of 85.6%, whereas CLR triple therapy had the lowest success rate of 63.5% [91]. A multicenter randomized controlled trial demonstrated that vonoprazan improves the efficacy of bismuth quadruple therapy as a rescue treatment [87]. Furthermore, a 14‑day vonoprazan-based dual therapy has been shown to exhibit high eradication rates and a low incidence of adverse reactions in retreated patients, indicating its effectiveness and safety as a rescue regimen [89].

For patients with persistent infection after multiple treatment failures, third‑line regimens should ideally be guided by antibiotic susceptibility testing. Frustratingly, however, In a meta-analysis of rescue therapy for H. pylori infection including 16 studies with 1,356 patients, Nyssen et al.[14] reported that the eradication rate in the susceptibility-guided group was numerically higher than that in the empirical group (85% vs. 76%), but the difference did not reach statistical significance (risk ratios : 1.07, 95%, confidence intervals: 0.97–1.18), with considerable heterogeneity across studies (I² = 78%). This lack of superiority was consistently observed when the analysis was restricted to randomized controlled trials (risk ratios: 1.15, 95%, confidence intervals: 0.97–1.36). These findings suggest that, in the rescue setting, empirical therapy with optimized regimens (e.g., bismuth-containing quadruple therapy) is not inferior to susceptibility-guided treatment, and the routine implementation of antimicrobial susceptibility testing in clinical practice remains insufficiently supported by current evidence. Given the comparable efficacy of empirical regimens, the clinically available rescue options include rifabutin‑based triple therapy, LEV‑based therapy, and FZD‑based regimens. Rifabutin‑containing triple therapy has shown high efficacy (80.7% to 100%) in patients with a history of multiple treatment failures [92]. Vonoprazan‑AMX dual therapy has also demonstrated effectiveness as a rescue regimen; a multicenter RCT reported eradication rates of 87.50% in retreated patients, with significantly lower adverse reactions compared to susceptibility‑guided individualized therapy [93]. Furthermore, vonoprazan‑TE dual regimen for 14 days achieved eradication rates of 90.6%, which were noninferior to BQT (89.3%), while substantially reducing adverse events (10.9% vs 45.7%, P<0.001) and improving adherence [94]. In general, compared with most PPIs, vonoprazan has a longer duration of activity, a stronger acid suppression effect, and better therapeutic effectiveness, as reported[95].

Despite its theoretical advantages, the clinical implementation of antimicrobial susceptibility testing -based therapy for H. pylori faces considerable obstacles. Conventional culture-dependent antimicrobial susceptibility testing methods are hampered by demanding laboratory conditions, subjective endpoint interpretation, prolonged turnaround times, and an inherent requirement for upper gastrointestinal endoscopy to obtain gastric biopsy specimens—a procedure that is not routinely indicated for all patients, except those at elevated risk of gastric malignancy[96]. Although molecular diagnostic platforms, particularly PCR-based assays, offer enhanced reliability in detecting specific resistance-conferring mutations, they still largely depend on endoscopically collected tissue samples. Encouragingly, non-invasive antimicrobial susceptibility testing strategies are currently under investigation, including RT-PCR applied to stool specimens, which hold promise for broadening the accessibility of resistance testing [97, 98]. Nonetheless, before antimicrobial susceptibility testing-guided tailored therapy can be recommended for routine clinical practice, further prospective studies are warranted to rigorously assess its overall clinical benefit and cost-effectiveness relative to empirical treatment regimens, particularly in diverse healthcare settings.

Perspectives for the Future

The escalating burden of antibiotic resistance in H. pylori, coupled with the inherent limitations of current diagnostic and therapeutic paradigms, underscores an urgent need for strategic innovation.

A priority direction is the development and deployment of affordable, user-friendly molecular diagnostic platforms capable of multiplex detection of clinically relevant resistance mutations, particularly those associated with CLR, LEV, and MTZ resistance. Such platforms would substantially lower the technical and financial barriers that currently restrict the widespread adoption of genotypic testing in primary care and resource-limited settings. Equally important is the establishment of regional or national resistance surveillance networks to generate real-time, locally relevant epidemiological data, which would not only inform regional empirical treatment guidelines but also enable early detection of emerging resistance trends, allowing timely adjustments to first-line regimens before widespread treatment failures occur. Furthermore, antimicrobial stewardship programs should be systematically implemented at both hospital and community levels, with particular emphasis on restricting the use of macrolides and fluoroquinolones for non-H. pylori indications.

Artificial intelligence and machine learning approaches offer novel opportunities to integrate genotypic resistance data with pharmacokinetic, pharmacodynamic, and host factors, and the synthesis of such multidimensional data holds promise for establishing predictive models of treatment outcomes[99]. Non-antibiotic adjunctive strategies, including probiotic supplementation, phage therapy, and therapeutic vaccines, also warrant further investigation, as their successful development could fundamentally reduce antibiotic dependence in H. pylori management. Additionally, the discordance between phenotypic and genotypic resistance remains a critical scientific challenge, and next-generation sequencing technologies hold promise for discovering novel resistance genes and improving the predictive accuracy of molecular tests. Antibiotic susceptibility research should also take into greater consideration the interactions within the gastric microenvironment, as in vitro susceptibility testing results may not fully reflect the in vivo conditions.

Collectively, the implementation of these measures would alleviate the current reliance on empirical therapy, reduce unnecessary antibiotic exposure, slow the progression of resistance.

Conclusions

In conclusion, the escalating global burden of antibiotic-resistant H. pylori has fundamentally challenged the sustainability of empirical eradication therapies, necessitating a paradigm shift toward molecular diagnostics and personalized treatment strategies. The marked geographical heterogeneity in resistance patterns, driven by divergent antibiotic consumption, socioeconomic conditions, and regulatory practices, underscores the critical need for region-specific surveillance and tailored therapeutic guidelines. While conventional phenotypic methods remain valuable for functional susceptibility assessment, molecular genetic techniques, particularly RT-PCR and NGS offer, superior capabilities for rapid, precise identification of resistance-conferring mutations, though their clinical adoption remains constrained by cost, infrastructure demands, and the invasive nature of sample acquisition. The emergence of potassium-competitive acid blocker-based regimens and bismuth-containing quadruple therapies has provided encouraging alternatives, yet their long-term efficacy hinges on antimicrobial stewardship and continuous resistance monitoring. Looking forward, the integration of affordable multiplex molecular platforms, resistance surveillance networks, and artificial intelligence-driven predictive models, alongside exploration of non-antibiotic adjunctive strategies, will be essential to transition from empirical to precision-based management, ultimately improving eradication outcomes while curbing further resistance propagation.

Author Contributions

All authors contributed to the study conception and design. WZ: Formal Analysis, Writing – Review & Editing JL: Data Curation, Writing– Original Draft, Funding Acquisition XZ: Writing – Review JY: Writing – Review YL: Funding Acquisition YJ: Project Administration, Writing – Review & Editing

Funding

This work was supported by Science and Technology Program of Shenzhen (JCYJ20230807151201004); Guangdong Drug Administration Science and technology innovation project (2025TDB64).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no conflicts of interest.