**4. Regional Characteristics of** *H. pylori* **Resistance**

*H. pylori* has regional resistance patterns. In Europe, resistance rates of *H. pylori* to clarithromycin are 18–21.4%, levofloxacin −11.0–16.3%, to metronidazole −39.1–56%. At the same time, in the countries of Central and Southern Europe these rates are significantly higher in comparison to the countries of Northern Europe (Figure 2) [36].

**Figure 2.** Antibiotic resistance rates (in %) in Europe.

There has been an increase in *H. pylori* resistance to clarithromycin, levofloxacin, and metronidazole in Europe (Figure 3).

**Figure 3.** Dynamics of H. pylori antibiotic resistance (%) in Europe. 1998: *n* = 1227 (22 centers, 17 countries); 2008: *n* = 1893 (32 centers, 18 countries); 2018: *n* = 1332 (24 centers, 18 countries).

In the USA, resistance to clarithromycin has been reported at the level of 10% [37]. In China, primary *H. pylori* resistance to clarithromycin, metronidazole, and levofloxacin is estimated at 28.9%, 63.8%, and 28%, respectively. Similar rates of *H. pylori* resistance are observed in South Korea [38,39].

General rates of antibiotic resistance in Southwest China are shown in Figure 4 [40].

**Figure 4.** Antibiotic resistance rates (%) of *H. pylori* in Southwest China.

A study was conducted to investigate the changes in antibiotic resistance over time among children in southeastern China. *H. pylori* was cultured from gastric biopsies obtained from children in the time period from 2015 to 2020. Sensitivity to clarithromycin (CLA), amoxicillin (AML), metronidazole (MTZ), furazolidone (FZD), tetracycline (TET), and levofloxacin (LEV) was evaluated. Previously reported data from 2012 to 2014 was used to compare temporal trends in antibiotic resistance. A total of 1638 *H. pylori* strains (52.7%) were isolated from biopsies of 3111 children. The resistance rates to CLA, MTZ, and LEV were 32.8%, 81.7%, and 22.8%, respectively (Figure 5). Single resistance was found in 52.9% of strains, double resistance in 28.7%, and triple resistance in 9.0%. The overall resistance rate and resistance rates to CLA, MTZ, LEV, CLA + LEV, and CLA + MTZ + LEV increased linearly every year. All types of resistance, except single resistance, clearly increased from 2015 to 2017 and from 2018 to 2020 compared with 2012–2014. Double resistance to CLA + MTZ increased significantly with age. The resistance rate to CLA and triple resistance to CLA, MTZ, and LEV were higher in children with previously treated for *H. pylori* infection in comparison to the ones who did not receive any treatment. The rates of antibiotic resistance of *H. pylori* were found to be at high levels in a large cohort of children in southeastern China from 2015 to 2020 [41].

**Figure 5.** *H. pylori* antibiotic resistance rates (%) in children in Southeast China.

The number of *H. pylori* strains studied in Russia is limited and most studies were carried out about 10 years ago. According to a recently published meta-analysis, in Russia, the level of *H. pylori* resistance to clarithromycin is 10%, to levofloxacin—20%, metronidazole— 34%, amoxicillin—1.35% and tetracycline—0.98% (95% CI) (Figure 6) [42].

Data on the low resistance of *H. pylori* to clarithromycin contradicts the data obtained from clinical practice, reflected in the European register of *H. pylori* infection management (Hp-EuReg). According to Hp-EuReg the effectiveness of classical triple therapy in Russia is only 80% with a prescription frequency of 56% [43]. Based on the data from the European registry Hp-EuReg, resistance of *H. pylori* to clarithromycin in Russia is 24%, to levofloxacin—27%, to metronidazole—29% [44].

During the revision of our article, a review paper on the evolution of *H. pylori* resistance to antibiotics by Lyudmila Boyanova was published [45]. The study included review articles on antibiotic resistance data from 14 countries, including Australia, Belgium, Bulgaria, Chile, China, Colombia, France, Italy, Iran, Russia, Spain, Taiwan, Vietnam, and the United States. The most commonly used susceptibility test in these studies was the E-test, the agar dilution method, disk diffusion whereas molecular methods were less common. Sensitivity criteria according to EUCAST and CLSI were used. The dynamics of resistance to amoxicillin, metronidazole, tetracycline, levofloxacin were evaluated. It has been shown that in some countries, such as Bulgaria, Belgium, Iran and Taiwan, there has been an

increase in *H. pylori* resistance to three or more antibacterial drugs over time, in France and Spain, on the contrary, resistance levels to most antibiotics have stabilized. The absence of antibiotic resistance growth and even a decrease in resistance levels were usually associated with a decrease in the consumption of that antibiotic in the country, adherence to the latest recommendations for the treatment of *H. pylori* infections, and strict antibiotic policies in countries such as France and the United States [46,47].

**Figure 6.** Antibiotic resistance rates (%) in Russia.

Table 3 presents a summary of studies regarding *H. pylori* genetic resistance in world populations. Colombia and South America are characterized by relatively high rates of *H. pylori* infection and stomach cancer. Antibiotic resistance was evaluated for 28 strains of *H. pylori* isolated from gastric biopsy samples from residents of two Colombia regions: with a high risk of gastric cancer (HGCR), and 31 strains from a region with a low risk of gastric cancer (LGCR). Mutations leading to antibiotic resistance were investigated by PCR for all isolates, and for 29 isolates whole genome sequencing was performed. None of the strains were resistant to amoxicillin, clarithromycin, or rifampin. One strain was resistant to tetracycline and had the A926G mutation in the *16S rRNA* gene. Levofloxacin resistance was observed in 12 of 59 isolates and was mainly associated with N87I/K and/or D91G/Y mutations in *gyrA*. Most of the isolates were resistant to metronidazole, and this resistance was significantly higher in the low-risk gastric cancer group (31/31) compared to the high-risk gastric cancer group (24/28). Mutations in the *rdxA* and *frxA* genes were present in almost all metronidazole-resistant strains [48].

Sequencing of the *H. pylori 23S rRNA* gene in the Iranian population showed that the most common mutations leading to antibiotic resistance to clarithromycin are the A2143G and A2142 mutations [49].

During the analysis of the *rdxA* and *frxA* gene in 12 metronidazole-resistant and 10 metronidazole-sensitive *H. pylori* strains in the Myanmar population, it was found that all twelve resistant strains had mutations in the *rdxA* gene, three of them contained mutations with a preliminary stop codon. The most common was the point substitution V175I (8/12, 66.7%), followed by S91P (5/12, 41.7%) and R16H/C (4/12, 33.3%). Mutations in the *frxA* gene were observed in 76.9% (10/13) of resistant strains, preliminary early stop codon was observed in only one strain. In the *frxA* gene, the most frequent point mutation was the L33M substitution (3/13, 23.1%) [50].

Several genetic determinants of *pbp-1* have been reported to be associated with amoxicillin resistance: S414R and N562Y [51]. Two mutations, S414R and V45I, were present in 67% of amoxicillin-resistant *H. pylori* strains [50,51].

In a study by P. Subsomwong et al. in the Myanmar population, almost all levofloxacinresistant *H. pylori* isolates had an amino acid substitution at position 91 (Asp-91 to Asn or Tyr). Interestingly, no mutation was identified at position 87, which is associated with fluoroquinolone resistance and is found in levofloxacin-resistant *H. pylori* strains in Myanmar's neighboring Southeast Asian countries such as Indonesia, Malaysia, and Cambodia. Both mutations are also found in Chinese and Turkish populations [50].


**Table 3.** Evaluation of genetic resistance of *H. pylori* in world populations.


**Table 3.** *Cont.*

In a large-scale study conducted by Tal Domanovich-Asor et al., the whole genome of *H. pylori* was sequenced (WGS) with the aim of studying the bacterium's phylogeny and genetic aspects of antibiotic resistance. A total of 1040 genomes of *H. pylori* isolates were analyzed. The study focused on identifying point mutations in genes associated with bacterial antibiotic resistance (*pbp1A, 23S rRNA, gyrA, rdxA, frxA*, and *rpoB*), as well as conducting phylogenetic analysis. As a result, a significant geographic clustering of *H. pylori* genomes was identified in different regions of the world. The resistance analysis showed that the most common point mutations leading to antibiotic resistance were S589G (*pbp1A*, 48.8% of perfectly aligned sequences), A2143G (*23S rRNA*, 27.4% of perfectly aligned sequences), N87 K\I\Y (*gyrA*, 14.7% of perfectly aligned sequences), R131K (*rdxA*, 65.7% of perfectly aligned sequences), and C193S (*frxA*, 62.6% of perfectly aligned sequences). These research results provide a greater understanding of the relationship between antibiotic resistance and changes in the *H. pylori* genome. Further analyses that combine WGS and phenotypic methods will provide a deeper understanding of the relationship between mutations and resistance [60,61].

A group of scientists conducted a 6-year study on the antibiotic susceptibility of *H. pylori* in Israel. The study included 540 *H. pylori* isolates obtained from gastric biopsy specimens, that were collected from 2015 to 2020. Antibiotic resistance to amoxicillin, clarithromycin, metronidazole, levofloxacin, rifampicin, and tetracycline, was evaluated using the E-test method. Generalized linear models were used to estimate differences in gross and adjusted mean MIC values and odds ratios (ORs) for each year compared to the baseline year of 2015, for each antibiotic and for multi-resistance. The results showed the highest resistance rates to clarithromycin and metronidazole: 46.3% and 16.3%, respectively. Patients over 18 years old had higher levels of resistance to rifampicin and multi-resistance (3.3% and 14.8%, respectively) compared to those under 18 years old (0.5% and 8.4%, respectively). Resistance rates to levofloxacin, rifampicin, and multi-resistance were significantly higher among Arab patients compared to Jewish patients. This study highlights the importance of continuous monitoring of *H. pylori* antibiotic resistance for increasing eradication rates of this bacterium. Therapy for *H. pylori* infection should be revisited and updated based on data on antibiotic resistance [62].

A study on the antibiotic resistance of *H. pylori* was conducted in the Tibetan Autonomous Region of China. The study included 397 patients, from whom 153 strains of *H. pylori* were isolated. The overall resistance rates were as follows: clarithromycin (27.4%), levofloxacin (31.3%), metronidazole (86.2%), amoxicillin (15.6%), tetracycline (0%), furazolidone (0.6%), and rifampicin (73.2%). 2.0% of the *H. pylori* isolates were susceptible to all tested antibiotics, with monoresistance, dual resistance, triple resistance, quadruple resistance, and quintuple resistance rates of 18.3%, 44.4%, 18.3%, 12.4%, and 4.6%, respectively.

The resistance rates to levofloxacin (40.5%) and amoxicillin (21.5%) in strains obtained from female patients were significantly higher than those in strains obtained from male patients (21.6% and 9.5%, respectively).

The conducted study indicates a very high level of *H. pylori* antibiotic resistance in the Tibetan region of China, which suggests a high risk of developing *Helicobacter*-associated complications in these patients. The high resistance to rifampicin demonstrates the need for further research of its derivative, rifabutin [63].

A group of scientists from Portugal conducted a meta-analysis of data on *H. pylori* antibiotic resistance in this Country. The analysis included eight cross-sectional studies evaluating the resistance of *H. pylori* to antibiotics. The overall frequency of resistance was as follows: clarithromycin (CLA) 42% (95% CI: 30–54), metronidazole (MTZ) 25% (95% CI: 15–38), ciprofloxacin (CIP) 9% (95% CI: 3–18), levofloxacin (LVX) 18% (95% CI: 2–42), tetracycline (TTC) 0.2% (95% CI: 0–1), and amoxicillin (AMX) 0.1% (95% CI: 0–0.2) (Figure 7). Multiple drug resistance was also evaluated and results for global resistance rates were as follows: CLA plus MTZ 10% (20% in adults (95% CI: 15–26) compared to 6% in children (95% CI: 4–9) and 2% for CLA plus CIP (primary resistance in the pediatric group). High rates of secondary resistance were found for all antibiotics. In relation to antibiotic resistance, the findings indicate that adults exhibited higher levels of resistance to all antibiotics, with the exception of clarithromycin (CLA), which demonstrated high resistance levels in both adults and children (42% 95% CI: 14–71 and 40% 95% CI: 33–47) [64].

**Figure 7.** Antibiotic resistance rates (%) of *H. pylori* in Portugal.

A study was conducted to investigate the resistance of *H. pylori* in a cohort of children in Jordan (*n* = 166). The age of the children ranged from 10 to 14 years, with 82.7% of them not having received anti-*Helicobacter* therapy. The authors noted that the detection rate of *H. pylori* infection by rapid urease test, histological, phenotypic, and molecular genetic methods was 93.9%, 89.6%, 61.7%, and 84.3%, respectively. The resistance rates obtained by the phenotypic method were 25.9% for clarithromycin, 50% for metronidazole, and 6.9% for levofloxacin. Interestingly, mutations in the clarithromycin resistance gene were detected in 26.1% of the samples, while mutations in the levofloxacin resistance gene were found in 5.3% of the samples. The authors have concluded that real-time PCR is a valuable alternative method for the identification of *H. pylori* and determination of antibiotic susceptibility [65].
