Since its discovery, Helicobacter pylori (H. pylori) has emerged as one of the most widespread microorganisms worldwide. The overall prevalence of H. pylori worldwide is reported to be 44.3%, with developing countries exhibiting a higher prevalence (50.8%) compared to developed countries (34.7%). (Zamani et al., 2018) In addition to its role in chronic active gastritis and peptic ulcers, H. pylori is the most significant identified risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma (MALT). Notably, in China, stomach cancer holds the third position for males and the second position for females in terms of malignant tumor occurrences. (Azzaya et al., 2020) Consequently, the infection caused by H. pylori has evolved into a substantial public health concern that requires immediate attention. (Shu et al., 2022) The escalation of antimicrobial resistance in recent times underscores the imperative to enhance the efficacy of H. pylori treatment, thereby emphasizing the urgency of addressing this pivotal concern (Kuo et al., 2021; Azrad et al., 2022).

The Maastricht VI/Florence consensus report advocates for the incorporation of routine antibiotic sensitivity testing prior to commencing H. pylori eradication treatment. This approach is recommended to facilitate the accurate selection of treatment strategies. (Malfertheiner et al., 2022) At present, a limited array of antibiotics, namely amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and furazolidone, remains effective in combatting H. pylori. Nevertheless, the extensive utilization of these constrained antibiotics has hastened the emergence of antibiotic resistance. (Lee et al., 2019; Jiang et al., 2022) China harbors a substantial population afflicted by H. pylori infection, revealing a complex landscape of antibiotic resistance (Xie and Lu, 2015).

The eradication regimen incorporating levofloxacin presents a promising approach. It not only stands as a primary therapeutic choice for addressing H. pylori infection but also assumes the role of a rescue program when needed. (Miyachi et al., 2006) A meta-analysis showed that 14-day levofloxacin-based sequential therapy was the most efficient therapy globally. (Zamani et al., 2022) However, notable variations exist in the susceptibility of H. pylori to levofloxacin across diverse global regions. A study reported that the overall mean prevalence of primary H pylori resistance was 18% for levofloxacin in the Asia-Pacific region. (Kuo et al., 2017) But in China, the prevalence of primary resistance to levofloxacin has reached 34.21%. (Zhong et al., 2021) Given the significant prevalence of levofloxacin resistance, it is crucial to address two fundamental aspects: firstly, gaining a comprehensive understanding of the phenotypic resistance variations to levofloxacin in the local region over recent years, and secondly, scrutinizing the patterns of gene mutations associated with levofloxacin resistance.

In light of the escalating drug resistance observed in H. pylori, there’s an immediate need to monitor and gather data within the local region consistently. Our center previously reported a cross-sectional study from 2018 to 2021, summarizing the drug resistance of H. pylori and genotypic resistance of clarithromycin in Nanjing. (Jiang et al., 2022) However, there is limited available data concerning the prevalence and underlying mechanisms of levofloxacin resistance within the local area. The present study delved into the variations in the percentage of phenotypic resistance exhibited by H. pylori towards six commonly administered antibiotics over the past five years. Our emphasis centered on investigating the phenotype resistance profiles specifically related to levofloxacin. Subsequently, we evaluated the influence of the treatment eradication history employing levofloxacin-based regimens and the frequency of eradication attempts on the rate of phenotypic drug resistance.

Our research also focuses on the genotypic resistance of levofloxacin, in addition to phenotypic resistance. The development of drug resistance in H. pylori is primarily attributed to mutations that modify crucial drug targets or hinder their effectiveness. Evidence indicates that the resistance of H. pylori to fluoroquinolones arises due to specific point mutations occurring in the quinolone resistance determining region (QRDR) of the gyrA/B gene. The gyrA/B gene encodes the A and B subunits of DNA gyrase. As of now, various mutation patterns within this gene have been documented. (Moore et al., 1995; Dascalu et al., 2023) The predominant mutation types observed in the gyrA gene of H. pylori in China are Asn87Lys, followed by Asp91Asn and Asp91Gly. (Zhong et al., 2021) The genotypic and phenotypic resistance of levofloxacin exhibit a strong correlation, thereby suggesting the potential for predicting the phenotypic resistance of levofloxacin through the detection of gyrA mutations. (Xiong et al., 2023) Enhancing the precision of the above prediction can be achieved by analyzing the various patterns of gyrA mutations observed in recent years and specifically investigating the differential effects of mutations at positions 87 and 91 on the phenotypic resistance of levofloxacin. In this study, a comprehensive analysis was undertaken to elucidate the specific mutations with substantial significance in conferring levofloxacin resistance within H. pylori.

The present study provides an up-to-date analysis of drug resistance patterns of H. pylori in the Nanjing region of China, specifically examining the phenotypic and genotypic resistance to levofloxacin. The findings of this research will provide assistance for the rapid diagnosis and personalized treatment of H. pylori.

As a notable contributor to the pathogenesis of upper gastrointestinal diseases, H. pylori assumes a critical role. The prevalence of H. pylori infection ranges from 25% to 50% in developed countries and can reach as high as 80% in developing countries. (Jiang et al., 2022) Particularly in mainland China, the overall occurrence rate of H. pylori is reported at 44.2%, affecting approximately 589 million individuals (Ren et al., 2022).

The escalating antibiotic resistance exhibited by H. pylori has elicited global concern. China, too, confronts a significant challenge of antibiotic resistance, which presents grave implications for the effective treatment of H. pylori infections. (Zhong et al., 2021; Xu et al., 2022) Despite recent studies suggesting that high-dose dual therapy (HDDT) demonstrates similar effectiveness and adherence compared to bismuth-containing quadruple therapy (BQT) or standard triple treatment, BQT remains the predominant treatment approach for H. pylori infection in China (Chey et al., 2022; Zou et al., 2022).

Nanjing, situated in eastern China, is a metropolis with a populace exceeding 10 million. The prevalence of H. pylori infection in this region is relatively high. The imperative need arises to monitor H. pylori drug resistance within the region continuously. Such monitoring is a vital resource for local healthcare professionals, aiding them in making well-informed antibiotic selections for effective treatments.

Based on these considerations, our center assessed the alterations in H. pylori isolate resistance to frequently employed antibiotics spanning 2018 to 2023. Considering that the levofloxacin based BQT is one of the predominant treatment strategies for eradicating H. pylori, it is crucial to promptly obtain current data on antibiotic resistance within the specific region, with a specific focus on the resistance rate of levofloxacin against H. pylori. Besides, many studies have already confirmed that the genotypic and phenotypic resistance of levofloxacin exhibit a strong correlation. The objective of this study is to enhance the precision of prognosticating phenotypic resistance to levofloxacin through gyrA mutations by examining diverse patterns of gyrA mutations observed in recent years and investigating the distinct impacts of mutations at positions 87 and 91 on phenotypic resistance to levofloxacin.

A previous study conducted by our center uncovered that within the timeframe spanning 2018 to 2021, the pre-treatment resistance rates for metronidazole, clarithromycin, levofloxacin, amoxicillin, furazolidone, and tetracycline in Nanjing were documented at 67.19%, 35.99%, 24.23%, 0.76%, 0.28%, and 0.09%, respectively. (Zhong et al., 2021) In our study, the most recent primary resistance rates for the aforementioned antibiotics were recorded at 77.23%, 37.24%, 27.72%, 0.52%, 0.19%, and 0.06%, respectively. Our research findings indicate a notable upsurge in the rate of metronidazole resistance in comparison to previously documented data. Additionally, a slight elevation was observed in the resistance rates for clarithromycin and levofloxacin, whereas a decline was noted in the rates of resistance to amoxicillin, furazolidone, and tetracycline. It is noteworthy to observe that the prevalence of multidrug-resistant H. pylori is progressively escalating over time, potentially attributable to the utilization of non-standard treatment approaches and the excessive use of antibiotics. For patients who experienced initial treatment failure, the resistance rates for clarithromycin, levofloxacin, and metronidazole significantly increased. Furthermore, it was revealed that an escalation in the number of eradication attempts was correlated with a corresponding rise in drug resistance rates. This exacerbates the already complex issue of multiple antibiotic resistance.

In the intricate landscape of drug resistance, we advocate the idea that all individuals diagnosed with H. pylori infection should ideally undergo antibiotic sensitivity testing to enable the formulation of targeted treatment plans. Nevertheless, implementing such an approach may not be feasible for all patients. For those initiating their treatment journey, a regimen incorporating levofloxacin or clarithromycin for eradication is a favorable choice in situations where sensitivity testing cannot occur. Because in many local secondary and community hospitals, levofloxacin and clarithromycin are commonly used due to their accessibility.

It has been documented that in China, the primary and secondary antibiotic resistance rates for levofloxacin in H. pylori were 34.21% and 61.58%, respectively. (Zhong et al., 2021) In contrast, the resistance rate of levofloxacin in Nanjing is higher. Our study has demonstrated a pronounced correlation between the history of eradication attempts using levofloxacin-containing regimens, the escalation of phenotypic drug resistance rates, and the prevalence of gyrA gene mutations. Consequently, if levofloxacin proves ineffective in eradicating H. pylori during the initial treatment, it is advisable to avoid its usage in subsequent treatment strategies. In light of the prevailing drug resistance observed in H. pylori, we recommend that local healthcare practitioners possess a comprehensive understanding of the specific drug resistance landscape within their region. Physicians should diligently inquire about patients’ medication history before making prescription decisions, allowing for the formulation of personalized treatment strategies that align with the individual’s unique circumstances.

Furthermore, the examination of levofloxacin’s genotypic resistance is also a central aspect of the present study’s discourse. Firstly, PCR exhibited a substantial correlation with levofloxacin susceptibility tests, indicating its potential utility in clinical settings for guiding the appropriate usage of levofloxacin. This outcome aligns with prior research findings, reinforcing the validity and applicability of PCR as a reliable method for guiding treatment decisions related to levofloxacin. (Trespalacios-Rangel et al., 2016; Hu et al., 2023) Consistent with previous studies conducted in our nation, (Zhong et al., 2021) this investigation primarily focused on identifying point mutations occurring at amino acids 87 (resulting in substitutions from Asn to Lys, Ile, Try, or Arg) and 91 (resulting in substitutions from Asp to Asn, Gly, or Tyr) within the strains under examination. However, distinct from earlier studies, our research documented an added dimension by identifying 53 cases of gyrA double mutations and one instance of triple mutation. Furthermore, our findings revealed that the occurrence of mutations in codon 87 surpassed that in codon 91. Again, among the sensitive strains, 1.77% displayed a point mutation in codon 87, while a more significant proportion of 8.37% exhibited sensitivity despite a point mutation in codon 91.

Except for the N87K+D91N mutation, all isolates carrying multiple gyrA mutations resisted levofloxacin. Most instances showing discrepancies between antibiotic sensitivity tests and gyrA mutation detection outcomes were observed in strains featuring point mutations in codon 91. Among these, the most pronounced difference arose in strains with the D91N mutation. An in vitro investigation suggested that mutations in codon 87 are more impactful than those in codon 91, and the effects of combined mutations are even more pronounced than single mutations. Moreover, the study found that mutations in codon 91 were reversible, (Wang et al., 2020) indicating potential instability. This inherent instability in codon 91 mutations might explain the variance between antibiotic sensitivity and gyrA mutation test outcomes.

Currently, various kits are in development for gyrA genetic testing, aiming to predict H. pylori resistance to levofloxacin. These kits predominantly target the identification of gyrA genes within feces, gastric mucosa, or even gastric juice samples. (Ierardi et al., 2017; Tsuda et al., 2022) Analyzing the specific types of gyrA mutations holds the potential to enhance the precision and effectiveness of PCR detection tools. Our study proposes that gyrA dual and triple mutations exhibit a significant level of precision in predicting the resistance phenotype of levofloxacin, except for N87K+D91N mutations. This information can contribute to refining PCR detection components, improving sensitivity and specificity within gyrA detection kits. The findings of our study have the potential to enhance the precision of clinical practitioners in forecasting levofloxacin phenotypic resistance through gene testing outcomes, consequently providing improved guidance for clinical medication.

Furthermore, a subset of levofloxacin-sensitive strains, totaling 6.38% (27/423), exhibited the presence of gyrA mutations at either Asn-87 or Asp-91. On the contrary, 3.22% (21/653) showed an absence of gyrA mutation among the levofloxacin-resistant strains. These observed phenomena likely stem from the coexistence of both mutant and wild-type strains. It’s well-established that a single patient can host diverse strains of H. pylori. (Kim et al., 2003) Hetero-resistance or genetic heterogeneity may manifest within a patient’s pair of biopsy samples. Furthermore, it is conceivable that a single sample could potentially encompass a mixture of two distinct phenotypic or genotypic populations. This is also the reason why double point mutation was found at the same locus in our study. According to a study conducted by Emmanuelle Cambau, a single biopsy specimen exhibited the presence of a double mutation at codon 91 (D91N+D91G or D91N+D91Y). (Cambau et al., 2009) Further, some reports suggest that N87 wild-type and N87K mutant-type may exist in the same biopsy sample. (Cambau et al., 2009; Binmaeil et al., 2021) The presence of a combination of susceptible and resistant genotypes has been associated with clinical failure. While infections with more than one strain may be associated with more severe disease (Sheu et al., 2009).

This study is not without its limitations. Firstly, the samples were sourced solely from a single center in Nanjing, which may introduce inherent biases to the results. Secondly, the conclusions drawn may not fully encapsulate the broader antibiotic resistance patterns of H. pylori across various anatomical locations within the stomach. Thirdly, the study did not determine minimum inhibitory concentrations (MICs) for H. pylori isolates, consequently inhibiting the establishment of a definitive link between the gyrA mutation patterns and levofloxacin MIC values.

In conclusion, the resistance of H. pylori in Nanjing has not improved compared to recent years, with a slight elevation observed in primary resistance to levofloxacin, clarithromycin, and metronidazole. The prevalence of multidrug-resistant strains is progressively escalating. The necessity for antibiotic sensitivity testing before employing these three antibiotics in eradication treatment is highlighted. It is not recommended to reuse the eradication plan containing levofloxacin on the same patient. For evaluating levofloxacin susceptibility in H. pylori, a DNA-based screening approach is a promising substitute for conventional culture methods. However, predicting phenotypic resistance of levofloxacin through PCR require more attention to the mutation type of gyrA to improve prediction accuracy. Vigilant levofloxacin resistance monitoring remains crucial to formulating effective strategies for eradicating H. pylori.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Ethics Committee of Nanjing First Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

WW: Data curation, Investigation, Writing – original draft, Writing – review & editing. ZW: Data curation, Investigation, Methodology, Writing – original draft. CL: Data curation, Investigation, Writing – review & editing. ZJ: Formal Analysis, Investigation, Writing – review & editing. ZZ: Conceptualization, Project administration, Supervision, Writing – review & editing. SW: Conceptualization, Project administration, Supervision, Writing – review & editing.