Introduction

Helicobacter pylori (H. pylori) is a type of microaerophilic, gram-negative bacterium that is present throughout the world, particularly in developing nations. It functions as a prevalent pathogenic microorganism and has been identified over the past thirty years as a notable contributor to gastrointestinal ailments, which encompass peptic ulcers, persistent gastritis, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The classification assigned by the World Health Organization is that of a class I carcinogen. Furthermore, H. pylori infection has been linked to irregularities within the nervous, circulatory, and hematopoietic systems. The eradication of H. pylori infection assumes a pivotal role in the prognosis of these conditions. Clinical investigations underscore that removing the infection yields tangible advantages by fostering the healing of gastric mucosa and reducing the factors predisposing to MALT lymphoma and gastric cancer. Thus, H. pylori eradication holds significant importance in clinical practice1,2,3,4,5,6.

The presently endorsed primary approach for eliminating H. pylori involves a triple therapy that centers around clarithromycin, coupled with a proton pump inhibitor (PPI) and either amoxicillin or metronidazole. This treatment protocol yields an eradication success rate ranging from approximately 70 to 85%. Nonetheless, instances of treatment ineffectiveness are primarily attributed to poor adherence to the regimen and the escalating incidence of antibiotic resistance, notably the rising levels of clarithromycin resistance on a global scale7. The utilization of multiple antibiotics in H. pylori treatment has spurred the emergence of bacterial antibiotic resistance, albeit with distinct patterns varying across regions8. The infection rates with H. pylori tend to be higher in developing countries compared to developed ones. Consequently, understanding resistance levels within individual countries and populations is crucial. The objective of this study is to discern the antibiotic resistance pattern of H. pylori in previously untreated patients in Palestine.

Methods

Study population

The study targeted all adult patients who underwent upper GI endoscopy for various medical reasons at NNUH between July 2016 and January 2017 and agreed to participate by signing the consent form.

  • Inclusion criteria:

    All participants were aged 18 years or older and had undergone upper endoscopy at An-Najah National University Hospital during the study period.

  • Exclusion criteria:

    Patients were excluded if they had made previous attempts to eradicate H. pylori, used antibiotics or proton pump inhibitors within the last 2 weeks before endoscopy, or had undergone previous gastric surgery.

Sampling

Ninety-one patients with diverse gastrointestinal symptoms representing various age groups and genders were included. Samples were collected from the Endoscopy Department of Al-Najah National University Hospital in Nablus. Prior informed written consent was obtained from each patient.

Sample size calculation

The sample size was calculated using Cochrane formula for sample size calculation in prevalence studies.

$${\text{n}}_{{\text{o}}} = \frac{{\left( {{\text{Z}}_{{\upalpha /2}}^{2} } \right){\text{p}}\left( {1 - {\text{p}}} \right)}}{{\Delta^{2} }}$$

Considering 95% confidence level (z = 1.96), estimation error (E = 0.10), and the prevalence (p = 0.5), the calculates sample size is n = 86 samples. Considering drop out 5% the required sample size is 90 samples.

Specimen collection

During endoscopic diagnosis, Dr. Qusay Abdoh, a gastroenterologist at An Najah National University Hospital, collected antral and corpus mucosal biopsies from the stomach. The antral biopsy was taken from the lower part adjacent to the pylorus, while the corpus biopsy was obtained from the stomach’s body region responsible for producing gastric juices. Biopsies were transported in 2 ml Brain–heart infusion broth with ice and processed within four hours of collection to ensure sample integrity.

Biopsy culturing

Biopsy samples were minced and homogenized in sterile petri dishes near a bunsen burner. The minced biopsy, along with 0.2 ml of transport media, was incubated on Brain–heart infusion agar media plates supplemented with 5% sheep blood and Helicobacter pylori selective supplement (Dent) for primary isolation. Cultures were incubated at 37 °C under microaerophilic conditions. Plates were examined for positive growth at 7 days intervals. Positive growth was characterized by tiny, glistering, translucent or gray colonies with intact edges.

Biochemical tests of H. pylori

  • Urease test:

    Grown colonies were inoculated on urea agar slants containing phenol red. The color change from yellow to pink was observed before and after 1 h and 24 h of incubation.

  • Catalase test:

    Hydrogen peroxide (3%) was added to grown isolated colonies on a sterile slide. The production of gas bubbles within 20–30 s indicated a positive reaction.

  • Oxidase test:

    An isolated colony was transferred into an oxidase strip. A positive reaction was indicated by an intense deep purple color appearing within 5–10 s.

  • Antibiotic susceptibility test:

    Positive H. pylori isolates were inoculated in brain–heart infusion broth supplemented with 5% sheep or human serum and incubated at 37 °C for 24 h. Inoculated broth was spread on Muller-Hinton agar plates supplemented with 5% sheep blood. Antibiotic disks were placed on the plates and incubated under microaerophilic conditions for 7 days. The diameters of inhibition zones were measured using a ruler(mm).

Statistical analysis

All statistical analyses were completed using SPSS version 27 (IBM Corp., Armonk, NY, USA) and R version 3.5.1 (R Foundation for Statistical Computing, Vienna, Austria). A p value lower than 0.05 was considered the threshold for statistical significance.

Ethical approval

Ethical approval was taken from the institutional review board (IRB) of An-Najah National University.

Informed consent

Informed consent was obtained from all individual participants included in the study. and conducted according to the Helsinki Declaration of Human Rights.

Results

A total of 91 individuals experiencing dyspepsia were enrolled, consisting of 49 females and 42 males. Their ages spanned from 19 to 82 years, with an average age of 55. These participants underwent diagnostic upper gastrointestinal endoscopy at the endoscopy department of Al-Najah National University Hospital in Nablus, Palestine. Multiple gastric biopsy samples were extracted from both the antrum and the body of the stomach. The identification of H. pylori within the gastric biopsy specimens was carried out using tests for urease production and cultural examination.

Detection of H. pylori occurrence among dyspeptic patients

The occurrence of H. pylori was observed in 38 out of the 91 patients (41.7%), with 23 cases from males and 15 cases from females. Table 1 illustrates the distribution of infection percentages among various diagnoses. Notably, the highest infection percentage was observed in patients with duodenal ulcers, where all three cases (100%) were infected. Among patients with gastritis, 20 cases (54%) were infected, followed by 12 cases (41.1%) of individuals with a normal endoscopic appearance, and 3 cases (30%) of patients with gastric ulcers. The remaining twelve patients with different endoscopic features did not exhibit H. pylori infection.

Table 1 Occurrence of H. pyloi among dyspeptic patients.
Full size table

Antibiotic sensitivity of H. pylori

The customary disk diffusion technique was utilized to evaluate the antibiotic resistance profile of H. pylori strains in relation to six distinct antibiotics. Table 2 reveals that ciprofloxacin and levofloxacin were the most effective antibiotics against H. pylori isolates, with all isolates showing sensitivity to them. Moxifloxacin followed with only one isolate showing resistance. In contrast, amoxicillin exhibited resistance in seven isolates. Conversely, metronidazole emerged as the least effective antibiotic, with all thirty-eight isolates being resistant to it. Clarithromycin followed with eighteen isolates exhibiting resistance.

Table 2 Antibiotic susceptibility of H. pylori.
Full size table

Discussion

The strategy for addressing H. pylori infection differs markedly from the procedures employed for other contagious ailments. For example, the treatment of H. pylori necessitates customization, ideally guided by local and individual antibiotic resistance profiles9. Our understanding of the underlying mechanisms driving treatment resistance in H. pylori has advanced considerably. This involves a range of mechanisms, including chromosomally encoded alterations and physiological changes that affect drug uptake, efflux, as well as biofilm and coccoid production10. In Palestine, gaining insights into the initial rates of resistance exhibited by H. pylori strains against antibiotics such as amoxicillin, metronidazole, clarithromycin, ciprofloxacin, levofloxacin, and moxifloxacin holds paramount importance. This is particularly significant given the significant public health concerns associated with antibiotic resistance concerning coThe increasing prevalence of multi-drug-resistant (MDR) H. pylori strains, resilient to various drug categories, presents a disconcerting challenge amid the global antibiotic resistance surge10. Helicobacter pylori infection affects over 50% of the global population, yet only a small fraction of those infected develop serious gastroduodenal conditions such as duodenal ulcer, gastric ulcer, and gastric adenocarcinoma. These MDR strains, constituting over 40% of infections in specific regions, confront effective treatment approaches11. The development of MDR in H. pylori arises from mechanisms including concurrent mutations conferring resistance to diverse drug families, leading to cumulative MDR profiles10,12. Additional mechanisms involve the activation of expulsion systems for drug removal and potential changes in membrane protein or lipopolysaccharide expression, affecting drug uptake. The formation of biofilms, structured bacterial communities enclosed in a matrix, amplifies resilience and resistance. Unlike free-floating bacteria, biofilm-linked H. pylori can endure, potentially facilitating antibiotic resistance evolution. While H. pylori predominantly exists as free-floating in the stomach, biofilms might contribute to resistance and alternative transmission routes10,13,14,15,16.

According to a recent clinical practice update study, the choice of treatment regimen for eradicating H. pylori infection should be influenced by the local prevalence of clarithromycin resistance and the patient's history of macrolide use17. For initial treatment, quadruple therapies such as bismuth quadruple and concomitant regimens are recommended. In regions with a low incidence of clarithromycin resistance and among individuals who haven’t previously used macrolides, a 14 days triple therapy containing clarithromycin is suggested. However, the effectiveness of sequential therapy against clarithromycin-resistant H. pylori strains is conflicting, leading to a general reluctance towards its use. In cases where first-line treatments fail, second-line options encompass levofloxacin-containing triple therapy and bismuth quadruple therapy. Incorporating probiotic supplementation aims to mitigate antibiotic-related adverse effects. Recent evidence endorses the existing guideline recommendations for Helicobacter pylori infection treatment, reinforcing their relevance and validity17.

The findings indicated that all the strains examined in this investigation displayed resistance to metronidazole18. This level of resistance aligns with observations from developing nations, where metronidazole resistance has been reported to range from 17 to 100%17. However, in developed countries, most reports indicate resistance rates of 15.8–40% for H. pylori strains18. Additionally, 18.4% of the isolates were resistant to amoxicillin. This increase in resistance rates to metronidazole and amoxicillin could be attributed to the increased colonization of the stomach with antibiotic-resistant plasmids transferred from other bacteria.

Clarithromycin is a macrolide antibiotic commonly employed as part of combination treatments for H. pylori infection. Nevertheless, the emergence of clarithromycin resistance has emerged as a primary cause of treatment ineffectiveness19. Resistance in 47.4% of our isolates resembles data from one European study19. In contrast, resistance rates were notably lower in the USA (29%), Mexico (28.2%), Japan (30%), China (38%), and Turkey (47.5%)19. Since clarithromycin is used to treat infections outside the gastric tract, its prevalence in H. pylori resistance is on the rise.

Resistance rates of ciprofloxacin, levofloxacin, and moxifloxacin in our isolates were 0%, 0%, and 2.6%, respectively19. Resistance to levofloxacin remains low globally, less than 19%20. According to prevailing global recommendations and an extensive meta-analysis, treatment approaches involving non-bismuth quadruple therapies lasting 10–14 days and vonoprazan-based triple therapies lasting 7 days are presently advised for H. pylori infection. These regimens achieve eradication rates of approximately 90%, even in areas where antimicrobial-resistant bacteria are prevalent21.

Conclusion

In this study carried out in Palestine researchers conducted an investigation, into the occurrence and susceptibility of H. Pylori among patients with digestive discomfort. The results unveiled a prevalence of H. Pylori infection at 41.7% within the study participants with an occurrence observed among individuals diagnosed with duodenal ulcers and gastritis. Notably the resistance rates to two prescribed antibiotics, metronidazole and clarithromycin were alarmingly high at 100% and 47.4% respectively. On the hand ciprofloxacin and levofloxacin exhibited effectiveness as all tested samples showed sensitivity to these antibiotics. Moreover, amoxicillin resistance was detected in 18.4% of cases providing insights into the landscape of resistance, in H. Pylori.

Based on these findings it is recommended to be cautious when treating individuals infected with H. Pylori, in Palestine. Due to the rates of resistance observed it is advised to exercise caution when prescribing metronidazole and clarithromycin as part of the treatment plan. Instead considering the effectiveness of ciprofloxacin and levofloxacin these antibiotics could be suitable options for combination therapy. Adding ciprofloxacin or levofloxacin to the treatment protocol with amoxicillin or other appropriate antibiotics may increase the chances of successful eradication. Additionally considering the concerning increase in resistance a comprehensive approach is necessary. It is crucial to monitor patterns of antibiotic resistance regularly evaluate treatment outcomes and prioritize patient education on adhering to prescribed regimens. This study underscores the importance of tailoring treatment approaches based on resistance profiles and emphasizes the critical need for ongoing research and vigilance to effectively mitigate the impact of antibiotic resistance on both H. pylori management and public health.

Limitation

In our study on Helicobacter pylori resistance at An-Najah University Hospital, we encountered challenges. Firstly, it was difficult to standardize the size of the inoculum because the bacterias growth rate was slow and it had requirements. Secondly although we carefully measured the zone of inhibition, we did not clearly specify the sizes and cutoff points which could impact how the results are interpreted. Thirdly our ability to measure Minimum Inhibitory Concentration (MIC) was hindered by the unavailability of the E test method. Moreover, despite our efforts incorporating ATCC strains for quality control purposes proved unsuccessful due, to contamination and degradation issues. These challenges underscore the importance of refining our methods and exploring approaches, in studies.