Evolution of Helicobacter pylori Resistance to Antibiotics: A Topic of Increasing Concern

Antibiotic resistance among Helicobacter pylori strains is the major cause of eradication failure. Resistance prevalence is dynamic and can greatly vary among countries over the years. We revealed H. pylori resistance trends for five antibiotics in 14 countries through articles predominantly published in 2018–2022, since the latest data can best show the most recent trends in resistance evolution. Amoxicillin resistance generally exhibited no evolution, yet it increased in Bulgaria, Iran, China, and Vietnam. Metronidazole resistance exhibited different trends, including an increase, a decrease and no evolution in six, three, and five studies, respectively. Clarithromycin resistance increased in Australia, Belgium, Bulgaria, Italy, Iran, and Taiwan, but remained stable in France, Spain, Russia, China, Chile, and Colombia. Tetracycline resistance was low and stable except in Iran. Levofloxacin resistance increased in four European and six other countries/regions, without significant increases in France, Spain, and Chile. In Chile, triple resistance also increased. In countries such as France and Spain, resistance to most antibiotics was stabilized, while in Bulgaria, Belgium, Iran and Taiwan, resistance to three or more agents was reported. Use of non-recommended regimens, national antibiotic consumption, patient’s compliance, host factors, strain virulence, migrations, and azithromycin overuse during the COVID-19 pandemic can influence resistance evolution. New drugs, eradication regimens and diagnostic methods, such as next-generation sequencing can improve H. pylori infection control.


Introduction
Helicobacter pylori is a Gram-negative, spiral-shaped and microaerophilic bacterial species closely adapted to humans [1]. It has great medical importance as one of the most frequent causative agents of human infections. In the systematic review and meta-analysis of Zamani et al. [2] encompassing 73 countries worldwide, it has been emphasized that H. pylori infects >44% of the global human population, including >1/2 of the inhabitants of developing countries and >1/3 of those in developed countries.
These bacteria cause chronic gastritis, gastric and duodenal ulcers, gastric cancer or mucosa-associated lymphoid tissue (MALT) lymphoma [3], and although the infection is asymptomatic in 80-90% of the infected subjects, it carries some risks of severe diseases. Gastric cancer is the second leading cause of cancer-related mortality worldwide, and H. pylori has been recognized as one of the most potent carcinogens by the World Health Organization (WHO) [3].
H. pylori infection is most often acquired in childhood and becomes chronic, frequently lifelong, if not successfully eradicated [4]. H. pylori eradication (negative results from repeated tests for H. pylori detection at least 4 weeks after treatment) is curative, however however it has been constantly decreasing over the years, mainly due to bacterial resistance [5]. Different composite regimens have been used to eradicate the infection; they have usually included a proton pump inhibitor (PPI) or (more recently) vonoprazan to increase gastric pH, plus two or three antibacterial agents, sometimes with the addition of bismuth preparations [5][6][7]. Amoxicillin, metronidazole, clarithromycin, tetracycline and levofloxacin are the most frequent antibiotics used in different combinations in eradication regimens.
Despite the complex treatment regimens, antibiotic resistance is the major cause of eradication failure. H. pylori resistance to antibiotics is most often due to chromosomally encoded mutations. However, it can also appear due to efflux systems, membrane permeability changes and biofilm formation [8][9][10]. Moreover, the lack of H. pylori susceptibility testing in most laboratories, as well as side effects of eradication regimens, can additionally hamper eradication success [7].
The aim of the present review was to determine the recent evolution of H. pylori resistance to five antibiotics of choice in eradication regimens in different countries worldwide.

Methods
We reviewed some recent data on the dynamics of H. pylori resistance to antibiotics that can cause H. pylori treatment failure. We considered data from recent studies providing details on patients' numbers and characteristics, study methods, breakpoints for resistance, and resistance rates in different years. Data from recent years were included as they can best show the most recent trends in resistance evolution. For this purpose, we evaluated H. pylori resistance evolution by searching PubMed, Science Direct, and Google Scholar for publications with the following keywords or word combinations in the title or abstract: "Helicobacter pylori", "H. pylori", "antibiotic", "antibacterial", "resistance", "primary", "secondary", "evolution", "prevalence", "rate", "trend", and "increase". We included publications published since 2018, with rare exceptions.
Data from several review articles and from 14 countries, including Australia, Belgium, Bulgaria, Chile, China, Colombia, France, Italy, Iran, Russia, Spain, Taiwan, Vietnam and USA were included and discussed ( Figure 1).   Most articles were in English and only few were in Spanish. The most commonly used susceptibility testing method in the studies was an E test, while the agar dilution method, disk diffusion method and molecular tests were less frequently applied. The most commonly used breakpoints for resistance were those of EUCAST, although some studies used CLSI breakpoints or others specified by the authors (see below).
We also included our recent findings on H. pylori resistance in strains from consecutive Bulgarian patients with gastroduodenal diseases during the first study period (2007-2014 for amoxicillin and 2010-2015 for the other antibiotics) and the second period (2015-2021 for amoxicillin and 2016-2022 for the other antibiotics) [11,12], (Boyanova, this study). Most data (83.6%) in our unpublished study are from 2018 to 2022. Amoxicillin is a beta-lactam antibiotic that binds to penicillin-binding proteins (PBPs) in bacterial periplasm and affects transpeptidase activity for cross-linking of peptidoglycan molecules, thereby impeding peptidoglycan synthesis and H. pylori growth [13]. Amoxicillin is a frequently used antibiotic for H. pylori eradication in various regimens, such as: triple amoxicillin-based regimens, concomitant quadruple regimen, high-dose dual therapy, sequential therapy, hybrid therapy, as well as in some vonoprazan-based regimes [14][15][16][17][18].

Results and Discussion
Amoxicillin resistance in H. pylori usually results from mutations leading to diminished binding of the agent to penicillin-binding protein PBP1A, and mutations in PBP2 or PBP3 may additionally increase the resistance [10]. In 2006-2016, H. pylori resistance to amoxicillin was rare (0-<10%) in the WHO regions [19].
The growing amoxicillin resistance in Vietnam was explained by the frequent use of amoxicillin/clavulanic acid for various infections in the country [35]. As for Bulgaria, consumption of antibiotics has been high and increasing. From 2012 to 2021, while a significant decline in the consumption of J01 antibiotics (antibacterials for systemic use) was detected by the European Union/European Economic Area population-weighted mean overall, a significant increase was observed in Bulgaria [36].
Although the evolution of H. pylori resistance can be accounted for using the same resistance breakpoints in the same longitudinal study, the percentage of resistance depends on both the methods and the resistance breakpoints used [12,37]. It should be mentioned that the EUCAST breakpoints for amoxicillin resistance are lower (>0.12 mg/L) than those used in some studies, such as in China [33], and that some authors used a disk diffusion method to assess resistance [31].
Amoxicillin resistance rates have remained low over time (0-<2%) in countries such as Belgium, France, Australia and Colombia [20][21][22]27,29,30]. However, the present results show the need to monitor the dynamics of the resistance over prolonged periods of time due to its increase in some countries. Moreover, amoxicillin resistance was found to be an independent risk factor of H. pylori eradication failure when using clarithromycin-based triple therapy [37].

Metronidazole
Metronidazole is a prodrug which becomes active when it is activated by the reduction of its nitro group, and thus causes bacterial DNA damage [38,39]. Metronidazole and sometimes tinidazole have comparable activity and are used in different H. pylori eradication regimens, such as triple regimens, bismuth quadruple therapy, sequential therapy and concomitant quadruple regimen [15,16].
Metronidazole resistance in H. pylori can result from different mechanisms, such as decreased prodrug activation by mutations in rdxA and frxA genes, mutations leading to RecA DNA repair upregulation and other mechanisms [10,40].
The systematic review and meta-analysis of Savoldi et al. [19] revealed that in 2006-2016, primary metronidazole resistance rates most often ranged from 23 to 56% worldwide, while secondary resistance reached >62% in the Eastern Mediterranean Region and the Western Pacific Region.
Recent data showed different trends in metronidazole resistance rates in H. pylori over time. An increase in resistance was observed in six studies, a decrease was found in three reports, and no evolution was detected in five studies.
An increase in 2010/13, followed by a plateau in 2013/16, was found in one of the two Italian studies [41].
Decrease in H. pylori metronidazole resistance was also detected, although less frequently. Resistance to metronidazole diminished in Chile and Spain, as well as in the study of nine European countries [24,31,42]. No significant evolution has been reported in an Italian study, in Australia, Colombia, Iran, and in Spanish children [25,27,29,30,33,34,43] ( Table 2).
However, it is a concern that high H. pylori metronidazole resistance was present in >75% of consecutive Colombian adults in 2009 and 2015, in consecutive Chinese adult patients in 1998-1999 and 2016-2017, as well as in treated patients in France (in 2014-2018) and Taiwan (in 2019) [22,29,30,32,33].
One of the reasons for the increase in the resistance may be the wide use of nitroimidazoles in some countries to treat H. pylori and other infections, such as dental and oral infections, anaerobic infections, bacterial vaginosis, pelvic inflammatory disease and parasitic diseases. In Belgium, the rise in metronidazole resistance was probably linked to the growing number of immigrants from Africa (by 18.7%) in the period 2010-2015 [20,21].
Importantly, increasing the therapeutic dose of metronidazole to 1500 mg and treatment duration up to 14 days in the bismuth quadruple regimen can either partially or completely overcome metronidazole resistance of H. pylori [7,22,23]. It is also important that the accuracy of susceptibility testing results for metronidazole depends on the appropriate redox potential of the media used [23].

Clarithromycin
Among all macrolides, clarithromycin is the most important agent for eradication therapy of H. pylori infection. Clarithromycin is a bacteriostatic antibiotic, acting by inhibiting bacterial protein synthesis through reversible binding to the 50S ribosomal subunit in the 23S rRNA gene of H. pylori [10].
Overall, primary clarithromycin resistance in H. pylori in 2006-2016 was >15% in the European region, reaching ≥33% in the Eastern Mediterranean Region and Western Pacific Region, and secondary resistance (15 to 67%) to the agent was found in the WHO regions [19].
Clarithromycin resistance was most often linked to A2142C, A2142G and A2143G mutations in V domain of 23S rRNA, and some minor mutations outside the domain [10].
As stated by Megraud et al. [23], macrolide consumption (of intermediate-acting agents such as clarithromycin, and long-acting agents such as azithromycin) in the community can strongly affect the primary clarithromycin resistance in H. pylori several years later. The lack of clarithromycin resistance evolution in France can be due to the decrease of macrolide consumption (by >46%) from 2000 to 2015, and its stability during the last years [23]. The decrease in secondary clarithromycin resistance in French patients has been explained by increasing the use of recommended quadruple therapies as a first-line regimen [22].
By contrast, the increase in overall clarithromycin resistance in H. pylori in Bulgaria can be associated with high macrolide, lincosamide and streptogramin (J01F) consumption (5.5 DDD per 1000 inhabitants per day) in 2021, compared to that of other European countries [36].
Several other factors can be associated with the increase in H. pylori clarithromycin resistance over the years. Apart from H. pylori-associated diseases, macrolides are also used to treat upper and lower respiratory tract infections, and sexually transmitted infections. H. pylori clarithromycin resistance was >15%, with the exception of studies in Russia and Colombia [26,29,30], and the highest resistance rates (50% or more) were found in consecutive Chinese adults, treated French adults, Spanish children, and treated adults in Taiwan [22,25,32,33]. Frequent use of a triple clarithromycin-based regimen in countries with high resistance to the agent can contribute to the increase in H. pylori resistance to clarithromycin. In countries where resistance prevalence to clarithromycin is high or increasing over time, the use of clarithromycin-based triple therapy may only be appropriate if susceptibility testing is performed, and isolates are found to be susceptible to the agent [29].
Overuse or misuse of azithromycin has increased since the beginning of the COVID-19 pandemic and can also influence H. pylori macrolide resistance [44]. In Australia, migration from countries with high resistance prevalence or exposure to macrolides in food has been suggested as a factor contributing to the increase in H. pylori clarithromycin resistance [27].
Detection of clarithromycin resistance before using the agent for treatment of H. pylori infection is highly important since risks of eradication failure by clarithromycin-based regimens were about 7-fold higher (odd ratio, 6.97) in the presence of clarithromycinresistant strains than for clarithromycin-susceptible strains [15,45].

Tetracycline
Tetracycline is a bacteriostatic agent which reversibly binds to the 30S subunit of H. pylori ribosomes containing 16S rRNA, thereby suppressing protein synthesis and bacterial growth [45].
Tetracycline is one of the agents used in bismuth quadruple therapy [7,46]. However, one of the main disadvantages of the bismuth quadruple therapy containing metronidazole and tetracycline has been adverse effects observed in half of the patients [7].
In 2006-2016, primary H. pylori resistance rates to tetracycline were low (≤10%) in most countries worldwide [19]. H. pylori resistance to tetracycline was associated with single, double and especially simultaneous triple point-mutations within both copies (rrnA/B genes) of 16S rRNA [47].
Importantly, in the French study of Mégraud et al. [22], no tetracycline resistance in H. pylori was detected in the untreated and treated patients despite the launch of the single triple capsule of bismuth subcitrate, metronidazole, and tetracycline (Pylera ® ).

Levofloxacin
Levofloxacin is a third-generation fluoroquinolone with bactericidal activity, suppressing DNA gyrase of H. pylori, since, unlike other bacteria, the species lacks genes for the topoisomerase [10]. The agent is used in eradication regimens such as a levofloxacin-based triple regimen, a sequential therapy regimen, and a concomitant bismuth-and levofloxacinbased therapy [48].
Levofloxacin resistance in H. pylori results from mutations in gyrA and gyrB genes encoding DNA gyrase subunits, and especially in codons 87 and 91 in QRDR (quinolone resistance-determining region) of GyrA [10]. Primary levofloxacin resistance was ≥11-15% in most WHO regions in 2006-2016 [19]. Secondary levofloxacin resistance during the period was high in the Eastern Mediterranean Region and Western Pacific Region (30%) [19].
Recent data showed that in contrast to tetracycline, H. pylori resistance to levofloxacin displayed an increase in many European studies, such as those from Belgium, Bulgaria, Italy (two studies), and Russia, [12,20,21,26,41,43] (Boyanova, this study). The rise in fluoroquinolone resistance was also common in non-European countries such as China, Iran, Taiwan (in both untreated and treated patients), and in the overall Western Pacific region [19,[32][33][34] (Table 5).   NA-not available or not appropriate. In Iran, ciprofloxacin was tested, in all other countries, susceptibility to levofloxacin was evaluated. Methods-DDM-disk diffusion method, ADM-agar dilution method. * Nine European countries-Italy, Spain, Norway, Greece, Slovenia, Israel, Russia, France, Ireland.
Primary levofloxacin resistance increased in Belgium, Italy, and Taiwan [20,21,32,43] but did not show evolution in other countries such as France, Spain, Chile, and in the study of nine European countries [22,24,25,31,42].
No significant evolution of H. pylori levofloxacin resistance was found in treated patients in France, whereas in Taiwan both primary and post-treatment (2nd line) resistance increased [22,32].
High levofloxacin resistance rates (>30%) were found in consecutive Chinese adults Levofloxacin resistance can be associated with quinolone (J01M) use. In Europe, a significant association was observed between H. pylori resistance to levofloxacin and consumption of second-generation quinolones, such as ciprofloxacin [23]. In Bulgaria, the increase in levofloxacin resistance correlated with higher J01M use (3.9 DDD per 1000 inhabitants per day) compared to that in France, where there was no significant rise in the resistance and the J01M consumption was 1.0 DDD [36]. In France, a decrease (by >25%) in quinolone consumption was detected between 2000 and 2015, and this can explain the lack of increase in levofloxacin resistance rates in the country [22,23].

Double and Multidrug Resistance
In the systematic review and meta-analysis of Savoldi et al. [19] in 2006-2016, primary double resistance to clarithromycin and metronidazole in H. pylori varied from <10 to 19% worldwide. Overall secondary resistance to both clarithromycin and metronidazole during the period was 18% in the European Region [19].
Multidrug resistance is simultaneous resistance to ≥3 antibiotics of different categories (classes). H. pylori multidrug resistance can result from several simultaneous mutations associated with resistance to different antibiotics, however, efflux pumps, diminished drug uptake and biofilm production, can also be involved [10]. Upregulated expression of TolC homologous genes, such as hefA that increases activity of efflux pumps, was observed in multidrug-resistant H. pylori strains [40].
According to the recent studies, increasing double resistance to both metronidazole and clarithromycin was found in some countries. The increase was observed in consecutive Bulgarian patients (1.6-fold increase), untreated Taiwanese adults  [24,[29][30][31]34,43] (Table 6).   Importantly, an increase in overall triple resistance (from 3.7% 2005-2007 to 18.0% in 2015-2017) was observed in strains from untreated children and adults in Chile [31].
Although there were not many recent reports on the evolution of multidrug resistance in H. pylori, in our previous review publication on the topic [49], primary multidrug resistance varied from <10% in most of the European countries, to >40% in some countries such as Peru, and overall resistance rates of >23-36% were found in half of the studies. In pediatric patients, multidrug resistance was also found, ranging from 3.8% in Slovenia in 2011-2014 and 6.6% in Bulgaria in 2012-2021, to >20% in untreated children in China [50].
Multidrug resistance in H. pylori is a hard challenge to overcome in treatment and although there has been no significant evolution of the resistance in many countries so far, the results emphasized both the importance of laboratory susceptibility testing of the isolates, and the search for new therapeutic drugs and/or regimens.

Factors for Resistance Evolution
H. pylori infection is common and affects <20%, up to 90% of the population in different countries [51]. Antibiotic resistance in H. pylori has been increasing over time. In the 1980s, clarithromycin resistance rates were from 0% to <9% according to the review article of Lahaie and Gaudreau [52], versus >20% in many patient groups and countries during the last five years.
Important factors of H. pylori resistance evolution are the use of currently non-recommended regimens, such as triple clarithromycin-based therapy in regions with high (>15%) resistance to the agent [15,53], national antibiotic consumption, patient compliance and other host factors. High antibiotic consumption in Bulgaria can be associated with an increase in H. pylori resistance to all antibacterials, except tetracycline [36] (Figure 2). In Italy and Spain, primary antibiotic resistance varied according to the patients' age and sex [42,43]. In Italy, female sex, age (>50 years), body mass index (>25) and smoking were associated with resistance to some of the antibiotics [43].
H. pylori virulence factors were also related to resistance of some antibacterials. The systematic review and meta-analysis of Karbalaei et al. [54] showed that less virulent (vacA s2m2) strains were associated with lower antibiotic resistance rates, possibly due to lower biofilm production or lower blood flow to the stomach compared to those of more virulent strains. Using antibiofilm agents is a strategy to improve therapy of the infection [55]. Heteroresistance (different susceptibility to specific antibiotics by H. pylori subpopulations in the same patient) has also been evaluated [56].
In addition, access to antibiotics over-the-counter (without prescription), mostly in some developing countries, can increase and spread antibiotic resistance [57,58]. Other factors such as azithromycin misuse or overuse since the beginning of the COVID-19 pandemic and migrations from countries with higher resistance rates may also be of importance for H. pylori resistance evolution [27,44].

Conclusions
Knowledge of H. pylori resistance evolution to the five most commonly used antibiotics in eradication regimens is necessary to limit treatment failure.
In some countries, such as Bulgaria, Belgium, Iran, and Taiwan, growing H. pylori resistance to three or more antibacterial agents has been observed over time, while in other countries, such as France and Spain, resistance to most antibiotics used for H. pylori eradication has been stabilized.
The lack of increase in antibiotic resistance and even a decrease in resistance rates were usually related to the decrease in the national antibiotic consumption of the given antibiotic, compliance with the latest guidelines for H. pylori infection management and strongly enforced antibiotic policy in some countries, such as France and the USA [22,28].
In 2017, the WHO included clarithromycin-resistant H. pylori in high priority bacteria for antibiotic research and development [59]. Current specialists and international guidelines recommend either bismuth-or non-bismuth-based quadruple therapy for 14 days as a first-line treatment for H. pylori in regions of high clarithromycin or metronidazole resistance rates [14][15][16][17][18]. In addition, most of the current eradication regimens recommend an extended treatment duration of 14 days. These recommendations are especially important for countries with increasing H. pylori antibiotic resistance. In Italy and Spain, primary antibiotic resistance varied according to the patients' age and sex [42,43]. In Italy, female sex, age (>50 years), body mass index (>25) and smoking were associated with resistance to some of the antibiotics [43].
H. pylori virulence factors were also related to resistance of some antibacterials. The systematic review and meta-analysis of Karbalaei et al. [54] showed that less virulent (vacA s2m2) strains were associated with lower antibiotic resistance rates, possibly due to lower biofilm production or lower blood flow to the stomach compared to those of more virulent strains. Using antibiofilm agents is a strategy to improve therapy of the infection [55]. Heteroresistance (different susceptibility to specific antibiotics by H. pylori subpopulations in the same patient) has also been evaluated [56].
In addition, access to antibiotics over-the-counter (without prescription), mostly in some developing countries, can increase and spread antibiotic resistance [57,58]. Other factors such as azithromycin misuse or overuse since the beginning of the COVID-19 pandemic and migrations from countries with higher resistance rates may also be of importance for H. pylori resistance evolution [27,44].

Conclusions
Knowledge of H. pylori resistance evolution to the five most commonly used antibiotics in eradication regimens is necessary to limit treatment failure.
In some countries, such as Bulgaria, Belgium, Iran, and Taiwan, growing H. pylori resistance to three or more antibacterial agents has been observed over time, while in other countries, such as France and Spain, resistance to most antibiotics used for H. pylori eradication has been stabilized.
The lack of increase in antibiotic resistance and even a decrease in resistance rates were usually related to the decrease in the national antibiotic consumption of the given antibiotic, compliance with the latest guidelines for H. pylori infection management and strongly enforced antibiotic policy in some countries, such as France and the USA [22,28].
In 2017, the WHO included clarithromycin-resistant H. pylori in high priority bacteria for antibiotic research and development [59]. Current specialists and international guidelines recommend either bismuth-or non-bismuth-based quadruple therapy for 14 days as a first-line treatment for H. pylori in regions of high clarithromycin or metronidazole resistance rates [14][15][16][17][18]. In addition, most of the current eradication regimens recommend an extended treatment duration of 14 days. These recommendations are especially important for countries with increasing H. pylori antibiotic resistance.
Newer agents, such as vonoprazan, bismuth, in addition to some triple eradication regimens, and evaluation of newer antibiotics with improved stability at low pH and/or increased antibacterial activity, such as delafloxacin, can be considered as well [6,45,48,60,61]. However, regular monitoring of antibiotic resistance rates is utterly important to determine the appropriate eradication regimens in a given country or region.
In addition to the classical susceptibility testing methods, the use of molecular techniques [62], non-invasive tests for detection of resistance or adjuvants, such as antibiofilm substances [49] and nanoparticles [63], as well as the development of vaccines, are strategies to control both H. pylori infection and antibiotic resistance.
Most importantly, stabilizing and even reducing H. pylori antibiotic resistance, which has already been reported in some studies, is an important and achievable goal for all countries, provided that antibiotic overuse and misuse are reduced, antibiotic policy is strictly followed, and the recent guidelines are complied with in practice.  Institutional Review Board Statement: Ethical review and approval were waived for this study, since it is a review, data for Bulgaria were collected by routine diagnostic practice.

Informed Consent Statement:
This is a review article. As for Bulgarian patients, informed consent was obtained from all patients involved in the study.
Data Availability Statement: Data are contained within the review.