Antibiotic Resistance, Susceptibility Testing and Stewardship in Helicobacter pylori Infection

Abstract

It is promising to observe a worldwide decrease in the prevalence of Helicobacter pylori (H. pylori), yet consistent efforts are required to enhance treatment regimens in accordance with antibiotic stewardship in the era of growing antibiotic resistance. Various molecular mechanisms are responsible for antibiotic resistance, and thanks to technological advancements, our understanding of these mechanisms has improved, which facilitated the assessment of antibiotic susceptibility to H. pylori. Progress made in next-generation sequencing technology could potentially herald a new era, encouraging the routine use of non-invasive tests to gather data on antibiotic resistance. It is crucial that studies and clinical guidelines ensure the proposed treatment can consistently meet an acceptable level of eradication rate, taking into account the variations in antibiotic resistance across different populations. Future investigations should strive to refine the duration and dosage and reduce side effects of treatment regimens. Through personalized medicine, better treatment strategies can be formulated. It is equally important to actively encourage patient adherence to these treatments through all available strategies. Collaboration among local, regional, and international bodies is necessary to reinforce antibiotic stewardship in H. pylori treatment.

1. Introduction

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that preferentially colonizes the antrum, but may infect any part of the stomach to cause gastritis. Although most patients infected with H. pylori are asymptomatic, approximately 10–15% develop peptic ulcer disease [1], and 1–3% develop stomach cancer and lymphoma [2, 3]. The carcinogenic evolution of H. pylori-induced chronic gastritis follows the Correa’s cascade [4] via a stepwise process from active gastritis, to precursor lesions of gastric cancer, and finally adenocarcinoma. Gastric cancer is the fifth most common cancer and the fourth leading cause of cancer-related mortality across the globe [5].

H. pylori infection usually lasts for life unless it is treated. The recognition of H. pylori as an infectious disease and its associated complications by the Kyoto consensus [6] and Maastricht/Florence Consensus [7], has led to a unanimous agreement on the necessity of treating all individuals diagnosed with H. pylori infection, irrespective of symptoms. Successful eradication of H. pylori aids not only in peptic ulcer healing [8, 9] but also plays an important role in the prevention of gastric cancer both primarily and secondarily [10], thus significantly reducing the incidence and mortality of gastric cancer [11, 12, 13].

Eradication of H. pylori requires a combination of gastric acid suppressants and antibiotics. Various treatment regimens have been adopted based on local antibiotic resistance patterns, drug availability, and patient acceptability. Standard triple therapy consisting of amoxicillin or metronidazole, clarithromycin, and a proton pump inhibitor (PPI) was the gold standard in the treatment of H. pylori infections during the 90s [14, 15]. Given the emergence of clarithromycin resistance, a substantial decline in its efficacy to levels below 80–85% has been observed [16, 17]. Bismuth-based quadruple therapy consisting of bismuth, PPI, metronidazole, and tetracycline, has been shown to achieve over 90% success rate [18, 19] and has been increasingly recommended as first-line treatment [7, 20, 21]. Resistance in metronidazole varies among different regions, with a resistance rate of 43.6% across the US [22] and 78% in China [23]. Increasing the metronidazole dose and duration may overcome antibiotic resistance [24]. While sequential regimen is discouraged [25], the marginal effect of metronidazole resistance in affecting therapeutic outcome allows quadruple therapy to remain as an effective eradication regimen [26].

Antibiotic stewardship, encompassing the judicious use of antibiotics, selecting optimal treatment regimens (including the appropriate antibiotics, dosing, and duration of treatment), and ensuring sustainable access to antibiotics, is increasingly advocated to put forward better clinical outcomes for patients and minimize subsequent resistance [27]. In order to provide information for designing optimal treatment regimens, routine susceptibility testing has been recommended to be performed even before first-line treatment in compliance with antibiotic stewardship [14, 15]. Recently, antibiotic susceptibility testing obtained via culture or molecular testing for H. pylori eradication has been more readily available [7, 20, 21]. This shifts the paradigm toward personalized medicine in which H. pylori treatment regimens can be tailored according to local resistance patterns and individual profiles.

This review will provide an overview of the current resistance patterns and mechanisms of H. pylori antibiotics, as well as different antibiotic susceptibility testing methods. We will emphasize the importance of antibiotic stewardship, and illustrate how susceptibility testing has become an integral part of this paradigm. We will also highlight the new direction of H. pylori eradication and the emergence of potential therapeutic options.

2. Antibiotic resistance in Helicobacter pylori

H. pylori treatment is challenged by high and continuously rising antibiotic resistance. There are two types of resistance—primary and secondary. Primary antibiotic resistance is identified in patients who are naïve to H. pylori eradication treatment, whereas secondary or acquired resistance emerges following one or more failed attempts at eradication [28]. Eradication of H. pylori infection now relies heavily on local antibiotic resistance patterns and patients’ prior antibiotic exposure.

A meta-analysis by Savoldi et al. in 2018 has reported a pooled prevalence of >15% in both primary and secondary resistance of H. pylori to key antibiotics (clarithromycin, metronidazole, and levofloxacin) in most World Health Organization (WHO) regions [28], which exceed the common threshold for choosing alternative empiric H. pylori eradication treatment [7, 21]. Resistance profile differs among regions. In the Asia-Pacific region, a meta-analysis found an overall prevalence of primary antibiotic resistance of H. pylori to clarithromycin, metronidazole, and levofloxacin between 1990 and 2022 to be 22%, 52%, and 25%, respectively [29]. Prevalence varied considerably across study periods. From 1990–2000 to 2016–2022, the prevalence increased from 7% to 30% for clarithromycin, 37% to 61% for metronidazole, and 9% to 35% for levofloxacin, but remained consistently low for both amoxicillin and tetracycline at less than 6% [29]. In the United States, the pooled resistance prevalence of clarithromycin, levofloxacin, and metronidazole from 2011 to 2021 was 31.5%, 37.6%, and 42.1%, respectively [30]. Similar trends have also been observed in Africa and Latin America [31, 32]. Conversely, primary resistance to key antibiotics showed a decreasing trend in Europe. From 2013–2016 to 2017–2020, the primary resistance of clarithromycin, metronidazole, and levofloxacin decreased from 25% to 20%, 33% to 24.5%, and 20.5% to 18%, respectively [33].

The resistance mechanisms that H. pylori evolved are multifarious. The exact nature of such genetic modifications varies depending on the specific class of antibiotics involved. Most often, it is due to chromosomally encoded mutations [34, 35, 36]. Clarithromycin resistance is frequently caused by 23S rRNA gene mutation [37, 38, 39, 40], while levofloxacin resistance is commonly due to gyrA mutations [37, 41]. For metronidazole resistance, the mutational changes display greater diversity, and the genotype-phenotype correlation is more complex [37]. Mutations in RdxA and FrxA gene, which encodes NADPH nitroreductase, are most frequently reported [42]. Levofloxacin resistance is primarily attributed to point mutations within the quinolones resistance-determining region (QRDR) of the gyrA gene, or, less often, the gyrB gene [43]. Amoxicillin resistance is mainly attributed to mutations in the penicillin-binding proteins (PBP), namely PBP1, PBP2, and PBP3 [44]. Beyond these genetic adaptations, H. pylori can also resist antibiotics through physiological defenses such as efflux pumps and biofilm formation, which can further complicate treatment [45].

Multidrug resistance (MDR) is defined as non-susceptibility to three or more antibiotics of different classes. The prevalence of MDR is characterized by a regional variation and appears to be changing over time. A significant concern is the concurrent resistance to both clarithromycin and metronidazole, with prevalence of approximately 8–15% in Europe [28, 33, 46], 6–11% in Asia [28], and 3–11% in the Americas [22, 28, 30]. In China, double, triple, quadruple, and quintuple resistance rates were reported to be 23, 20, 6, and 4%, respectively between 1998 and 2007 [47]. A study in the Tibet Autonomous Region of China has further demonstrated a high resistance rate where only 3 (2.0%) out of the 153 H. pylori strains isolated were susceptible to all the seven tested antibiotics [48]. In Spain, triple resistance decreased from 3.3% in 2013 to 1.8% in 2015, and then increased to 2.4% in 2017 [49]. The exact mechanism of MDR is not well known, but various mutations that simultaneously induce resistance to different classes of antibiotics are believed to confer a cumulative MDR profile. Apart from biofilm formation and increased efflux pump commonly described in single drug resistance, additional mechanisms include coccoid formation. The coccoid form is an inactive form of H. pylori that slows metabolism to facilitate survival [50], which substantially increases the minimum inhibitory concentrations (MIC) of different antibiotics. Subsequent ultrastructural modifications in the cell membrane and metabolic pathways contribute to the development of MDR by reducing drug target exposure and drug penetration [51].

Heteroresistance is a term indicating the diverse responses to specific antibiotics by bacterial subpopulations in the same patient, which do not respond to antibiotic therapy in vitro or in vivo [52]. Currently, there are no recommendations for the treatment of H. pylori infections caused by heteroresistant strains in the clinical setting. Given the great interest in developing new strategies to enhance the eradication rate of H. pylori eradication therapy, apart from strategies tackling the single- or multi-drug resistance, future guidelines should include a personalized aspect in the protocol to address heteroresistance to H. pylori.

3. Antimicrobial susceptibility testing for Helicobacter pylori

H. pylori treatment regimen is considered to be acceptable if it achieves at least 90% cure rate [17], while an optimized regimen should reliably achieve ≥95% cure rates [53]. H. pylori’s resistance to key antibiotics like clarithromycin, levofloxacin, or metronidazole is very common. The increasing resistance of H. pylori to antibiotic therapy necessitates local availability of susceptibility tests for individuals, as well as regional and national monitoring programs, to develop eradication strategies. Antimicrobial susceptibility testing (AST) for H. pylori can be primarily categorized into two types: culture-based techniques and molecular-based methods. The details of each approach, including their application, performance, advantages, and disadvantages, will be discussed below.

3.1 Culture-based techniques

Culture-based techniques are considered the gold standard for AST of H. pylori. These techniques employ the phenotypic identification of susceptibility and resistance based on the minimal inhibitory concentration (MIC) of the tested antibiotic [54, 55]. Traditional culture-based techniques can be categorized into quantitative or qualitative methods. Qualitative methods, for instance, the disc diffusion method, allow broad categorization of antibiotic susceptibility or resistance without an exact MIC value. Quantitative methods include agar dilution, gradient diffusion susceptibility testing (also known as the E-test), and broth dilution method, which express results numerically. Details of each method are outlined below.

3.1.1 Disk diffusion method

In the disc diffusion test, filter paper discs impregnated with various pre-defined concentrations of antibiotics are placed on an agar plate that has been pre-swabbed uniformly with pure bacterial culture. The agar plate is then incubated at a certain temperature optimal for the growth of a microorganism. The incubation time is dependent on the natural growth rate of the tested microorganism. During the incubation, the antibiotic diffuses from the disk to the agar, with the concentration decreasing as the distance to the disk increases. If the microorganism is susceptible to an antibiotic in the disk, there is a clear inhibition zone around the disk. The size of the inhibition zone indicates categorial results, for instance, susceptible, intermediate, and resistant, and can be related to MIC. The disk diffusion method is the simplest and most economical routine susceptibility testing. However, it is generally not recommended for H. pylori, a slow-growing bacterium. Disk diffusion has been validated in France to detect macrolide resistance [56]. However, this method has not been validated for the other antibiotics.

3.1.2 Agar dilution method

The agar dilution method, which is considered the reference method to compare other techniques, was first proposed by the Clinical Laboratory Standard Institute (CLSI) as the method to be used for H. pylori clarithromycin susceptibility testing, which later extended to testing of other antibiotics such as amoxicillin, rifabutin, ciprofloxacin, etc. Maastricht VI Florence consensus report has affirmed the agar dilution method as the gold standard for AST. Metronidazole, however, is a special case in which inter- and intra-laboratory reproducibility is lacking, and a correlation between the susceptibility results and H. pylori eradication is not evident [57]. Therefore, since the Maastricht III 2005 consensus report, routine metronidazole susceptibility testing has been discouraged.

3.1.3 E-test

E-test is a method derived from the disk diffusion test that provides quantitative MIC results. In the E-test, a commercially available thin plastic test strip with a continuous gradient of antibiotic is placed on an agar plate, which has been pre-swabbed uniformly with pure bacterial culture. The agar plate is then incubated during which a continuous gradient of concentrations of antibiotic is created at the intermediate vicinity of the test strip. Since the concentration gradient remains stable for at least 18–24 hours, the long enough duration allows most fastidious and non-fastidious microorganisms, including H. pylori, to be tested. After overnight incubation, a clear ellipse-shaped inhibition zone appears along the test strip where the MIC value could be read from the scale point, taken as the intersection between the edge of the ellipse and the strip. The E-test method has the advantage of being a quantitative method with a direct expression of MICs. Except for metronidazole, a good correlation has been found between the E-test and the standard agar dilution method.

3.1.4 Broth dilution method

In the broth dilution test, a known number of microorganisms are inoculated into tubes containing dilution of antibiotics where MIC will be measured. The broth dilution method has the advantage of being adaptable to automation. It is typically used to test pure isolates of non-fastidious aerobic microorganisms, which are easily grown by overnight incubation. However, it is not commonly used for H. pylori because of the difficulty in growing this bacterium in broth. Supplementation of media such as brucella broth or Mueller Hinton broth allows a good correlation with the E-test, except for metronidazole.

Susceptibility testing is currently available from a number of diagnostic laboratories. Given the slow-growing nature of H. pylori, the broth dilution method is not routinely used as previously mentioned [37, 58, 59]. For routine testing, the minimum is to test a macrolide. The disk diffusion method, with an erythromycin disk, is the simplest cost-effective strategy. To quantify the exact MIC, an E-test or agar dilution method can be used. The agar dilution method has proven reliable and can be adapted for simultaneous testing of multiple H. pylori strains [55, 60]. E-test quantifies the disc diffusion method and strongly correlates with the agar dilution method for most antibiotics. The only exception is metronidazole due to the absence of anaerobic preincubation of plates used [58, 59], lack of reproducibility, and low predictive value for treatment efficacy. If necessary, the best method is agar dilution or its simplified version, known as the break-point method. The breakpoint susceptibility test consists of inoculating antibiotic concentration equal to the breakpoint concentration, which defines resistance to classify the strains as susceptible, intermediary, or resistant. Compared to the agar diffusion method for the metronidazole susceptibility test, a correlation of 94% has been shown [61], although reproducibility was not studied.

Culture and susceptibility testing are performed using fresh or frozen gastric biopsies obtained via endoscopy. Adequate tissue sampling and proper handling are needed for accurate antimicrobial analysis. At least 2 biopsies (1 from the corpus and 1 from the antrum) should be taken, preferably using large cup forceps. Samples collected have to be placed in a transport medium (e.g., Brucella broth with 20% glycerol, Portagerm pylori, or an equivalent). Biopsy specimens should be transported and processed for culture as soon as possible, ideally within six hours. If processing is delayed, refrigeration is recommended. Biopsies frozen at −70°C in Brucella broth with glycerol remain stable for months [62].

The efficacy of these methods greatly depends on the conditions under which the tests are performed, and the quality of the samples provided. Factors such as the biopsy site, the number of samples taken, the quality of the sample, the time interval between sampling and culture, and even transport conditions like temperature and air exposure can significantly affect the reliability of these methods [63, 64]. Consequently, the process becomes costly, labor-intensive, and time-consuming, often taking up to two weeks to yield results [37, 63]. H. pylori culture, an integral part of this process, is complex due to the bacterium’s requirement for a microaerophilic environment and a specialized medium that doesn’t interfere with the AST. In specific to metronidazole, redox variations in the medium can affect metronidazole susceptibility testing [35, 64]. Furthermore, the interpretation of results is subjective and highly dependent on experimental conditions [65]. A recent systematic review underscored these limitations by revealing a failure of bacterial growth in about 20% of the cases studied [66]. The study also found that, although culture-tailored treatment achieved an optimal eradication rate of over 90% when used before first- and second-line regimens [7, 53], it was ineffective after two or more eradication failures [66]. To tackle the time, cost, and intensive labor required for culture-based AST methods, molecular (or genotypic AST) is recommended as an alternative for the detection of AST.

3.2 Molecular-based techniques

Molecular-based techniques employ the genotypic detection of resistance to the tested antibiotics. These methods facilitate the identification of specific mutations that lead to antibiotic resistance. Molecular-based tests can be categorized into polymerase chain reaction (PCR) assays and next-generation sequencing (NGS).

3.2.1 PCR-based assay

The PCR assay uses nucleic acid amplification to detect specific genes responsible for antibiotic resistance. The microorganisms isolated from specimen samples are lysed with their DNA extracted and fragmented. Targeted genes subject to antibiotic resistance are identified from genomic analysis of the amplified DNA fragments.

Common PCR-based assays include real-time polymerase chain reaction (RT-PCR), hybridization with oligonucleotide probes, and analysis of restriction fragment length polymorphism (RFLP). RT-PCR is a repetitive cycle of target DNA synthesis that allows the determination of the presence of H. pylori and mutation types, as well as its quantification. Fluorescence in situ hybridization (FISH) is used to detect H. pylori antibiotic resistance via histological preparations, where the result is interpreted under a fluorescent microscope. The PCR-RFLP method is based on the amplification of DNA gene regions and selective cleavage of PCR products using restriction endonucleases that recognize mutation sites.

Most available PCR-based assays primarily focus on testing common point mutations in 23S rRNA for clarithromycin resistance and in gyrA for levofloxacin resistance [37, 63, 67]. However, the mechanism of metronidazole resistance is not well understood, and thus, a definitive panel of point mutations accounting for metronidazole resistance has not yet been described [68].

Recent advancements in assays for detecting clarithromycin resistance can be applied to both biopsy and stool specimens, enabling simultaneous detection of the presence of H. pylori and clarithromycin resistance. Some assays can even differentiate between high and low resistance levels based on the point mutation [69, 70]. However, the sensitivity of molecular assays in detecting H. pylori using stool samples varies—some studies have reported high sensitivity up to 93.8% [71], and others have found lower sensitivity ranging from 63% to 84% compared to gastric biopsy specimens [72, 73, 74]. This discrepancy may be explained by variable DNA extraction methods and the molecular assays selected in the testing process. For instance, the use of FFPE gastric biopsy samples may lead to false negatives, as the DNA can be broken into smaller fragments by the fixative [75, 76, 77]. The accuracy of these assays can be influenced by factors such as the sample’s quality and the condition and purity of the DNA. Lately, quantitative PCR using gastric fluid collected via string-test has been proposed for the detection of H. pylori and its resistance against clarithromycin and levofloxacin [78].

Gastric fluid collection via string test was done by asking the patient to swallow and retain a gelatin capsule containing a cotton string absorbed with water for one hour. Compared to conventional culturing techniques, string-qPCR test allows resistance test to be done in a non-invasive, low-cost, simple, and rapid manner [79]. The high eradication rate greater than 90% indicates its feasibility and applicability in clinical practice [80].

3.2.2 Next-generation sequencing

The Sanger sequencing method is the analysis of the nucleotide sequence of the studied DNA section. Instead of requiring two primers from opposite strands, which is required in PCR amplification, Sanger sequencing allows amplification using only one primer. It has the advantage over PCR methods in reducing errors associated with improper DNA amplification and subsequent interpretation. With Sanger sequencing, sequences up to 1000 base pairs long could be read in one cycle with a high accuracy of 98% [81]. The precision and robustness of Sanger sequencing contribute significantly to the scientific basis of clinical investigations. However, the inability to detect large deletions or duplications of the sequence has moved Sanger sequencing (also known as first-generation sequencing) to next-generation sequencing.

Next-generation sequencing (NGS), a step beyond Sanger and Maxam-Gilbert sequencing, is a potential substitute for culture-based methods and current PCR-based assays. These novel methods enable the identification of complex genetic variants involved in antibiotic resistance, offering an advantage through their ability to perform massively parallel sequencing of DNA and RNA at a relatively lower cost [82]. Modern sequencers have a capacity of more than 15 billion base pairs per run, a maximum read length of more than 600 base pairs, and the ability to analyze up to 96 samples per run [83]. One significant application of NGS technology is whole-genome sequencing (WGS). This technique, which sequences an organism’s entire genome, has revolutionized the prediction of antibiotic resistance merely based on point mutations identified on target genes [35]. Furthermore, it enables the detection of novel genetic mutations in clinical isolates, which addresses the limitations of conventional PCR-based assays that can only detect well-known and prevalent mutations. The applicability of WGS extends beyond detecting novel resistance-related mutations in clarithromycin and levofloxacin [36, 84], reaching also to metronidazole [85] and amoxicillin [36, 86]. A study conducted recently in Shenzhen, China, discovered a novel Gln31Arg mutation in the fliJ gene in both clarithromycin- and levofloxacin-resistant H. pylori strains, and a Ser176Thr/Ala mutation in the cheA gene in levofloxacin-resistant strains [84]. Another study in China discovered new mutations in metronidazole-resistant H. pylori strains, specifically N118K in Fur, Q24K in Ribf, and K219Q/N and H705fs in Omp11 [85]. Two other studies revealed a total of seven novel hypothetical genotypes in pbp1A, potentially contributing to amoxicillin resistance [36, 86]. The identification of new candidate genes might offer a better genotypic-phenotypic correlation than currently known genes, enhancing our understanding and management of resistance.

The accuracy of NGS in predicting antibiotic resistance has been found to be satisfactory and largely equivalent to culture-based methodologies, which has been widely accepted as the gold standard for AST. Hulten et al. have illustrated a strong concordance between NGS and agar dilution methods with both clinical isolates and FFPE gastric biopsies, in particular for clarithromycin (k = 0.900 and k = 0.812 respectively, p < 0.01) and levofloxacin (k = 0.782 and k = 0.750 respectively, p < 0.01), albeit less satisfactory for metronidazole and amoxicillin [87]. Building upon these findings, Moss et al. have delved deeper into the accuracy of NGS in predicting antibiotic resistance in stool samples. They found a 91.4% concordance rate between the results from stool samples and those from FFPE gastric biopsies, and a 92.2% concordance rate with fresh gastric specimens [88]. The agreement between stool and fresh gastric samples was substantial for clarithromycin (k = 0.94), levofloxacin (k = 0.88) and metronidazole (k = 0.89). These results highlight the advantage of NGS over conventional PCR assays, which may not be sensitive enough when performed on stool samples, thus positioning NGS as a potentially robust, non-invasive tool for AST.

However, NGS is not without its limitations. Concern about the potential inaccuracy of WGS when applied to gastric biopsies was raised due to the predominance of human DNA and the relatively low content of bacterial DNA [36, 89, 90]. As a result, careful selection and application of appropriate DNA extraction methods are critical. Moreover, the genetic makeup of an organism and the actual observable traits may not always correlate. This discrepancy necessitates further research to validate the predictive accuracy of new genotypes identified by NGS. Such verification can be done using phenotypic outcomes and clinical observations or by retrospectively analyzing the sequencing data [37, 91, 92]. Moreover, current evidence on the accuracy of NGS or WGS in predicting antibiotic resistance mainly lies on clarithromycin, levofloxacin, amoxicillin, and metronidazole. Studies on other antibiotics like tetracycline and rifabutin are limited by small sample sizes due to the low prevalence of antibiotic resistance [35, 87]. Therefore, more comprehensive studies featuring broader gene coverage, larger sample sizes, and multi-center designs spanning different geographical regions are warranted. To facilitate the analysis and application of NGS data in routine clinical practice, there is also a pressing need for the development of standardized, user-friendly computational software and tools [36, 37].

Molecular-based methods provide several advantages over culture-based approaches. They offer a higher degree of standardization and reproducibility, which provides the opportunity for rapid analysis and enables same-day diagnosis. It is more cost-effective as multiple samples can be tested together, and evaluation is simpler without culture. Culture and susceptibility testing require fresh or frozen gastric biopsies. Molecular testing can be performed on the same material and, in addition, can utilize specimens that do not require immediate processing or that are obtained non-invasively [63], including formalin-fixed paraffin-embedded (FFPE) gastric biopsy samples, stool samples, or even gastric juice [37]. Furthermore, molecular test has the advantage over culture-based tests in detecting heteroresistance, which is particularly important in genetic clarithromycin resistance. De Francesco has reported that H. pylori infection was cured less frequently in patients with pure resistant strains (46%) than in those infected with hetero-resistant strains (78.5%) or susceptible strains (94.5%) [93].

However, one shoe does not fit all. It must be noted that the majority of molecular tests do not detect resistance based on uncommon genetic mechanisms. Rare mutations within the rrl and gyrA gene regions, resistances of H. pylori strains that lie outside the rrl and gyrA genes, or other possible mechanisms of clarithromycin and quinolone resistance should also be considered. Also, molecular-based tests cannot distinguish between live and dead cells [94]. Despite the fact that NGS may detect uncommon mutations, the correlation and significance between genotypic resistance and clinical outcomes are yet to be verified. Therefore, results from phenotypic AST approaches are considered more reliable in general, and it was still the gold standard in detecting antibiotic resistance in the current guidelines.

4. Antibiotic stewardship in the treatment of Helicobacter pylori infection

The emergence of antibiotic resistance to H. pylori is attributed to subtherapeutic levels of an antibiotic, which promotes the survival and natural selection of resistant strains [37]. Antibiotic stewardship is increasingly recognized as a solution to this problem. A series of measures have been advocated to emphasize the judicious use of antibiotics, to encourage the selection of optimal treatment regimens, and to guarantee sustainable access to antibiotics for those who require them [53, 95, 96].

4.1 Issues of the “better-than approach” in H. pylori treatment and research

Traditional methods for the treatment of H. pylori adopted a trial-and-error approach in which treatment regimens are chosen based on their relative effectiveness compared to conventional empirical regimens, rather than on the absolute cure rate [53, 96]. The conventional “better-than” approach used in clinical trials poses a concern. In cases where none of the treatment arms reach the optimal success rate as defined by antibiotic stewardship, the value of the trial may be compromised [96]. Ethical issues might also arise, as participants may not be informed of potentially suboptimal eradication rates, which have been anticipated based on previous experience [16, 96, 97, 98]. Furthermore, overemphasizing relative comparisons in meta-analyses may potentially skew the results, given the inherent heterogeneity among the included clinical trials, such as geographical variations in resistance patterns [96]. Antibiotic stewardship encourages a shift toward therapies with reliably high cure rates [53].

4.2 Susceptibility-guided therapy vs. empirical therapy in achieving antibiotic stewardship

The core principle of antibiotic stewardship is that antibiotics should only be employed to which the bacteria show susceptibility. To achieve an optimal efficacy, treatment regimen should either be guided directly based on AST or indirectly based on test-of-cure results. Yet, the customary empirical therapies employed by physicians in treating H. pylori often do not reflect the latest knowledge of local antibiotic resistance patterns, likely due to the unavailability of such data or its delay in dissemination to clinicians. Five meta-analyses have demonstrated that susceptibility-guided therapy (SGT) outperforms empirical regimens in first-line treatment [99, 100, 101, 102, 103]. However, it should be noted that these studies exhibited substantial heterogeneity. The differences might not be statistically significant when each empirical regimen is scrutinized independently. The most recent meta-analysis revealed that SGT, by either culture- or PCR-based method, outperformed the empirical clarithromycin-containing triple therapy as a first-line treatment. This was proven in areas with high clarithromycin resistance (>20%) (RR: 1.13, 95% CI: 1.03–1.25), as well as in regions with low clarithromycin resistance (RR: 1.24, 95% CI: 1.15, 1.32) [100]. However, no significant difference was noted in second- or third-line treatments, nor in first-line quadruple therapy [100]. With regard to NGS, recent evidence shows that treatment derived from the routine evaluation of mutations associated with antibiotic resistance using NGS on gastric biopsies offered 4.4 times greater odds of eradication compared to regimens adopted from recommendations provided by NGS-based susceptibility testing reports [104]. However, comparative studies on NGS using other samples, such as stool, are sparse.

The cost-effectiveness of a susceptibility-guided strategy is also an important consideration in H. pylori eradication. Much like the cases with efficacy, studies evaluating the cost-effectiveness of susceptibility-guided strategies to empirical treatments have not reached a consensus [64]. Although the continuous refinement of molecular-based methods holds promising results in reducing costs compared to culture-based testing, further studies are necessary to substantiate this claim [7, 105]. Two Korean studies reported that dual priming oligonucleotide (DPO)-PCR-based SGT would only be more cost-effective than empirical triple therapy if the eradication rate of the latter drops below 75–80% [106, 107]. Nonetheless, a study from Korea observed lower total medical costs for SGT as compared to empirical treatments, including triple therapy, sequential therapy, and BQT. The cost of implementing PCR-based SGT in these studies ranges between USD$90.3 [108] and USD$503.5 [109]. The integration of NGS in the design of SGT for routine clinical practice is currently impeded by several factors, including the need for specialized machinery, software, capability in managing extensive genomic data, and personnel trained in the operation of such systems. These collectively contribute to the high cost of NGS, which can reach up to USD$2000–3000 per sample merely for pathogen detection alone [110]. Therefore, routine NGS usage may not be feasible in the short term due to these financial considerations.

It may be conceivable that empirical regimens given according to updated local antibiotic susceptibility patterns are more cost-effective than SGT. Nevertheless, it is essential to continually monitor the performance of these empirical regimens, ensuring the up-to-date adaptation to the ever-changing antibiotic susceptibility patterns.

4.3 Measures to achieve antibiotic stewardship in H. pylori treatment

Routine analysis of clinical records comprising diagnosis, treatment, and tests in confirmation of treatment success is an indirect way to evaluate antibiotic susceptibility [53, 96, 111]. Despite the economic advantage and simplicity, such measure is not frequently utilized. The most recent Maastricht VI Florence consensus report has recommended the routine use of susceptibility tests, even before prescribing first-line treatment, with respect to antibiotic stewardship [7]. This recommendation sparks a contrast to earlier guidelines, which suggested that AST was only necessary for patients with failed eradication attempt [112]. Given that molecular methods for AST are increasingly more accessible and affordable, the ultimate goal of shifting toward personalized medicine is one step forward. This individualized strategy aims to maximize eradication rates and minimize the use of inappropriate antibiotics [53, 96, 113].

Implementing antibiotic stewardship in the treatment of H. pylori demands a concerted effort from multiple stakeholders. The study showed that compliance was the factor most associated with eradication rates across all evaluated treatment regimens [17]. The importance of drug compliance must be emphasized to patients by their physicians during consultations or by educational campaigns. Physicians should also receive education on the latest trends in antibiotic resistance patterns and relevant guidelines regarding antibiotic use. Local healthcare authorities should take the initiative to establish antibiotic stewardship programs that incorporate routine AST in clinical settings, monitor antibiotic prescriptions and resistance patterns, and provide treatment guidelines tailored to their local resistance pattern. At the international level, consensus reports and guidelines play a vital role in promoting antibiotic stewardship measures and providing evidence-based treatment recommendations. Existing antibiotic surveillance programs, both locally and internationally, should include H. pylori if it is not already incorporated. The collective contributions from all stakeholders are essential to ensure the successful application of antibiotic stewardship in the treatment of H. pylori infection.

5. Adjuncts and new agents in H. pylori eradication

The changing profile of H. pylori antibiotic resistance greatly affects the efficacy of empirical therapies. Despite the appropriate eradication regimens, persistent infection was found in around 10% of patients. Continuous development of better treatment regimens and discovery of novel agents against H. pylori infection are required [114].

Recently, vonoprazan, a potassium-competitive acid blocker (P-CAB), which demonstrates greater gastric acid-suppressive potency than PPIs, has been incorporated into eradication treatments. Stronger acid suppression by keeping the gastric acidity at a pH greater than 6 is the key in H. pylori eradication. Such a microenvironment promotes bacterial growth and renders antibiotics to be more efficient. Several clinical trials conducted in Asia have highlighted the potential of vonoprazan in achieving eradication rates of over 90% [115, 116, 117, 118, 119, 120, 121, 122]. As such, Maastricht VI consensus has recommended using P-CAB–antimicrobial combination treatments in patients with evidence of antimicrobial-resistant infections.

Probiotics also play a role as an add-on to eradication. It can enhance antibiotic activity and inhibit resistance by altering gastric microenvironment pH, modulating immunity, producing antioxidants, and affecting H. pylori colonization [123]. Probiotics, mainly Lactobacillus, Saccharomyces boulardii, or Bacillus clausii, were studied. A meta-analysis found that patients treated with probiotics had a higher eradication rate than those not treated with probiotics (80.3% vs. 72.2%). A growing number of systematic reviews and meta-analyses have also observed decreased side effects, such as diarrhea caused by H. pylori eradication therapies [123].

Few novel drugs have been proposed to treat H. pylori infection. Finafloxacin, a novel fluoroquinolone, was shown to have high antimicrobial efficacy against H. pylori in an acidic environment at pH = 5 [124]. Flavodoxin inhibitor, an agent that targets the unique flavodoxin component of an essential metabolic pathway of H. pylori, was shown to have bactericidal activity against H. pylori cells [125]. Further evaluation is needed to ensure the safety and adequate effectiveness of these agents.

Anti-H. pylori vaccine development may be a new strategy to reduce the prevalence of H. pylori infection. A phase III randomized controlled trial reported a satisfying result of vaccination success in 71.8% of Chinese children within the first year [126]. However, more data is needed to support its use where epitope mapping, determination of antigen choice, genomic approach, and safety assessment have to be evaluated.

6. Conclusion

To combat the growing threat of H. pylori resistance, there is an imperative need for a multifaceted approach, including the development of new antibiotics, the implementation of stewardship programs to prevent overuse, the improvement of diagnostic methods to tailor therapy to individual resistance profiles, and increased funding for research in alternative therapies and the development of standardized, user-friendly computational software and tools. The implementation of antibiotic stewardship is crucial to maintaining the efficacy of current treatment protocols. A key aspect of antibiotic stewardship involves carrying out antibiotic susceptibility testing prior to the initiation of empirical treatment. The major technological strides have paved the way for molecular-based sequencing techniques, enabling swift and convenient detection of antibiotic resistance. In particular, stool-based molecular tests may prove to be an integral part of routine clinical practice going forward due to their ease of use and prompt results. Additionally, WGS holds the potential to uncover novel genetic mutations related to antibiotic resistance. However, these findings must be substantiated through rigorous validation studies to ensure their clinical relevance and accuracy (Table 1 and Figure 1).

	Prevalence	Resistance mechanisms
Clarithromycin	Europe: 18%; America: 10% [28]; Southeast Asia: 10% [28]; African: 29.2% [31]	Point mutations in 23S rRNA gene
		Increased antibiotic efflux
		Alteration of outer membrane proteins
Metronidazole	Europe: 32%.; America: 23%; China: 87.8%; Oceania: 50% [127]	Mutations in metronidazole-reducing enzymes coding genes, mainly RdxA coding gene, frxA or fdxB gene
		Increased antibiotic efflux
Levofloxacin	Europe: 20% [33]; America: 14% [28]; Asia: 18% [128]; Africa: 17.4% [31]	Point mutations in quinolones resistance-determining region (QRDR)
Amoxicillin	Europe: 0.4% [33]; Asia: 3% [128]; Africa: 72.6% [31]	Mutations in the penicillin-binding proteins
		Increased antibiotic efflux
		Production of β-lactamase
Tetracycline	Europe: <1% [33]; America: 0.87% [30]; China: 1.18% [129]	Point mutations in 16S ribosomal primary binding site
		Increased antibiotic efflux

Table 1.

Resistance rate and resistance mechanism of antibiotics commonly used in Helicobacter pylori eradication therapies.

Figure 1.

Comparison of culture-based and molecular-based antibiotic susceptibility testing (AST) methods.

Funding

This research received no external funding.

Conflicts of interest

The authors declare no conflict of interest.

Data availability statement

No new data were created or analyzed in this study.