Abstract
The emergence of antibiotic resistance in Helicobacter pylori led to a sharp decline in eradication rates, but there are limited data regarding the prevalence and genetic mechanisms of antibiotic resistance in Shandong. This study aims to assess the prevalence and molecular mechanisms of H. pylori antibiotic resistance to commonly used antibiotics in Shandong populations. Antimicrobial susceptibility testing was performed for clarithromycin, levofloxacin, metronidazole, tetracycline, rifampicin, amoxicillin, azithromycin, and moxifloxacin using E-test method. PCR amplified resistance-associated genes for all the strains, and 32 were whole-genome sequenced. 62 H. pylori strains were obtained and the infection rate was 30.1% (62/206). No resistance to amoxicillin was observed. Two and four strains were resistant to tetracycline and rifampicin, respectively, and no resistance-associated mutations were found in the 16S rRNA and rpoB genes. The resistance rates of azithromycin, clarithromycin, levofloxacin, and moxifloxacin were 56.45% (35/62), 33.87% (21/62), 48.39% (30/62), and 56.45% (35/62), respectively. The percentages of dual, triple, and quadruple resistance were 32.26% (20/62), 24.19% (15/62), and 1.61% (1/62), respectively, and only one isolate was resistant to quadruple antibiotics. Phenotypic resistance associated with the A2143G mutations in 23S rRNA for azithromycin and clarithromycin (kappa coefficient, 0.808 and 0.671, respectively), and gyrA mutations (N87K/N87I/D91G/D91Y/D91N) for levofloxacin and moxifloxacin (kappa coefficient, 0.526 and 0.442, respectively). Metronidazole resistance was observed in 43.55% (27/62) isolates and most of the metronidazole-resistant strains had truncated rdxA and frxA. The detection of a truncated rdxA gene correlated with phenotypic resistance results (kappa coefficient, 0.728). There was no association between whole-genome phylogeny and antibiotic resistance profiles. Our results revealed the high resistance rates and multiple resistance of H. pylori to commonly used antibiotics. Continued surveillance of H. pylori antibiotic resistance is crucial in Shandong to establish effective eradication therapy for this population.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-025-16264-2.
Keywords: Shandong, Helicobacter pylori, Antimicrobial susceptibility testing, Genetic mutation, Whole-genome sequencing
Subject terms: Gastrointestinal diseases, Antimicrobial resistance
Introduction
Helicobacter pylori (H. pylori) colonizes approximately half of the world’s population, causing chronic gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. It has been classified as a class I carcinogen by the World Health Organization1. Moreover, H. pylori infection might be associated with the development of extraintestinal diseases, including cardiovascular disease, neurological disorders, iron deficiency anemia, vitamin B12 deficiency, or even allergic diseases2. The eradication of H. pylori plays an important role in treating and preventing these diseases3. However, in recent years, antibiotic resistance has led to H. pylori eradication failure, and the resistance rates vary widely around the world4. Without effective measures, it is estimated that the annual death related to antibiotic resistance will reach approximately 10 million people worldwide by 20505. Therefore, selecting an efficient therapeutic regimen holds tremendous significance in clinical practice.
The bismuth-containing quadruple therapy, combining two antibiotics with a proton pump inhibitor (PPI), was recommended as the main empirical therapy for H. pylori eradication in the Maastricht VI and Chinese Consensus Report6. It has been reported that bismuth-containing quadruple therapy has a high eradication rate ranging from 85 to 94%7. However, H. pylori resistance rates to some antibiotics, such as clarithromycin (CLR), metronidazole (MTZ), and levofloxacin (LEV), are increasing, with potential regional variations8. The study in China demonstrated H. pylori resistance rates to CLR, MTZ, and LEV at 55.2%, 71.3%, and 18.4% respectively, with an increasing trend9. Moreover, the resistance rates varied between 11%–60% for CLR and 14.9%–80% for MTZ in other countries10. According to the recommendation, if the triple therapy containing CLR, MTZ, and LEV is considered, a drug susceptibility test should be performed when the CLR resistance rate exceeds 15% or the LEV resistance rate reaches 10%11. The eradication efficacy of alternative regimens, such as quadruple, sequential, concomitant, and levofloxacin-containing triple regimens, varies widely6.
Considering different point mutations, antibiotic resistance mechanisms in H. pylori have been identified. Mutations in the 23S rRNA (A2142G, A2142C, and A2143G), 16S rRNA (AGA926-928TTC), and rpoB genes are responsible for macrolide, tetracycline (TET), and rifampin (RIF) resistance, respectively. Additionally, mutations, including frameshift mutation, insertions, and deletions of the frxA and rdxA genes, confer resistance to MTZ12,13. Furthermore, mutations in the quinolone resistance-determining region (QRDR) of the gyrA and gyrB genes account for fluoroquinolone resistance14. Studies reported that A2143G mutation was the most common in CLR-resistant strains, followed by A2142G and A2142C, while Asn87 along with Asp91 of the gyrA mutation point were the most frequent in the LEV-resistant strains15. Moreover, in MTZ-resistant strains, mutations in the rdxA and frxA genes can confer resistance to MTZ10. Understanding these mutations can guide more rational antibiotic combinations and treatment options.
The prevalence of H. pylori infection varied among different regions within a country and between countries, accompanied by differences in the antimicrobial resistance levels16. Shandong province, situated in the east of China, reported an incidence of malignant tumors in 2016 at 277.40 per 100,000 persons, with gastric carcinoma ranking as the second most common cause of cancer mortality, presenting an incidence rate of 34.58 per 100,000 persons17. The imperative for H. pylori eradication is strongly recommended due to the high prevalence of H. pylori infection and the elevated risk of gastric carcinoma. Although quadruple therapy has been widely employed in the infected population, limited studies have explored the prevalence of H. pylori antibiotic susceptibility and the presence of genetic mutations associated with antibiotic resistance. Thus, this study aims to examine the susceptibility of Shandong H. pylori isolates to 8 commonly used antibiotics using the E-test method and further to explore the antibiotic resistance molecular mechanisms of H. pylori.
Results
H. pylori infection rate and endoscopic findings
We performed endoscopy for 206 dyspeptic patients. The prevalence of H. pylori infection determined via histological examination was 34% (70/206) (Table S1 in Additional file 1). Clinical diagnoses were 191 cases of non-ulcer dyspepsia (NUD) and 15 cases of peptic ulcer disease (PUD) (8 gastric ulcers and 7 duodenal ulcers). A total of 62 H. pylori isolates (62/206, 30.1%) were successfully obtained from gastric biopsies of 206 dyspeptic patients. Of these, there were 45 males (age range, 32–78 years; mean age, 57.62 ± 9.30 years) and 17 female patients (age range, 36–73 years; mean age 54.06 ± 10.81 years). Among the isolates, 53 (85.48%) were isolated from subjects with NUD, and 9 (14.52%) with PUD (3 gastric ulcers and 6 duodenal ulcers).
Phenotypic antimicrobial resistance of H. pylori isolates
Antimicrobial susceptibility tests were investigated in 62 H. pylori isolates, and the results were listed in Table 1. Cross-resistance was observed between azithromycin (AZM) and CLR in 21 isolates, as well as between LEV and moxifloxacin (MXF) in 28 isolates. MTZ resistance was identified in 43.55% (27/62) isolates, with MICs for MTZ ranging from 0.016 to 256 µg/mL. Resistance to RIF was observed in 6.45% (4/62) isolates (MIC range, 0.006 to 32 µg/mL), and only two isolates exhibited resistance to TET. All isolates were susceptible to amoxicillin (AML), and the MICs were concentrated in the low MIC intervals (98.54% for 0.016 µg/mL or less). Antimicrobial resistance rate did not differ among different genders, ages, and clinical outcomes (P > 0.05) (Tables 2 and 3). Among all the isolates, the percentage of dual, triple, and quadruple resistance was 32.26% (20/62), 24.19% (15/62), and 1.61% (1/62), respectively, and only one isolate was resistant to quadruple antibiotics (Table 4). The average age of single, dual, triple, and quadruple resistant strains was 58.31, 56.50, 54.87, and 64 years, respectively, without a statistically significant difference in age between single and multiple resistant strains (P = 0.632).
Table 1.
Prevalence of antimicrobial susceptibilities of 62 H. pylori isolates.
| Antimicrobial | MIC Range (µg/mL) |
MIC Mean (µg/mL) |
Resistance | Susceptible | ||
|---|---|---|---|---|---|---|
| No. | % | No. | % | |||
| AZM | 0.016-256 | 162.936 | 35 | 56.45 | 27 | 43.55 |
| CLR | 0.016-256 | 18.254 | 21 | 33.87 | 41 | 66.13 |
| LEV | 0.002-32 | 13.916 | 30 | 48.39 | 32 | 51.61 |
| MXF | 0.006-32 | 14.638 | 35 | 56.45 | 27 | 43.55 |
| MTZ | 0.016-256 | 37.063 | 27 | 43.55 | 35 | 56.45 |
| RIF | 0.006-32 | 0.880 | 4 | 6.45 | 58 | 93.55 |
| TET | 0.016-4 | 0.234 | 2 | 3.22 | 60 | 96.77 |
| AML | 0.016–0.064 | 0.019 | 0 | 0 | 62 | 100 |
AZM, azithromycin; CLR, clarithromycin; LEV, levofloxacin; MXF, moxifloxacin; MTZ, metronidazole; RIF, rifampicin; TET, tetracycline; AML, amoxicillin.
Table 2.
The distribution of antimicrobial resistance of H. pylori isolates by gender and age.
| Antimicrobial | All patients (n = 62) | Gender | Age (years) | |||||
|---|---|---|---|---|---|---|---|---|
| Male (n = 45) | Female (n = 17) | < 40 (n = 4) | 40–49 (n = 8) | 50–59 (n = 27) | 60–69 (n = 17) | > 70 (n = 6) | ||
| AZM | 35(56.45) | 24(53.33) | 11(64.71) | 3(75) | 6(75) | 13(48.15) | 10(58.82) | 3(50) |
| CLR | 21(33.87) | 13(28.89) | 8(47.06) | 2(50) | 4(50) | 6(22.22) | 6(35.29) | 3(50) |
| LEV | 30(48.39) | 21(46.67) | 9(52.94) | 3(75) | 3(37.5) | 14(51.85) | 8(47.06) | 2(33.33) |
| MXF | 35(56.45) | 26(57.78) | 9(52.94) | 3(75) | 4(50) | 15(55.56) | 11(64.71) | 2(33.33) |
| MTZ | 27(43.55) | 21(46.67) | 6(35.29) | 2(50) | 3(37.5) | 10(37.04) | 8(47.06) | 4(66.67) |
| RIF | 4(6.45) | 3(6.67) | 1(5.88) | 0(0) | 0(0) | 4(14.81) | 0(0) | 0(0) |
| TET | 2(3.23) | 1(2.22) | 1(5.88) | 0(0) | 0(0) | 1(3.70) | 1(5.88) | 0(0) |
| AML | 0(0) | 0(0) | 0(0) | 0(0) | 0(0) | 0(0) | 0(0) | 0(0) |
Table 3.
The summary of resistant H. pylori isolates in NUD and PUD patients.
| Parameter | AZM (MIC > 4 µg/mL) |
CLR (MIC > 1 µg/mL) |
LEV (MIC > 1 µg/mL) |
MXF (MIC > 1 µg/mL) |
MTZ (MIC > 8 µg/mL) |
|||||
|---|---|---|---|---|---|---|---|---|---|---|
| Disease | NUD | PUD | NUD | PUD | NUD | PUD | NUD | PUD | NUD | PUD |
| Range (µg/mL) | 0.016-256 | 0.016-256 | 0.016-256 | 0.016-12 | 0.002-32 | 0.002-32 | 0.006-32 | 0.016-32 | 0.016-256 | 0.75–256 |
| No. | 30/53 | 5/9 | 20/53 | 1/9 | 27/53 | 3/9 | 31/53 | 4/9 | 23/53 | 4/9 |
| % Resistance | 56.60 | 55.56 | 37.74 | 11.11 | 50.94 | 33.33 | 58.49 | 44.44 | 43.40 | 44.44 |
| P value* | 0.953 | 0.088 | 0.328 | 0.432 | 0.953 | |||||
NUD, non-ulcer dyspepsia; PUD, peptic ulcer disease.
Table 4.
Multi-resistance of 62 H. pylori isolates.
| Multi-resistance pattern | Number (%) | Total (%) | |
|---|---|---|---|
| Dual resistance | AZM + MTZ | 2(3.23) | 20(32.26) |
| MXF + MTZ | 2(3.23) | ||
| AZM + MXF | 2(3.23) | ||
| AZM+ (LEV MXF)a | 3(4.84) | ||
| (AZM CLR) b+MXF | 1(1.61) | ||
| (AZM CLR) b+LEV | 1(1.61) | ||
| (AZM CLR) b+MTZ | 1(1.61) | ||
| (LEV MXF) a+MTZ | 2(3.23) | ||
| (AZM CLR) b+ (LEV MXF)a | 3(4.84) | ||
| (LEV MXF) a+TET | 1(1.61) | ||
| MXF + RIF | 1(1.61) | ||
| MTZ + RIF | 1(1.61) | ||
| Triple resistance | AZM + LEV + MTZ | 1(1.61) | 15(24.19) |
| (AZM CLR) b+MTZ + RIF | 1(1.61) | ||
| AZM+ (LEV MXF) a+RIF | 1(1.61) | ||
| AZM+ (LEV MXF) a+MTZ | 3(4.84) | ||
| (AZM CLR) b+ (LEV MXF) a+MTZ | 9(14.52) | ||
| Quadruple resistance | (AZM CLR) b+ (LEV MXF) a+MTZ + TET | 1(1.61) | 1(1.61) |
aBoth LEV and MXF resistant.
bBoth AZM and CLR resistant.
Determination of virulence genes and antimicrobial resistance genes in H. pylori
The detection results of virulence genes can be found in our previous study18. Antimicrobial resistance-related genes for AZM, CLR, LEV, MXF, MTZ, TET, and RIF from all 62 isolates were amplified by PCR, followed by Sanger sequencing to identify gene mutations associated with resistance phenotypes. None of the previously reported antibiotic resistance-associated mutations in the 16S rRNA and rpoB genes were present in our isolates19. Mutations of the 23S rRNA gene in macrolide-resistant isolates are shown in Table 5. The A2143G mutations in 23S rRNA were demonstrated in 82.86% (29/35) and 95.23% (20/21) isolates phenotypically resistant to AZM and CLR, respectively. Interestingly, the mutation A2143G was found in nine CLR-susceptible isolates, and conversely, no A2143G mutation was observed in six AZM-resistant isolates with high MIC values (> 256 µg/mL). The kappa coefficients (κ = 0.808) and (κ = 0.671) suggested a good level of concordance between the A2143G mutations and phenotypic E-test results for AZM and CLR, respectively (Table 6). Additionally, five mutations G2241A, C2195T, T2247C, G2269A, and G2304A were present only in macrolide-resistant isolates, while none of the macrolide-susceptible isolates exhibited these mutations (Table S2). The QRDR of gyrA (N87K/N87I/D91G/D91Y/D91N) was determined from 37 quinolone-resistant isolates (Table 5). The mutations in gyrA gene were confirmed in 70% (21/30) and 71.42% (25/35) isolates phenotypically resistant to LEV and MXF, respectively. However, we identified 8 and 6 isolates that were resistant to LEV and MXF, respectively, by sequencing but that were phenotypically sensitive (Table 5). Other mutations, including A88P, R130K, R140K, D143E, V172I, A183V, and V199A, were found only in quinolone-resistant strains (Table S3). No mutations associated with quinolones were observed in the gyrB gene. The gyrA mutations (N87K/N87I/D91G/D91Y/D91N) correlated with phenotypically resistance results for LEV and MXF (kappa coefficient, 0.526 and 0.442, respectively), suggesting moderate agreement between phenotypic and genotypic approaches to predict quinolone resistance in these isolates (Table 6).
Table 5.
H. pylori isolates MIC values and resistance-associated mutations for AZM, CLR, LEV, MXF, and MTZ.
| Strains | Disease | AZM | CLA | 23S rRNA | LEV | MXF | gyrA | MTZ | rdxA | frxA |
|---|---|---|---|---|---|---|---|---|---|---|
| JN1 | NUD | > 256 | 0.5 | 3 | 4 | 1.5 | 211ELONGATED | |||
| JN2 | NUD | > 256 | 8 | A2143G | 0.016 | 0.047 | 0.5 | |||
| JN3 | NUD | > 256 | 0.19 | A2143G | > 32 | > 32 | D91Y | > 256 | 11STOP | 85STOP |
| JN4 | NUD | > 256 | 8 | A2143G | 0.125 | 0.094 | 0.38 | 95frameshift, 96STOP | ||
| JN5 | NUD | > 256 | 0.5 | A2143G | 0.016 | 0.016 | N87K | 0.5 | 105frameshift, 108STOP | |
| JN6 | PUD | 0.5 | 0.25 | 2 | 2 | 1 | 154STOP | |||
| JN7 | NUD | 0.016 | 0.064 | 0.75 | 3 | 0.094 | ||||
| JN8 | NUD | 0.125 | 0.016 | > 32 | > 32 | N87K | 0.094 | 17frameshift, 38STOP | ||
| JN9 | NUD | 0.016 | 0.016 | 6 | 6 | N87K | 6 | |||
| JN10 | NUD | > 256 | 0.19 | A2143G | > 32 | > 32 | N87K | 0.064 | ||
| JN11 | NUD | > 256 | 24 | A2143G | 2 | 3 | N87K | > 256 | 184frameshift, 209STOP | |
| JN12 | NUD | > 256 | 8 | A2143G | > 32 | 32 | N87K | 12 | 64frameshift, 72STOP | 105frameshift, 108STOP |
| JN13 | NUD | > 256 | 32 | A2143G | 0.125 | 0.094 | D91Y | 2 | 53frameshift, 56STOP | |
| JN14 | NUD | > 256 | 3 | A2143G | > 32 | 0.016 | N87K | 0.032 | ||
| JN15 | NUD | 0.016 | 0.016 | 0.002 | 0.047 | > 256 | 74STOP | 9STOP | ||
| JN16 | NUD | > 256 | 0.047 | > 32 | 0.023 | 12 | 64frameshift, 72STOP | 69frameshift, 73STOP | ||
| JN17 | NUD | > 256 | 0.19 | A2143G | 0.016 | 6 | N87K | 0.38 | ||
| JN18 | NUD | 0.016 | 0.016 | 0.016 | 0.016 | N87I | 6 | |||
| JN19 | NUD | > 256 | 0.75 | A2143G | 0.016 | 0.016 | 24 | 48STOP | ||
| JN20 | NUD | > 256 | 32 | A2143G | > 32 | > 32 | 8 | 123STOP | 69frameshift, 73STOP | |
| JN21 | PUD | > 256 | 0.016 | A2143G | > 32 | > 32 | 24 | 65frameshift, 73STPOP | 69frameshift, 73STOP | |
| JN22 | PUD | 0.016 | 0.016 | 0.75 | 0.023 | 0.75 | 164frameshift, 175STOP | |||
| JN23 | NUD | > 256 | 0.5 | > 32 | > 32 | N87I | 32 | 49STOP | 69frameshift, 72STOP | |
| JN24 | NUD | 0.016 | 0.016 | > 32 | > 32 | N87K | 1.5 | |||
| JN25 | NUD | > 256 | > 256 | A2143G | 0.125 | 0.19 | 12 | |||
| JN26 | NUD | 0.016 | 0.016 | > 32 | > 32 | N87I | 1 | |||
| JN27 | NUD | > 256 | 24 | A2143G | > 32 | > 32 | N87I | 16 | 51STOP | |
| JN28 | NUD | > 256 | 1 | A2143G | 0.016 | 0.012 | 0.016 | 17frameshift, 38STOP | ||
| JN29 | NUD | > 256 | 0.016 | A2143G | > 32 | > 32 | D91G | 1.5 | 74STOP | |
| JN30 | PUD | > 256 | 0.016 | A2143G | > 32 | > 32 | N87K | 1 | 196STOP | |
| JN31 | NUD | 0.016 | 0.016 | 0.016 | 0.016 | 0.032 | ||||
| JN32 | NUD | 0.016 | 0.016 | 0.016 | 0.064 | 48 | 88STOP | 69frameshift, 73STOP | ||
| JN33 | PUD | > 256 | 0.016 | 0.125 | 0.125 | D91G | > 256 | 64frameshift, 72STOP | ||
| JN34 | NUD | 0.016 | 0.016 | 0.016 | 0.016 | 0.5 | 17frameshift, 53STOP | |||
| JN35 | NUD | 0.016 | 0.016 | 0.75 | 0.023 | 1 | ||||
| JN36 | PUD | > 256 | 12 | A2143G | 0.75 | 0.016 | 64 | 85STOP | ||
| JN37 | NUD | 0.016 | 0.016 | > 32 | > 32 | N87K | 12 | 48STOP | ||
| JN38 | PUD | > 256 | 0.016 | 0.002 | 0.032 | 0.38 | ||||
| JN39 | NUD | 0.016 | 0.016 | 0.016 | 0.016 | 0.5 | ||||
| JN40 | NUD | 0.094 | 0.016 | 0.064 | 0.064 | 12 | 37frameshift, 58STOP | 115STOP | ||
| JN41 | NUD | > 256 | > 256 | A2143G | > 32 | > 32 | D91G | 24 | 123STOP | |
| JN42 | NUD | 0.016 | 0.032 | 0.75 | 0.047 | 0.5 | ||||
| JN43 | NUD | 0.25 | 0.016 | 0.19 | 0.094 | N87K | > 256 | 64frameshift, 72STOP | 69frameshift, 72STOP | |
| JN44 | PUD | 0.094 | 0.25 | 0.75 | 3 | N87I | > 256 | 57STOP | 69frameshift, 73STOP | |
| JN45 | NUD | > 256 | 1 | A2143G | > 32 | > 32 | 16 | |||
| JN46 | NUD | > 256 | 4 | A2143G | > 32 | > 32 | N87K | 12 | 71STOP | |
| JN47 | NUD | > 256 | 1 | A2143G | > 32 | > 32 | D91N | 12 | 131frameshift, 136STOP | |
| JN48 | NUD | 0.064 | 0.016 | 0.125 | 0.064 | 12 | ||||
| JN49 | NUD | > 256 | 0.016 | A2143G | 0.002 | 4 | 0.5 | 20STOP | ||
| JN50 | NUD | 0.016 | 0.016 | > 32 | > 32 | 16 | ||||
| JN51 | NUD | > 256 | > 256 | > 32 | > 32 | D91G | 16 | 30STOP | ||
| JN52 | NUD | 0.016 | 0.032 | 0.75 | 0.047 | 0.25 | 69frameshift, 73STOP | |||
| JN53 | NUD | > 256 | 12 | A2143G | 0.5 | 2 | N87K | 0.5 | ||
| JN54 | NUD | > 256 | 32 | A2143G | > 32 | > 32 | D91G | 64 | 48STOP | 74STOP |
| JN55 | NUD | > 256 | 12 | A2143G | > 32 | > 32 | N87K | 2 | ||
| JN56 | NUD | 0.094 | 0.016 | 0.032 | 0.032 | 4 | ||||
| JN57 | NUD | 0.032 | 0.016 | 4 | > 32 | 3 | ||||
| JN58 | NUD | > 256 | 12 | A2143G | 3 | > 32 | 6 | 69frameshift, 73STOP | ||
| JN59 | NUD | > 256 | 6 | A2143G | 3 | > 32 | N87I | 3 | ||
| JN60 | NUD | 0.016 | 0.016 | 0.016 | 0.016 | 4 | ||||
| JN61 | NUD | 0.023 | 0.064 | 0.38 | 1.5 | 16 | 145STOP | |||
| JN62 | NUD | 0.032 | 0.125 | 0.75 | 1.5 | 0.38 |
Numbers in boldface type indicate isolates with phenotypically resistant MIC values.
Table 6.
Agreement between genotypic resistance and phenotypic MIC values.
| Antibiotics | MIC breakpoint (µg/mL) | Genotype | Phenotype | Kappa coefficient (95% CI) | P value | |
|---|---|---|---|---|---|---|
| Sensitive | Resistant | |||||
| AZMa | > 1 | Sensitive | 27 | 6 | 0.808 (0.66–0.95) | < 0.0001 |
| Resistant | 0 | 29 | ||||
| CLRa | ≥ 1 | Sensitive | 32 | 1 | 0.671 (0.49–0.85) | < 0.0001 |
| Resistant | 9 | 20 | ||||
| LEVb | > 1 | Sensitive | 24 | 9 | 0.526 (0.31–0.74) | < 0.0001 |
| Resistant | 5 | 21 | ||||
| MXFb | > 1 | Sensitive | 21 | 12 | 0.442 (0.22–0.66) | < 0.001 |
| Resistant | 6 | 23 | ||||
| MTZc | > 8 | Sensitive | 33 | 7 | 0.728 (0.56–0.90) | < 0.0001 |
| Resistant | 1 | 20 | ||||
aGenetic resistance was defined as an A2143G mutation in 23S rRNA.
bGenetic resistance was defined as mutations N87K/N87I/D91G/D91Y/D91N in gyrA.
cGenetic resistance was defined as a truncation in rdxA.
The mutations in rdxA and frxA genes of 27 MTZ-resistant isolates were analyzed in this study compared to 35 MTZ-sensitive isolates (Table S4 and Table S5). Most of the rdxA of MTZ-resistant isolates contained nonsense mutations that resulted in a premature stop codon (21/27, 77.78%, Table 5). These mutations led to the synthesis of truncated rdxA genes, making the predicted products of ≤ 209 compared to 210 residues of the rdxA in the reference strain 26,695. Moreover, rdxA genes of 8 MTZ-resistant isolates (29.63%) contained nucleotide deletion or insertion that resulted in frameshift mutations. A similar pattern was observed in frxA of MTZ-resistant isolates, which also contained nonsense mutations and frameshift mutations (15/27, 55.56%, and 8/27, 29.63%, respectively). The truncated frxA genes yielded predicted products of ≤ 123 residues versus 217 residues for frxA found in strain 26,695 (Table 5). Twenty-three resistant isolates had at least one truncated rdxA or frxA gene, except for four isolates (JN25, JN45, JN48, JN50). Twelve of the 35 MTZ-susceptible isolates had truncated frxA genes but intact rdxA sequences, suggesting that rdxA may be more important in terms of the MTZ resistance mechanism in some H. pylori isolates. There was a statistical association between truncated rdxA gene and MTZ phenotypic resistance results (P < 0.0001), and the kappa coefficient had a substantial agreement of 0.728 (95% CI: 0.56 to 0.90) (Table 6).
Whole-genome sequencing analysis of H. pylori isolates
Whole-genome sequencing was conducted on 32 isolates (24 NUD and 8 PUD) to further elucidate mechanisms of antimicrobial resistance (Table S6). The WGS results identified identical resistance mutations as those detected by Sanger sequencing for all genes examined in this study. Chromatiblock analysis revealed chromosomal structural variation and genetic diversity at the whole-genome level, displaying the arrangement and presence of the syntenic blocks encoding of 23S rRNA, gyrA, rdxA, and frxA genes, which may lead to the emergence of antimicrobial resistance in these genes (Fig. 1). The whole genomes of 32 isolates (24 NUD and 8 PUD) were also used to analyze phylogenetic origins. Interestingly, antimicrobial resistance profiles did not correlate with whole-genome phylogeny. The whole-genome phylogenetic tree, based on core gene sequences, demonstrated that isolates clustered according to clinical outcomes (Fig. 2).
Fig. 1.
Whole-genome alignments of syntenic blocks of 32 H. pylori isolates versus the reference strain 26,695. (Top) Global alignment view. (Bottom) Alignment difference view.
Fig. 2.
Whole-genome phylogenetic tree based on the multisequence alignment of concatenated core gene sequences of 32 H. pylori isolates and the reference strain 26,695. Local support values are indicated on the branches. R, resistance; S, susceptible.
Genotypic and phenotypic correlation of the multidrug-resistant H. pylori isolates
We examined the phenotypic and genotypic correlation of the multidrug-resistant H. pylori in our study. As shown in Table 5, the genotypic and phenotypic correlations in eight isolates (JN3, JN11, JN12, JN27, JN41, JN46, JN47, and JN54) were consistent for AZM or CLA (mutation of 23S rRNA), LEV or MXF (mutation of gyrA), and MTZ (mutations of frxA and rdxA). Meanwhile, the genotypic results in nine isolates (JN5, JN10, JN14, JN17, JN29, JN30, JN53, JN55, and JN59) were consistent with the phenotypic results of AZM or CLA, and LEV or MXF resistance. These samples harbored mutations of the resistance genes 23S rRNA and gyrA, which is important in antibiotic resistance. Additionally, the mutations of the resistance gene that conferred resistance to MTZ were not detected in three isolates (JN25, JN45, and JN50), although these isolates were resistant to MTZ at the phenotypic level (Table 5).
Relationship between virulence genes and antimicrobial resistance in H. pylori isolates
The virulence genes and antimicrobial resistance profiles of H. pylori isolates were analyzed. The results of the analysis showed that there was no correlation between virulence genes and antimicrobial resistance (Table S7).
Discussion
This study evaluated antimicrobial resistance in 62 H. pylori isolates cultured from gastric biopsies of patients in Shandong. Given the high prevalence of H. pylori infection, effective antibiotic use is crucial for eradicating H. pylori and reducing the risk of gastroduodenal ulcers and gastric cancer. In addition to determining the prevalence of antibiotic resistance to eight antibiotics commonly used in therapeutic procedures, we also evaluated the mutation profiles of antibiotic resistance-related genes in H. pylori isolates with phenotypic susceptibility and resistance to these antibiotics, and to provide a basis for attaining a better eradication regimen.
Given the importance of antibiotic susceptibility testing for H. pylori, it is necessary to choose a highly accurate detection method. According to the M45-A3 guidelines of the Clinical and Laboratory Standards Institute (CLSI), the agar dilution method is the gold standard for H. pylori susceptibility testing20. However, the agar dilution method is too laborious for daily clinical practices4. There have been studies using the E-test and agar dilution methods to determine the susceptibility of H. pylori to common antibiotics21–23. They found that although there were some discrepancies, the E-test presented a better agreement with agar dilution, and they recommended the E-test as a reliable and alternative method for H. pylori susceptibility testing to a wide range of antimicrobial agents in clinical practice. Some studies found discrepancies for metronidazole when the E-test was compared with agar dilution24,25, however, Chen JN et al. found no discrepancies when metronidazole was studied26. Therefore, in the present study, we used a relatively simple method, the E-test method, to detect the antibiotic susceptibility for H. pylori.
Currently, the AML resistance rate is rare (0%−5%) and it can be reused after the failure of H. pylori treatment11. There was no AML resistance strain in our study, which is in contrast with a study of antibiotic resistance in Yunnan that reported the prevalence of AML resistance to be 11.59%27. The main reasons for the low resistance to AML may be the inherent genetic stability of H. pylori, limited horizontal gene transfer, genetic diversity and heteroresistance, and limited antibiotic use in non-gastrointestinal infections. The mechanisms of resistance to AML are complex, and a phenomenon called AML tolerance has been described, that is, AML resistance is easily lost once the culture is frozen at −80℃28. Considering this, AML susceptibility testing should be performed as early as possible before the strain is frozen. AML is the preferred choice for H. pylori eradication therapy due to its strong anti-H. pylori effect and low resistance to H. pylori. In our study, only 2 (3.23%) and 4 (6.45%) isolates were resistant to TET and RIF, respectively, with no mutations in the 16S rRNA and rpoB genes. The prevalence of TET and RIF resistance in 13 Chinese provinces between 2010 and 2016 was reported as 1.9% and 1.5%29. Globally, TET resistance is less than 10%, except for the Eastern Mediterranean region4. According to the recommendation, TET is used as an antibiotic in a bismuth-containing quadruple therapy and maybe the main empirical therapy for H. pylori eradication11.
The CLR resistance rate in our study differed from previous studies (52.6% in Beijing, 17.76% in Zhejiang, and other countries (from 7 to 72.44%)30,31. Additionally, a study on H. pylori resistance to CLR in children and adults from China concluded increased resistance rates for CLR, especially in the group of adults, and lower resistance rates to CLR in patients aged between 10 and 24 years32. CLR is a key antibiotic in the H. pylori eradication therapy, and a high prevalence of CLR resistance has been considered as the main reason for eradication failure. According to the recommendation, if the CLR resistance rate is more than 15%, bismuth-containing quadruple, sequential, or hybrid therapies are preferred11. Our results indicated that CLR-based eradication regimen may be insufficient as first-line treatment for H. pylori eradication in Shandong without a susceptibility test. AZM was widely used for lower respiratory infections in China and became an important risk factor for cross-resistance to CLR33. Strong cross-resistance of AZM and CLR was observed in this study, consistent with previous studies that resistance to one macrolide becomes resistance to all others34.
The mutations, A2143G, A2142G, and A2142C, are the most frequent and account for nearly 90% of CLR resistance10. The mutant genotype of CLR in H. pylori varies among countries or regions. In China, previous studies described the frequency of A2143G mutation ranging from 34.78 to 98.4%35–37. A study conducted by Bui et al.38 reported that the frequency of A2143G and A2142G was 85% and 6.7%, respectively, through high-throughput next-generation sequencing by using 123 H. pylori isolates directly extracted from gastric biopsies of patients in Vietnam. In Malaysia, studies described the frequency of A2143G mutation ranging from 50 to 90.5%39,40. In addition, in this study, only the A2143G mutation was detected, with a detection rate of 95.23% and 82.86% in CLR- and AZM-resistant isolates, respectively. Notably, 9 (21.95%) CLR-susceptible isolates had A2143G mutations, and 6 (20.69%) AZM-resistant isolates lacked 23S rRNA mutations. One plausible explanation for the presence of the A2143G mutation in CLR-susceptible isolates may be attributed to the phenomenon of heteroresistance within the H. pylori population. Conversely, the absence of the A2143G mutation in certain AZM-resistant isolates with high MIC values may indicate alternative resistance mechanisms or genetic variations influencing the phenotypic resistance41. Factors such as environmental conditions, drug dosage, or strain-specific genetic mutations could contribute to this discordance between genotypic and phenotypic results17. Further exploration into the genetic diversity and specific mutations within the bacterial population is warranted to elucidate the intricacies of antibiotic resistance in H. pylori. Our analysis suggested the presence of mutations had good correlations with phenotypic resistance to AZM and CLR, consistent with the previous study42. None of our isolates harbored the A2115G, G2141A, C2147G, T2190C, A2144T, or G1939A mutation, which have previously been associated with CLR resistance43. However, we identified several novel mutations, G2241A, T2247C, G2269A, and G2304A. It may be that compensatory mutations and gene expression regulation play crucial roles in determining the genotype of resistant strains, leading to genetic diversity that can impact their evolution and interaction with their environment. The role of these novel mutations in antibiotic resistance remains to be further investigation. The T2182C mutation in almost all isolates (Table S3) was not associated with CLR- and AZM-resistance.
The resistance rate for quinolones varied in China, which was relatively high in North (21.2%−54.8%) and East (15.4%−40%) China, and low in Central (17.7%−20.9%) and Southwest China (23.36%−30.4%)29,44. In Shandong, our data showed resistance rates for LEV and MXF at 48.39% and 56.45%, respectively, higher than those in East China29. Our results found that 45.16% (28/62) H. pylori isolates were resistant to LEV and MXF, indicating cross-resistance between the quinolones. We suggested that other quinolones should be avoided empirically to eradicate H. pylori after the failure of LEV-containing regimen therapy. Resistance to LEV and MXF is mainly caused by the specific mutations N87K/I/G/Y/N and D91G/N/Y/A in the QRDR of the gyrA gene, which encodes DNA gyrase14. In this study, the genotyping outcomes suggested moderate agreement values for resistance to LEV and MXF, with kappa coefficients of 0.526 and 0.442, respectively. Regarding the antibiotic target genes, not only the well-known mutations in gyrA (N87K/I and D91G/N/Y) but also several other mutations (A88P, R130K, R140K, etc.) associated with antibiotic resistance were detected, and the latter may be the genetic diversity of H. pylori caused by compensatory mutations, which may arise from the accumulation of mutations in response to selective pressures40. Thus, further research is required.
The mutations in frxA and rdxA, such as nonsense mutations, frameshift and truncation mutations, have been reported to confer resistance to MTZ45. These mutations play a significant role in conferring resistance to MTZ by disrupting the reductase function that is crucial for MTZ activation. Mutations in frxA and rdxA can lead to loss of function through frameshift or nonsense mutations, further diminishing the bacteria’s ability to activate the drug. The combined effect of mutations in both frxA and rdxA allows bacteria to effectively evade the lethal effects of MTZ, resulting in a robust resistance mechanism46,47. In our study, MTZ resistance prevalence was 43.55%, with no statistical association between clinical outcomes and antibiotic resistance. However, a significant association was found between resistance phenotype and a truncated rdxA gene among all isolates. In addition to truncation, mutations at positions A16T, S43A, I44F, F72S, G73S, N111D, and C193S in the rdxA were present in most isolates (Table S4). These mutations in rdxA have been previously associated with MTZ resistance, but the results were inconsistent in another study48. Recently, a study in Beijing revealed that the MTZ resistance rate had remained stable at 63.5%−66.8% from the year 2009 to 201449. In addition, multidrug-resistant strains of H. pylori have been reported in many countries, which can lead to a significant decline in the H. pylori eradication rate50. In our study, triple resistance to macrolides, quinolones, and MTZ was the most common multiple antibiotic-resistance pattern in Shandong, indicating that these antibiotic-based standard triple therapies were not useful as a first-line option for H. pylori eradication.
In this study, a comprehensive analysis of the eight antibiotics-related H. pylori drug resistance showed that a high rate of multidrug-resistant strains was observed. The dual, triple, and quadruple antibiotic-resistant rates were 32.26% (20/62), 24.19% (15/62), and 1.61% (1/62), respectively, and only one isolate was resistant to quadruple antibiotics. Multidrug resistance in H. pylori strains has been reported in many countries40,51. Multidrug resistance significantly reduces the eradication rate of H. pylori, increasing treatment failure risks52. Although the mechanisms of resistance to individual antibiotics in H. pylori vary, synergistic interactions between these resistance pathways often amplify multidrug resistance53. The correlation between phenotypic and genotypic antibiotic resistance was found in AZM or CLA, LEV or MXF, and MTZ resistance. Interestingly, a discrepancy in the genotypic and phenotypic correlation was discovered in MTZ resistance. Three H. pylori isolates were susceptible to MTZ at the phenotypic level, although the frxA and rdxA gene mutations that are associated with MTZ were not detected. This finding suggests that the MTZ resistance mechanism in H. pylori can occur through other mechanisms, which still needs further investigation.
In our study, we applied Illumina sequencing technology to detect selected H. pylori isolates to complement studies on antimicrobial resistance mechanisms. Unlike Sanger sequencing, WGS provides a more detailed analysis approach for detecting and identifying the genes and variations within the genome, which allows for a thorough understanding of the antibiotic resistance mechanisms of H. pylori, including the types and distribution of resistance genes. Moreover, WGS can help identify new resistance genes or variations that may not be detectable using traditional methods. Thus, in addition to identifying resistance genes and resistance-associated specific mutations, we also analyzed whole-genome phylogenetic relationships and chromosomal rearrangements. Phylogenetic analysis based on core gene sequences indicated differences between H. pylori isolates from PUD and NUD, with antimicrobial resistance phenotypes not correlating with whole-genome phylogeny. Chromosomal rearrangements were evident among the genomes, possibly due to selective pressure from antibiotic use or adaptive evolutionary selection between the strain and the host.
In summary, our study found that the H. pylori strains isolated from Shandong exhibited high resistance rates and multiple resistance. Furthermore, we found that antibiotic resistance phenotypes were associated with mutations in genes previously reported in H. pylori drug resistance. Despite the valuable insights obtained, this study still has some limitations. First, the sample size of isolates was too small, which limited the study’s statistical power and may not fully capture the genetic and phenotypic diversity of H. pylori in Shandong. Second, our study lacked data on eradication failures that would help clarify whether resistance patterns were linked to previous treatments, particularly to multidrug resistance. Third, the MIC values of different colonies from the same patient were not tested, which may lead to the possibility of bias in the experimental results. Future studies should aim to overcome these limitations and thoroughly detect and characterize antibiotic resistance in H. pylori from Shandong to establish effective eradication therapy for this population.
Methods
Study participants and H. pylori culture
Gastric mucosal biopsy specimens were taken from 206 dyspeptic patients (147 males and 59 females; age range, 32–78 years; mean age, 57.49 ± 9.11 years) who underwent upper endoscopy. Exclusion criteria included individuals who had received anti-H. pylori eradication treatment in the month preceding the gastroscopy examination. Two biopsy samples were taken from the antrum of each patient for H. pylori culture and histological examination. Gastric biopsy specimens for culture were stored in the brain heart infusion broth (BHI, CM1135, Oxoid) with 20% glycerin. Biopsies were homogenized and inoculated onto the Karmali agar (CM0935, Oxoid) plate supplemented with 5% defibrinated sheep blood, 10 µg/mL vancomycin, 5 µg/mL trimethoprim, 5 µg/mL cefsulodin, and 5 µg/mL amphotericin B under microaerobic atmosphere (5% O2, 10% CO2, and 85% N2) for up to 10 days. H. pylori isolates were identified by colony morphology, Gram staining, and oxidase, catalase, and urease reactions54.
Antimicrobial susceptibility testing
All antibiotics were purchased from Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy (Liofilchem, Italy). Antimicrobial susceptibility to CLR, LEV, MTZ, TET, RIF, AML, AZM, and MXF were tested by the E-test method using Mueller-Hinton agar medium (Oxoid) supplemented with 5% sheep blood and incubated in microaerophilic conditions for 72 h55. The experiments were performed according to the M45-A3 guidelines of CLSI20. The antibiotic resistance breakpoints were determined following the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, version 13.1; available at http://www.eucast.org/). Isolates were resistant for the minimum inhibitory concentrations (MICs) > 8 µg/mL for MTZ, 0.125 µg/mL for AML, and 1 µg/mL for LEV, AZM, MXF, RIF, and TET. The resistance breakpoints of CLR were based on CLSI criteria (≤ 0.25 µg/mL, susceptible; 0.5 µg/mL, intermediate; and ≥ 1.0 µg/mL, resistant)20. The quality control reference strain was performed using H. pylori ATCC 43,504. Multidrug resistance is defined as resistance to two or more classes of antibiotics.
Analysis of resistance genes and virulence genes
The DNA was extracted with the TIANamp Bacteria DNA Kit (TIANGEN, China) according to the manufacturer’s instructions. The gyrA/gyrB and 23S rRNA genes conferring resistance to macrolide and quinolone antibiotics, respectively, were amplified. Additionally, the rdxA/frxA, 16S rRNA, and rpoB genes were also amplified for MTZ, TET, and RIF, respectively. The PCR primers and the nucleotide sequences used in this study were summarized in Table S8 as described previously4,55–57. Genes cagA and vacA were amplified as described previously18. The amplification was as follows: 35 cycles of initial denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 55 to 60℃ for 30 s, and extension at 72℃ for 40 s, followed by a final extension of 10 min at 72℃. The PCR products were purified using the E.Z.N.A.® Gel Extraction Kit (OMEGA, USA) and then sequenced with Sanger sequencing (Sangon Biotech, Shanghai, China).
Whole-genome sequencing
DNA was extracted from 32 (24 NUD and 8 PUD) of the 62 isolates using the TIANamp Bacteria DNA Kit (TIANGEN, China). The DNA sequencing was performed by an Illumina HiSeq PE150 platform (Illumina Inc., San Diego, CA, USA) in Novogene Bioinformatics Technology Co., Ltd (Beijing, China). For genome sequencing, a 350 bp paired-end Library was constructed, and then 150 bp reads were generated using NEBNext® UltraTM DNA Library Prep Kit for Illumina (NEB, USA). The Library quality was assessed on the Agilent 5400 system (Agilent, USA) and quantified by qPCR (1.5 nM). Fastp (https://github.com/OpenGene/fastp) software was applied to perform basic statistics on the quality of the raw reads. Low-quality reads were discarded if the proportion of low-quality (Phred quality < 5) bases was over 50% in either one read. The de novo assembly of the 32 genomes was performed using SOAPdenovo (http://soap.genomics.org.cn/soapdenovo.html). Genomes were predicted and annotated using the RAST (https://rast.nmpdr.org/) and PATRIC (https://www.patricbrc.org/). Roary was used to search for concatenated core gene sequences, followed by MAFFT for multi-sequence alignment and FastTree (http://www.microbesonline.org/fasttree/) to construct maximum likelihood phylogenetic trees. Chromatiblock was used to create a linear visual representation of structural variation available for alignments of large numbers of genomes58. Genomes were analyzed using the Center for Genomic Epidemiology (http://www.genomicepidemiology.org) to identify antibiotic-resistance genes.
Statistical analysis
Statistical data were analyzed using SPSS software (version 20, Chicago, USA) using the chi-square test and Fisher’s exact test. The one-way analysis of variance (ANOVA) test was used to analyze the association between ages and multi-resistance. A P-value < 0.05 was considered statistically significant. Cohen’s kappa coefficient was used to determine the agreement between phenotypic resistance and the mutations of antimicrobial resistance. A value of < 0.4 was considered low agreement, a value of 0.4 to 0.6 was considered moderate agreement, a value of 0.61 to 0.8 was considered substantial agreement, and a value of 0.81 to 1.0 was considered nearly perfect or perfect agreement.
Ethical approval and consent to participate
All experimental protocols were approved by the Ethical Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention (approval number: ICDC-2013001), and informed consent was obtained from all subjects. We confirmed that all experiments were performed in accordance with relevant guidelines and regulations.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We thank the Endoscopy Center of Jinan Central Hospital for collecting gastric mucosa samples and information.
Author contributions
Z.X. developed the idea, designed the study, and drafted the manuscript, Q.Z., F.P., Y.G., and F.W. collected the samples, Y.W., Q.C., and Y.L. performed the DNA extraction, Q.X., J.T., and Y.L. analyzed the data. YZ and GS supervised the study and revised the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the Natural Science Foundation of Shandong Province (ZR2021QC219), the Shandong Medical and Health Science and Technology Development Plan Project (202202080452), the Jinan Clinical Medical Science and Technology Innovation Program (202328036), the Natural Science Foundation of Shandong Province (ZR2024MH159), and the Jinan Special Fund for High level Talents in the Medical and Health Industry (202412).
Data availability
The nucleotide sequence data have been deposited in the National Center for Biotechnology Information (accession numbers: PP101335-PP101396 and PP129348-PP129499). The genomes have also been deposited into GenBank with BioProject number PRJNA1060242. Please contact the corresponding author for further information if necessary.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Zhijing Xue, Email: 573799518@qq.com.
Yan Zheng, Email: 8793822@qq.com.
Guohai Su, Email: gttstg@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The nucleotide sequence data have been deposited in the National Center for Biotechnology Information (accession numbers: PP101335-PP101396 and PP129348-PP129499). The genomes have also been deposited into GenBank with BioProject number PRJNA1060242. Please contact the corresponding author for further information if necessary.