Minimum inhibitory concentrations of commonly used antibiotics against Helicobacter Pylori: A multicenter study in South China

Aim To determine the minimum inhibitory concentrations (MICs) of commonly used antibiotics against Helicobacter Pylori (H. pylori) in South China and compare their resistance rates by using EUCAST breakpoints and other breakpoints. Methods Patients who had not previously received H. pylori treatment in clinical centers in South China were enrolled in this study from 2017 to 2020. Gastric biopsies were obtained for H. pylori culture. The MICs of amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), tetracycline (TET) and furazolidone (FZD) were tested by broth microdilution method and assessed by two different breakpoints. ATCC43504 standard strain served as a control. Results A total of 208 H. pylori strains were isolated from patients’ biopsy samples. The MICs of AMX, CLA, MTZ, LEV, TET and FZD for H. pylori were 0.0156-256mg/L (MIC50 0.125mg/L, MIC90 4mg/L), 0.0156- >256 mg/L (MIC50 0.0312mg/L, MIC90 64mg/L), 0.0156- >256mg/L (MIC50 8mg/L, MIC90 256mg/L), 0.0156-256mg/L (MIC50 0.25mg/L, MIC90 16mg/L), 0.0156-256mg/L (MIC50 0.0625mg/L, MIC90 4mg/L), and 0.0156- >256mg/L (MIC50 0.0312mg/L, MIC90 2mg/L), respectively. The MICs of AMX, CLA, MTZ, LEV, TET and FZD for ATCC43504 strain were 0.25mg/L, 0.0625mg/L, 64mg/L, 0.5mg/L, 1mg/L and 0.25mg/L, respectively. The resistance rate of FZD was 11.05%. The overall resistance rates according to EUCAST breakpoints and other breakpoints were 57.21% and 14.90% for AMX (p<0.001), 38.94% and 38.94% for CLA (p = 1), 39.42% and 50.96% for MTZ (p<0.001), 12.98% and 10.58% for TET (p = 0.025), 35.10% and 35.10% for LEV (p = 1), respectively. Conclusions Our results demonstrate that AMX, FZD, and TET, but not MTZ, CLR or LEV, showed good anti-H. pylori activity in vitro in South China. When different breakpoints were used, similar results were found with CLA, and LEV, but not with AMX, MTZ, or TET.


Introduction
Helicobacter pylori (H. pylori) is a spiral-shaped, gram-negative, and microaerophilic bacterium, which specifically colonizes the gastric epithelia and infects approximately half of the population worldwide [1]. In general, the infection rate of H. pylori is higher in developing countries compared to developed countries [2]. H. pylori is an important etiology of global health problems because it can cause chronic gastritis, dyspepsia, peptic ulcers, gastric malignancies, and extragastric diseases [3,4]. Eradication of H. pylori can effectively alleviate symptoms in functional dyspepsia, heal peptic ulcers, and improve atrophic gastritis and intestinal metaplasia [5]. Specifically, since 1994, H. pylori has been identified as a class-A carcinogen by the International Agency for Research on Cancer (IARC) [6]. Eradication of H. pylori has a preventive effect on gastric cancers [7] and has been proven to be efficient in treating nearly 75% of early mucosa-associated lymphoid tissue (MALT) lymphoma [8]. Therefore, early treatment of H. pylori is recommended for all symptomatic patients [9].
The first-line treatment for the eradication of H. pylori consists of 1 proton pump inhibitor (PPI) and 2 antibiotics (amoxicillin, clarithromycin or metronidazole) [1]. Unfortunately, the eradication rate using the above-mentioned treatment plan has declined over the last decade to less than 90% [10,11], mainly due to a rapid increase in antibiotic resistance, especially against clarithromycin and metronidazole [12,13]. The prevalence of H. pylori antibiotic resistance significantly varies from country to country and between regions within the same country [14,15]. Therefore, it is essential to conduct local surveillance of antibiotic resistance.
Before implementation of this study, there were limited data regarding the minimum inhibitory concentrations (MICs) of H. pylori resistance to antibiotics in South China. As to breakpoints proposed for commonly used antibiotics against H. pylori, there are different guidelines including European Committee on Antimicrobial Susceptibility Testing (EUCAST), British Society for Antimicrobial Chemotherapy (BSAC), Clinical and Laboratory Standards Institute (CLSI), et al. In this study, we aimed to investigate the MICs of commonly used antibiotics to H. pylori isolated from patients in South China and compare the resistance rates when using different breakpoints to provide guidance on prescription of antibiotics.

Patients
Patients who had dyspeptic symptoms and received gastroscopy in the following 4 hospitals in South China from January 4 th , 2017 to October 1 st , 2020 were enrolled in this study: Fujian Provincial Hospital (Fuzhou, Fujian, China), the Fifth Affiliated Hospital of Sun Yat-Sen University (Zhuhai, Guangdong, China), Xiamen Hospital of Traditional Chinese Medicine (Xiamen, Fujian, China), and Pucheng Hospital (Nanping, Fujian, China). Exclusion criteria were as follows: (1) patients who had used bismuth salts, proton pump inhibitors, antibiotics, or H2 receptor antagonists a month before entrance of the study; (2) patients who had undergone surgery; (3) patients who had malignant tumors; (4) patients who had contraindications for gastroscopy.

Isolation, identification, and antibiotic susceptibility tests of H. pylori
One gastric mucosal specimen was taken from the antrum of the stomach in each patient during gastroscopy to detect the presence of H. pylori with a rapid urease test. If the rapid urease test was positive, another specimen from the antrum was collected, stored, and then cultured in the liquid medium for H. pylori containing brain heart immersion broth and 10% calf serum (Zhuhai Yeoman Bioengineering Products Factory). Organisms were identified as H. pylori if isolates demonstrated curved gram-negative rods along with positive urease, catalase, and oxidase reactions. The MICs of H. pylori to commonly used antibiotics including amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), tetracycline (TET) and furazolidone (FZD) were determined by broth microdilution method. The standard strain ATCC43504 (provided by Shanghai Bioplus Biotechnology Company) served as a control. The culture suspension was inoculated onto plates with turbidity adjusted to McFarland standard of 2.0. The antibiotic powders of AMX, CLA, and LEV were purchased from BioDuly Biotechnology Co.Ltd. (Nanjing, China), and MTZ, TET and FZD were from Meilun Biotechnology Co. Ltd. (Dalian, China).

Statement of ethics
This study was approved by the independent Ethics Committees of the Fujian Provincial Hospital (Approval No. K2016-11-028) and performed following the World Medical Association Declaration of Helsinki. Written informed consent was obtained from each patient. Authors had access to information that could identify individual participant during and after data collection.

Statistical analysis
Data were analyzed using SPSS 22.0 software (IBM, Armonk, NY. USA). Percentages were used to describe the antibiotic resistance rates of H. pylori isolates. The Chi-Square test was used to compare the drug resistance rates calculated by two breakpoint systems. A P-value of < 0.05 was considered statistically significant.

Impact of diseases on antibiotic resistance of H. pylori in South China
A significant difference was detected in the resistance of H. pylori to LEV among different disease groups (p <0.05). The MIC of LEV was highest in erosive gastritis (log2 μ = 0.47), followed by chronic superficial gastritis (log2 μ = -1.16), chronic atrophic gastritis (log2 μ = -1.42), and peptic ulcers (log2 μ = -1.81). Additionally, the MICs of erosive gastritis were also highest among the 4 disease groups for the other 5 antibiotics, although they were not statistically different (p >0.05) (Fig 2).

Impact of age on antibiotic resistance of H. pylori in South China
Patients were divided into two groups according to age (<45 years old vs. �45 years old). No significant difference in the resistance of H. pylori to all tested antibiotics including AMX, CLA, MTZ, LEV, TET, and FZD between the two age groups (p >0.05) (Fig 3).

Impact of gender on antibiotic resistance of H. pylori in South China
There was no significant difference in resistance to AMX, CLA, MTZ, LEV, TET and FZD among H. pylori strains isolated between the two gender groups (p>0.05) (Fig 4).

Discussion
Resistance of H. pylori to the commonly used antibiotics has posed an increasing threat to the standard therapeutic regimens worldwide [11]. Therefore, it is extremely important to continuously monitor the resistance of H. pylori in various regions to find effective antibiotics to eradicate H. pylori. Various methods have been reported to assess antibiotic susceptibility of H. pylori in vitro, including Kirby-Bauer, agar dilution, and broth dilution methods. Kirby-Bauer antibiotic test is semi-quantitative and unreliable. Agar and broth dilution methods, the most commonly used methods, are quantitative and can provide more accurate results [21]. Agar dilution is recognized as the gold standard of susceptibility testing. However, this method is labor-intensive and expensive. Epsilometer test (E-test) is an agar diffusion method and demonstrates an excellent correlation with the agar dilution method [22,23]. However, antibiotics has to be supplied by specified manufacturers, making this method costly for screening. The broth dilution method is also highly accurate and comparable to agar dilution [24], and can test more than one antibiotic at one time. Broth microdilution method uses microdilution plates with a capacity of 500 μL/well to perform the broth dilution. The results of the ampicillin and clarithromycin disk diffusion assays for H. pylori showed 100% correlation with those of broth microdilution [25]. The E-test tends to overestimate the resistance of metronidazole for H. � Other breakpoints were as follows: � 2 mg/L for AMX, � 1 mg/L for CLA, � 8 mg/L for MTZ, �2 mg/L for LEV, �4 mg/L for TET, and > 2mg/L for FZD.  pylori. Therefore, the results of metronidazole using E-test should be confirmed by agar dilution or broth microdilution [25,26]. All discrepancies occurred when the E-test MIC values were at the range of 8 and 32 mg/L [25]. Therefore, the broth microdilution method was used in our study to monitor the drug resistance rates. Breakpoints were used to classify the results of antimicrobial susceptibility by agar dilution or broth dilution methods into sensitive, intermediate, and resistant to a specific antibiotic. The CLSI only recommends breakpoints for clarithromycin to treat H. pylori and proposes agar dilution method as the standard for antimicrobial susceptibility testing. Different studies use different breakpoints, for example, the breakpoints of amoxicillin ranges from 0.125 to 2 μg/ml [27][28][29][30], making it hard to compare the results of different studies. Alarcón et al. [31] conducted antimicrobial susceptibility in H. pylori clinical isolates by using EUCAST breakpoints and compared to other breakpoints. They found similar results lied in the most frequently tested antibiotics, e.g. metronidazole, clarithromycin, tetracycline, and levofloxacin, with exception of amoxicillin and rifampicin. Our results agreed with these findings. The overall resistance rate for amoxicillin was 60.98% by using EUCAST breakpoints and 12.80% by using other breakpoints in our study. There were distinct differences between the two breakpoints for simple AMX, MTZ, and TET resistance. However, there was no significant difference for CLA and LEV. In addition to CLSI, EUCAST, and BSAC breakpoint systems, a breakpoint system appropriate for China, especially for AMX, MTZ and TET, is desired.
Resistance rates of CLA, MTZ, and LEV for H. pylori were all high in South China. While the MIC 90 of AMX, TET, and FZD were low, the MIC 90 of MTZ was high, 128 times higher than that of FZD and 64 times higher than those of AMX and TET. Our results indicate that FZD, AMX, and TET remain good choices for the eradication of H. pylori in South China, consistent with the results observed in other regions in China and abroad [20, 28,32]. However, CLA, MTZ, and LEV should be avoided in empiric treatment of H. pylori in South China. CLA resistance has been indicated to account for the sharply decreased eradication rate by 70% (from 87.8% to 18.3%) in the regimen of PPI-clarithromycin-amoxicillin and 47% (from 97% to 50%) in the regimen of PPI-clarithromycin-metronidazole [33]. Resistance to CLA and MTZ has compromised the therapeutic efficacy. Malfertheiner et al. [1] recommended to use the bismuth quadruple as one of the first-line medications in areas with high dual resistance to CLA and MTZ.
MTZ is the classic nitroimidazole antibiotic. It reaches a high concentration in the stomach and its bactericidal activity is not affected by low pH in the stomach. H. pylori resistance to MTZ and CLA has been globally reported with regional differences and has been on the rise. While the resistance rate of H. pylori to MTZ is relatively low in Southern Europe, Taiwan, and Japan (less than 20%) [34][35][36], it is high in Africa (75.8%) [13] and China (78.2%) [37]. The main reasons for the high MTZ resistance are its previous prescription for parasitic or gynecological infections [38], extensive availability, and affortability. In majority of countries, the resistance rate of CLA is greater than 20% [32,33,37]. From 1997 to 2008, the resistance rate of CLA in Japan gradually increased from 8.7% to 34.5% [11].
We found that the MICs of H. pylori were highest in patients with erosive gastritis. This may be due to the resistant H. pylori isolates are virulent strains and often cause gastric erosion. Our study failed to detect significant difference in the resistance rate of H. pylori to tested antibiotics in different age and gender groups.
There were at least two limitations in our study. Firstly, drug resistance and sensitivity determined by antibiotic susceptibility test in vitro cannot fully represent the condition in vivo. And secondly, the selected samples from our clinical centers could not represent the whole population in South China.

Conclusions
Our results demonstrate that AMX, FZD, and TET, but not MTZ, CLR or LEV, showed good anti-H. pylori activity in vitro in South China. When different breakpoints were used, similar results were found with CLA, and LEV, but not with AMX, MTZ, or TET.