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Whole-genome sequencing analyses and antibiotic resistance situation of 48 Helicobacter pylori strains isolated in Zhejiang, China

Gut Pathogens volume 16, Article number: 62 (2024) Cite this article

Abstract

Purpose

In the Zhejiang region, research on Helicobacter pylori is lacking. The purpose of this study was to assess the extent of antibiotic resistance in H. pylori in this region, explore alternative methods for predicting the resistance patterns of H. pylori, and investigate the colonization of native gastric mucosa by other clades of H. pylori in the structure population of this bacterium.

Methods

Strains were cultured under microaerobic conditions, and antimicrobial susceptibility testing (AST) was performed via agar dilution. Whole-genome sequencing (WGS) was performed via next-generation sequencing (NGS) technology. Epidemiological data including data from this study and reported articles from Zhejiang, China, were included. Further analyses based on AST, WGS, and epidemiological date include virulence genes, antibiotic resistance-related mutations, and phylogenetic trees based on 7 housekeeping genes and core-genome single nucleotide polymorphisms (SNPs).

Results

The bacterial isolates in this study presented higher antibiotic resistance rates than previously reported, especially against levofloxacin and clarithromycin. The point mutation A2147G in 23 S rRNA is specific to clarithromycin resistance. Mutations at position/s 87 and/or 91 of the gyrA gene amino acid sequence are highly consistent with levofloxacin resistance highly. The point mutations C1707T in 23 S rRNA and E463K in the gyrB gene have not been previously documented in China. All the bacterial isolates belong to Asian branches in the structure population. The resistance rate to clarithromycin of isolates from hosts born after January 1, 1977 is statistically higher than that of hosts born before 1977.

Conclusion

Eradication therapy based on AST results is urgently needed in Zhejiang. The point mutation A2147G in 23 S rRNA and point mutations in the gyrA gene at amino acid/s 87 and/or 91 are sufficient for predicting resistance to clarithromycin and levofloxacin, respectively. The isolate with the mutation E463K in the gyrB gene represents a significant contribution to the field. Mutations in 23 S rRNA may offer valuable insights into the dynamics of H. pylori transmission among hosts.

Introduction

Helicobacter pylori is a gram-negative bacterium, categorized as a class I carcinogen that affects nearly half of the world’s population, especially in developing countries [1,2,3]. H. pylori transmitted through multiple pathways, including human‒human, animal‒human, food‒borne, and occupational exposure [1]. Once a human is infected with H. pylori, the status of infection can last for a lifetime without standardized treatment [4]. The incidence of gastric cancer in H. pylori-infected patients with peptic ulcers, dyspepsia, or gastric atypical hyperplasia is greater than that in H. pylori-negative patients [5]. Most individuals are infected with H. pylori during childhood (1–6 years of age, especially before 4 years of age) [6]. Therefore, eradication treatment is necessary when H. pylori infection first begins.

The combination of multiple antibiotics, proton pump inhibitors, and/or bismuth accounts for the dramatic increase in resistance to key antibiotics in H. pylori eradication efforts [7, 8]. Nevertheless, some of the epidemiological mechanisms of antibiotic resistance in H. pylori remain unclear [6]. Owing to the alarming prevalence of antibiotic resistance in H. pylori, the therapeutic effects are unsatisfactory, and the universal recurrence, recrudescence, and reinfection rates of H. pylori infection have increased [9, 10]. In China, clarithromycin and levofloxacin are used as initial-line and second-line antibiotics for H. pylori eradication, which means that the resistance rate of H. pylori to these two antibiotics affects therapeutic outcomes. To enhance the eradication of H. pylori, analyses should encompass antibiotic resistance, novel mutations and modified genes, with a focus on clarithromycin and levofloxacin. Subsequent clinical drug reform can be performed based on sensitivity and resistance to antibiotics according to the 2022 Chinese national clinical practice guidelines [11,12,13,14]. However, the majority of research on H. pylori in Zhejiang Province primarily focuses on antibiotic sensitivity and basic demographic characteristics of infected individuals, such as age and sex. Only a limited number of studies include results from polymerase chain reaction (PCR) analyses. Literature on whole-genome sequencing (WGS) data analysis is rare in Zhejiang. We attempted to obtain more current WGS data for H. pylori by performing a deep-dive analysis of the strains. In the long run, the experimental data in our study can provide reusable data for more subsequent studies in H. pylori.

To investigate the relationships among the whole-genome sequences of H. pylori strains, antibiotic resistance patterns, and their host organisms, as well as to determine whether there have been changes in the population structure of H. pylori within the native population, our research plan was designed. In our study, we isolated 48 strains of H. pylori from gastroscopy-harvested gastric mucosa at a teaching hospital in Zhejiang Province, China. After obtaining stable cultures of H. pylori strains, AST, WGS, and epidemiological data were performed in conjunction. The results of our study highlight the recent occurrence of antibiotic resistance in H. pylori, and explore potential mechanisms and subsequent developments. Furthermore, we uploaded the WGS data collected from these strains, providing important data for subsequent studies on H. pylori genetics in Zhejiang, China.

Materials and methods

Collection, culture, and storage of isolated strains

Our study collected H. pylori strains cultured from gastric mucosa samples for clinical testing purposes from August 2021 to August 2022 at a tertiary care hospital. Colonies formed after 3–5 days of incubation on Columbia blood agar media under microaerobic conditions (5% O2, 7.5% CO2, 7.5% H2, 80% N2, 37 °C) in clinical cultures. Positive isolates were detected by gram staining and antigen tests with the H. PYLORI ANTIGEN RAPID TEST produced by Abbott. Afterward, positive isolates were purified, amplified, and stored at -80 °C. Basic patient information was collected from clinical diagnostic reports. This study was approved by the Ethic Committee of the First Affiliated Hospital of Zhejiang University School of Medicine: Expedition review No. 0535 in 2023.

AST of H. Pylori isolates

By using the H. pylori standard strain NCTC11637 (ST415) as the quality control strain, minimum inhibitory concentrations (MICs) of six common clinical antibacterial drugs were calculated, including amoxicillin (AMX, S ≤ 0.125 mg/L, R > 0.125 mg/L), levofloxacin (LEV, S ≤ 1 mg/L, R > 1 mg/L), clarithromycin (CLR, S ≤ 0.25 mg/L, R > 0.25 mg/L), tetracycline (TCY, S ≤ 1 mg/L, R > 1 mg/L), metronidazole (MZ, S ≤ 8 mg/L, R > 8 mg/L), and rifampicin (RI, S ≤ 1 mg/L, R > 1 mg/L), according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Breakpoint tables for interpretation of MICs and zone diameters (Version 13.1, valid from 2023-06-29). The isolated H. pylori strains were cultured and detected via the agar dilution method according to the Clinical and Laboratory Standard Institute (CLSI, M45 3rd Edition, 2015) [15].

Genomic DNA extraction and WGS

To analyze the genomic background of 48 H. pylori isolates, genomic DNA extraction was carried out via the Fast DNA™ Spin Kit for Soil following the manufacturer’s protocols. The sequencing libraries were prepared via the Nextera XT Kit (Illumina), and genome sequencing was performed via the HiSeq X 10-PE150 platform (Illumina). The quality of the raw sequencing reads was assessed through FastQC v.0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). After trimming and quality filtering the adapters via fastp v0.20.1, the processed paired-end reads were assembled via the shovill v1.1.0 pipeline (https://github.com/tseemann/shovill; SPAdes v3.14.1) with default settings [16]. The assembled sequences (contigs) for each strain were exported and saved in FASTA format.

Phylogenetic tree

After uploading the assembled contigs to the PubMLST website (sequence query at pubmlst.org), we identified seven pairs of housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, and yphC) along with their corresponding sequence types (STs). We then conducted multilocus sequence typing (MLST) on these strains by comparing the seven housekeeping genes against those in the PubMLST database. After downloading and randomly selecting 429 bacterial steward gene strains with clear structure populations from the PubMLST website, these data were combined with the steward genes of the 48 isolated H. pylori strains. Phylogenetic tree analysis was conducted, and similar phylogenetic relationships were determined by via PHYLOViZ version 2.0. (https://ngstar.canada.ca/). The tree-building results were labeled via the online software ITOL (https://itol.embl.de/) to complete the tree-building process. This comparison helped us identify the geographic type of each strain, and consequently, we were able to construct a phylogenetic tree.

Detection of virulence genes and antibiotic resistance-related mutations in strains

The virulence genes of H. pylori strains were detected via Abricate v1.0.0 (https://github.com/tseemann/abricate) with the VFDB core database, with cutoff values of 80% identity and 80% query coverage. To identify possible drug resistance-related mutation loci in the strains, we compared the whole-genome sequences of the strains with CARD through RGI v6.0.2 (https://github.com/arpcard/rgi) with default settings. This allowed us to understand the resistance patterns of the strains. Furthermore, we correlated drug resistance phenotypes and drug resistance mutation loci.

Core-genome-based phylogenetic tree and further analyses related to population mobility

By constructing a core-genome-based tree for the 48 strains in this study and incorporating factors such as drug resistance phenotypes, virulence genes, and birth year, correlation analysis was performed on the data. Roary v3.13.0 was used for pangenome analysis and core genome alignment of all strains. SNP sites v2.5.1 was used to extract SNP alignments from the core genome alignment, and RAxML v8.2.11 was employed to construct a phylogenetic tree using the ASC_GTRGAMMA substitution model with 1,000 bootstrap replications. The iTOL web page (https://itol.embl.de/) was used to generate a heatmap of birth year, clarithromycin resistance, A2147G mutation in 23 S rRNA, levofloxacin resistance and gyrA gene mutation. Using a time cutoff of 0–6 years of age prior to January 1, 1979, we evaluated the differences in the resistance rates to clarithromycin among H. pylori strains isolated from patients.

Collection of data from published literature in Zhejiang Province

The Pubmed terms “((Helicobacter pylori) AND (Zhejiang China)) AND ((antibiotic resistance) OR (antimicrobial resistance))” were searched. Only the original strains were isolated from Zhejiang Province.

Collection of other H. Pylori genomic data

We downloaded other genome sequences of H. pylori for comparison analysis from the NCBI database (helicobacter pylori - Assembly - NCBI (nih.gov), 4460 strains, last updated: December 12, 2023) in the union of GenBank.

Statistical analysis

SPSS Statistics version 24.0 software was used to analyze the correlation of the data. Methodology consistency was compared via the kappa test, and differences in grouped data were analyzed via the chi-square test. A kappa value of ≥ 0.75 indicates good consistency between the two methods. A P value of < 0.05 for chi-square tests was considered to indicate a significant difference.

Results

Phenotypic resistance and clinical data

During the study period, no gastric cancer patients underwent H. pylori culture. A total of 158 gastric mucosa specimens were cultured in the hospital, 57 (36.08%) of which were positive for H. pylori. From these, 50 strains were purified, amplified, and preserved, whereas 48 strains were successfully revived and tested after preservation. The positive rate of culture in our study was comparable to that reported in other studies involving general patients of various ages in Zhejiang Province (Table 1) [17,18,19,20,21].

Table 1 Comparison of H. Pylori culture and antibiotic sensitivity between the present study and reported studies from Zhejiang Province
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The eradication histories of patients from whom 48 strains were isolated are detailed in Supplementary Table 1. Of these, 25 patients (Group 1) had previous unsuccessful attempts at H. pylori eradication. In Group 1, 22 patients followed a clinical antimicrobial susceptibility testing (AST)-based eradication plan. Of these, six patients returned for a recheck, which confirmed eradication success, while the remaining 16 have not returned. The other three patients in Group 1 did not return for further treatment. Conversely, the remaining 23 patients (Group 2) had no documented history of prior H. pylori eradication attempts or their histories were not recorded. Of these, 10 patients received eradication plans based on clinical AST results; six returned for a recheck, confirming eradication success, whereas the other four have not returned. The last 13 patients in Group 2 have no medical records of eradication attempts or follow-up rechecks. Resistance to clarithromycin was higher in Group 1 (24/25) compared to Group 2 (14/23), although resistance rates for other antibiotics were similar across both groups. Due to the substantial lack of clinical data regarding patients’ eradication histories, further analysis in this area is not feasible in our study.

The detailed minimum inhibitory concentrations (MICs) for the 48 H. pylori strains are presented in Supplementary Tables 2, and the phenotypic antibiotic resistance rates are shown in Table 2. Multidrug-resistant (MDR) strains, which refer to isolates resistant to three or more antibiotics tested simultaneously, accounted for 70.83% of the strains. Three strains exhibited resistance to five antibiotics simultaneously, and 25% of the strains were concurrently resistant to clarithromycin, levofloxacin, and metronidazole (Table 3). Resistances rates to clarithromycin and levofloxacin among the isolates in the present study were higher than those historically reported in articles from Zhejiang Province. The amoxicillin resistance rate in our study was 37.50% based on the EUCAST standard (S ≤ 0.125 mg/L, R > 0.125 mg/L). However, resistance breakpoints for minimum inhibitory concentrations (MICs) vary between studies [17, 18], ranging from > 0.125 mg/L to > 1 mg/L [17, 18]. Additionally, 17 strains in our study had MICs within this range. Given these variations, it is not appropriate to compare amoxicillin resistance rates across different studies in Zhejiang Province.

Table 2 Antimicrobial sensitivity results of the 48 H. pylori strains
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MIC, minimum inhibitory concentration; S, susceptible; R, resistant.

AMX, amoxicillin; CLR, clarithromycin; LEV, levofloxacin; TCY, tetracycline; RI, rifampin; MZ, metronidazole.

Table 3 Antibiotic resistance patterns
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Antibiotic resistance-related mutations using WGS

Comparison of the whole-genome sequences of the strains with CARD mutations revealed antibiotic-associated genetic point mutations. Further analyses of antibiotic resistance results and genetic point mutations revealed that resistance to clarithromycin and levofloxacin was highly consistent with resistance-associated gene mutations (Tables 4 and 5).

Among the 48 isolates, 37 strains with 23 S rRNA point mutations type (A2147G, C1707T, A2144G) were resistant to clarithromycin. The remaining 11 strains (C1707T, A2144G) were almost completely susceptible (90.91%) to clarithromycin. The consistency in predicting clarithromycin resistance between the A2147G point mutation and the agar dilution method was high (Kappa = 0.939, P < 0.001). The point mutation C1707T has not been previously documented in China, and all the isolates in our study had this mutation.

Table 4 Consistency between point mutations and antimicrobial resistance phenotype
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Mutations in the DNA gyrase A (gyrA) gene (Tables 4 and 5), including N87I, N87K, D91N, D91G, and D91Y, confer resistance to levofloxacin, and the resistance rate of 27 H. pylori strains with gyrA gene mutations to levofloxacin was 100%. Among these strains, one levofloxacin-resistant strain harbored mutations at D91G and E463K in the gyrA and the gyrB genes, respectively. In contrast, among the 21 isolates without gyrA/B gene mutations, 80.95% were sensitive to levofloxacin. The consistency in predicting levofloxacin resistance between the amino acid/s point mutation/s 87 and/or 91 of amino acid in the gyrA gene and the agar dilution method was high (Kappa = 0.827, P < 0.001). The point mutation (E463K) in the gyrB gene has never been reported in China before.

Table 5 Different mutations in 23 S rRNA and DNA gyrase gene families
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In our study, point mutations in at least one of the penicillin-binding proteins (PBPs)—including PBP1, PBP2, and PBP3—were identified in each of the 48 strains, as detailed in Supplementary Table 3. Among these strains, 8 exhibited simultaneous mutations in PBP1, PBP2, and PBP3; 17 strains had mutations in both PBP1 and PBP2; 6 had mutations in both PBP2 and PBP3; and 17 strains exhibited mutations solely in PBP2. We assessed the relationship between these point mutations in the PBPs and amoxicillin resistance. Intriguingly, both amoxicillin-resistant and -sensitive strains displayed various degrees of mutations in the PBPs. Notably, three strains with triple mutations in PBP1 (T593A + G595S + A474T, N562Y + G595S + T593G, and N562Y + T593A + G595S) were resistant to amoxicillin, whereas other strains with double, single, or no mutations in PBP1 showed comparable resistance rates to amoxicillin.

Due to the limited number of strains in our study resistant to tetracycline or rifampicin, or susceptible to metronidazole, we did not analyze the correlation between point mutations associated with antibiotic resistance and their respective resistance phenotypes. This limitation in sample size restricts our ability to draw reliable conclusions about these specific correlations.

Structure population analysis and STs of strains

Genomic analysis of all H. pylori strains in our study showed genome sizes ranging from 1.50 Mb to 1.69 Mb and GC content between 38.51% and 38.96%. These findings align with the average genome size and GC content for H. pylori reported in the pubMLST database (1.5–1.8 Mb) and documented strains in the GenBank database (GCA_025998455.1 and GCA_003050665.1) with GC percentages of 38.5% and 39%, respectively. The strains isolated in our study were all novel STs (Supplementary Table 4). The MLST minimum spanning tree (Fig. 1) revealed that the population to which the strains belong was highly clustered by region. Among the 48 isolated H. pylori strains, 47 were classified as belonging to the Asian group (including hspEAsia, hpEAsia, hpAsia2, and hpEastAsia), whereas only one was located in the mixed area between the Asian and European groups (most of the strains in this mixed area are from East Asian and Southeast Asian countries), which is consistent with our geographical location.

Fig. 1
figure 1

The MLST minimum spanning tree. China: Zhejiang Province, strains isolated in this study; Asia, structure population (including hspEAsia, hpEAsia, hpAsia2, and hpEastAsia); Amerind, structure population of hspAmerind; Africa, structure population (including hpAfrica, hpAfrica1, hpAfrica2, hpNEAfrica, hpWAfrica, hspSAfrica, hspSAfrica1, hspWAfrica and hspWAfrica1); Europe, structure population of hpEurope; Sahul, structure population of hpSahul; Maori, structure population of hspMaori; Africa-Europe, structure population of hpAfrica-Europa

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Core-genome-based phylogenetic tree and relationship with population mobility

The overall genetic relationship of the strains we isolated was relatively close, whereas the distribution of drug resistance on the phylogenetic tree was relatively scattered (Fig. 2). There was a statistically significant difference in the clarithromycin resistance rates of H. pylori strains isolated from hosts born before and after January 1 of the 1978, 1977, and 1974 cutoffs (Table 6). These three years corresponded to 1, 2, and 5 years of age in 1979. Among these, the dividing line from January 1, 1977, presented the greatest significant difference (c2 = 7.277, P = 0.019, Supplementary Table 4).

Fig. 2
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The results of the core-genome-based phylogenetic tree

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Table 6 Relationship between hosts’ born year and clarithromycin resistance
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Detection of the cagA gene and vacA gene

The positive rate of H. pylori by culture in the present study was 36.08% (57/158) among general patients, whereas Yonglin Li et al. reported a rate of 98.67% (222/225) among gastric cancer (GC) patients [22]. A comparison of the genome sequences of these 48 strains from the VFDB core database (standard strain H. pylori 26695) revealed that 18 strains contain the vacuolating cytotoxin A (vacA) gene and that 47 strains contain the cytotoxin-associated gene A (cagA). On the other hand, carriage rates of the vacA gene and cagA gene were 100% (222/222) and 97.75% (217/222), respectively, among GC patients detected via PCR in Wenzhou in 2019 [22]. Owing to significant differences in vacA carrying rates between the two groups, we obtained H. pylori genome data from the NCBI database and compared the genomes to those in the VFDB core database. The detection rate of the vacA gene in the genomes downloaded from NCBI was 54.51% (2431/4460).

Discussion

H. pylori lives in the human stomach and has a population structure akin to that of its host [23]. Infection with this bacterium can lead to gastritis, peptic ulcers, GC, and other extra-gastric diseases [24,25,26]. The success of eradicating H. pylori infection is significantly correlated with antibiotic resistance. Our study revealed that clarithromycin resistance rates and levofloxacin resistance rates, mainly due to point mutations in 23 S rRNA (A2147G) and mutations in the gyrA gene respectively, have reached historically high levels in Zhejiang. Furthermore, the point mutations C1707T in 23 S rRNA and E463K in the gyrB gene found in our study have not been documented previously in China. Epidemiologically, the clarithromycin resistance rate of H. pylori is significantly different between hosts born before and those born after 1977. This difference may be caused by the massive migration during the Chinese economic Reform and Opening-up and the pressure of antibiotic regimen.

Clarithromycin serves as the initial-line therapy for H. pylori infection. The continuous increase in clarithromycin resistance is the most common reason for H. pylori treatment failure [27]. In the present study, we found that the prevalence of resistance to clarithromycin (79.17%) was greater than thar reported in Zhejiang [17,18,19,20,21]. The wide use of this agent for various other infections, such as respiratory tract and urinary infections, might contribute to an increase in the resistance of H. pylori. The binding of clarithromycin to domain V of 23 S rRNA inhibits the growth of bacteria. Therefore, point mutations in this region inhibit the binding of clarithromycin and 23 S rRNA, leading to clarithromycin resistance [28]. The point mutations A2147G, A2146G, and A2146C (which are also referred to as A2143G, A2142G, and A2142C, respectively) are considered major mutations implicated in clarithromycin resistance [29]. All strains with the A2147G mutation in the present study were resistant to clarithromycin. Among the clarithromycin-resistant strains 37/38, had the A2147G mutation. The point mutation A2147G in 23 S rRNA is highly specific for clarithromycin resistance in this region. The point mutations C1707T and A2144G were identified in all 48 strains in this study. Among these, 11 strains had the combined mutation type (C1707T, A2144G), with only one strain exhibiting resistance to clarithromycin. The A2144G mutation, reported in various geographical regions, is linked to the clarithromycin resistance phenotype [30, 31], but such strains typically represent only a small fraction of the local bacterial population. However, in our study, strains with the A2144G mutation constitute a significant majority of the local population. Previous research has shown that A2144G is associated with lower clarithromycin minimum inhibitory concentrations (MICs) compared to another common mutation, A2147G [32, 33]. This indicates that clarithromycin resistance mechanisms in H. pylori strains with the A2144G mutation in the 23 S rRNA may differ across geographic regions. The C1707T mutation has only been previously reported in a single isolate from Mongolia, also linked to clarithromycin resistance [34]. Given the limited data, it remains uncertain whether the C1707T mutation is specifically associated with clarithromycin resistance.

Levofloxacin serves as the second-line eradication treatment for H. pylori infection after amoxicillin and clarithromycin therapy fails. The prevalence of resistance to levofloxacin reached 64.58% in our study. The levofloxacin resistance rate in the present study was higher than that reported previous studies conducted inside or outside our hospital in Zhejiang [17,18,19,20,21]. The causes of the higher resistance rate to levofloxacin was similar to that of clarithromycin. It is generally believed that point mutations in the quinolone-resistance-determining region (QRDR) of the gyrA gene lead to levofloxacin resistance [35]. Point mutations of gyrA often occur at amino acid/s 87 and/or 91, such as N87K/I, and D91G/N/Y in fluoroquinolone-resistant H. pylori strains [36, 37]. In the present study, the gyrA mutation was present in 27/31 strains; mutations in the gyrA gene at position/s 87 and/or 91 are highly specific for levofloxacin resistance. The gyrB mutation is rarely reported and is not considered a common mechanism of fluoroquinolone resistance. However, a mutation at position 463 (Glu to Lys) of gyrB leading to levofloxacin resistance was proposed [38]. One levofloxacin resistant strain in our study contained both the gyrA and gyrB mutations D97G and E463K, respectively. It is difficult to ensure that the gyrB mutation E463K leads to levofloxacin resistance; however, the point mutation E463K has not been previously documented in China, and this strain can be used as a material strain for knockout validation in subsequent resistance studies.

Amoxicillin is a pivotal antibiotic for eradicating H. pylori infections [39], yet its resistance rates vary across different geographical regions [40,41,42,43]. In our study, the resistance rate to amoxicillin in H. pylori was 37.5% (18/48). Notably, three strains harbored triple point mutations in PBP1 (T593A + G595S + A474T, N562Y + G595S + T593G, and N562Y + T593A + G595S) and were all resistant to amoxicillin. However, the small sample size of these specific strains precludes definitive conclusions without further research. The other strains, exhibiting various degrees of point mutations in the PBPs, displayed similar amoxicillin resistance rates. Previous studies have suggested that mutations in PBP1, PBP2, and PBP3 may have synergistic effects on amoxicillin resistance [44]. Furthermore, the patterns of resistance and PBP mutations in our study align with recent findings by Zhou, Y., et al. [45]. These observations suggest that additional factors—including geographical variation, host interactions, and strain transmission, along with amino acid substitutions in PBP motifs—should be considered to fully understand the mechanisms of amoxicillin resistance in H. pylori.

The H. pylori strains isolated in the present study are all novel STs, with high mutation rates of the H. pylori housekeeping genes in different isolates. The structure population analyzation of these strains conforms to the racial characteristics and geographical distribution of population subgroups. There is a clear correlation between the genotype of H. pylori and factors such as geographical distribution, ethnicity, and population migration [46]. Our isolated strains are from the same progenitor line. In addition, H. pylori is highly stable in structure population typing, which characterizes the origin of the host well. All these factors lead to the conclusion that H. pylori spreads only in limited regions with restricted transmission chains, which indicates geographical limitations in the use of clinical medication.

The spread of H. pylori can be attributed to human migratory fluxes and genetic variation in the genome sequence of H. pylori shows a phylogeographic pattern similar to that of its host [47, 48]. During 1958 to 1978, there was a period of strict control of population migration in China. Subsequently, China entered a period of large-scale population migration from 1979 (the Reform and Opening-up) to the present. Statistical analysis of the clarithromycin resistance rate and birth period revealed significant differences between the populations at the dividing lines 1974, 1977 and 1978. Individuals born in those three years were 1, 2 and 5 years old in 1979. These ages corresponded to the changes in the prevalence of H. pylori infection rates in children aged 1–6 years in the study of M Constanza Camargo et al. [49]. On the other hand, the positive rate of culture in our study was similar to that reported in other studies of general patients of different ages [17,18,19,20,21]. All the data above indicate that H. pylori infection occurs in childhood, which explains why H. pylori is highly stable in structural population typing. Different clades of H. pylori can exist in a single gastric biopsy, and genetic recombination within clades is a continuous, ongoing process that lasts for at least 12 years [50]. On the basis of these findings, we hypothesized that with large-scale population migration after the Reform and Opening-up, the genetic intermingling of different clades of H. pylori also greatly increased. In children, this progress of migration may be accompanied with the progress from absence to absent of H. pylori infection. Till the year 1993, clarithromycin was initially used, for the treatment of H. pylori infection and various other infections such as respiratory tract and urinary infections, which included children in China. We speculate that H. pylori infected in childhood was selected for resistance by antimicrobial treatment. Among the antibiotic-resistant mutations, mutations in 23 S rRNA on the large subunit of the ribosome were the easiest to disseminate under pressure of clarithromycin, leading to a significantly higher rate of clarithromycin resistance in H. pylori carried by populations born after 1977 than in those carried by populations born before 1977. In this context, mutations in 23 S rRNA may offer valuable insights into the dynamics of H. pylori transmission among hosts.

The presence of cagA is associated with greater risk of GC development in Western countries, whereas in East Asia almost all H. pylori strains harbor cagA [51]. Considering that the majority of H. pylori strains isolated in East Asia contain the cagA gene irrespective of disease status, cagA cannot be considered a useful marker of gastrointestinal disease [52]. Consistent with previous studies, 47/48 isolates harbored the cagA gene in the present study. In a previous study in Zhejiang patients with GC, the positive rate of H. pylori infection was 98.67%, and all of the positive samples had the vacA gene [22]. In our study, the positive rate of H. pylori was 36.08%, and 37.5% of the analyzed strains were vacA gene positive among general patients. Both the positive rate of H. pylori and the carriage rate of vacA gene are differed greatly between the two studies. Although, the positive rate of the culture method is affected by the sampling position of the stomach and is lower than the actual positive rate, the results of the difference between the two groups still revealed a potential relationship between H. pylori and GC. The sequence of the vacA gene, with a length of 3873 bp, was obtained from the H. pylori 26,695 strain and was stored in the NCBI database and VFDB core database. WGS analysis of strains from both in the present study and the NCBI database revealed a lower prevalence of the vacA gene than its theoretical value [53]. Most vacA gene detection methods involve the following methods: vacA s1/s2 nucleic acid length 259/286 bp, vacA m1/m2 nucleic acid length 570/645 bp, and vacA i1/i2 nucleic acid length 426/432 bp [54,55,56]. However, even if the presence of a vacuolating cytotoxin gene sequence for subtype identification is demonstrated in H. pylori by PCR, it should not be equated with the existence of a functionally complete vacA gene. A study by Cover TL et al.. revealed substantial differences between the vacA sequences of tox-positive and tox-negative strains in vitro, which may explain the lower prevalence of the vacA gene detected by WGS in the present study [57]. The relationship between the genetic sequence and the cytotoxin activity of the vacA gene still needs to be researched further.

With a total population of approximately 1.4 billion, the number of H. pylori hosts in China is anticipated to be large. Owing to the complex geographical environments, numerous ethnic groups, and large differences in population mobility in different regions, different clades of H. pylori have their own unique genetic evolution. As a result, the treatment of H. pylori should be adapted to local conditions. Recent reports have shown different eradication rates and antibiotic resistance patterns of H. pylori within different regions of China [40,41,42,43]. In our study, the resistance rates of the H. pylori isolates were 64.58% and 79.17% for levofloxacin and clarithromycin, respectively. The resistance rate for amoxicillin (EUCAST standard) approached 37.50%, but the rate for tetracycline was approximately 10%. These results indicate that the resistance rate to commonly used initial/second-line drugs for the eradication of H. pylori is continuously increasing in Zhejiang. Additionally, in our study, the resistance rate to clarithromycin among H. pylori strains isolated from patients with a history of failed eradication attempts was higher compared to those from patients without a documented eradication history. Therefore, we recommend adjusting the commonly used drugs for patients receiving secondary treatment for H. pylori infection in clinical practice on the basis of AST results. The development of treatment strategies based on the analysis of antibiotic resistance genes in H. pylori and the resistance analysis of antibiotics (especially clarithromycin) is becoming commonplace [58]. As Maastricht VI guidelines suggest, the use of an antimicrobial susceptibility guidance strategy is the first choice for eradication therapy [59]. In addition, our method of WGS, combined with the correlation of the drug resistance phenotype and drug resistance mutation loci, was an effective way to analyze H. pylori strains when culture methods are not suitable.

WGS analysis of H. pylori is still necessary for subsequent studies on antibiotic resistance, population mobility, cytotoxin genes, and clinical therapies. To make our findings more applicable to clinical applications, uniform and standardized diagnostic gastroscopy and pathological diagnoses should be included in subsequent studies. On the other hand, the relationship between vacA gene sequence analysis and the in vitro cytotoxin activity of H. pylori should be systematically investigated. An isolated strain with the gyrB gene mutation E463K is rare, and can be used in subsequent studies of antibiotic resistance.

The present study has a few limitations: the sample size of 48 strains in this study was insufficient to fully elucidate some of the findings. Additionally, H. pylori is not the sole cause of gastric cancer or other gastric diseases, which means that the conclusions drawn from comparisons between different disease groups depending on H. pylori alone may not be comprehensive.

Conclusion

In conclusion, the present study emphasizes the importance of conducting region-specific surveillance of H. pylori resistance and customizing treatment strategies accordingly. The point mutations, A2147G in 23 S rRNA and at amino acid/s 87 and/or 91 in the gyrA gene, can effectively predict phenotypic resistance to clarithromycin and levofloxacin in the native population. We found that one H. pylori strain with rare point mutation E463K in the gyrB gene can be used in subsequent resistance studies. Mutations in 23 S rRNA have the potential to serve as indicators of host mobility.

Data availability

The whole-genome sequences of H. pylori isolated in this study have been deposited in the China National Microbiology Data Center (NMDC) under the BioProject accession number NMDC10018501. URL is https://nmdc.cn/resource/en/genomics/project/detail/NMDC10018501. All data relevant to the study are included in the article and supplementary files, and are available from the corresponding author upon request.

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Acknowledgements

This work was supported by the “Fundamental Research Funds for the Central Universities (2022ZFJH003)”; and “Research Project of Jinan Microecological Biomedicine Shandong Laboratory (NL-2022044D)”.

Funding

This work was supported by the “Fundamental Research Funds for the Central Universities (2022ZFJH003)”; and “Research Project of Jinan Microecological Biomedicine Shandong Laboratory (NL-2022044D)”.

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Author notes

  1. Yunhui Fang and Shiman Jiang contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, National Medical Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Rd, Hangzhou City, 310003, China

    Yunhui Fang, Shiman Jiang, Wangxiao Zhou, Xinrong Jiang, Yunbo Chen & Lanjuan Li

  2. Jinan Microecological Biomedicine Shandong Laboratory, Jinan, Shandong, China

    Yunhui Fang & Lanjuan Li

  3. Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China

    Xinxin Zhou

  4. Department of Medical Engineering and Material Supplies, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China

    Lifeng Chen

  5. Zhejiang University of Finance & Economics, Hangzhou, Zhejiang Province, China

    Mengting Wang

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Contributions

L. L, Y. F, S. J and Y. C conceived of and supervised the work. W. Z and Y. F performed the combination of sequencing results and the data analysis. X. Z and X. J analyzed the clinical datasets. Y. F, W. Z, and X. J performed the experiments, including the agar dilution tests and DNA extraction. L. C is responsible for the commissioning of instruments related to this study. M. W conducted the translation of Japanese materials of H. pylori in the preliminary stage of the study. Y. F and S. J wrote the main manuscript and prepared the figures, with support from L. L and Y. C. All authors contributed to the general discussion.

Corresponding authors

Correspondence to Yunbo Chen or Lanjuan Li.

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This study has been approved by the Ethic Committee of the First Affiliated Hospital of Zhejiang University School of Medicine: Expedition review No. 0535 in 2023.

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Fang, Y., Jiang, S., Zhou, X. et al. Whole-genome sequencing analyses and antibiotic resistance situation of 48 Helicobacter pylori strains isolated in Zhejiang, China. Gut Pathog 16, 62 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13099-024-00656-2

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