Antibiotic Resistance of Helicobacter Pylori and Inuencing Factors in Yangzhou, China: a Cross-sectional Study

Background: The Helicobacter pylori (H. pylori) antibiotic resistance pattern differs geographically, knowledge of local antimicrobial resistance pattern is key to successful eradication. The current study was performed to investigate the resistance of H. pylori to 5 commonly used antibiotics in Yangzhou. Methods: A total of 461 H. pylori strains were collected from April 2018 to September 2019 in Yangzhou and tested for their susceptibility to clarithromycin, levooxacin, metronidazole, amoxicillin, tetracycline using gene chip technology. Results: The resistance rates of H. pylori to clarithromycin, levooxacin, metronidazole, amoxicillin and tetracycline were 41.0%, 44.9%, 38.8%, 6.3% and 1.1%. In addition, sixteen multiple resistance patterns were detected, and fortunately there were no strains resistant to all ve antibiotics. The vacA s1 allele was detected in each strain, 64.0% of the isolates were s1/m2 genotype, 36.0% s1/m1 genotype. In multivariate analysis, history of chronic supercial gastritis was signicantly associated with clarithromycin resistance, strains isolated from subjects with gastrointestinal symptoms had a signicantly higher likelihood of metronidazole resistance, and antibiotic resistance of H. pylori was independent of both sex and age. Conclusion: The resistance rate of H. pylori to clarithromycin, levooxacin and metronidazole were very high in Yangzhou, the results will help in selecting effective eradication regimens.


Introduction
Helicobacter pylori (H. pylori) infection is a common worldwide infection, half of the world's population has been suggested infected with H. pylori [1]. Although most people with H. pylori infection are asymptomatic, it has been considered associated with chronic gastritis, peptic ulcers, lymphoid tissue lymphoma, and gastric cancer [2,3]. H. pylori eradication treatment can help cure chronic gastritis and reduce the incidence of gastric cancer [4,5]. Standard triple therapy with a proton pump inhibitor (PPI) and 2 antimicrobial agents, such as amoxicillin, clarithromycin, or metronidazole, has been considered di cult to achieve the desired eradication rate because of compliance, high gastric acidity, high bacterial load, and bacterial strains, but the key is the increasing H. pylori resistance to antibiotics [4,6]. Nowadays, bismuth quadruple therapy is recommended as the main empirical treatment therapy in China [7], but it still faces the risk of treatment failure.
The H. pylori resistance to antibiotics shows a growing trend over time especially in developing countries, and is related to antibiotic consumption rate in the population. In Korea, the levo oxacin resistance rate of primary strains showed an amazing increase from 4.5% in 2003-2005 to 62.2% in 2017-2018 [8]. The clarithromycin resistance rate increased from 39.9% in 2009-2010 to 52.6% in 2013-2014 in Beijing [9].
Various measures have been taken to face with this severe situation, such as changing therapies, increasing dose, monitoring drug susceptibility. Extensive studies [10][11][12] have demonstrated that tailored therapy may lead to reduced eradication failures. Current methods to test H. pylori resistance to Page 4/16 antibiotics can be divided into drug sensitivity testing and PCR-based molecular methods. It is well known that strain culture is more di cult to achieve due to time-consuming and technical di culties, rapid and effective molecular biology methods [13] are increasingly recommended in clinical applications and scienti c research. Gene mutation has been regarded as the main reason of antibiotic resistance,which is the basis of molecular methods. It has been reported that point mutations in the peptidyltransferase region encoded in domain V of 23S rRNA gene are responsible for clarithromycin resistance, of which the A2143G and A2142G mutations are the most frequent [14]. Resistance to uoroquinolones which act by inhibiting the H. pylori DNA gyrase, is due to point mutations in the Quinolones Resistance-Determining Region (QRDR) of the gyrA gene [15]. As for metronidazole resistance, the most prominent claim is that mutations in the gene encoding nitroreductase, such as rdxA, frxA, and frxB, have been reported to be the most important mutation sites [16]. Concerning amoxicillin, mutations of the pbp1 gene are considered as the main cause, the degree of amoxicillin resistance gradually increases with the accumulation of pbp1 mutations [17]. Tetracycline is also one of the antibiotics commonly used in H. pylori eradication, which acts by affecting the 30S subunit of the ribosome and blocking aminoacyl-tRNA binding, leading to impaired protein biosynthesis. Tetracycline resistance of H. pylori is also caused by mutations in 16S rDNA, particularly at positions 926-928 [18,19]. In our study, it was considered as drug-resistant H. pylori strain if the gene mutations mentioned above were detected based on gene chip technology [20].
Several important virulence factors play an important role in initial colonization and tissue destruction of H. pylori such as agella-mediated motility, urease, babA2, oipA, cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) [21,22]. The vacA gene exists in almost all H. pylori strains and composed of s-region (s1, s2) and m-region (m1, m2) [23]. Li et al. [24] illustrated a close relationship between vacA and both peptic ulcer and gastric cancer. VacA s1 genotype leads to a stronger protein production than vacA s2 genotype [25], the m1 allele, but not m2, is associated with higher levels of toxin activity and more severe gastric epithelial damage [26]. Moreover, it has been reported a possible relationship between vacA and antibiotic resistance, a study [27] conducted in Iranian showed strains with vacA s1/m2 genotype were detected more frequently in resistant isolates.
Our knowledge about resistance of H. pylori to antibiotics always comes from other regions' study, no relevant reports have been found in Yangzhou, a comprehensively monitoring of H. pylori antibiotic resistance is urgently required. This study aimed to assess the antibiotic resistance pattern of H. pylori strains, which will help in selecting effective eradication regimens of H. pylori in Yangzhou in the future.

Materials And Methods
Isolation of H. pylori strains Fresh antrum biopsy specimens were taken during endoscopy from citizens with a positive 13 C-urea breath test ( 13 C-UBT). All participants were part of the endoscopic screening program in Yangzhou between April 2018 and September 2019. Informed consent was obtained beforehand. Frozen specimens were thawed rst and inoculated onto Columbia agar plates supplemented with 10% calf blood, antibiotic selective supplement (5mg/L trimethoprim, 10mg/L vancomycin, 0.38mg/L polymyxin B). These plates were incubated at 37°C for 3-5 days under microaerophilic conditions (5% O2, 10% CO2 and 85% N2). H. pylori colonies were initially identi ed by their typical morphology, then transferred the positive growth to a fresh Columbia blood agar plate and incubated for 48-72 hours. The isolates were frozen at -80°C in brain-heart infusion storage medium containing 20% glycerol until assayed.
H. pylori strains DNA extraction Isolated strains were unfrozen at room temperature. Following the removal of supernatant, the cell precipitation was fully resuspended in liquid with 200-µl physiological saline and 20-µl pathogen DNA extracting protease K. Nucleic acid extraction was performed according to instruction provided by reagent manufacturer, and isolated DNA was stored at 4°C.
Polymerase chain reaction DNA ampli cation was performed under the following steps: uracil-DNA glycosylase enzyme reaction at 50°C for 10 minutes and pre-denaturation for 10 minutes at 95°C, then 1 cycle of denaturation at 95°C for 30 seconds, extension at 56°C for 30 seconds, and a further extension for 30 seconds at 72°C by 45 cycles. Followed by the nal cycle, primer extension for 5 minutes at 72°C. The nal product of the reaction was stored at 4°C till it was analyzed.
Gene chip detection of H. pylori H. pylori-related gene probes distributed on membrane strips are shown in Table 1. The membranes were put into the 24-well plate sequentially and 1mL liquid A added, preheated in boiling water for 20 minutes, the PCR products were added and hybridized at 48℃ for 1.5 hours. Aspirated the reaction solution, added 1mL pre-warmed liquid B, and gently shook at 48°C for 15 minutes. The incubation liquid was then discarded. Then gently shook again for 30 minutes at room temperature with incubation solution, and 5 minutes twice with liquid A, afterwards washed with liquid C for 1-2 minutes at room temperature. Membranes were then in ltrated in the chromogenic solution, kept in darkness at room temperature for 10 minutes, nally rinsed with pure water to observe the results.  (Fig. 1).
The pattern of antibiotic resistance is shown in    Risk factors associated with antibiotic resistance Results of univariate analysis of factors associated with antibiotic resistance are summarized in Table 4. We didn't perform the analysis of risk factors associated with amoxicillin and tetracycline resistance, due to their low resistance rates. Gastrointestinal symptoms, history of super cial gastritis and endoscopic ndings (ulcer or cancer) were signi cantly associated with clarithromycin resistance. Strains isolated from people who drunk regularly were more commonly resistant to levo oxacin. What's more, gastrointestinal symptoms, previous history of super cial gastritis and rst-degree relatives with gastric cancer were signi cantly associated with levo oxacin resistance. Metronidazole resistance was found signi cantly frequent among subjects who had gastrointestinal symptoms. Previous history of super cial gastritis was also associated with metronidazole resistance. Risk factors for resistance to any antibiotic included vacA genotype, alcohol consumption, presence of gastrointestinal symptoms, history of chronic super cial gastritis, and family history of gastric cancer in rst-degree relatives. Clarithromycin, levo oxacin, metronidazole, and any antibiotic resistance had no correlation with sex or age.
Multiple logistic analysis showed that resistance to clarithromycin was higher in those with previous history of super cial gastritis. In addition, clarithromycin resistance rate was lower in those diagnosed with peptic ulcer or tumor. In multivariate analysis, drinking, previous history of super cial gastritis and family history of gastric cancer in rst-degree relatives were signi cantly associated with levo oxacin resistance. Gastrointestinal symptoms were signi cantly associated with resistance to any antibiotics, and this persisted after adjustment by sex, age, vacA genotype, gastrointestinal symptoms, history of super cial gastritis, and family history of gastric cancer in rst-degree relatives (Table 5).

Discussion
Yangzhou is located in the middle of Jiangsu province, which has a high stomach cancer incidence of 44.05/100000 in 2014 [28]. H. pylori infection is regarded as the major risk factor of stomach cancer, a long-term cohort study [29] from Taiwan, China showed that eradicating H. pylori can reduce gastric cancer incidence and mortality. Unfortunately, the high prevalence of H. pylori infection and the increasing antibiotic resistance constitute the main challenge for current treatment. The main reason for us to conduct this work is that a disturbing phenomenon was found that a large proportion of people failed to eradicate H. pylori during the follow-up investigation, local resistance analysis is urgently required.
First-line eradication treatment is important in China because the rate of H. pylori reinfection after successful treatment is low (1.5% per person-year) [30], while the global annual reinfection rate of H. pylori was 3.1% [31]. It suggests we should choose the most effective therapy to improve rst-line eradication rate. Antibiotic resistance rate of H. pylori varies among different countries or regions. In Italy in 2016, resistance rate to clarithromycin was 35.9% [32], and rate in Korea was 43.7% [8]. The present study showed that the resistance to clarithromycin (41.0%) is slightly higher than that in Zhuanghe (31%) [33] and signi cantly higher than the reported rate (22.1%) in the study included a large number of strains from 13 provinces or cities in China [34]. The signi cantly higher rate of clarithromycin resistance in Yangzhou may be caused by the long-time and wide use in clinical practice which can promote the emergence of drug-resistant bacteria. In addition, unhealthy lifestyle (no handwashing before meals, sharing utensils, etc.) could lead to the transmission of H. pylori among the population as well [35,36]. According to consensus recommendations [7], levo oxacin-containing regimen is generally suggested to be avoided as an initial treatment, but as an alternative for rescue therapy because of the high rate of drug resistance. Resistance rate to levo oxacin was 38.8% in Taiwan, China in 2019 [37], and 56% in an area of China with a high risk of gastric cancer [33]. It is the highest of levo oxacin resistance rate in our study, maybe due to the widely use of quinolones owing to respiratory and urogenital infections. Metronidazole is a 5-nitroimidazole drug that is widely used for general anaerobic infections worldwide. The rate of metronidazole resistance was found to be 38.8% in this work, which is lower than the average rate (61%) of China in a meta-analysis [38].
The prevalence of resistance to amoxicillin was low in this study, concerning with its low rate of adverse reactions, amoxicillin-containing bismuth quadruple therapy can be used as the preferred choice for H. pylori eradication therapy. In addition, high-dose dual therapy is becoming familiar to doctors for great e cacy and lower side effects. The study conducted by Song et al. reported that dual therapy (esomeprazole and amoxicillin four times daily) was non-inferior to, and even superior to triple plus bismuth therapy as rst-line H. pylori eradication treatment [39]. However, the ndings are not yet consistent and remains to be con rmed with further studies. Tetracycline also presented a high sensitivity, and it is recommended to replace amoxicillin for people who are allergic to penicillin. Our study showed that 42.3% of the strains were resistant to at least two antibiotics. The main resistance patterns were clarithromycin + levo oxacin (14.3%), clarithromycin + metronidazole (9.8%), clarithromycin + levo oxacin + metronidazole (6.7%). In addition, we also nd several quadruple resistance patterns, and no strains resistant to all ve antibiotics tested.
The vacA gene is the main virulence factor of H. pylori, strains with vacA s1/m1 genotype are the most cytotoxic, followed by s1/m2, and s2/ m2 strains were virtually non-toxic [40]. VacA s1 allele was detected in all strains in our study and was predominant in s1/m2 and s1/m1, is similar to the study conducted by Wang et al [41]. The results suggests that the strains in Yangzhou are more pathogenic and more likely to cause clinical disease. However, our statistics showed that the vacA genotype had no association with any gastric diseases, maybe for the reason of a small sample, which needs to be explored in further studies.
Multivariate analysis showed that history of chronic super cial gastritis was associated with clarithromycin resistance. H. pylori is the major cause of chronic gastritis, long -term H. pylori infection and the formation of bacterial bio lms [42] may lead to antibiotic resistance. Subjects with an endoscopic diagnosis of peptic ulcer or cancer have a lower risk of resistance to clarithromycin, which is consistent with a previous study in France [43]. Levo oxacin resistance was signi cantly associated with alcohol consumption. It is possible that physicians do not prescribe levo oxacin to patients who drink alcohol because of a disul ram-like reaction, which might contribute to a low levo oxacin resistance rate among drinkers. In addition, in our study, people with family history of rst-degree relatives with gastric cancer have a lower possibility of resistance to levo oxacin, it cannot be well explained at this time and needs to be further clari ed by other studies. Gastrointestinal symptoms were signi cantly associated with levo oxacin and any antibiotics resistance, due to these individuals were infected by H. pylori early but ignore it. Unfortunately, the vacA s1/m2 genotype has not been shown to be associated with antibiotic resistance, although it was found more frequently in resistant strains.
Our study has some limitations. Firstly, we only performed endoscopic screening program in two small towns of Yangzhou city and the participants in our study ranged from 32 to 75 years old, with 80.5% of the population aged 50 years and above, it may not be adequately re ective of the general population. Secondly, our study was conducted using gene chip technology to detect known mutation sites to determine H. pylori antibiotic resistance, which may underestimate the antibiotic resistance. In addition, there are no follow-up investigation on H. pylori eradication in positive populations, and we will further strengthen observations.

Conclusions
In conclusion, Clarithromycin, metronidazole and levo oxacin resistance rates were very high and serious multi-drug resistance cases are detected in Yangzhou. In order to achieve > 90% eradication rate of H. pylori in rst-line treatment, it is suggested to avoid the combination of the above three antibiotics, individualized therapy likely be the best strategies locally. We should strengthen monitoring antibiotic resistance of H. pylori, as well as educate local residents in the next screening activities, to maintain a good lifestyle and seek medical attention in case of uncomfortable symptoms.

Availability of data and materials
All data generated or analyzed throughout this research are included in this published article.
Authors' Contributions BD and YBD: study concept and design, obtained funding. BD, WMX and GTL: critical revision of the manuscript for important intellectual content. YZ and XYF: performed the experiments, analysis of data, statistical analysis, wrote the paper. LJB, YZ, QL, QS and YMX: acquisition of data, analysis of data, statistical analysis. YZ, XYF, and CWY: analysis and interpretation of data. YZ and XYF contribute equal to this work. All authors read and approved the nal manuscript.

Ethics approval and consent to participate
The protocol was approved by the Ethics Committee of The A liated Hospital of Yangzhou University.

Consent for publication
Not applicable.